INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

AGING IS ASSOCIATED with a number of specific physiological and metabolic changes affecting several organs and systems. The aged cardiovascular system exhibits diminished responsiveness to -adrenergic receptor (-AR) stimulation in the rat aorta (5), the mesenteric and pulmonary arteries (10, 16), the rabbit and guinea pig aorta (26), and the human dorsal hand veins (31). Multiple mechanisms have been implicated in this phenomenon, including changes in the affinity of -AR (40), coupling efficacy of G proteins such as G_s (25, 34), function of effector enzymes such as adenylyl cyclase (24), and/or activity of -AR-receptor kinases (15, 33). However, at this time, the exact nature of the age-induced impairment of the -AR pathway remains unknown.

Vascular tone is produced via a complex interaction among a variety of vasoactive agents that affect vascular smooth muscle cells. These vasoactive agents activate (or deactivate) an elaborate network of signal transduction pathways that modulate blood pressure (41). Agents such as isoproterenol (Iso) activate -ARs that stimulate adenylyl cyclase to increase intracellular cAMP, which activates protein kinase A (PKA) and ultimately elicits vasorelaxation (29). In contrast, agents such as angiotensin II (ANG II) elicit increases in intracellular calcium, which lead to activation of myosin light chain kinase and thus vasoconstriction (37).

A recent study (2) has shown a novel action of ANG II in that it enhanced cAMP-mediated vasorelaxation via ANG II type 1 (AT₁) receptors. This enhancement was endothelium independent and was also observed when other receptors, besides -AR, upstream from adenylyl cyclase were activated (prostaglandin I₂ receptor). However, ANG II did not enhance the relaxant effect of dibutyryl-cAMP. This suggests that this effect of ANG II is upstream from PKA in the -AR signaling cascade and points to cAMP production as the amplification mechanism by which ANG II exerts its effect on vasorelaxation. This hypothesis is further supported by in vitro studies, which have shown that ANG II enhances -AR-mediated cAMP production in cultured aortic vascular smooth muscle cells (22, 30, 43) as well as in preglomerular microvascular smooth muscle cells (19, 27).

It has been suggested that the ANG II enhancement of agonist-induced cAMP production and subsequent augmented vasorelaxation may preserve vascular smooth muscle from the provasoconstrictive and proproliferative effects of ANG II (27). Therefore, this regulatory mechanism, if malfunctioning, could contribute to cardiovascular diseases such as hypertension and atherosclerosis (19). Indeed, it is conceivable that ANG II could possibly correct the age-related decline in vascular -AR function. Such a finding could have important clinical implications and may provide new therapeutic options. Also, understanding whether cross-talk between ANG II signaling cascades and -AR signaling cascades changes with age would yield critical information about the molecular basis of aging in the cardiovascular system.

Consequently, we examine the interaction among aging, -AR-mediated vasorelaxation, and ANG II. We question whether the provasorelaxing effect of ANG II is preserved during aging. The experiments are conducted with aortas from young (6 wk and 6 mo) and old (12 and 24 mo) male Fischer 344 rats, an animal model widely used in experimental aging research (9).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Chemicals were obtained from Sigma (St. Louis, MO) unless indicated. All vasoactive chemicals were diluted in deionized water. Composition of phosphate-buffered saline (PBS) is as follows (in mM): 114 NaCl, 4.7 KCl, 1.15 KH₂PO₄, 1.1 Na₂HPO₄, 1.18 MgSO₄, 15 NaHCO₃, 1.25 CaCl₂, and 5.0 glucose.

Animals. Male Fischer 344 rats in four age groups (6 wk and 6, 12, and 24 mo old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). Rats were euthanized by pentobarbital sodium sedation and exsanguination in accordance with the procedures approved by the Institutional Animal Care and Use Committee at the Portland VA Medical Center. Thoracic aortas were quickly removed and cleaned of the adhering fat and connective tissue in ice-cold PBS gassed with 95% O₂-5% CO₂.

