REVIEW
Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide

¹ Department of Medicine and Therapeutics, Western Infirmary, University of Glasgow, G11 6NT Glasgow, United Kingdom; ² Laboratory of Experimental Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada H2W 1R7; ³ Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912; and ⁴ Department of Medicine, Boston University, Boston, Massachusetts 02118

	ABSTRACT

TOP ABSTRACT INTRODUCTION PHYSIOLOGICAL ROLE OF ANG... ANGIOTENSIN RECEPTORS PHYSIOLOGICAL ACTIONS OF... INTRACELLULAR SIGNALING THROUGH... PHYSIOLOGICAL AND... CERAMIDE SIGNALING CERAMIDE AND ANG II AREAS FOR FUTURE INVESTIGATION REFERENCES

Angiotensin II (ANG II) is a pleiotropic vasoactive peptide that binds to two distinct receptors: the ANG II type 1 (AT₁) and type 2 (AT₂) receptors. Activation of the renin-angiotensin system (RAS) results in vascular hypertrophy, vasoconstriction, salt and water retention, and hypertension. These effects are mediated predominantly by AT₁ receptors. Paradoxically, other ANG II-mediated effects, including cell death, vasodilation, and natriuresis, are mediated by AT₂ receptor activation. Our understanding of ANG II signaling mechanisms remains incomplete. AT₁ receptor activation triggers a variety of intracellular systems, including tyrosine kinase-induced protein phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species activities, and fluxes in intracellular Ca²⁺ concentrations. AT₂ receptor activation leads to stimulation of bradykinin, nitric oxide production, and prostaglandin metabolism, which are, in large part, opposite to the effects of the AT₁ receptor. The signaling pathways of ANG II receptor activation are a focus of intense investigative effort. We critically appraise the literature on the signaling mechanisms whereby AT₁ and AT₂ receptors elicit their respective actions. We also consider the recently reported interaction between ANG II and ceramide, a lipid second messenger that mediates cytokine receptor activation. Finally, we discuss the potential physiological cross talk that may be operative between the angiotensin receptor subtypes in relation to health and cardiovascular disease. This may be clinically relevant, inasmuch as inhibitors of the RAS are increasingly used in treatment of hypertension and coronary heart disease, where activation of the RAS is recognized.

renin-angiotensin system; angiotensin receptor antagonist; second messenger

	INTRODUCTION

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IN THIS REVIEW, we compare and contrast the mechanisms of action and vascular effects of the angiotensin type 1 (AT₁) and type 2 (AT₂) receptors. The physiology of angiotensin II (ANG II) continues to be a major field of investigation. Recently reported mechanisms of AT₁ receptor activation, such as receptor transactivation of tyrosine kinase receptors and stimulation of reactive oxygen species (ROS) production, suggest that ANG II has growth factor and cytokine-like properties in addition to its vasoconstrictor actions. The AT₂ receptor has only recently been identified, and its mechanisms of action continue to be elaborated. Therefore, we consider the emergent physiological systems that are activated by the AT₂ receptor.

One such pathway is the interaction between ANG II and ceramide. Ceramide is a lipid second messenger that is involved in a variety of physiological pathways, including cytokine-induced apoptosis and vasodilation. We summarize the recently described novel interaction of ANG II and ceramide and consider the potential importance of ceramide as an intracellular second messenger of the AT₂ receptor. Finally, we discuss the physiological antagonism and cross talk that may exist between the two angiotensin receptor subtypes in relation to health and cardiovascular disease.

	PHYSIOLOGICAL ROLE OF ANG II

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ANG II, an octapeptide hormone, is the active component of the renin-angiotensin system (RAS). It plays an important physiological role in the regulation of blood pressure, plasma volume, sympathetic nervous activity, and thirst responses. ANG II also has a pathophysiological role in cardiac hypertrophy, myocardial infarction, hypertension, and atherosclerosis. It is produced systemically via the classical RAS and locally via tissue RAS. In the classical RAS, circulating renal-derived renin cleaves hepatic-derived angiotensinogen to form the decapeptide angiotensin I (ANG I), which is converted by angiotensin-converting enzyme (ACE) in the lungs to the active ANG II (59, 228, 278). ANG I can also be processed into the heptapeptide ANG-(1-7) by tissue endopeptidases (75).

Although the RAS was originally regarded as a circulating system, many of its components are localized in tissues, indicating the existence of a local tissue RAS as well (48, 66). ACE exists in plasma, in the interstitium, and intracellularly. Tissue ACE is present in all major organs, including the heart, brain, blood vessels, adrenals, kidney, liver, and reproductive organs (115), and is already functional in utero (73). All components of the RAS, except renin, have been demonstrated to be produced in the vasculature (66, 198). Because vascular renin is absent, local generation of ANG II in the interstitium is regulated by tissue ACE that is probably dependent on circulating renin. In addition to ACE-dependent pathways of ANG II formation, non-ACE pathways have been demonstrated. Chymotrypsin-like serine protease (chymase) may represent an important mechanism for conversion of ANG I to ANG II in the human heart (307), kidney (115), and vasculature (115, 289) and may be particularly important in pathological conditions, such as coronary heart disease (227).

	ANGIOTENSIN RECEPTORS

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In mammalian cells, ANG II mediates its effects via at least two plasma membrane receptors: AT₁ and AT₂ receptors. Both receptor subtypes have been cloned and pharmacologically characterized (149, 201, 205, 251). The ANG II receptors can be distinguished according to inhibition by specific antagonists. AT₁ receptors are selectively antagonized by biphenylimidazoles, such as losartan, whereas tetrahydroimidazopyridines specifically inhibit AT₂ receptors (8). The AT₂ receptor may also be selectively activated by CGP-42112A. This is a hexapeptide analog of ANG II, which may also inhibit the AT₂ receptor, depending on concentration (46). Two other angiotensin receptors have been described: AT₃ and AT₄ subtypes (20, 39, 285). However, the pharmacology of AT₃ and AT₄ receptors has not been fully characterized, and therefore these receptors are not included in a definitive classification of mammalian angiotensin receptors as defined by the International Union of Pharmacology Nomenclature Subcommittee for Angiotensin Receptors (50, 51).

AT₁ Receptor

The AT₁ receptor belongs to the seven-membrane-spanning G protein-coupled receptor family and typically activates phospholipase C (PLC) through the heterotrimeric G_q protein, although it may also signal through G_i, G_11/13, and G_s (19, 51, 201, 205, 251). The human AT₁ receptor gene is mapped to chromosome 3 (47). AT₁ receptors are widely distributed throughout the cardiovascular, renal, endocrine, and nervous system in humans (6). Allen et al. (7) and Zhuo and colleagues (344) demonstrated that, in the human vasculature, AT₁ receptors are present at high levels in smooth muscle cells and at relatively low levels in the adventitia. Freeman et al. (82) and Pueyo et al. (231) reported that AT₁ receptors are also expressed in cultured rat aortic endothelial cells. In studies undertaken in human myocardial biopsies, Regitz-Zagrosek et al. (238) demonstrated that, in the heart, AT₁ receptors are present in atrial and ventricular myocytes and in fibroblasts. Using radioligand binding techniques, they also demonstrated that the abundance of the AT₁ receptor protein is reduced in patients with heart failure, which may be due to a reduction in the abundance of myocardial AT₁ receptor mRNA (237).

In rodents, the AT₁ receptor has two functionally distinct subtypes, AT_1A and AT_1B, with >95% amino acid sequence homology (126, 134). On the basis of the cDNA sequence, the AT₁ receptor is composed of 359 amino acids (250). It is a glycoprotein and contains extracellular glycosylation sites at the amino terminus (Asn⁴) and the second extracellular loop (Asp¹⁷⁶ and Asn¹⁸⁸) (54). The transmembrane domain at the amino-terminal extension and segments in the first and third extracellular loops are responsible for G protein interactions with the receptor (113). Internalization of G protein-coupled receptors involves receptor phosphorylation, which may be mediated, in part, via caveola (123).

Although G protein-coupled receptors do not contain intrinsic kinase activity, they are phosphorylated on serine and threonine residues by members of the G protein receptor kinase (GRK) family (293, 294). AT₁ receptors may be phosphorylated in the basal state and in response to ANG II stimulation (147). Threonine and serine residues between Thr³³² and Ser³³⁸ of the cytoplasmic tail are essential for receptor internalization (for review see Ref. 124). The AT₁ receptor may also be phosphorylated at tyrosine residues. Potential tyrosine phosphorylation sites within the AT₁ receptor include amino acids 302, 312, 319, and 339 within the carboxy terminal (19, 127). Tyr³¹⁹ is important, inasmuch as it is part of the motif Tyr-Ile-Pro-Pro, which is analogous to an Src homology 2 (SH2) binding motif in the platelet-derived growth factor (PDGF) receptor (PDGFR), Tyr-Ile-Pro, and in the epidermal growth factor (EGF) receptor (EGFR), Tyr-Leu-Pro-Pro (74). In EGFR and PDGFR, these motifs are target sequences for tyrosine phosphorylation. The question of agonist-induced tyrosine phosphorylation of the AT₁ receptor and the possible effects that may be consequent on this remain controversial (293, 294). Initial studies in vascular smooth muscle cells (VSMC) reported that agonist-induced phosphorylation of the AT₁ receptor was mediated by tyrosine and serine kinase-mediated phosphorylation (147). However, studies by Thomas et al. (294) demonstrated that a serine/threonine-rich segment of the carboxy terminus was essential for phosphorylation and internalization of this receptor. Other studies in cells transfected with the AT₁ receptor (218) and VSMC (133) determined that GRKs, such as GRK2 and GRK5, and second-messenger-activated kinases, such as protein kinase C (PKC), mediate predominantly serine phosphorylation, which results in desensitization of the agonist-occupied receptor.

AT₂ Receptor

The second major angiotensin receptor isoform is the AT₂ receptor. The gene of this receptor is localized as a single copy on the X chromosome (161). The AT₂ receptor is a seven-transmembrane-type, G protein-coupled receptor comprising 363 amino acids. It has low amino acid sequence homology (~34%) with AT_1A or AT_1B receptors (128, 201). Although the exact signaling pathways and the functional roles of AT₂ receptors are unclear, these receptors may antagonize, under physiological conditions, AT₁-mediated actions (42, 334) by inhibiting cell growth and by inducing apoptosis and vasodilation (84, 107, 118, 119, 273, 306). The exact role of AT₂ receptors in cardiovascular disease remains to be defined.

The AT₂ receptor is ubiquitously expressed in human fetal mesenchymal tissues. However, the expression of this receptor rapidly declines after birth (208). In adults, AT₂ receptor expression is detectable in the pancreas, heart, kidney, adrenals, brain, and vasculature (162, 222, 295, 299, 318, 324, 325). In blood vessels, the AT₂ receptor has been detected in the mesenteric blood vessels of adult rats, but this receptor is not detectable in all vascular beds (6).

The distribution of the AT₂ receptor in the human cardiovascular system is poorly characterized. AT₂ receptor mRNA has been demonstrated in cultured coronary artery endothelial cells and in the medial layer of interlobular renal arteries (170). Most recently, Malendowicz et al. (181), using RT-PCR techniques, investigated whether AT₂ receptor mRNA could be detected in vastus lateralis muscle biopsies obtained from healthy individuals or patients with severe chronic heart failure treated with an ACE inhibitor or an AT₁ receptor antagonist. Control studies identified the presence of von Willebrand factor mRNA in muscle biopsy homogenates. This was important to confirm the presence of endothelial cells and, therefore, vascular tissue in these biopsies. Although AT₁ receptor transcripts were detected in these homogenates, the AT₂ receptor was undetectable in any biopsy other than that obtained from a human fetus. Although these data suggest that the AT₂ receptor is absent in human skeletal muscle vasculature, further investigations to detect AT₂ receptor protein are required to confirm or refute this thesis.

Studies in ex vivo human cardiac tissue have demonstrated the AT₂ receptor to be present in tissue from healthy (327) and failing human hearts (10, 108). The AT₂ receptor is present in human cardiac myocytes (10) and fibroblasts (304). In healthy human myocardium, the AT₂ receptor predominates (327). In heart failure, although expression of the AT₂ receptor gene in cardiomyocytes, as measured by competitive RT-PCR, may be unchanged, the overall abundance of the receptor protein (and also that of the AT₁ receptor) falls (108).

