SPECIAL TOPICS
Regulation of Cardiovascular Signaling by Kinins and Products of Similar Converting Enzyme Systems
Prologue: Kinins and related systems. New life for old discoveries

¹ Department of Pharmaocolgy, University of Illinois College of Medicine, Chicago, Illinios 60612; ² Institut National de la Santé et de la Recherche Médicale U367, Paris VI-University, Broussais Medical School, 75005 Paris, France; and ³ Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

	ARTICLE

TOP ARTICLE REFERENCES

The current Special Topic section of the AJP: Heart and Circulatory Physiology presents papers on the theme: "Regulation of Cardiovascular Signaling by Kinins and Products of Similar Converting Enzyme Systems." The topic is broad because of the diverse nature of research on the kallikrein-kinin system and because of its numerous interactions with other peptide systems and signaling pathways, most notably the renin-angiotensin system. Given the pivotal role of angiotensin-converting enzyme (ACE) in connecting these two pathways and the widespread clinical use of its inhibitors, it is appropriate that we dedicate this issue to Dr. Ervin G. Erdös in honor of his lifetime commitment to research in this area and his seminal contributions that have been instrumental in driving the field forward. In addition to being a leader in the field and winner of many awards for his contributions, Dr. Erdös has been a highly esteemed colleague and mentor to the editors as well as numerous other scientists over the years. Despite recently achieving a certain age milestone, his youthful curiosity and enthusiasm for research and vigor continue unabated. This attitude is reflected in a sentence he wrote several years ago in response to a request to summarize his contributions to cardiovascular research: "It is far better to delegate achievements where they belong, to the past, and to keep looking forward to next week's findings in the laboratory."

Ervin Erdös's initial forays into kinins came in 1952 when he joined the laboratory of Eugen Werle as a postdoctoral fellow [E. Werle and E. K. Frey were the founding fathers of the kallikrein-kinin field and established many of the basic tenets of the cascade in their pioneering work starting in the 1920s and 1930s (61)]. This research experience stimulated a life-long interest in peptides and peptidases that has now spanned over 50 years. His initial investigations into the inactivation of bradykinin in blood and tissues led to the discovery of both an aminopeptidase that removes the NH₂-terminal Arg¹ of bradykinin or Lys¹ of kallidin and a carboxypeptidase (named kininase I or carboxypeptidase N) that removes the COOH-terminal Arg⁹. Continuation of these studies led to the discovery of a different enzyme in blood and tissues that inactivated bradykinin by removing the COOH-terminal Phe⁸-Arg⁹ dipeptide, which was called kininase II. Further investigation by Erdös and colleagues showed that kininase II was a peptidyl dipeptidase that was identical with ACE, leading to the concept (controversial at the time) that the same enzyme could activate a vasopressor (angiotensin) and inactivate a vasodilator (bradykinin) (10, 14). These seminal studies set the stage for the development of ACE inhibitors, which have become highly successful drugs for the treatment of hypertension and a variety of other cardiovascular and renal diseases. In addition to continued work on these enzymes, Erdös and colleagues also discovered additional peptidases and new functions for known peptidases, including prolylcarboxypeptidase (angiotensinase C), neutral endopeptidases 24.11 (neprilysin; NEP), carboxypeptidase M, and deamidase-cathepsin A. More recently, his group has uncovered three unique mechanisms for activation of kinin receptors: 1) ACE inhibitors can resensitize or potentiate the effects of ligands on B₂ receptors by inducing protein-protein crosstalk between ACE and the receptor; 2) ACE inhibitors directly activate the B₁ kinin receptor; and 3) kallikrein directly activates B₂ receptors independent of kinin generation. The latter discovery introduces the concept of a "shunt" in the kallikrein-kinin system in which the normal multistep cascade to release bradykinin is effectively bypassed by kallikrein causing direct receptor activation. It is perhaps fitting that this recent discovery provides new insight into the function of kallikrein because it brings the research full circle, back to the initial component of the kallikrein-kinin system, which was detected in urine almost 100 years ago (1) and isolated and characterized by Frey and Werle over 70 years ago (61). It is impossible to do justice to the numerous contributions of Ervin Erdös in the space allowed here, therefore, the reader is referred to three recent reviews that summarize his contributions and provide interesting historical perspectives on the development of the kallikrein-kinin and renin-angiotensin fields (14, 15, 17). His large and diverse body of work touches on many aspects of the studies reported in this issue.