Vascular reactivity studies. Isometric tension development was determined as described (5). Aortic rings (3-4 mm wide) were suspended between a fixed support and a Grass Instruments (Quincy, MA) FT.03 force transducer in a muscle bath system containing PBS at 37°C. Rings were stretched to their optimal length and allowed to equilibrate for 90 min. After equilibration, the aortic rings were challenged three times with 45 mM KCl to confirm responsiveness before testing. Rings that responded to KCl were used to create a dose-response relationship (1 nM-10 µM) to phenylephrine (PE) by cumulative addition. After washout and if experimental conditions dictated, we added losartan (10 µM; an AT₁-receptor antagonist) and/or PD-123319 (10 µM; an AT₂-receptor antagonist). Vessels were then precontracted to 70% of the maximum level of PE by addition of appropriate dose of PE (~0.3 µM). Once a stable level of precontraction was achieved, 0.05 µM isobutylmethylxanthine (IBMX; a nonspecific phosphodiesterase inhibitor) was added. This low dose of IBMX did not alter tension. Subsequently, either 0.1 µM ANG II or appropriate control was added. ANG II evoked a transient contraction that returned to the initial level of PE-induced tension after ~15 min. Thereafter, a dose-response relationship was determined for Iso, forskolin (FSK; a direct adenylyl cyclase activator), IBMX, or dibutyryl-cAMP (a nonhydrolyzable, cell-permeable cAMP analog). Figure 1 shows the complete experimental paradigm.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Representative tracing of isolated ring preparation. Aortic rings were precontracted with phenylephrine (PE; 70% of maximal effect of PE), and, after the PE-induced contraction reached plateau, 0.05 µM isobutylmethylxanthine (IBMX) was applied. Five minutes after the addition of IBMX, ANG II (0.1 µM) was added, which induced a transient (~15 min) contraction that returned to the initial level of PE-induced tone. Subsequently, a dose-response relationship to isoproterenol (Iso; 0.001 µM-10 µM) was achieved via cumulative addition. If experimental design dictated, losartan (LO; 10 µM) or PD-123319 (PD; 10 µM) were added as indicated.

cAMP determination. Aortas were collected from rats of each age group (n = 7 rats/age group) and cut into five equal-sized rings. Each ring was placed into a gas-tight tube containing gassed PBS and allowed to equilibrate for 10 min at 37° C. Each ring was then exposed to a different treatment cumulatively as follows (see Fig. 3B): 1) Basal treatment (vehicle only for 30 min); 2) IBMX treatment (1 µM IBMX for 30 min); 3) IBMX + ANG II treatment (IBMX for 30 min and then 0.1 µM ANG II starting 10 min after the addition of IBMX and continuing through 30 min); 4) IBMX + Iso treatment (IBMX for 30 min and then 1 µM Iso 20 min after the addition of IBMX and continuing through 30 min); and 5) IBMX + ANG II + Iso treatment (IBMX for 30 min, ANG II starting 10 min after the addition of IBMX, and Iso 20 min after the addition of IBMX and continuing through 30 min). Immediately after treatment, rings were immersed into ice-cold 0.1 N HCl, finely minced, and then homogenized in a glass-glass motor-driven Kontes tissue homogenizer. Homogenates were centrifuged at 500 g for 15 min at 4°C, and the resulting cleared supernatant was analyzed for cAMP content with a commercially available enzyme-immunoassay kit per the manufacturer's instructions (Assay Designs, Ann Arbor, MI). The protein concentration of the pellet was determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL). Concentration of cAMP is expressed as picomoles of cAMP per milligrams of protein.