The expression of both angiotensin receptor types is tightly regulated. The AT₁ receptor may be subject to "negative feedback" by ANG II (1), whereas expression of the AT₂ receptor is upregulated by sodium depletion (222) and is inhibited by ANG II and growth factors such as PDGF and EGF (125).

	PHYSIOLOGICAL ACTIONS OF ANGIOTENSIN RECEPTORS

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Physiological Effects Mediated by the AT₁ Receptor

ANG II mediates its effects by acting directly through ANG II receptors, indirectly through the release of other factors, and via cross talk with intracellular signaling cascades of other vasoactive agents, growth factors, and cytokines. Integrated responses to ANG II are the result of combined AT₁- and AT₂-mediated actions. The established cardiovascular effects of AT₁ and AT₂ receptor activation in humans are shown in Table 1 (8, 129). AT₁ receptor activation stimulates vasoconstriction, vascular cell hypertrophy and hyperplasia, and sodium retention. Other more recently described physiological effects of this receptor include stimulation of ROS production (23, 94, 234, 234) and induction of inflammatory (202), thrombotic (315), and fibrotic processes (26, 211, 280).

View this table: [in this window] [in a new window]   Table 1.   Physiological effects of AT1 and AT2 receptors

Some of the pathophysiological effects of ANG II may be mediated through activation of the transcription factor nuclear factor-B (NF-B) (247), which participates in a variety of inflammatory responses (13). For example, studies in experimental rats overexpressing the human renin and angiotensinogen genes (double-transgenic rats) suggest that AT₁ receptor-coupled NF-B activation may be of pathological importance (202). Chronic treatment of these animals with the antioxidant pyrrolidine dithiocarbamate was associated with reductions in blood pressure, cardiac hypertrophy, macrophage tissue infiltration, and albuminuria. Furthermore, electrophoretic mobility shift assay demonstrated that pyrrolidine dithiocarbamate inhibited NF-B binding activity in heart and kidney.

Increasingly, ANG II is recognized to trigger diverse effects, some of which may be opposite to what might be anticipated (84, 246, 247). For example, under some circumstances, AT₁ receptor activation is reported to lead to apoptosis in some cell types (57, 169).

Physiological Effects Mediated by the AT₂ Receptor

Most of the biological actions of ANG II are thought to be mediated by the AT₁ receptor. However, recent evidence suggests that the AT₂ receptor may have a physiological role in the regulation of blood pressure and renal function, counterbalancing the vasoconstrictor and antinatriuretic actions of ANG II. The significance of AT₂ receptors in blood pressure regulation was recently demonstrated by Tsutsumi et al. (303), who selectively overexpressed the AT₂ receptors in VSMC of transgenic mice. In animals overexpressing the AT₂ receptor, ANG II infusion did not cause a pressor response, which was present in wild-type mice. Furthermore, in the presence of AT₁ receptor blockade, ANG II infusion decreased blood pressure not only in transgenic, but also in wild-type, mice. These findings suggest that AT₂ receptors do regulate blood pressure, probably by modulating vasoconstrictor responses. In general, cardiovascular effects of the AT₂ receptor appear to be opposite to those of the AT₁ receptor (189) (Table 1). The vasodilator, antigrowth, and apoptotic actions of the AT₂ receptor are in contradistinction to those of the AT₁ receptor. Other novel, physiological effects attributed to the AT₂ receptor include modulation of thirst, behavior, and locomotor activity (110, 126).

Are all the effects of the AT₂ receptor beneficial? Paradoxically, AT₂ receptor activation may have proinflammatory effects, such as in induction of NF-B (244), and trophic effects, leading to vascular (36, 220) and cardiac (30, 177, 265, 304) hypertrophy. These observations underline the complexity and our limited understanding of the angiotensin receptor systems.

	INTRACELLULAR SIGNALING THROUGH ANGIOTENSIN RECEPTORS

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AT₁ Receptor-Mediated Signaling Events

AT₁ receptors are coupled to multiple, specific signaling cascades, leading to diverse biological actions. The signaling processes are multiphasic with distinct temporal characteristics. The trophic effects of ANG II are mediated by activation of pathways that involve tyrosine phosphorylation and enhanced gene expression (19, 65, 67, 98, 127) (Fig. 1). Processes involved in these and other ANG II-stimulated pathways have only recently been elucidated. In this section, mechanisms of AT₁ receptor-induced activation of tyrosine kinase and phospholipase pathways, prostaglandin (PG) metabolism, and transcription factor and ROS activities are discussed.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Signaling mechanisms of the angiotensin type 1 (AT1) and type 2 (AT2) receptors, interactions with ceramide, and physiological effects. NOS, nitric oxide synthase; PLA2, PLB, and PLD, phospholipase A2, B, and D, respectively; PGF2, prostaglandin F2; DAG, diacylglycerol; PA, phosphatidic acid.

Ligand-receptor binding results in activation of heterotrimeric G proteins through exchange of GTP for GDP, resulting in the release of G-GTP and complexes, which induce downstream actions (102). These intracellular responses are dependent on the identity of the G protein subunits. Activation of G_i and related subunits results in cGMP, which is sensitive to pertussis toxin, whereas G_s and G_olf activate adenylate cyclases and G_q leads to activation of PLC (102). AT₁ receptor-G_i activation inhibits adenylate cyclase, leading to a reduction in cAMP (8).

AT₁ receptor-induced phosphorylation by tyrosine kinase. Vascular cell growth involves non-receptor- and receptor-associated tyrosine kinase-mediated intracellular protein phosphorylation (19). Activation of these pathways is important for the physiological growth and contractile responses consequent on AT₁ receptor activation (19). This is supported by studies demonstrating that inhibition of tyrosine kinases attenuates ANG II-induced hypertrophic (163), proliferative (299), and contractile (114) responses in cultured VSMC.

In VSMC, the nonreceptor tyrosine kinases (non-RTKs) include PLC-₁, Src family kinases, Janus kinases (Jak and Tyk), focal adhesion kinase (FAK), Ca²⁺-dependent tyrosine kinases (e.g., PYK2), p130^Cas (a Crk-associated substrate), and phosphatidylinositol 3-kinase (PI3K) (19, 67, 246). The receptor tyrosine kinases (RTKs) involved in vascular ANG II signaling include EGFR, PDGFR, and insulin-like growth factor I. In this section, we discuss recent developments relating to ANG II signaling and tyrosine kinases and consider the emerging evidence of ANG II transactivation of RTKs.

ACTIVATION OF NONRECEPTOR TYROSINE KINASES. Src family kinases. ANG II induces rapid phosphorylation of c-Src, measured by autophosphorylation or kinase activity toward enolase (132, 225, 226, 302). This appears to be a redox-sensitive process, as recently demonstrated by Ushio-Fukai et al. (310). Src plays an important role in ANG II-induced phosphorylation of PLC- and inositol trisphosphate formation. Src, intracellular Ca²⁺, and PKC regulate ANG II-induced phosphorylation of p130^Cas, a signaling molecule involved in integrin-mediated cell adhesion (246, 254) (Fig. 2). Src has also been associated with ANG II-induced activation of PYK2 (56, 245) and with phosphorylation of extracellular signal-regulated kinases (ERKs) (132) as well as activation of other downstream proteins including pp120, p125^Fak, paxillin, Jak2, signal transducer and activator of transcription 1 (STAT1), G, caveolin, and the adapter protein Shc (49, 172). From a physiological viewpoint, phosphorylation of these proteins is important. For example, Shc is a linker protein involved in mediating intracellular signaling of G protein-coupled receptors (GPCRs) (19). Studies in VSMC isolated from human resistance arteries suggest that c-Src may also be important in the regulation of ANG II-stimulated Ca²⁺ mobilization (132). Furthermore, c-Src mediates ANG II regulation of plasminogen activity in bovine aortic endothelial cells (16).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   AT1 receptor (AT1R)-induced tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1) is mediated by binding of Janus kinase 2 (Jak2) by this receptor at a YIPP motif in the COOH-terminal domain of this receptor. Activation of Jak2 may occur independent of any physical association with the AT1 receptor. p59fyn serves as a docking protein for Jak2 and STAT1. Phosphorylation of STAT1 results in homo/heterodimerization and translocation to the nucleus, where binding with specific promoter elements leads to enhanced gene transcription. STAT1 phosphorylation is negatively regulated by MKP-1, a nuclear mitogen-activated protein kinase phosphatase. VSMC, vascular smooth muscle cell.

Although there is increasing evidence that Src family kinases are functionally linked to the AT₁ receptor (132, 183, 302, 310), the exact mechanisms whereby AT₁ receptors associate with Src are unclear. The interaction between the G-subunits, their associated kinases, and kinase substrates could provide the signaling complex that activates and binds c-Src (345). It has also been suggested that Src interacts indirectly with the receptor, via other proteins such as Jak2 (255) or possibly via -arrestin (179).

JANUS FAMILY KINASES, TYROSINE KINASE, AND STAT ACTIVATION. Similar to classical cytokine receptors, the AT₁ receptor stimulates Jak2 and Tyk2, members of the Janus family kinases (185). Using immunoprecipitation and immunoblotting techniques with an antiphosphotyrosine antibody, Marrero et al. (185) demonstrated that AT₁ receptor activation leads to rapid phosphorylation (within minutes) of the intracellular kinases Jak2 and Tyr2 (Fig. 3). This pathway involves an association between Jak2 and the AT₁ receptor, which is dependent on a YIPP sequence in the carboxy-terminal intracellular domain of this receptor (5). In addition, it has recently been shown that Jak2 autophosphorylates on tyrosine in response to ANG II, which is important for the interaction between Jak2 and STAT1, a process that seems to be independent of physical binding to the AT₁ receptor (4).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Non-receptor kinase AT1 receptor-dependent tyrosine phosphorylation. The AT1 receptor-dependent interaction of Src and PYK2 (also known as CADTK or CAK) involves a PYK2 kinase-mediated autophosphorylation, which is sensitive to Ca2+ and protein kinase C. This may in turn result in Src-mediated Shc (adapter protein) phosphorylation, resulting in the association of this complex with Grb2 and Sos and with Ras-Raf-MEK-ERK1/2 activation. This then stimulates JNK activation, leading to activator protein 1 (AP-1) DNA binding, gene expression, and protein synthesis. AT1 receptor activation may also lead to mitogen-activated protein kinase (MAPK) activation by Ras-independent pathways. MEK, MAPK kinase; ERK, extracellular signal-regulated kinase.

Venema et al. (316) demonstrated in rat VSMC that ANG II induced tyrosine phosphorylation of STAT1, which could be prevented by cotreatment with a Jak2 inhibitor or p59^Fyn kinase (a member of the Src family of kinases) inhibitor. These authors proposed that neither of these kinases were "upstream" but, rather, that p59^Fyn acted as a docking protein for Jak2 and STAT1 (316), which facilitates Jak2-mediated phosphorylation of STAT1, resulting in nuclear translocation of this transcription factor (185). By contrast, treatment of VSMC with sense or antisense MKP-1, a nuclear mitogen-activated protein kinase (MAPK) phosphatase, demonstrated that this enzyme induced dephosphorylation of STAT1 (316). Gene expression of this phosphatase is stimulated by treatment with ANG II, suggesting a negative-feedback mechanism in Jak-STAT signaling. Electroporation of antibodies against STAT1 and STAT3 abolished VSMC proliferative responses to ANG II, but not to other growth factors, implicating an essential role of STAT proteins in ANG II-induced cell proliferation (184). The Jak-STAT signaling pathway activates early growth response genes and may be a mechanism whereby ANG II influences vascular and cardiac growth, remodeling, and repair (19, 109).