The kallikrein-kinin field has relevance to numerous aspects of cardiovascular and renal function, blood coagulation and fibrinolysis, inflammatory conditions, and cancer. A comprehensive summary of the field in 1970, published as a volume of the Handbook of Experimental Pharmacology and edited by Erdös, already contained almost 800 pages (11) and a similarly sized volume, containing only updates to the initial publication, was published in 1979 (12). Since then, the field has continued to prosper, fueled by the advent of molecular techniques and the development of reagents and animal models to dissect the roles of the various components and discoveries of connections to many other systems and signaling pathways. Consequently, the papers presented in this issue only provide a very limited snapshot of this diverse field. Nevertheless, many current trends in this area emerge from the studies reported here. Most of the contributions fit into three broadly defined areas: 1) ACE; 2) kinin receptors; and 3) enzymes involved in generating, inactivating, or modulating kinin activity.

In the last several years, angiotensin-(1-7) has received increasing attention as an alternate product of the renin-angiotensin system, which has effects generally opposite to those of angiotensin II (22). Angiotensin-(1-7) can be generated by several pathways, including cleavage of angiotensin I by prolylendopeptidase (22) or angiotensin II by the newly described ACE2 (59). Interestingly, two other potential pathways of angiotensin-(1-7) generation were elucidated by the earlier work of Erdös and colleagues. In 1968, Yang et al. (64) reported the identification of a new angiotensinase C (prolylcarboxypeptidase) that removed the COOH-terminal Phe⁸ of angiotensin II to produce angiotensin-(1-7). Because it is primarily a lysosomal enzyme with an acidic pH optimum (5.0), it was not considered to be relevant to the generation of angiotensin-(1-7) in the blood. However, prolylcarboxypeptidase maintains 20-50% of its activity at neutral pH with physiologically relevant peptide substrates (41). In addition, endothelial cells contain very high amounts of prolylcarboxypeptidase (56), and recently it was unexpectedly identified as the prekallikrein activator on the surface of human endothelial cells (48). Erdös and his colleagues (23) were also first to demonstrate the ability of neutral endopeptidases 24.11 to generate angiotensin-(1-7) directly from angiotensin I. Although generation of angiotensin-(1-7) was initially considered to be an inactivation pathway, it is now clear that angiotensin-(1-7) has numerous physiological effects (22). In this issue, Sampaio et al. (44) report the ability of very low doses of angiotensin-(1-7) (110 fmol/min for 10 min) to increase regional blood flow to the kidney, mesentery, brain, and skin as well as cause an increase in cardiac index and a reduction in total peripheral resistance. Interestingly, a 100-fold higher dose produced the opposite effects, likely due to activation of AT₁ receptors. Many of the low-dose effects were blocked by the angiotensin-(1-7) antagonist, D-Ala⁷-angiotensin-(1-7) (A-779), indicative of a direct effect on an as-yet unidentified receptor. The effects on cerebral blood flow, cardiac index, and total peripheral resistance were only partially attenuated (44). In addition to its direct effects, angiotensin-(1-7) can work through enhancement of bradykinin that can be blocked by B₂ receptor antagonists (22). Erdös and colleagues (8, 31) showed that angiotensin-(1-7) is cleaved by the N-domain of ACE but is a potent and specific inhibitor of the C-domain active site (22) and, apart from its effects on inhibiting the hydrolysis of bradykinin, angiotensin-(1-7) can resensitize the B₂ receptor and/or potentiate the effects of bradykinin on its receptor, as do other ACE inhibitors.