Statistical analysis. Results are expressed as mean values ± SE. The experimental unit was the number of animals.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of ANG II on Iso-stimulated vasorelaxation in young and old rats. We examined the influence of ANG II on Iso-induced relaxation in 6-wk-old and 6-, 12-, and 24-mo-old animals to determine the effect of age on this phenomenon. A representative tracing is shown in Fig. 1. Isolated rings achieved a sustained contraction ~7 min after exposure to PE. Addition of a low dose of IBMX (0.05 µM) after the PE-mediated contraction reached plateau had no effect. Subsequent addition of ANG II (0.1 µM) evoked further contraction over that produced by PE. However, the ANG II effect was transient, and tension returned to that produced by PE by ~15 min. Control aortic rings were time matched and received ANG II vehicle (deionized H₂O). Neither the amplitude nor the time course of the contraction produced by ANG II were significantly different among age groups (data not shown). Cumulative addition of Iso (1 nM-10 µM) produced dose-dependent vasorelaxation. Regardless of presence or absence of ANG II, age-related differences were detected in Iso-ED₅₀ and Iso-ED_max (Table 1). The presence of ANG II significantly reduced Iso-ED₅₀ in 6-wk-old and 6-mo-old animals but not 12- or 24-mo-old animals (Fig. 2). ANG II also significantly enhanced the ED_max effect of Iso in 6-mo-old animals. Iso-ED₅₀ was reduced in the aortas from 6-wk-old and 6-mo-old animals by 43.7 ± 1.1 and 28.1 ± 1.5%, respectively, but increased by 9.1 ± 1.1 and 2.9 ± 0.9% in 12- and 24-mo-old animals, respectively. The ED_max effect of Iso was enhanced in 6-mo-old animals only (Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effect of ANG II on Iso-mediated vasorelaxation in 6-wk-old (A), 6-mo-old (B), 12-mo-old (C), and 24-mo-old (D) animals. Aortic rings were contracted with PE, incubated in ANG II (0.1 µM; ) or vehicle (), and then relaxed with a cumulative addition of Iso after the ANG II-mediated contraction returned to its initial tone. Data are expressed as a percentage of PE-induced contraction versus dose of Iso. Each point represents the mean response to Iso ± SE (n = 12 animals/age group). *P < 0.05 via 2-way ANOVA (± ANG II by dose of Iso) with Bonferroni's post hoc comparison. The efficacy of Iso alone (solid bars) versus ANG II + Iso (open bars) effect on the concentration of the relaxing agent (Iso) producing a 50% maximal effect (Iso-ED50; E) and the concentration of the relaxing agent (Iso) producing a maximum effect (Iso-EDmax; F) is also shown. ANG II significantly decreased the Iso-ED50 in 6-wk-old and 6-mo-old animals, whereas ANG II increased the EDmax effect of Iso in 6-mo-old animals. *P < 0.05 via 2-way ANOVA (± ANG II by age) with Bonferroni's post hoc comparison.

Effect of ANG II on Iso-stimulated cAMP accumulation in young and old rats. To establish a mechanism for ANG II-Iso interaction, aortic rings from 6-wk-old and 6-, 12-, and 24-mo-old animals (n = 7 rats/age group) were treated as illustrated in Fig. 3B, and cAMP accumulation was determined. There were no age-related differences in either basal, IBMX-, or IBMX + ANG II-stimulated cAMP accumulation (Fig. 3A). A significant decline in IBMX + Iso-stimulated cAMP accumulation was observed with age (275.3 ± 34.1, 165.5 ± 17.8, 125.6 ± 16.8, and 95.3 ± 19.7 pmol/mg for 6-wk-old and 6-, 12-, and 24-mo-old animals, respectively). Finally, ANG II significantly altered the stimulatory effect of Iso on cAMP accumulation in 6-wk-old and 6-mo-old animals but not in 12- or 24-mo-old animals (+44.6 ± 3.1, +63.6 ± 7.5, 6.4 ± 1.3, and 7.3 ± 3.3%, respectively, for each age group; Fig. 3A).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of ANG II (0.1 µM) on Iso (1 µM)-mediated cAMP production in aortas (n = 7 rats/age group) from 6-wk-old and 6-, 12-, and 24-mo-old animals (A) as stimulated in the protocol depicted in B. An age-related decline in cAMP production was detected in response to Iso. aP < 0.05 compared with 6 wk old; bP < 0.05 compared with 6 mo old. ANG II (0.1 µM) enhanced the effect of Iso on cAMP production in aortas from 6-wk-old and 6-mo-old animals only. *P < 0.05 compared with the effect of Iso alone to ANG II + Iso. Results determined by 2-way ANOVA (±ANG II by age) with Bonferroni's post hoc comparison. Notice discontinuous ordinate. EQUILIB, equilibrium.