FAK. ANG II promotes cell migration and induces changes in cell shape and volume by activating FAK-dependent signaling cascades (120, 164, 337). Focal adhesion complexes, specialized sites of cell adhesion, act as supramolecular structures for the assembly of signal transduction mediators. The best-characterized tyrosine kinase localized to focal adhesion complexes is a 125-kDa protein, FAK (99). p125^Fak and the related cytoplasmic tyrosine kinase PYK2 are nonreceptor kinases associated with the cytoskeleton (34, 259, 342). Ligand induction of FAK autophosphorylation, such as by ANG II, requires the enzyme to associate with cell surface integrins. By contrast, inhibition of FAK induces cytoskeletal disassembly (93). FAK exhibits extracellular matrix-dependent tyrosine autophosphorylation and physically associates with two non-RTKs, c-Src and Fyn (pp59), via their SH2 domains (256). FAK autophosphorylation may also result in physical associations with PI3K, which is a "downstream" tyrosine kinase involved in trophic cellular responses (41). As a consequence of its association with c-Src, FAK undergoes further tyrosine phosphorylation, which results in FAK binding to Grb2, an association with the GDP-GTP exchange protein Sos and Ras. This in turn leads to ERK1/2 activation (259) (Figs. 2 and 4).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   AT1 receptor-receptor kinase transactivation. AT1 receptor activation may stimulate intracellular protein tyrosine phosphorylation, although the AT1 receptor has no intrinsic tyrosine kinase activity. Reactive oxygen species and Ca2+-dependent activation of PYK2 and Src lead to activation of, for example, epidermal growth factor receptor (EGFR) kinase and phosphorylation of Shc. This in turn triggers downstream activation of Ras-Raf-mediated MAPK pathways, c-fos/c-jun expression, and increased protein synthesis. PKB, protein kinase B; FAK, focal adhesion kinase; PLC-1, phospholipase C-1; PI3K, phosphatidylinositol 3-kinase.

FAK is abundant in developing blood vessels, and elevation of its phosphotyrosine content in VSMC is a rapid response to ANG II (215, 230). ANG II-induced activation of FAK causes its translocation to sites of focal adhesion with the extracellular matrix and phosphorylation of paxillin and talin, which may be involved in the regulation of cell morphology and movement. The functional importance of ANG II-induced FAK activation in VSMC has recently been investigated by Govindarajan et al. (93). In these studies, FAK activation was inhibited by treatment with an actin depolymerizing agent, cytochalasin D, and by transfection of these cells with an adenovirus encoding FAK-related nonkinase (FNKNK) (93). Both of these treatments attenuated ANG II-induced ERK1/2 activation, c-fos mRNA expression, and new protein synthesis in these cells. These observations implicate FAK as an upstream promoter of ANG II-induced hypertrophic responses in VSMC. The link between the AT₁ receptor and FAK is unknown, but the Rho family of GTPases may be important (11, 242).

P130^CAS. p130^Cas is an ANG II-activated tyrosine kinase that plays a role in cytoskeletal rearrangement (242). This protein serves as an adapter molecule, because it contains proline-rich domains, an SH3 domain, and binding motifs for the SH2 domains of Crk and Src (Fig. 2). In cultured VSMC, ANG II induces a transient increase in p130^Cas tyrosine phosphorylation (254). Sayeski et al. (254) found that this phosphorylation is dependent on Ca²⁺, c-Src, and PKC and that it requires an intact cytoskeletal network. Other investigators reported that ANG II-induced activation of p130^Cas is Ca²⁺ and PKC independent (287). Although the exact functional significance of ANG II-induced activation of p130^Cas is unclear, it might regulate -actin expression, cellular proliferation, migration, and cell adhesion (210, 214, 246, 254).

PI3K. PI3Ks, a large family of intracellular signal transducers that phosphorylate inositol lipids to generate the 3-phosphoinositides phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-diphosphate, and phosphatidylinositol 3,4,5-trisphosphate, are heterodimeric proteins composed of 85- and 110-kDa subunits (166). These kinases influence cell survival, metabolism, cytoskeletal reorganization, and membrane trafficking and have recently been identified to play an important role in the regulation of VSMC growth (166, 253). PI3K, characteristically associated with tyrosine kinase receptors, is also activated by AT₁ receptors (253). PI3K inhibition by pharmacological agents completely blocks ANG II-stimulated hyperplasia in cultured rat VSMC, suggesting the important regulatory role of this non-RTK in cell growth (253). Several molecular targets for PI3K have been identified, including the protein serine/threonine kinase Akt/protein kinase B (PKB) (330). Akt/PKB regulates protein synthesis by activating p70 S6 kinase (p70^S6K) (43, 69), and it modulates ANG II-mediated Ca²⁺ responses in aortic VSMC by stimulating Ca²⁺ channel currents (263). Akt/PKB has also been implicated to protect VSMC from apoptosis and to promote cell survival by influencing Bcl-2 and c-Myc expression and by inhibiting caspases (43). Mechanisms whereby the AT₁ receptor mediates activation of PI3K-dependent Akt/PKB are unclear, but redox-sensitive pathways and c-Src may be important (292, 309).

SMALL GTP-BINDING PROTEINS AND MAPK ACTIVATION BY TYROSINE KINASES. Small GTP-binding proteins include Ras, a cell membrane protein, and Cdc42, Rho, and Rac, which are cytosolic proteins (100). On activation by GPCR, tyrosine kinase receptors, or cytokine receptors, these pleiotropic GTP proteins participate in signaling pathways that result in a variety of cell functions, such as differentiation, proliferation, and contraction (100) (Figs. 2 and 4).

MAPKs, a family of serine/threonine protein kinases, mediate nuclear transduction of extracellular signals by intracellular protein phosphorylation, leading to a cascade of transcription factor activation, enhanced gene expression, and trophic cellular and vascular responses (19, 19, 186, 192). Furthermore, these AT₁ receptor systems are causally implicated in the pathophysiology of vascular disease (112, 159).

Mammalian MAPKs are grouped into six major subfamilies: 1) ERK1/2 (also known as p42^MAPK and p44^MAPK, respectively), 2) c-Jun NH₂-terminal protein kinases/stress-activated protein kinases (JNK/SAPK), 3) p38, 4) ERK6 (p38-like MAPK), 5) ERK3, and 6) ERK5 (also called Big MAPK1) (78, 240, 296, 300). ERK1/2 is activated in response to growth and differentiation factors, whereas JNKs and p38^MAPK are usually activated in response to inflammatory cytokines and cellular stress (78, 130, 157, 199, 213, 240). ANG II differentially activates the three major members of the MAPK family: ERK1/2, JNKs, and p38^MAPK (156, 164, 297, 298). Induction of MAPK activation typically involves phosphorylation by an MAPK kinase, also known as MEK (45). MEK is, in turn, regulated by other MEK kinases, including Raf-1 (157). Although activated by similar stimuli, the signaling processes leading to JNK and p38^MAPK activation are quite different. The best-characterized MAPK cascade is the Raf-Ras-MEK-ERK1/2 pathway (Fig. 4).

ERK1/2 is a proximal kinase that phosphorylates and activates numerous transcription factors, such as Elk-1, leading to c-fos protooncogene expression and formation of the activator protein-1 (AP-1) complex. This is a heterodimeric transcriptional factor, formed by binding of the Fos and the Jun family gene products, which mediates cell growth (132, 186). ANG II-induced activation of ERK1/2 is an important step in the induction of VSMC trophic responses by this hormone in rat (331) and human VSMC (296). Studies by Xi et al. (331) in rat VSMC demonstrated that inhibition of ERK1/2, by treatment with an MEK inhibitor or by transfection of these cells with ERK1 and ERK2 antisense oligodeoxynucleotides, was associated with reductions in AT₁ receptor-dependent ERK1/2 activation, c-fos induction, DNA synthesis, and VSMC migration.

Touyz et al. (296) examined the importance of AT₁ receptor-induced MEK/ERK1/ERK2 activation for Ca²⁺ handling and contraction in cultured human VSMC that had been obtained from isolated small resistance arteries. They demonstrated the involvement of ERK1/2 in AT₁ receptor-mediated stimulation of Ca²⁺ currents and VSMC contraction.

Boffa et al. (26) recently explored the role of ERK1/2 in ANG II-induced tissue fibrosis. In initial studies in transgenic mice that overexpressed the ₂-chain of the collagen I gene, induction of hypertension by inhibition of nitric oxide (NO) synthesis was associated with renal and vascular fibrosis, which could be prevented by cotreatment of these animals with an AT₁ receptor. Treatment of ex vivo aortic and renal cortical sections with ANG II was associated with increased expression of c-fos and increased abundance of collagen I-₂ gene mRNA (291). These effects were inhibited by treatment with an AT₁ receptor antagonist, by blockade of the MAPK-ERK cascade, and by an inhibitor of the transcriptional factor AP-1 (291). Furthermore, inhibition of transforming growth factor- (TGF-) abolished the ANG II-induced effect on collagen I gene expression, implicating TGF- and ERK activation in this pathway.

There is considerable interest in dissecting the role of GTP-binding proteins in ANG II-stimulated MAPK activation. GTP-binding proteins are intermediary factors in the activation of ERK1/2 and JNK/stress-activated protein kinases (SAPK) (70, 249). In studies undertaken in rat VSMC, Eguchi et al. (70) demonstrated that AT₁ receptor activation stimulates a rapid, Ca²⁺-calmodulin, tyrosine kinase-dependent increase in the binding of GTP to p21^Ras. Activation of Ras by binding GTP is one important event in AT₁ receptor-G_q-induced activation of MAPK in cultured VSMC (70). Furthermore, the activation of Ras appears to involve a signaling cascade via c-Src (258). AT₁ receptor activation of Ras involves the phosphorylation of Shc linker protein, which then binds the adapter protein, Grb2, via an SH2 domain (249). The guanine nucleotide exchange protein Sos then stimulates GTP binding by Ras, leading to formation of the activated Shc-Grb2-Sos-Ras adapter protein complex. Raf may then be recruited into the plasma membrane, inducing MEK phosphorylation, which in turn triggers phosphorylation and activation of ERK1/2 (19).

Interestingly, in studies using VSMC treated with an adenovirus dominant-negative mutant of Ras, Takahashi et al. (286) observed that AT₁ receptor-stimulated MAPK activation and stimulation of protein synthesis were preserved. This suggests that AT₁ receptor activation may stimulate MAPK and VSMC hypertrophy by Ras-independent pathways. By contrast, other studies by Eguchi et al. (69) demonstrated that Ras was required for AT₁ receptor-induced ERK activation in these cells.

ANG II-stimulated ERK1/2, JNK, and p38^MAPK activation involves cell-specific signaling pathways. For example, Kudoh et al. (156) reported that, in neonatal rat cardiomyocytes, ANG II-induced activation of the transcription factor AP-1 is mediated by ERK1/2- and JNK-dependent pathways, which is PKC and AT₁ receptor dependent. Molloy et al. (196) demonstrated that ANG II induced rapid tyrosine phosphorylation of ERK1/2 and hyperphosphorylation of Raf in VSMC. Furthermore, Rac and Cdc42 GTP proteins mediated JNK activation in these cells (193). By contrast, in VSMC, AT₁ receptor-induced activation of p38^MAPK appears to be tyrosine kinase and PKC independent (260, 317). Whereas ERK1/2 and p38^MAPK are rapidly phosphorylated in response to ANG II, JNK activation is delayed, indicating differential regulation of MAPK in VSMC by ANG II (260, 297, 317).

Schmitz et al. (260) demonstrated that p21-activated kinase (PAK) is an upstream mediator for ANG II-induced activation of JNK in cultured rat VSMC. Rac and Cdc42 GTP-bound proteins associate with PAK, suggesting that ANG II induces activation of the Rac and Cdc42. Importantly, ANG II activation of PAK involved a Ca²⁺-dependent kinase other than Src. This suggests that multiple tyrosine kinase pathways may exist for the AT₁ receptor/GPCR-induced activation of small GTP-binding proteins.