High-molecular-weight kininogen (HK), originally identified as a precursor molecule for bradykinin, plays additional important roles in fibrinolysis, thrombosis, and inflammation (6). However, it was not clear whether the cleaved form of the molecule (HKa), generated after release of bradykinin, might also have important functions. In recent reports, HKa was anti-angiogenic and induced apoptosis of endothelial cells (5, 66). In the current Special Topic, Wang et al. (60) explored the mechanism of this action and found that HKa significantly upregulates the expression of both Cdc2 kinase and cyclin A, which corresponded with an increase in Cdc2 activity. Cdc2 and cyclin A are important regulators of the cell cycle but have also been associated with apoptosis. It seems that increased Cdc2 activity alone is not sufficient to induce apoptosis. In control cells that did not become apoptotic, Cdc2 activity increased after 48 h without a concomitant increase in protein expression (60). The apoptotic response likely requires an unregulated increase in Cdc2 activity at an inappropriate time in the cell cycle. Further studies will be required to completely unravel this interesting finding.

Four articles in this Special Topic deal with ACE, the major point of intersection between the renin-angiotensin and kallikrein-kinin pathways. Despite the fact that ACE inhibitors have been successfully used clinically for decades, new mechanisms of action continue to be discovered. For example, in a series of studies, Erdös and colleagues have provided evidence that ACE and the B₂ kinin receptor physically interact on the cell membrane (16, 32). The inhibitor binding to ACE likely induces a conformational change that is transmitted to the receptor causing additional or alternate signaling pathways to be activated (16, 32). In addition, ACE inhibitors directly bind to the B₁ kinin receptor, which is dependent on the presence of a unique HEXXH motif in the second extracellular loop (26). Recent studies have indicated that ACE inhibitor treatment causes upregulation of B₁ receptors in the cardiovascular system (33), which are usually expressed only in response to inflammation, injury, or cytokines (29). In this issue, Ongali et al. (42) investigate the effect of ACE inhibitors or losartan on B₁ and B₂ kinin receptors in the rat spinal cord. They find that the effects are age dependent. B₁ receptors are only present and upregulated by ACE inhibitors in young animals (8 wk old with 4 wk of treatment), whereas B₂ receptors are primarily upregulated by inhibitor treatment in older animals (16 and 24 wk). The effect of the ACE inhibitor enalapril on endothelin 1-induced hypertension in rats was investigated by Elmarakby et al. (9). Enalapril abolished the hypertensive effect of a 60-min infusion of endothelin 1, whereas an angiotensin AT₁ receptor blocker had no effect. However, a B₂ receptor antagonist completely reversed the effect of enalapril, indicating the ACE inhibitor enhanced the effects of bradykinin. Although it was suggested that increased kinin survival after enalapril was responsible for the effect, kinin levels were not measured. It is also possible that enalapril potentiated the effects of kinins on the receptor by inducing ACE-B₂ receptor crosstalk as mentioned above. Interestingly, the combined ACE-NEP inhibitor omapatrilat had no effect. Because this inhibitor should have blocked degradation of bradykinin to at least the same extent as enalapril, it was concluded that NEP inhibition prolonged the half-life of endothelin and increased its levels, overwhelming the bradykinin vasodilation. This possibility was not tested directly (9).

ACE is considered a cell surface marker of endothelial cells (10, 52). Marchetti et al. (30) report in this Special Topic that angiotensin I stimulates intracellular calcium increases in rat glomerular afferent arterioles, thin efferent arterioles, or muscular efferent arterioles. An ACE inhibitor was only able to block the effect of angiotensin I in the afferent arterioles and muscular efferent arterioles but not the thin efferent arterioles. This surprising finding indicates that thin efferent arterioles lack sufficient ACE to convert angiotensin I to II, leading to the possibility that a different enzymatic pathway is involved. However, several additional protease inhibitors also had no effect, indicating either a direct effect of angiotensin I or a novel proteolytic pathway generating angiotensin II. These observations further document the heterogeneity of the distribution of ACE along the vascular tree, a phenomenon that may have important consequences for the regulation of regional blood flow.