Determination of ANG II receptor type involved in enhanced Iso-stimulated vasorelaxation. Two receptor types for ANG II have been identified: AT₁ and AT₂, both of which have been found in vascular tissue, including the rat aorta (39). Accordingly, both AT₁ and AT₂ receptors have distinct signal transduction pathways and subsequent physiological effects (reviewed in Ref. 17). To establish whether AT₁ and/or AT₂ receptor stimulation was responsible for ANG II/Iso enhancement of vasorelaxation, aortic rings from 6-wk-old and 6-, 12-, and 24-mo-old animals (n = 8 rats/age group) were examined in the presence or absence of losartan and/or PD-123319. Losartan blocked the contractile effect of ANG II in each age group. However, PD-123319 did not significantly change the force or duration of ANG II-mediated vasoconstriction in any age group (data not shown). The enhancing effect of ANG II (in terms of reduced Iso-ED₅₀ and increased Iso-ED_max) was blocked by losartan in 6-wk-old and 6-mo-old animals, whereas PD-123319 did not significantly alter Iso-ED₅₀ or Iso-ED_max when ANG II was present. Neither losartan nor PD-123319 affected Iso-ED₅₀ or Iso-ED_max in 12- or 24-mo-old animals (Fig. 4).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Effect of LO and or PD-123319 on Iso-ED50 (A) and Iso-EDmax (B) on ANG II-enhanced, Iso-stimulated vasorelaxation in 6-wk-old and 6-, 12-, and 24-mo-old animals (n = 8/age group). *P < 0.05 compared with Iso alone within each age group.

Effect of ANG II on FSK-, dibutyryl-cAMP-, or IBMX-stimulated vasorelaxation in young and old rats. Experiments were performed to localize the signal transduction targets in the ability of ANG II to enhance agonist-induced vasorelaxation. Aortic rings from 6-wk-old and 12-mo-old animals (n = 10/age group) were treated as shown in Fig. 1 except vessels were relaxed with either FSK (1 nM-10 µM), dibutyryl-cAMP (5 µM-1 mM), or IBMX (1 nM-10 µM). ANG II significantly enhanced the effect (both ED₅₀ and ED_max) of FSK in 6-wk-old but not 12-mo-old animals (Fig. 5). In 6-wk-old animals, the ED₅₀ for FSK was reduced 48.6 ± 6.5% by ANG II pretreatment (35.4 ± 5.6 vs. 18.8 ± 4.1 nM). The maximal effect of FSK was enhanced 9.1 ± 0.8% via ANG II pretreatment in 6-wk-old animals (100.2 ± 1.1 vs. 110.9 ± 1.5%). In 12-mo-old animals, the ED₅₀ was 33.5 ± 6.9 versus 29.9 ± 6.2 nM, whereas the ED_max was 106.3 ± 1.3 versus 104.5 ± 1.3% with and without ANG II pretreatment, respectively. Efficacy of dibutyryl-cAMP and IBMX were not altered with ANG II pretreatment (data not shown). Finally, no age-related changes were detected in the efficacy of either FSK (Fig. 5) or IBMX (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of ANG II on forskolin (FSK)-mediated vasorelaxation in 6-wk-old (A) and 12-mo-old (B) animals. Aortic rings were contracted with PE, incubated in ANG II (0.1 µM; ) or vehicle (), and then relaxed with a cumulative addition of FSK after the ANG II-mediated contraction returned to its initial tone. Data are expressed as a percentage of PE-induced contraction versus dose of FSK. Each point represents the mean response to FSK ± SE (n = 10 animals/age group). *P < 0.05 via 2-way ANOVA (± ANG II by dose FSK) with Bonferroni's post hoc comparison. Efficacy of FSK alone (solid bars) versus ANG II + FSK (open bars) effect on FSK-ED50 (C) and FSK-EDmax (D). ANG II significantly decreased FSK-ED50, whereas ANG II increased the FSK-EDmax in 6-wk-old animals only. *P < 0.05 via 2-way ANOVA (±ANG II by age) with Bonferroni's post hoc comparison.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study examines the effect of ANG II on agonist-induced vasorelaxation and cAMP production in the aortas from rats of different ages. Our results show that this effect of ANG II on agonist-induced vasorelaxation is limited to young (6 wk old) or adult (6 mo old) rats and is absent in aged (12 and 24 mo old) animals. In addition, ANG II only enhances vasorelaxation to agonists that function upstream of PKA in the -AR signaling cascade, such as Iso and FSK. ANG II does not enhance the vasodilatory effect of IBMX or dibutyryl-cAMP. With agents that ANG II enhances efficacy, the effect appears to be mediated by AT₁ receptors because it is blocked by losartan but not PD-123319. ANG II appears to amplify vasorelaxation in the aortas from 6-wk-old and 6-mo-old animals via enhanced production of cAMP. The mechanism(s) surrounding this phenomenon are discussed below and presented schematically in Fig. 6.