Inactivation of ANG II-stimulated MAPKs occurs via MKP-1-induced dephosphorylation of tyrosine and threonine on MAPKs. Inhibition of MKP-1 results in sustained activation of MAPK in response to ANG II, suggesting that this enzyme is primarily responsible for the termination of the MAPK signal (63, 64). In VSMC, ANG II modulates MKP-1 activity. MKP-1 expression is stimulated by ANG II, and activities of MKP-1 as well as tyrosine phosphatase (PTP-1C) and serine/threonine phosphatase 2A (PP2A) are increased by ANG II (15, 118, 149). Various studies have shown that these effects may be mediated, at least in part, via the AT₂ receptor subtype, which has been associated with inhibition of cell growth and apoptosis (15, 77, 118). It is thus possible that AT₁ receptors induce growth via stimulation of ERK-dependent signaling pathways, whereas AT₂ receptors oppose these effects by stimulating MKP-1 activity to inhibit ERK activity and to arrest the cell growth signal. Termination of ANG II-stimulated MAPK activity may also involve activation of protein kinase A (PKA), which inhibits the phosphorylation of Raf-1 (44).

RECEPTOR TYROSINE KINASES. RTKs mediate a variety of cellular growth responses. Ligand binding to the RTK results in activation of an intrinsic kinase, which in turn results in autophosphorylation and formation of new binding sites (e.g., for SH2 or phosphotyrosine binding domains). This results in RTK binding of adapter proteins, such as SHC, or tyrosine phosphorylation of other proteins, such as Src, PLC-₁, and PI3K. RTK may also be activated by a variety of nonligand stimuli, such as ROS (235), and ultraviolet radiation (178).

Recent evidence suggests that mitogenic responses to GPCR, such as the AT₁ receptor, may also be mediated by activation of RTKs, such as the EGFR (71, 204). Receptor transactivation may be defined as that process whereby ligand stimulation of one receptor leads to activation of another, distinct receptor. Three mechanisms have been proposed for ANG II-induced RTK transactivation in VSMC (67): tyrosine kinase phosphorylation (71, 204), ROS activation, or cleavage of the EGFR (67).

MECHANISMS OF AT₁ RECEPTOR-INDUCED RECEPTOR KINASE TRANSACTIVATION. Studies by Murusawa et al. (204) in cardiac fibroblasts demonstrated that inhibition of EGFR activity by a dominant-negative EGFR mutant or by treatment with a specific EGFR antagonist abrogated ANG II-induced ERK1/2 activation, induction of c-fos gene expression, and DNA synthesis. This mechanism does not involve production of autocrine factors (71) but appears to be mediated by a Ca²⁺-dependent kinase, such as PYK2, in VSMC (71) and cardiac fibroblasts (204).

Recent studies in cultured rat aortic VSMC by Bokemeyer et al. (27) demonstrated that ANG II-induced EGFR transactivation depends on c-Src. ANG II also resulted in EGFR kinase-induced phosphorylation of p52 and p66 isoforms of Shc adapter protein, leading to formation of an EGFR-Shc complex. These findings suggest an obligatory role for EGFR kinase in ANG II-induced signaling through the Shc adapter protein (Fig. 4).

Ushio-Fukai et al. (310) recently explored the possibility that ROS may mediate ANG II-induced EGFR transactivation. In these studies, pretreatment of VSMC with antioxidants prevented ANG II-induced tyrosine phosphorylation of the EGFR, but not EGF-induced phosphorylation of its own receptor. Alternatively, direct treatment of these cells with H₂O₂ and the superoxide-generating compound LY-83583, in the absence of any other ligand, was associated with a concentration-dependent increase in EGFR phosphorylation. These observations suggest that ROS may induce EGFR phosphorylation through activation of an upstream intermediary, rather than activation of EGFR kinase. In this case, redox-sensitive candidates include Ca²⁺ (284), PYK2 (80), and c-Src (67, 96). Further studies by Ushio-Fukai et al. in VSMC demonstrated that EGFR transactivation could be prevented by inhibition of tyrosine kinases, c-Src kinases, or Ca²⁺ chelation, but not by Jak2 kinase or PI3K inhibition. In addition, transfection of these cells with an adenovirus containing DNA for a kinase-inactive form of c-Src led to inhibition of the activity of c-Src compared with inactive (Ad.LacZ) control transfected cells. These data suggest that c-Src is an upstream effector for ANG II-induced EGFR transactivation by tyrosine phosphorylation.

Similarly, in VSMC, ANG II may transactivate the PDGF -receptor independent of autocrine PDGF production (173). Treatment of VSMC with ANG II leads to tyrosine phosphorylation of Shc proteins, resulting in subsequent complex formation between Shc proteins and the PDGFR. This in turn is associated with Src activation. Moreover, these events could be inhibited by treatment with an AT₁ receptor antagonist. Other studies have also demonstrated that ANG II induces rapid transactivation of the mitogenic insulin-like growth factor I receptor in VSMC (61). This effect involves autophosphorylation of the -subunit of the tyrosine kinase receptor and phosphorylation of insulin receptor substrate-1.

EFFECTS OF AT₁ RECEPTOR KINASE TRANSACTIVATION. AT₁ receptor-elicited tyrosine phosphorylation and activation of EGFR result in downstream activation of ERK1/2 and VSMC hyperplasia (Fig. 4) (27). In rat VSMC, ANG II-induced nuclear protooncogene expression and increase in c-Fos protein were prevented by treatment with an MEK or an EGFR kinase inhibitor (68). By contrast, this mechanism is not involved in ANG II-stimulated c-Jun expression in these cells (68). Alternatively, ANG II-induced expression of this growth-response gene may be mediated by PYK2 (260).

ANG II-mediated EGFR transactivation is also reported to be operative in other growth-promoting signaling pathways (69, 288). The p70 ribosomal protein S6 kinase (p70^S6K), which is activated on phosphorylation (233), is a major component of the cellular machinery involved in protein synthesis (319). ANG II may stimulate protein synthesis in rat VSMC through p70^S6K activation (87). Studies by Eguchi et al. (69) demonstrated that transfection of a dominant-negative mutant of Ras inhibited AT₁ receptor-induced p70^S6K Ser⁴¹¹ phosphorylation, implicating an Ras-PI3K-PKB cascade, rather than the Raf-MEK-ERK system, in the activation of p70^S6K.

Could AT₁ receptor transactivation participate in vascular pathobiology? In studies of cultured VSMC isolated from hypertensive rats, ANG II-induced increase in TGF- mRNA abundance was mediated through ERK1/2 activation, enhanced c-Fos/c-Jun expression, and increased activation of NF-B and AP-1 transcription factors (101). These observations suggest that AT₁ RTK transactivation may be involved in cellular processes associated with vascular remodeling.

Phospholipid second-messenger signaling. AT₁ receptor-G_q protein stimulation leads to activation of phospholipase A₂ (PLA₂), PLC-, and PLC- (8). The AT₁ receptor also activates phospholipase D (PLD) by a mechanism that involves the G protein subunit G and smaller G proteins (194, 309). PLA₂ activation results in catabolism of arachidonic acid (AA) and the production of PG metabolites (248).

PLC ACTIVATION BY AT₁ RECEPTORS. One of the earliest detectable events resulting from ANG II stimulation of VSMC is a rapid, PLC-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (3, 18, 95, 97). The PLC family includes three related enzymes: PLC-, PLC-, and PLC-, which are regulated by G proteins  and in the case of PLC- (290), by tyrosine phosphorylation in the case of PLC- (152), or by Ca²⁺ in the case of PLC- (19, 239). PLC-₁, PLC-₁, and PLC-₁ have been identified in VSMC (182). AT₁ receptor activation results in a rapid production of inositol 1,4,5-trisphosphate and a more sustained release of diacylglycerol (DAG; Fig. 5A) (127), which are involved in Ca²⁺ mobilization from the sarcoplasmic reticulum (20) and stimulation of PKC (320), respectively. ANG II-stimulated inositol trisphosphate generation may also be mediated, in part, via tyrosine kinase-dependent pathways (92). Increased intracellular Ca²⁺ results in VSMC contraction (60), whereas PKC activation regulates intracellular pH through the Na⁺/H⁺ exchanger (313). PLC activation correlates temporally with initiation of contraction in isolated VSMC, as well as in intact small resistance arteries and, most likely, constitutes the early signaling pathway for initiation of the Ca²⁺-dependent, calmodulin-activated phosphorylation of the myosin light chain that leads to cellular contraction (158, 252, 296, 299, 321).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   A: AT1 receptor-induced activation of PLC. IP3, inositol trisphosphate. B: AT1 receptor-induced activation of PLD.

ACTIVATION OF PLA₂ AND AA METABOLISM BY ANG II. ANG II stimulates PLA₂ activity, which is responsible for release of AA from cell membrane phospholipids (29, 53, 236). PLA₂-derived eicosanoids influence vascular and renal mechanisms important in blood pressure regulation (212). In VSMC and endothelial cells, these effects are mediated via AT₁ receptors (82, 231), whereas in neonatal rat cardiac myocytes, neuronal cells, and renal proximal tubule epithelial cells, ANG II-induced activation of PLA₂ occurs via AT₂ receptors (65, 176, 241, 343). ANG II-elicited activation of vascular PLA₂ is dependent on intracellular Ca²⁺ concentration, Ca²⁺-calmodulin-dependent protein kinase II, and MAPKs (206, 207). Activated PLA₂ and its metabolites in turn activate Ras/MAPK-dependent signaling pathways, amplifying PLA₂ activity and releasing additional AA by a positive-feedback mechanism (206). In renal epithelial cells, ANG II activates PLA₂ via an AT₂-mediated Ca²⁺-independent mechanism (14, 135). Renal-derived arachidonate phosphorylates the adapter protein Shc and stimulates its association with Grb2 and Sos1 (65). ANG II-generated eicosanoids regulate vascular contraction and growth, possibly by activating MAPKs and redox-sensitive pathways (65, 212). Thromboxanes are involved in ANG II-induced contraction, whereas vasorelaxant PGs such as PGE₂ and PGI₂ attenuate ANG II-mediated vasoconstriction in some vascular beds (328). Lipoxygenase-derived eicosanoids also influence ANG II-mediated actions in VSMC. 12-Hydroxyeicosatetraenoic acid (HETE) facilitates the stimulatory actions of ANG II on Ca²⁺ transients in cultured cells. Lipoxygenase inhibitors attenuate the vasoconstrictor action of ANG II and decrease blood pressure in spontaneously hypertensive rats (221, 281). Some of these effects may be mediated via modulation of the oxidative state of the cell (339).

PLD ACTIVATION BY ANG II. Hydrolysis of phosphatidylcholine by PLD leads to the production of phosphatidic acid and subsequent generation of DAG by phosphatidic acid phosphohydrolase (24, 55, 89, 158). DAG contributes to prolonged activation of PKC. This pathway probably represents the major cascade by which ANG II-induced activation of PKC remains sustained in VSMC. The downstream pathways associated with ANG II-induced activation of PLD in VSMC are PKC independent (81) but involve intracellular Ca²⁺ mobilization (81) and Ca²⁺ influx that is tyrosine kinase dependent (283). ANG II-induced PLD signaling has been implicated in cardiac hypertrophy as well as in proliferation of VSMC (55, 200). PLD-dependent signaling cascades also influence cardiac and vascular contraction (332). These effects are mediated via phosphatidic acid and other PLD metabolites (25, 55, 329) that influence vascular generation of superoxide anions by stimulating NADH/NADPH oxidase (89, 94, 309), activate tyrosine kinases and Raf, and modulate intracellular Ca²⁺ signaling (25, 72, 89). The long-term signaling pathways associated with ANG II-stimulated growth and remodeling in the cardiovascular system are dependent, in part, on PLD-mediated responses.

Molecular mechanisms coupling AT₁ receptors to PLD have recently been identified. AT₁ receptor-induced PLD activation involves a G_q/11- and G_i/_O-independent mechanism (Fig. 5B). Using immunoprecipitation techniques in astrocytoma cells, Mitchell et al. (194) demonstrated that AT₁ receptor activation resulted in translocation of ARF/RhoA to the plasma membrane, thereby forming an AT₁ receptor-GTP-binding protein functional complex. This in turn resulted in PLD activation. This pathway appears dependent on a specific Asn-Pro-XX-Tyr amino acid sequence within the AT₁ receptor. In summary, these observations confirmed that the AT₁ receptor and other GPCR can physically associate with intracellular proteins other than G_q/11, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors (194, 246). These observations are supported by studies by Ushio-Fukai et al. (309), which demonstrated that ANG II-induced stimulation of PLD was inhibited by electroporation of anti-G, anti-G₁₂, anti-c-Src, and anti-Rho antibodies, whereas anti-G_i and -G_aq/11 antibodies had no effect. These results implicate G and G₁₂ subunits in AT₁ receptor-induced PLD activation through a c-Src-RhoA-mediated pathway.