ACE knockout animals exhibit a complex phenotype with a marked decrease in blood pressure, impaired urine concentrating ability, renal morphologic defects, decreased hematocrit, and reduced male fertility (19, 27). To determine the possible contribution of bradykinin to these phenotypes, Xiao et al. (63) generated double knockout mice lacking both the ACE gene and the gene for the B₂ kinin receptor (4). The blood pressure and renal phenotype of the double knockout was essentially identical with that of the ACE knockout mouse, indicating that enhancement of bradykinin effects did not play a significant role in the phenotype of the ACE knockout animal. One disadvantage of this approach is the possible upregulation of compensatory mechanisms that obscure the function of the gene being knocked out. For example, the B₁ receptor can be upregulated in B₂ receptor knockout animals. Although they did not measure B₁ receptor expression, the authors argue against this possibility by showing a lack of a blood pressure response to the injection of bradykinin. Although it would be expected that plasma or cellular kininase I-type carboxypeptidases would convert the bradykinin to des-Arg⁹-bradykinin B₁ agonist, a more direct test would have been to inject a B₁ agonist. In addition, the low blood pressure of these knockout animals could have precluded further decreases due to bradykinin. These results are nevertheless consistent with the observation that tissue kallikrein knock-out mice have normal blood pressure. However, tissue kallikrein or B₂ receptor-deficient animals have endothelial dysfunction and display a markedly reduced flow-dependent vasodilatation, indicating an important role for the kallikrein-kinin system in arterial function (2, 36). The ACE-B₂ receptor knockout animals represent an interesting model system to investigate the interplay between angiotensins and kinins in the control of vascular function (10, 16) and to determine whether a constitutive angiotensin II deficiency can counteract the consequences of B₂ receptor deficiency and improve the arterial phenotype.

The enzymatic metabolism of bradykinin continues to attract attention, despite over 50 years of study since the original work of Erdös and colleagues (13, 54). Numerous peptidases beyond the original kininase I and II have been shown to inactivate bradykinin, at least in vitro. These include aminopeptidases, serine- and metallocarboxypeptidases, and several endopeptidases (50, 54). Because bradykinin is partly responsible for the cardioprotective effects of ACE inhibitors (28), there has been interest in developing inhibitors of other enzymes that metabolize bradykinin, for example, the dual ACE-NEP inhibitor omapatrilat. Two articles in this issue explore the role of two related enzymes, endopeptidase 24.15 and endopeptidase 24.16, in the metabolism of bradykinin (39, 40). These enzymes cleave bradykinin at the Phe⁵-Ser⁶ bond in vitro (38, 43). There was initial excitement generated several years ago by a report that a specific inhibitor of endopeptidase 24.15 potently lowered blood pressure in normotensive rats via inhibition of bradykinin degradation (24); however, later studies showed the activity of this inhibitor was due to its metabolism in vivo by NEP into an ACE inhibitor (65). In vivo studies using a recently developed stable inhibitor of both endopeptidase 24.15 and 24.16 (JA-2) have shown that it can potentiate the hypotensive effects of bradykinin in the absence of any effects on ACE (57). Norman et al. (39) show that JA-2 can potentiate bradykinin-induced increases in microvascular permeability in the brain. These effects were similar in magnitude to those produced by an ACE inhibitor, indicating that endopeptidase 24.15 and/or 24.16 play important roles in bradykinin metabolism in the brain. Both endopeptidase 24.15 and 24.16 are present in a transformed human umbilical vein endothelial cell line as well as ovine aortic endothelial cells, although endopeptidase 24.16 plays a more predominant role (40). Although primarily cytosolic enzymes, small amounts of both endopeptidases are associated with the plasma membrane (40). This is a common finding with many intracellular enzymes, but the mechanism(s) of translocation and interaction with the plasma membrane is unknown and is an obvious area for further study.

The first plasma kininase discovered by Erdös and Sloan (18) was carboxypeptidase N, which inactivates bradykinin by removal of the COOH-terminal Arg residue. However, with the discovery of the B₁ kinin receptor that specifically binds kinins with the COOH-terminal Arg removed, it became clear that this metabolic step generates an active agonist much like ACE conversion of angiotensin I to II. The discovery of two membrane-bound kininase I-type carboxypeptidases M and D (51, 53, 55) with a similar substrate specificity indicated that this conversion could take place at the cell membrane in the vicinity of the B₁ receptor (49). However, until now, their roles in generating B₁ agonists have not been directly addressed experimentally. Sangsree et al. (45) found that stimulation of human lung microvascular endothelial cells with the cytokines interleukin-1 and interferon- led to upregulation of B₁ receptors and also increased expression of carboxypeptidase M and D activity. When bradykinin was applied to the cells, a prolonged (>20 min) release of nitric oxide was measured with a porphyrinic microsensor, which was significantly reduced by either a B₁ receptor antagonist or a specific kininase I-type carboxypeptidase inhibitor. In control cells, bradykinin stimulated primarily a transient increase (~5 min) in nitric oxide production. These data indicate that cellular kininase I-type carboxypeptidases function to enhance kinin signaling and nitric oxide production by converting B₂ agonists into B₁ agonists, especially in inflammatory conditions.