View larger version (46K): [in this window] [in a new window]   Fig. 6.   Schematic representation of discussed interacting signal transduction pathways. -Adrenergic receptors (-AR) are activated by Iso, which induces coupling between the G protein (Gs) and adenylyl cyclase (AC). Upon activation by Gs, AC catalyzes the conversion of ATP to cAMP. cAMP activates protein kinase A (PKA), which ultimately promotes vasorelaxation. ANG II activates ANG II type 1 receptors (AT1-R), which couple to Gq and result in activation of phospholipase C (PLC), which promotes increases in intracellular calcium and vasoconstriction. Calcium also activates protein phosphatase 2b (PP2b). Pathways 1, 2, and 3 represent hypothetical interactions that may explain the ability of ANG II to enhance -AR-mediated responses and are reviewed in DISCUSSION.

Aging is associated with a pronounced decline in -AR-stimulated cAMP production and subsequent function (6, 10, 16, 23-25, 34). Generally, there is an age-related decrease in -AR responsiveness in the blood vessels, heart, brain, parotid gland, and lung to both circulating and pharmacological -AR agonists (21, 26). Experiments with human vessels, including in vivo studies of the dorsal hand vein and in vitro studies of the saphenous vein, found decreased -AR-mediated relaxation with age (18, 31).

The aortas from Fischer 344 rats exhibit impaired -AR-mediated vasorelaxation with age, whereas FSK-mediated relaxation is normal (5). -AR density in whole artery preparations is unaltered (40) or declines only very slightly with age (16), suggesting that age-impaired vasorelaxation is not related to -AR downregulation. However, Gurdal et al. (16) reported a complete loss of high-affinity receptors with age. The age-related loss of vasorelaxation appears to be explained by a deficiency in cAMP production but not PKA activity. Tissue cAMP accumulation to Iso stimulation is proportional to relaxation in young and old age groups, and both FSK and dibutyryl-cAMP relax both ages of vessels normally (6, 10, 20). Together, these data suggest that the age-related decline in -AR-mediated signaling may be due in part to changes in the -AR affinity state and, therefore, changes in the ability of -ARs to transduct signal with increasing age. The results of the present study confirm that there are age-related declines in cAMP production (Fig. 3) and -AR-mediated vasorelaxation (Fig. 2). The results further confirm that FSK- and IBMX-mediated vasorelaxation are unaffected by advancing age (Fig. 5).

The age-related effects of ANG II signaling are equivocal (11). Some investigators (4) have shown decreased vascular responsiveness with increasing age. Conversely, others (13) have shown an increased contractile effect to ANG II with increasing age. Still others (3) have reported no differences across age to the constrictor effect of ANG II. The differences in these reports may be explained by preparation differences (the aorta, cardiac artery, or saphenous vein), species differences (dogs, rats, rat strains, or humans), or analytical techniques (in vivo, in vitro and organ bath with tension development, or in vitro and perfused pressure development). The present study found no age-related changes in the time course or the maximal contractile effect of a single dose of ANG II.