AT₁ receptor-mediated transcription factor activation. Transcription factors, such as NF-B and AP-1, are important upstream mediators of ANG II trophic effects (156, 202, 312). In its inactive form, NF-B is a heterotrimeric, cytoplasmic protein that is bound to inhibitory-B (IB) (13). Activation of NF-B involves release from IB when the latter undergoes phosphorylation, which occurs because of the activity of cytokine-inducible oxidant-sensitive kinases (138, 151, 244). On activation, NF-B enters the nucleus and binds to the promoter region of target, inflammatory genes such as intercellular adhesion molecule and vascular cell adhesion molecule (13).

ANG II-induced activation of NF-B has been causally implicated in the inflammatory vasculopathy in rats made hypertensive by chronic blockade of NO synthesis (312) or by chronic infusion of ANG II (202). Treatment with an AT₁ receptor antagonist or an antioxidant inhibited these effects. In other studies in rat VSMC, Ruiz-Ortega et al. (244) demonstrated that ANG II treatment stimulated degradation of cytosolic IB- binding protein, which was paralleled by translocation of the activated heterotrimeric protein form of NF-B, p50/p65, to the nucleus. These effects were attenuated by AT₁ receptor and phosphotyrosine kinase inhibition. Furthermore, AT₁ receptor inhibition abrogated NF-B-induced gene transcription.

AT₁ receptor-mediated generation of ROS. ANG II increases VSMC superoxide (O·) production by activation of a membrane-bound NAD(P)H oxidase, suggesting that enhanced ROS activity may be involved in the vasoactive effect of this hormone (94, 160, 234). Zafari et al. (340) demonstrated that the ANG II-stimulated activation of NAD(P)H oxidase occurred through release of AA metabolites, triggering PKC activation. This in turn led to phosphorylation of the phox subunits of NAD(P)H oxidase and activation of this enzyme. Ushio-Fukai et al. (311) demonstrated that transfection of antisense p22^phox cDNA into cultured rat VSMC abrogated any ANG II-stimulated increase in O· concentrations or hypertrophy of these cells. These observations implicated the p22^phox subunit as a key element in NAD(P)H oxidase-dependent O· production. Further work in rat VSMC treated with diethyldithiocarbamate, a superoxide dismutase inhibitor, demonstrated that O· conversion to H₂O₂ was important for ANG II-stimulated VSMC hypertrophy (339). In studies undertaken by Pagano's group, cotreatment of ANG II-treated aortae with actinomycin D, an inhibitor of transcription, and cycloheximide, an inhibitor of protein synthesis, attenuated ANG II-stimulated O· production in these arteries (323). These data suggest that ANG II augments NAD(P)H oxidase-mediated O· production by enhancing the abundance of mRNA via transcriptional and nontranscriptional pathways (223, 322). Interestingly, the AT₁ receptor antagonist losartan inhibited ANG II-stimulated O· production in rat thoracic aortae; in similar studies in rabbits, losartan had no such action (323). This suggests that important species differences may exist in this pathway, inasmuch as ANG II-stimulated O· production in rabbits could be inhibited by the nonspecific receptor antagonist [Sar¹,Thr⁸]ANG II (224). Berry et al. (23) demonstrated that ANG II stimulates O· production in human internal mammary arteries by an AT₁ receptor-dependent, NAD(P)H oxidase-mediated pathway. They also found that the AT₂ receptor activation does not contribute to ANG II-stimulated O· production in these arteries (22).

ROS are involved in modulating a variety of intracellular signaling pathways for vascular cell growth regulation (80, 131). ROS are second messengers for AT₁ receptor activation, such as ANG II-induced EGFR transactivation (310). Schieffer et al. (257) recently investigated the possibility that ROS may act as signaling messengers for AT₁ receptor activation of Jak and STAT factors in rat aortic VSMC. Treatment of these cells with ANG II (10 µmol/l) stimulated an increase in the concentrations of O· and the cytokine interleukin (IL)-6. Both of these effects were abolished by cotreatment with the AT₁ receptor antagonist losartan or the flavoprotein inhibitor diphenyleneiodonium or by inhibition of p47^phox by electroporation of p47^phox antibodies into these cells. Similarly, treatment of these cells with ANG II led to Jak2, STAT1/, and STAT3 tyrosine phosphorylation, which could also be inhibited by treatment with losartan, diphenyleneiodonium, or electroporation of p47^phox antibodies. In other studies, these investigators demonstrated that treatment of rat VSMC with AG-940 (10 µmol/l), a selective antagonist of Jak2, or STAT1/ antisera prevented ANG II-induced synthesis of IL-6. These studies demonstrated that, in rat VSMC, ANG II-induced NAD(P)H oxidase-dependent O· production may be important for activation of the Jak-STAT cascade, which in turn stimulates an increase in the synthesis of IL-6.

In other studies in rat VSMC, Viedt et al. (317) reported that AT₁ receptor-induced ROS production stimulated JNK and p38^MAPK, but not ERK1/2, leading to an increase in AP-1 DNA binding. Inhibition of p22^phox activity by treatment with a specific antibody or antisense DNA abolished AT₁ receptor-induced JNK and p38^MAPK activation and reduced AP-1 DNA binding. In this study, ANG II induced ERK1/2 activation by a tyrosine kinase-, PKC-, and MEK-dependent pathway. Ushio-Fukai et al.(308) also demonstrated that ANG II-induced ERK1/2 activation may be ROS independent, whereas other studies by Frank et al. (79) demonstrated that NAD(P)H oxidase inhibition inhibits ERK1/2 activation. Taken together, these observations suggest that ANG II may activate tyrosine kinase pathways, such as ERK1/2, by ROS-dependent and ROS-independent pathways.

The activity of NF-B is also regulated by ROS activity (13, 202, 312, 312), suggesting the possibility that AT₁ receptor-induced ROS production (234) leads to activation of NF-B (23). This thesis has been recently investigated by Pueyo et al. (232), who demonstrated that AT₁ receptor-induced activation of NF-B, which was associated with enhanced vascular cell adhesion molecule-1 expression, is a redox-sensitive pathway.

AT₂ Receptor

The signaling pathways involved in AT₂ receptor activation are not fully understood but appear to involve G protein-dependent (106, 341) and -independent (31) pathways (32, 84, 116). Immunoselection studies in the rat fetus demonstrated that AT₂ receptors are associated with G_i2 and G_i3, rather than AT₁ receptor-G_q/11 (341). This raises the possibility that this receptor may be G protein coupled (Fig. 1).

Hansen et al. (106) recently investigated the pharmacology of the AT₂ receptor in relation to interactions of this receptor with G proteins. Studies with cells transfected with plasmids encoding the AT₂ receptor, G_i2, G₂, and G₁ sequences, facilitated the selective investigation of AT₂ receptor ligands and G protein activation. In other studies, NIH/3T3 cells, which express native AT₂ receptor, were also used. They found that the AT₂ receptor may catalyze the exchange of GDP for guanosine 5'-O-(3-thiotriphosphate) on G_i or G₀, but not G_q or G_s. Their other findings included evidence that the pseudopeptide CGP-41112A is a partial agonist of the AT₂ receptor.

The AT₂ receptor-G protein hypothesis is supported by studies in other cell types. For example, in cultured hypothalamic neurons, the AT₂ receptor-mediated delayed rectifier K⁺ current is abolished by treatment with a selective anti-G_i binding protein or pertussis toxin, a selective G_i protein inhibitor (150). Furthermore, in biochemical studies in cultured neurons, AT₂ receptor-stimulated activation of serine/threonine PP2A has also been shown to be selectively inhibited by pertussis toxin (121, 122). PP2A activation results in dephosphorylation and inactivation of growth factor-activated MAPK and, in particular, inactivation of ERK1/2. In studies in transgenic mice, cardiac overexpression of AT₂ receptor was associated with an inhibition of AT₁ receptor-mediated MAPK activation, which may have been involved in the AT₂ receptor-mediated negative chronotropic effect in these animals (187).

Using cultured fibroblasts that selectively express the AT₂ receptor but not the AT₁ receptor, Tsuzuki et al. (305) demonstrated that EGF-induced cell proliferation was attenuated by cotreatment of these cells with ANG II. This growth-retardant response was enhanced by the specific AT₂ receptor agonist CGP-42112A and inhibited by the AT₂ receptor antagonist PD-123319. Again, the mechanism for this appeared to involve activation of PP2A, supporting the thesis that AT₂ receptor-induced PP2A activation inhibits cell proliferation by counterregulation of MAPK phosphorylation.

Yamada et al. (333) demonstrated that AT₂ receptor activation may also lead to programmed cell death. In PC12W cells, a rat pheochromocytoma cell line that selectively expresses AT₂ receptors rather than AT₁ receptors, ANG II activation antagonized the growth-promoting effects of nerve growth factor and resulted in apoptosis of these cells. This mechanism is G protein coupled, inasmuch as pertussis toxin treatment inhibited ERK1/2 activation and apoptosis. Bcl-2 is an intracellular membrane protein that, when phosphorylated, inhibits apoptosis (262). In further studies in PC12W cells, Horiuchi et al. (118) demonstrated that the activity of Bcl-2 could be modulated by MAPK-induced phosphorylation and MAPK phosphatase-1 (MKP-1) dephosphorylation. AT₂ receptor activation inhibited nerve growth factor-induced Bcl-2 phosphorylation, whereas pretreatment of these cells with antisense oligonucleotide of MKP-1 abrogated this proapoptotic effect. The AT₂ receptor, therefore, promotes apoptosis through stimulation of ERK phosphatase, which is in turn associated with dephosphorylation and inhibition of MAPK and Bcl-2 (116).

Bedecs et al. (15) recently examined the possibility that AT₂ receptor-mediated tyrosine kinase dephosphorylation may be mediated by non-G_i/G_q pathways. In NIE-115 neuroblastoma cells, which selectively express AT₂ receptors, inhibition of G_i/G_q regulatory proteins or PP2A fails to abolish the rapid AT₂ receptor-mediated inhibition of ERK1/2 but, instead, could be inhibited by treatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor (15). With the use of immunoprecipitation and immunoblotting techniques, this PTP was identified as SHP-1. Furthermore, AT₂ receptor activation did not involve enhanced MKP-1 expression or other transcription-based mechanisms in these cells. Brechler et al. (32) also explored AT₂ receptor activation in relation to G protein-independent pathways. In cultured PC12W cells, removal of G proteins from solubilized membranes failed to inhibit AT₂ receptor-induced intracellular tyrosine phosphatase activation (32).

Interestingly, AT₂ receptor effects on MAPK are cell specific. For example, in renal proximal tubular epithelial (65) and COS-7 (106) cells, AT₂ receptor activation results in membrane-associated PLA₂ activation or PTP inhibition, respectively, which in turn leads to MAPK activation. Reasons for this variable effect of the AT₂ receptor on MAPK may include differences between the association of AT₂ receptor, G proteins, and GDP-GTP exchange between cell types. Touyz et al. (296) recently demonstrated that the AT₂ receptor is not involved in activation of the Ras-Raf-MEK-ERK pathway in human VSMC.

Signaling mechanisms of the AT₂ receptor: cardiovascular and renal physiology. BRADYKININ-NO-GUANOSINE 3',5'-CYCLIC MONOPHOSPHATE CASCADE. Stimulation of the AT₂ receptor is associated with increased generation of bradykinin (274, 276), NO (274), and cGMP (270), all of which have vasodilatory properties (38). Thus it is possible that AT₂ receptor stimulation leads to vasodilation through a signaling pathway that involves bradykinin, NO, and cGMP.