The cloning and sequencing of the B₂ (35) and B₁ (37) kinin receptors represent a significant advance in the kallikrein-kinin field and allow the application of genetic and recombinant approaches to the study of receptor function. One confounding factor in studies using a knockout approach is the genetic background of the mice. Schanstra et al. (46) studied B₂ receptor knockout mice with a homogeneous genetic background and bred in pathogen-free conditions to eliminate this variable. Under these conditions, the mice had normal blood pressures, renal hemodynamics, and morphology as previously reported. However, the mice exhibited a reduced renal nitrite excretion and glomerular cGMP content associated with a reduced glomerular capillary surface area, indicating that kinins influence glomerular trophicity. The relevance of these findings to the reported increased susceptibility of these mice to pathological insult will require further investigation.

The regulation of kinin receptor desensitization following ligand binding has demonstrated that B₂ receptors are rapidly desensitized and sequestered, whereas B₁ receptors are not (7, 21, 34). In this issue, Faussner et al. (20) show that B₂ kinin receptor sequestration is highly dependent on receptor expression levels, with high expressing cells exhibiting low sequestration compared with low expressing cells. This anomaly is overcome by the use of low ligand concentrations so that the internalization machinery does not become saturated by high numbers of receptors. This is an important observation because expression of recombinant receptors and mutants is a common technique used for the study of kinin as well as other G protein-coupled receptors. Whereas most studies have focused on the immediate consequences of ligand binding on cell surface receptors, Blaukat et al. (3) investigated the long-term (i.e., up to 24 h) effects of ligand exposure on B₂ receptor expression. They found a 50% reduction in surface binding sites that was paralleled by a similar decrease in total B₂ receptor protein. This was regulated posttranslationally and involved a reduction of receptor protein synthesis and stability. It will be of interest to determine the relationship of this response to the initial internalization/sequestration event upon ligand binding and to determine the consequences of this long-term downregulation in situations where kinin levels are elevated, for example, during ACE inhibition.

The synthesis of specific B₂ receptor antagonists represented a landmark development for investigations in the kallikrein-kinin field (58). Although the modified peptide antagonist developed by Hoescht, HOE 140, is a commonly used potent antagonist (25, 62), detailed analysis of its effects on B₂ receptors in various species and cell systems have revealed a puzzling variety of effects. Depending on the system studied, it can act as a full agonist, partial agonist, reverse agonist, or a full antagonist. The article by Schroeder et al. (47) represents an important advance in our understanding of this phenomenon. The authors took advantage of the unusual properties of the chicken or "ornithokinin" receptor (for which HOE 140 is a full agonist but bradykinin is not) and constructed chimeric human/chick receptors. Their elegant studies on these receptors revealed that replacement of the amino-terminal portion of the human B₂ receptor (amino acids 1-127) with the corresponding segments of the ornithokinin receptor confers full agonistic activity of HOE 140 to the chimeric human receptor without apparent loss of potency of the authentic ligand, bradykinin. The profile of HOE 140 agonism/antagonism results from differences in the affinity of HOE 140 for the G protein-uncoupled state of the receptor. These interesting results relate not only to kinin receptors but have broader implications for the functioning of other G protein-coupled receptors as well.

These 13 articles in this Special Topic represent a portion of our current understanding of the importance of kinins, ACE, and components of this pathway in cardiovascular physiology and cardiovascular pathophysiology. These studies have clarified controversies, described the importance of new and old pathways, and indicated areas that need additional research. With this knowledge, we should follow the advice of Dr. Erdös and "keep looking forward to next week's findings in the laboratory."

	FOOTNOTES

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.

10.1152/ajpheart.00164.2003

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