A vasotonic role for bradykinin production during AT₂ receptor activation has been reported in numerous animal models (88, 272). In the stroke-prone spontaneously hypertensive rat, the attenuation in ANG II-induced pressor response by cotreatment with losartan was associated with a rise in aortic cGMP concentrations, which could be inhibited by coinfusion of the bradykinin B₂ receptor antagonist icatibant (PD-123319, an AT₂ receptor antagonist) or nitro-L-arginine methyl ester (L-NAME) (88). These data suggest that AT₂ receptor activation during AT₁ receptor blockade is associated with an increase in bradykinin production, which in turn stimulates NO generation and counterregulatory vasodilation.

Siragy and Carey (270) made the original observation that, in rats, ANG II stimulated cGMP production in renal interstitial fluid (RIF), which could be inhibited by cotreatment with PD-123319 or L-NAME (271), suggesting the possibility that AT₂ receptor activation leads to increased renal NO production (271). Lo et al. (175) showed that AT₂ receptor activation was involved in the regulation of pressure natriuresis in vivo. Further studies by Madrid et al. (180) suggested that this mechanism occurred through AT₂ receptor-induced NO production. Moreover, the fact that expression of AT₂ receptor is increased in response to sodium depletion supports the concept that this receptor may be involved in salt and water homeostasis (222).

The role of AT₂ receptor modulation of vasomotor tone in vivo (88) was demonstrated in ANG II-infused AT₁-null mice (216, 217, 303). In these animals, AT₂ receptor activation was associated with increased renal production of cGMP and hypotension (37). AT₂-null mice have mildly elevated blood pressure, which is associated with low basal concentrations of RIF cGMP (275). In other studies in a transgenic mouse model that overexpresses the AT₂ receptor, Tsutsumi et al. (303) demonstrated that the pressor response to infused ANG II observed in wild-type animals was absent in AT₂ receptor transgenic mice. Furthermore, cotreatment with a bradykinin type 2 receptor antagonist or L-NAME restored the ANG II pressor response in these mice.

In vitro studies in aortic homogenates demonstrated that kininogenase activity was increased in the AT₂ receptor transgenic mice compared with wild-type mice; this occurred as a result of intracellular acidification due to an AT₂ receptor-induced inhibition of the amiloride-sensitive Na⁺/H⁺ exchanger (303). These observations suggest that AT₂ receptor activation may lead to vasodilation; the mechanism for this is activation of vascular kininogenase, which in turn leads to increased bradykinin release and NO production. Taken together, studies in animal models support the hypothesis that AT₂ receptor-stimulated renal and vascular NO production can be involved in the vasoactive responses of the RAS.

The role of ANG II as a vasodilator in human blood vessels is less well understood. Using isometric tension techniques, Garcha et al. (86) failed to detect any important vasorelaxant effect of ANG II in isolated human resistance arteries that had been pretreated with the AT₁ receptor antagonist losartan. More recently, Ytterberg and Edvinsson (336) failed to identify any AT₂ receptor mRNA in human subcutaneous resistance arteries obtained from healthy subjects. Furthermore, AT₂ receptor antagonism, by cotreatment of arteries (that had been denuded of endothelium) with PD-123319 (1 and 10 nmol/l), had no effect on ANG II concentration-response curves (336). Taken together, these in vitro observations suggest that in human resistance arteries, at least in healthy subjects, the AT₂ receptor may have little functional importance. The lack of supportive data for the presence and the distribution of AT₂ receptor protein in human blood vessels limits any further conclusions.

PROSTAGLANDINS. AA metabolites also appear to contribute to the vasoactive effects of AT₂ receptor activation. In the isolated microperfused rabbit kidney preglomerular afferent arteriole, which is important for the regulation of glomerular hemodynamics, treatment with ANG II led to vasoconstriction that was attenuated by cotreatment with an AT₁ receptor antagonist (9). During AT₁ receptor inhibition, however, ANG II-induced dose-dependent vasodilation was abrogated by endothelial denudation or by cotreatment with PD-123319 or an inhibitor of cytochrome P-450 AA metabolism, but not by treatment with an NO synthase (NOS) or cyclooxygenase inhibitor (9). The observation that inhibition of the synthesis of HETE, a metabolite of the cytochrome P-450 pathway, abolished this vasodilation implicates AA metabolites in AT₂ receptor-induced arteriolar vasodilation.

In AT₂ receptor-null mice, blood pressure is only marginally elevated compared with wild-type mice (277). This has been attributed, in part, to increased production of the vasodilator prostanoids PGE₂ and PGI₂. RIF concentrations of PGE₂, PGF₂, and cAMP, which are elevated in AT₂-null mice, are attenuated by treatment with an AT₁ receptor antagonist. However, when these mice were treated with indomethacin, AT₂ receptor-null mice became hypertensive compared with wild-type mice, suggesting a role for prostacyclins in this process.

Dulin et al. (65) provided further evidence in support of HETEs as second-messenger effectors for AT₂ receptor activation. In this case, AT₂ receptor-induced HETE activation led to phosphorylation of ERK1/2 in cultured rabbit renal proximal tubule epithelial cells (RPTEC). That AT₂ receptor coupling to MAPK activation is unique to this cell type suggests that the pharmacological properties of the AT₂ receptor in RPTEC may be different from those in other cell types. However, data from other studies in support of this thesis are lacking. Furthermore, Jiao et al. (141) demonstrated that, in RPTEC, membrane-bound PLA₂ activation and release of AA were important for AT₂ receptor-induced activation of p21^Ras.

Taken together, these findings suggest that, in addition to the vasodilator signaling cascade that includes bradykinin, NO, and cGMP, AA acts as an important source of lipid second messengers that are involved in AT₂ receptor-mediated cardiovascular events.

	PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL EFFECTS OF THE AT2 RECEPTOR: NOVEL CONCEPTS

TOP ABSTRACT INTRODUCTION PHYSIOLOGICAL ROLE OF ANG... ANGIOTENSIN RECEPTORS PHYSIOLOGICAL ACTIONS OF... INTRACELLULAR SIGNALING THROUGH... PHYSIOLOGICAL AND... CERAMIDE SIGNALING CERAMIDE AND ANG II AREAS FOR FUTURE INVESTIGATION REFERENCES

The physiological and pathophysiological significance of AT₁ receptors has recently been reviewed (154, 301). The role of AT₂ receptors in the cardiovascular system remains unclear and is the focus of this section.

Role of the AT₂ Receptor in Tissue Development and Cardiovascular Remodeling

Using in situ hybridization techniques, Shanmugam et al. (268) reported that AT₂ receptor mRNA was detectable in the large arteries, in the mesenchymal tissues, such as the kidney and the urogenital tract, and variably in the cardiomyocytes of fetal rats. Vascular AT₂ receptor mRNA was most abundant in late gestation and in the early postnatal period, becoming undetectable in the cardiovascular system of adult rats.

This is in contrast to the AT₁ receptor, where expression of AT₁ receptor mRNA was continuously expressed in the rat cardiopulmonary system from fetal life to adulthood (267). In these in vitro studies, AT₂ receptor mRNA was undetectable and may have been below the limits of detection of the assay. In biological terms, however, the expression of protein is most important. Ozono et al. (222) used immunohistochemical techniques to identify AT₂ receptor protein within the kidneys of fetal, newborn, and adult rats. The pronounced staining for AT₂ receptor in the fetal renal and adrenal cortex and, in particular, in undifferentiated mesenchymal cells and renal blood vessels declined with age but remained detectable in the adult adrenal gland, renal glomeruli, and nephrons.

More recently, Yamada et al. (333) compared the time-dependent differentiation of aortic VSMC from AT₂ receptor knockout mice with that from wild-type mice. In these studies, VSMC-specific contractile proteins increased from birth to 8 wk of age, but to a lesser extent in AT₂ receptor-null mice. Furthermore, the mRNA expression of one of these proteins, calponin, was temporally related to the expression of AT₂ receptor mRNA in wild-type mice. Calponin mRNA expression was significantly delayed in AT₂ receptor-null mice. By contrast, aortic mRNA expression of -smooth muscle actin, a protein that is not thought to be developmentally regulated, was not different between strains. Studies by Akishita et al. (2) in VSMC from wild-type and AT₂ receptor-null mice suggest that basal and serum-stimulated ERK activity may be higher and that this enhanced activity contributed to the enhanced proliferative responses observed in AT₂ receptor-null VSMC. Taken together, the high expression of AT₂ receptor mRNA and protein in fetal life (6, 222, 267) suggests a role for this receptor in the regulation of vascular growth and differentiation and organogenesis.

Interestingly, in the studies by Ozono et al. (222), sodium depletion in the adult rat was associated with an increased abundance of AT₂ receptor protein within the kidney. This suggests that homeostatic adaptations to changes in salt and water balance, which may trigger activation of the RAS, may result in increased expression of the AT₂ receptor.

The AT₂ receptor may also regulate cell growth and vascular remodeling. For example, treatment of quiescent rat coronary endothelial cells with an AT₂ receptor antagonist stimulated proliferation of these cells (282). In further in vitro studies, Goto et al. (90) demonstrated that AT₂ receptor expression in mesangial cells was modulated by the stage of growth of these cells, such that once the cells were confluent, AT₂ receptor expression was observed to be greatly increased. This suggests a growth-retardant effect of the AT₂ receptor on cell proliferation. Furthermore, Goto et al. also demonstrated that AT₂ receptor expression was greater in mesangial cells from normotensive than from hypertensive rats, implicating the reduced expression of the AT₂ receptor in the proliferative response of these cells in the hypertensive rat. Further in vitro evidence of an antiproliferative effect of the AT₂ receptor has been reported by Dudley and Summerfelt (62) in a mouse fibroblast cell line and by Nakajima et al. (209) in VSMC.

These in vitro data raise the question as to whether the AT₂ receptor may modulate growth responses in vivo. In rats fed a high-salt diet, infusion of a subpressor dose of ANG II was associated with AT₁ receptor-mediated cremaster muscle angiogenesis (203). Cotreatment with an AT₂ receptor antagonist was associated with increased microvascular density and an increase in blood pressure in these animals. This suggests that the AT₂ receptor may tonically modulate new vessel growth and blood pressure.

Role of the AT₂ Receptor in Regulation of Blood Pressure

In a recent study by Moore et al. (197), healthy female Sprague-Dawley rats received a 7-day renal interstitial infusion, via a surgically implanted osmotic minipump, of AT₂ receptor antisense oligodeoxynucleotide or scrambled antisense control. The contralateral kidney served as an anatomic control. Fluorescent imaging techniques demonstrated that infusion of AT₂ antisense resulted in delivery to the renal cortex and medulla. This was associated with reductions in AT₂ receptor protein and RIF concentrations of bradykinin and cGMP and increases in blood pressure and the pressor response to systemic infusion of ANG II. This in vivo study implicates the AT₂-bradykinin-cGMP cascade in the renal regulation of blood pressure.

Functional Role of the AT₂ Receptor in Cardiovascular Disease

The AT₂ receptor may play a homeostatic role in the regulation of blood pressure in animal models of hypertension (12, 88, 203). In one recent study in rats made hypertensive by infusion of ANG II, the observed increases in vascular cGMP concentrations induced during cotreatment with losartan were attenuated by pretreatment with the AT₂ receptor antagonist PD-123319 (88). This suggests that AT₂ receptor-stimulated increase in NO may contribute to the physiological and therapeutic effects of AT₁ receptor antagonists. In other studies in an ANG II-dependent rat model of hypertension, Wang et al. (325) showed that AT₁ receptor abundance in total kidney protein was reduced compared with AT₂ receptor abundance, whereas AT₂ receptor abundance was reduced only in kidneys made ischemic by clipping of the renal artery. These observations suggest differential regulation of the two receptors in renovascular disease.

Further studies from the same laboratory in a different rat model of renal vascular hypertension [2-kidney, 1-figure-8 wrap (Grollman)] demonstrated a functional role for counterregulatory vasodilation by the AT₂ receptor (272). Treatment of these animals with losartan normalized systolic blood pressure and increased renal function and interstitial fluid concentrations of bradykinin, NO metabolites, and cGMP in the contralateral nonischemic kidney. Treatment with PD-123319 alone or in combination with losartan attenuated these effects. These observations may be clinically relevant, inasmuch as AT₁ receptor inhibition is associated with secondary activation of the RAS (35). In this case, increased circulating and tissue concentrations of ANG II may stimulate the AT₂ receptor, thereby contributing to the vasoinhibitory effects of AT₁ receptor antagonism.

More recently, Barber et al. (12) infused genetically hypertensive and normotensive rats with the AT₁ receptor candesartan or the AT₂ receptor agonist CGP-42112 in the presence or absence of the AT₂ receptor antagonist PD-123319. In the hypertensive rats, AT₁ receptor inhibition, even with low-dose candesartan treatment, was associated with a reduction in mean arterial pressure that was further enhanced by coinfusion of the AT₂ receptor agonist. This latter effect was inhibited by cotreatment with the AT₂ receptor antagonist.

Further evidence in support of a putative functional role for AT₂ receptor activation during AT₁ receptor blockade has been provided by recent investigations using animal models of heart failure (136, 174). In studies in rats with heart failure induced by coronary artery ligation, treatment with an AT₁ receptor antagonist was associated with improvements in left ventricular (LV) systolic function, LV end-diastolic diameter, and LV end-systolic volume. The beneficial effects on cardiac dimensions, but not function, were prevented by cotreatment with an AT₂ receptor antagonist (174). In a pig (136) coronary artery ligation model, the beneficial hemodynamic effects of treatment with an AT₁ receptor antagonist were also attenuated by cotreatment with an AT₂ receptor antagonist. Interestingly, in both of these studies, bradykinin inhibition attenuated the improvements in LV function, suggesting that bradykinin may also mediate some of the clinically useful effects of AT₂ receptor activation in vivo.

Recent studies in human skeletal muscle biopsies (which included vascular cells) from healthy subjects and patients with severe chronic heart failure treated with an ACE inhibitor or AT₁ receptor antagonist failed to identify any AT₂ receptor mRNA (181). These data, therefore, argue against any role for AT₂ receptor activation in peripheral tissues in heart failure patients treated with either of these therapies. Further clarification of this question may be provided by future functional studies.

Are All the Physiological Effects of the AT₂ Receptor Beneficial?

Although the activation of the AT₂ receptor promotes natriuresis and vasodilation, the AT₂ receptor appears also to have other effects in the vasculature. Levy et al. (168) infused ANG II into normotensive rats that had been pretreated with either vehicle, the AT₁ receptor antagonist losartan or the AT₂ receptor antagonist PD-123319, for 3 wk. The pressor response to ANG II was attenuated by losartan, but not by PD-123319. By contrast, chronic AT₂ receptor inhibition was associated with a reduction in aortic collagen accumulation, hypertrophy, and fibrosis. Interestingly, treatment with the AT₂ receptor antagonist was not associated with a rise in plasma ANG II concentrations, as was the case with AT₁ receptor inhibition (168).

In spontaneously hypertensive rats, Otsuka et al. (220) observed that chronic treatment with PD-123319 was associated with an increase in AT₁ receptor mRNA and a reduction in aortic AT₂ receptor mRNA, hypertrophy of media smooth muscle cells, aortic collagen content, and aortic cross-sectional area. AT₂ receptor inhibition had no effect on blood pressure. The differences between these findings and those of Levy et al. (168) may relate to an upregulation of vascular AT₁ receptor activity, as evidenced by an increase in abundance of aortic mRNA with chronic AT₂ receptor inhibition in these animals. Other discrepancies between these two studies may relate to differences in methodologies or drug concentrations.

In rats made hypertensive by infusion of ANG II for 14 days, cotreatment with an AT₁ or an AT₂ receptor antagonist inhibited the ANG II-stimulated increase in mesenteric resistance artery weight and wall-to-lumen ratio (36). ANG II-induced hypertension was also prevented by treatment with an AT₁, but not an AT₂, receptor antagonist (36). The thesis that the AT₂ receptor may be involved in hypertrophic responses is strengthened by the observations by Brilla et al. (33), who reported that, in cultured rat cardiac fibroblasts, ANG II stimulates collagen synthesis through AT₁ and AT₂ receptor-dependent mechanisms.

Most recently, Mifune et al. (191) also demonstrated a prosynthetic action of AT₂ receptors in cultured rat VSMC. In these studies, treatment with the AT₂ receptor agonist CGP-42112A was associated with an increase in collagen synthesis, which could be blocked by cotreatment with the AT₂ receptor antagonist PD-123319 or the G_i antagonist pertussis toxin or G_i antisense oligonucleotides. These effects were replicated in AT₂ receptor-expressing mesangial cells but did not occur in an embryonal AT₂ receptor-expressing fibroblast cell line. Although these observations occurred in vitro, they do suggest that AT₂ receptor physiology is heterogeneous and may vary between different tissues.

AT₂ receptor activation has also been recently reported to mediate some of the proinflammatory effects of ANG II. For example, although AT₁ receptor activation is reported to stimulate NF-B-mediated transcription in rat aortic VSMC, this may also be the case with AT₂ receptor activation (244). In these studies, NF-B activation was also associated with increased expression of monocyte chemoattractant protein-1 and angiotensinogen, which are proinflammatory genes implicated in the pathobiology of inflammation, atherosclerosis, and restenosis (247). Ruiz-Ortega et al. (244) demonstrated that AT₁ receptor activation of NF-B occurred through a phosphotyrosine kinase-dependent pathway, whereas AT₂ receptor-induced activation of NF-B could be prevented by antioxidants or inhibitors of ceramide synthase. More recently, investigators from the same laboratory explored the possibility that other angiotensin-related peptides may trigger activation of transcription factors and gene expression. Treatment of cultured renal and mononuclear cells with the NH₂-terminal ANG II metabolite ANG III was associated with activation of NF-B and AP-1 transcription factors and increased monocyte chemoattractant protein-1 gene expression and protein levels (243).

The activation of proinflammatory genes by ANG II could, theoretically, lead to activation of leukocytes, leading to adhesion of these cells to the vascular endothelium and their migration into the vessel wall. This thesis was recently investigated by Piqueras et al. (229), who demonstrated that AT₁ and AT₂ receptor inhibition attenuated ANG II-stimulated leukocyte migration into rat mesenteric venules. These observations provide further evidence of a potential proatherogenic effect of the AT₂ receptor.

Studies in ex vivo human cardiac tissue also provide evidence to suggest that the AT₂ receptor may play a role in the pathophysiology of cardiovascular remodeling. In studies in failing human hearts, expression of AT₂ receptor protein was evident, even when AT₁ receptor proteins were downregulated (10, 108). Furthermore, in the failing human heart, AT₂ receptor gene expression and protein abundance are increased in cardiac fibroblasts present in areas of fibrosis (304). These observations suggest that the AT₂ receptor may promote fibrosis and ventricular remodeling in human heart failure.

Taken together, these findings conflict with those from other studies in cultured vascular cells in which AT₂ receptor activation led to growth inhibition and apoptosis. In attempting to explain these observations, Sadoshima (247) hypothesized that the trophic effects of AT₂ receptor activation in vivo may be due to the induction of transcription factors and growth pathways that may not be operative in cultured cells. Furthermore, these more recent observations suggest that the AT₂ receptor may have physiological effects beyond vasodilation and natriuresis, some of which may be deleterious.

Cross Talk Between the AT₁ and AT₂ Receptors: Physiological Antagonism

Many of the actions of AT₂ receptor activation are diametrically opposite to those of the AT₁ receptor. In teleological terms, high expression of the AT₂ receptor in early fetal life facilitates rapid cell turnover and differentiation (161). In later life, differences in receptor tissue distribution may well confer ANG II with a variety of tissue-specific effects (6).

What evidence is there for functional cross talk between angiotensin receptor subtypes? Many of the recent advances in our understanding of this subject have arisen through targeted manipulation of the expression of the AT₂ receptor gene or its second messengers (119). Nakajima et al. (209) demonstrated in the cat carotid artery subjected to balloon injury that there was attenuated smooth muscle cell proliferation in those arteries transfected with an AT₂ receptor overexpression vector compared with untreated controls. Using in vitro techniques, Horiuchi et al. (117) demonstrated that enhanced AT₂ receptor activity in rat VSMC transfected with AT₂ receptor cDNA resulted in an inhibition of AT₁ receptor-mediated tyrosine and serine phosphorylation of STATs and a decrease in c-fos expression. These data suggest that the balance between AT₁ and AT₂ receptor tissue activities may regulate cellular growth and hypertrophy.

Physiological antagonism has been demonstrated to exist in a variety of tissues. AT₁ receptor-induced rat cardiomyocyte hypertrophy is augmented by AT₂ receptor inhibition, suggesting that this receptor may exert a tonic inhibitory effect on cardiac hypertrophy (30). Clearly, these effects will be a function of the relative expression of angiotensin receptors in the myocardium. In the kidney, AT₁ receptor activation leads to sodium retention, whereas natriuresis may be modulated by activation of the AT₂ receptor (180). By contrast, intestinal AT₂ receptor activation results in salt and water retention, whereas this is opposed by the AT₁ receptor (142). In cultured neurons from the rat hypothalamus and brain stem, AT₁ receptor induction of ERK1/2 is under tonic control by the AT₂ receptor (121).

Do counterregulatory mechanisms exist between the angiotensin receptor subtypes in vivo? In the AT₂ receptor knockout mouse, for example, there is hypertension (110, 126) and an enhanced pressor response to ANG II (127). Analysis of AT₁ receptor mRNA expression in the aortae of these mice by competitive RT-PCR techniques shows the increased expression of this receptor compared with controls. Furthermore, pretreatment of these mice with the AT₁ receptor antagonist losartan normalized vascular responses to ANG II (127). Other supportive evidence of a functional antagonism between the two angiotensin receptor subtypes is provided from studies in mice with cardiac-specific overexpression of the AT₂ receptor, in which the chronotropic and pressor response to AT₁ receptor activation is attenuated (187).

Any contribution of AT₂ receptor to the effects of ANG II appears to be dependent on tissue expression and distribution of this receptor. Accordingly, in healthy rats, infusion of ANG II was associated with an increase in systemic vascular resistance that was attenuated by cotreatment with losartan (261). However, treatment with the AT₂ receptor antagonist PD-123319 had no effect on basal or ANG II-induced hemodynamic responses (261). This suggests that, in health, the AT₂ receptor may not have any important role in the regulation of vascular responses (86).

This situation may differ, however, in disease states where there is chronic activation of the RAS. For example, Siragy et al. (273) demonstrated that in rats fed a low-sodium diet the hypotensive effect of the AT₁ receptor antagonist valsartan was associated with increased RIF concentrations of bradykinin, NO metabolites, and cGMP. These effects were significantly inhibited by the coadministration of PD-123319.

Cross talk between signaling mechanisms of the angiotensin receptor does, therefore, appear to modulate the physiological effects of ANG II. Intriguingly, ANG II has recently been shown to activate other intracellular second-messenger systems that are non-G protein coupled and independent of tyrosine kinase activation. One example of this is the lipid second messenger ceramide (85, 167). In CERAMIDE SIGNALING, we review the physiology of ceramide and its interaction with ANG II. We also explore the hypothesis that the interaction of ANG II with ceramide might be one further example of how differences in the intracellular signaling mechanisms of the angiotensin receptor may contribute to their divergent effects.

	CERAMIDE SIGNALING

TOP ABSTRACT INTRODUCTION PHYSIOLOGICAL ROLE OF ANG... ANGIOTENSIN RECEPTORS PHYSIOLOGICAL ACTIONS OF... INTRACELLULAR SIGNALING THROUGH... PHYSIOLOGICAL AND... CERAMIDE SIGNALING CERAMIDE AND ANG II AREAS FOR FUTURE INVESTIGATION REFERENCES

Ceramide belongs to a family of lipids known as sphingolipids, characterized by a sphingoid backbone and distinct head groups (104, 105). Synthesis of ceramide occurs via two predominant pathways: catabolic and anabolic. Catabolic synthesis of ceramide occurs through hydrolysis of membrane sphingomyelin by sphingomyelinase, resulting in generation of ceramide and phosphocholine (190). Sphingomyelinase belongs to a superfamily of phospholipases that includes PLC and PLA₂ (266). Several forms of sphingomyelinase exist, including a magnesium-dependent, neutral-operating sphingomyelinase and an acidic-operating sphingomyelinase (279). Until recently, it was generally held that activation of sphingomyelinases represented the primary mechanism by which agonists such as cytokines stimulate ceramide synthesis.

Anabolic synthesis of ceramide occurs through cellular uptake of serine and its condensation with palmitoyl-CoA to form 3-ketosphinganine, which is reduced and hydrolyzed to form ceramide (Fig. 6). Traditionally, this de novo pathway has been considered the primary means for generating basal levels of ceramide. However, Lehtonen et al. (167) showed recently that, in a rat pheochromocytoma cell line (PC12W) that overexpresses the AT₂ receptor, stimulation with ANG II results in ceramide synthesis that does not coincide with a decrease in membrane sphingomyelin. The relevance of this particular pathway to the vasculature is discussed in more detail below.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Ceramide biosynthesis pathways.

Ceramide as a Second Messenger

Ceramide is structurally very similar to DAG, a well-known lipid second messenger generated through hydrolysis of inositol 4,5-bisphosphate by PLC to form DAG and inositol trisphosphate (21). In vascular smooth muscle, DAG is important in mediating contractile responses to agonists such as norepinephrine, vasopressin, and ANG II.

Characterization of ceramide as a second messenger initially occurred through studies examining cytokine signal transduction (266). Cytokines such as tumor necrosis factor- (TNF-), IL-1, and interferon- have been linked to ceramide generation (105). Many studies have demonstrated that ceramide generation in response to cytokines is due to activation of neutral sphingomyelinase (153). TNF--induced ceramide synthesis has been reported extensively, but the precise mechanism behind activation of sphingomyelinase by the 55-kDa TNF receptor has not been elucidated. Some studies have shown that activation of the 55-kDa TNF receptor stimulates sphingomyelin hydrolysis by sphingomyelinase through a PLA₂-dependent mechanism (140). This observation has been substantiated in vascular smooth muscle, indicating a role for ceramide signaling in the vasculature (144).

Downstream targets of ceramide have been poorly characterized, although several candidates have been identified. Ceramide has been shown to activate a PP2A (ceramide-activated protein phosphatase), a serine/threonine protein kinase (ceramide-activated protein kinase), and may interact with certain isoforms of PKC (58, 146, 188). Some studies have proposed interaction of these or other targets of ceramide with signaling pathways such as MAPK and/or the stress-activated protein kinase cascades (139, 326).

Ceramide as a Vascular Second Messenger

Recent studies have implicated ceramide as a possible vasodilatory second messenger (144, 145, 171). Initial studies probing the effect of ceramide on vascular contractility demonstrated that application of cell-permeant analogs of ceramide or exogenous bacterial sphingomyelinase to preconstricted vascular segments results in concentration-dependent relaxation (144). Given the causal relationship between cytokines and ceramide synthesis, the underlying hypothesis for most studies examining ceramide-induced vasodilation is that this signaling pathway may mediate the vasodilatory effects of cytokines. Indeed, TNF- has been shown to stimulate ceramide synthesis in a number of cell types, including VSMC (104, 105, 145).

Although the precise mechanism for ceramide-induced vasodilation has not been elucidated, recent studies have produced candidate downstream effectors for ceramide. In nonvascular cells, it has been shown that ceramide has an inhibitory effect on PKC (146, 165). Recent studies support a role for PKC inhibition as a mechanism for ceramide-induced vasodilation (144). Other possible mechanisms for ceramide-induced vasodilation include activation of PP2A phosphatases and regulation of cAMP (5, 26), but further investigation into this relationship is warranted.

Ceramide and Cell Proliferation

The antiproliferative effects of ceramide are well documented in nonvascular cell types (103-105, 269). Although a distinct role for ceramide-meditated regulation of cell proliferation in the vasculature has not been well characterized, preliminary evidence suggests that ceramide inhibits VSMC proliferation in culture (143) and may increase the incidence of apoptosis. This, coupled with the observation that TNF- stimulates ceramide synthesis in VSMC, supports a role for this signaling pathway in regulation of not only vascular contractility, but also cell proliferation.

The importance of ceramide-mediated regulation of cell proliferation/apoptosis can be further deduced from observations of cell proliferative behavior during atherogenic vascular remodeling. Bondjers et al. (28) demonstrated that, at the site of an atherosclerotic plaque, VSMC proliferation is increased. Others have shown increased apoptosis in VSMC derived from atherosclerotic coronary arteries (17). Increased cell proliferation coupled with increased apoptotic cell death implies increased cell turnover, a hallmark of vascular remodeling.

Most recently, Hernandez et al. (111) provided compelling evidence in support of the involvement of ceramide as a signal transducer of reoxygenation injury, a state associated with increased apoptosis (91). In their studies, cultured rat cardiac myocytes were subjected to a period of hypoxia and then reoxygenated. This was associated with activation of neutral sphingomyelinase and an increase in intracellular ceramide, which in turn led to an activation of c-Jun kinase (c-JNK). Ceramide accumulation and c-JNK activation were inhibited by pretreatment of these cells with the antioxidant N-acetylcysteine. This finding implicates ROS, which are a damaging product of reoxygenation, in activation of neutral sphingomyelinase. Taken together, these observations suggest that ceramide is an important stress-activated signal transducer in ischemia-reperfusion injury (264).

In a rabbit model of carotid artery balloon injury, local delivery of C₆-ceramide, a ceramide analog, was associated with a substantial reduction in neointimal stenosis compared with control (40). Further studies demonstrated that this effect was due to a reduction in the number of VSMC entering the cell cycle, but without induction of apoptosis. This fall in the number of VSMC correlated with a reduction in trauma-associated phosphorylation of ERK and activation of PKB/Akt. These latter observations are substantiated by other mechanistic studies that have demonstrated PKB/Akt and ERK to be operative in the transduction of ANG II-induced VSMC growth and migration (331). This study represents, therefore, the first step toward utilizing ceramide signaling components as a therapeutic intervention for cardiovascular disease.

Although the majority of ceramide research in the vasculature has focused on the role of ceramide in mediating the vascular effects of cytokines such as TNF-, other vasoactive substances such as ANG II are emerging as potential activators of this novel signaling pathway.

	CERAMIDE AND ANG II

TOP ABSTRACT INTRODUCTION PHYSIOLOGICAL ROLE OF ANG... ANGIOTENSIN RECEPTORS PHYSIOLOGICAL ACTIONS OF... INTRACELLULAR SIGNALING THROUGH... PHYSIOLOGICAL AND... CERAMIDE SIGNALING CERAMIDE AND ANG II AREAS FOR FUTURE INVESTIGATION REFERENCES

Ceramide, as an intracellular lipid second messenger, is known to be mechanistically involved in the vasodilator effects of certain cytokines, such as TNF-. Interestingly, in recent in vitro studies, AT₂ receptor activation has been demonstrated to increase intracellular concentrations of ceramide (85, 167). This suggests that AT₂ receptor-stimulated ceramide production may contribute to some of the physiological effects of ANG II.

In studies in PC12W cells, Lehtonen et al. (167) demonstrated that AT₂ receptor activation leads to an increase in intracellular concentrations of ceramide and apoptosis of these cells. The mechanism for this appeared to be independent of any functional alteration in sphingomyelinase, inasmuch as activities of this enzyme, sphingomyelin concentrations, and cellular glycolipid composition were unchanged. Alternatively, the increase in ceramide appeared to result from an ANG II-stimulated activation of serine palmitoyltransferase. The accumulation of ceramide and the resultant apoptosis of these cells were inhibited by antagonists of the de novo pathway for sphingomyelin synthesis, -chloro-L-alanine and fumonisin B₁. These data demonstrate that, in PC12W cells, ANG II induces sphingomyelin synthesis, which in turn leads to an increase in intracellular ceramide and apoptosis. In similar work, Gallinat et al. (85) confirmed this effect to be mediated by the AT₂ receptor, inasmuch as apoptosis of these cells could be inhibited by coincubation with the specific AT₂ receptor antagonist PD-123177.

Interestingly, ANG II has been demonstrated to stimulate TNF- production in several cell types (76, 83, 335). The mechanism by which ANG II stimulates TNF- synthesis is not clear but may involve ANG II-stimulated activation of NF-B (137, 155, 219). Kranzhofer et al. (155) suggest that activation of NF-B by ANG II in human monocytes is dependent on activation of the AT₁ receptor. However, it is difficult to extrapolate such observations to the vasculature, given the varying expression patterns and cell type-specific effects of AT₁ and AT₂ receptors. That TNF- is such an effective stimulus for ceramide synthesis introduces the possibility that ANG II might indirectly stimulate ceramide synthesis via TNF- induction, thereby adding another means of regulation of vascular function.

Ferreri et al. (76) examined the possibility that TNF- synthesized in response to ANG II might represent a compensatory mechanism to counterregulate the increase in blood pressure seen with ANG II infusion. In that study, administration of anti-TNF- antiserum to ANG II-infused rats caused an increase in blood pressure beyond that observed with ANG II alone. In this particular context, given the vasodilatory effects of ceramide and sphingomyelinase (144, 145), it is plausible that TNF-, induced by ANG II, could stimulate ceramide synthesis through activation of sphingomyelinase in the peripheral vasculature. This would therefore result in a counterregulatory vasodilator or anticonstrictor response to counteract the increase in blood pressure. Interestingly, the antiproliferative effects of ceramide could also counteract the cell proliferative response of the vasculature to ANG II.

These observations raise the possibility that ceramide may be involved in AT₂ receptor-induced growth inhibition and apoptosis in vascular and cardiac tissues. We might hypothesize, therefore, that given the role of ceramide as an intracellular signal for vasorelaxation and its involvement in AT₂ receptor signaling pathways, ceramide may also be involved in effecting AT₂ receptor-induced vasodilation. We believe that this hypothesis merits further investigation.

	AREAS FOR FUTURE INVESTIGATION

TOP ABSTRACT INTRODUCTION PHYSIOLOGICAL ROLE OF ANG... ANGIOTENSIN RECEPTORS PHYSIOLOGICAL ACTIONS OF... INTRACELLULAR SIGNALING THROUGH... PHYSIOLOGICAL AND... CERAMIDE SIGNALING CERAMIDE AND ANG II AREAS FOR FUTURE INVESTIGATION REFERENCES

Angiotensin receptor signaling mechanisms remain a focus for laboratory investigation. Future possible studies may investigate whether selective inhibition of specific MAPK may attenuate neointimal and vascular hypertrophy in animal models of atherosclerosis. Furthermore, the possibility that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors may inhibit ANG II-stimulated GTPase-dependent tyrosine kinase pathways also merits investigation.

There are few data on the presence and distribution of AT₂ receptor protein in the human vasculature. In humans, chronic treatment with an AT₁ receptor antagonist is associated with increased circulating concentrations of ANG II, which may hypothetically lead to activation of the AT₂ receptor (52). It remains plausible that, for example, AT₂ receptor activation may mediate some of the beneficial effects of these treatments. This thesis merits further study.

Emergent experimental intervention studies with ceramide analogs have provided data suggestive of an antistenotic effect of this agent (40). We might hypothesize, therefore, that AT₂ receptor-stimulated ceramide activation may contribute to the vasoactive effects of this receptor and, possibly, those of AT₁ receptor inhibition. We believe that further investigations are warranted into these potential interactions, given the emerging importance of the RAS and its therapeutic inhibition in human cardiovascular disease (314, 338).

	ACKNOWLEDGEMENTS

We thank Drs. A. F. Lever, R. Jones, A. Frater, and L. Work for contributing to this work.

	FOOTNOTES

C. Berry is supported by a Medical Research Council Clinical Training Fellowship. D. G. Johns is supported by Whitaker Cardiovascular Institute Training Fellowship 7224 from the National Heart, Lung, and Blood Institute. This work is also supported by British Heart Foundation Programme Grant RG97009 to A. F. Dominiczak.

Address for reprint requests and other correspondence: C. Berry, Dept. of Medicine and Therapeutics, Western Infirmary, University of Glasgow, 44 Church St., G11 6NT Glasgow, UK (E-mail: colin.berry{at}clinmed.gla.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 June 2000; accepted in final form 24 July 2001.

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