ANG II has been shown to potentiate the effect of agents that elevate cAMP, including Iso, which is a -AR agonist. To our knowledge, the first report of this interesting phenomenon was from Nabika et al. (30) in a cultured vascular smooth muscle cell model. ANG II, functioning through activation of G_q-linked receptors, stimulates the activation of protein kinase C (PKC) and release of intracellular calcium (12). Kubalak and Webb (22) showed that when cultured vascular smooth muscle cells were exposed to ANG II before treatment with Iso, the ANG II-exposed cells had enhanced cAMP generation. This effect was partially blocked by staurosporine (an inhibitor of PKC), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, an intracellular calcium chelator), and W-7 (a calmodulin inhibitor), suggesting that ANG II exposure enhanced Iso-mediated cAMP production through either PKC, calmodulin, or changes in intracellular calcium. In a subsequent study, Zhang et al. (43) extended these results to another G_q-linked receptor agonist, arginine vasopressin, thus establishing the importance of the G_q-linked receptor pathway rather than an effect of a specific agonist. Finally, Brizzolara-Gourdie and Webb (2) tested this paradigm in whole vascular tissue and studied vasorelaxation. Their data show that when vessels were initially exposed to ANG II and then treated with G_s-linked receptor agonists, Iso, or iloprost (prostaglandin receptor agonist), there was enhanced vasorelaxation. Enhanced vasorelaxation occurred regardless of the precontracting agent (KCl, PE, or endothelin) and was not affected by the presence or absence of the endothelium. Furthermore, ANG II had no effect on relaxation stimulated by nitroprusside.

Cross-talk between G_q-linked receptors and -ARs have been studied by others. Winstel et al. (42) found that activation of PKC by G_q-linked agonists directed G protein receptor kinases (GRKs) to the membrane, enhancing -AR phosphorylation. Also, it has been shown that GRK-2 is more effective at desensitizing -ARs after its activation by PKC (8). Finally, Shih and Malbon (35) used antisense technologies to knockout PKC expression and function and determined that this manipulation produced enhanced -AR agonist-induced desensitization rather than the expected attenuation result. They also found that this effect was linked to phosphatase activity (36). These findings suggest that PKC might also be involved with -AR resensitization through interaction with a phosphatase. Therefore, phosphorylation/dephosphorylation and desensitization/resensitization of -ARs can be induced from a number of stimuli and certainly through ANG II-mediated signaling. Determining how the aging may affect the phosphorylation process is necessary.

Mechanisms of action to explain the effect of ANG II on -AR signaling are equivocal. One explanation for the effect of ANG II on -AR-mediated signaling is provided by Zhang et al. (43), as discussed above, and by Fig. 6 (pathway 1). They suggest that ANG II, functioning through AT₁ receptors, elicits increases in intracellular calcium, which activates calcium-dependent adenylyl cyclases that produce more cAMP upon -AR activation. The present data (Fig. 5) and the literature (6) suggest that there are no age-related differences in adenylyl cyclase function with aging. FSK-stimulated cAMP production or FSK-mediated vasorelaxation are unaffected with advancing age (5). However, this adenylyl cyclase hypothesis could explain the results of the present study if there were an age-related shift in the ratio of calcium-sensitive to noncalcium-sensitive cyclases. In the aortas from 6-wk-old animals, there was enhanced FSK-mediated relaxation with ANG II pretreatment. This effect of FSK disappears in the aortas from 12-mo-old animals (Fig. 5). An age-related change in adenylyl cyclase subtypes is supported in the literature. Tobise et al. (38) found that concentrations of cardiac type V adenylyl cyclase remained constant, whereas type IV adenylyl cyclase declined with advancing age.

Likewise, ANG II could somehow modify the function of the G protein G_s (Fig. 6, pathway 2). Kubalak and Webb (22) found that ANG II potentiates the ability of not only cholera toxin (directly activates G_s) but also prostaglandin I₂, adenosine, and vasopressin (all of which activate G_s-linked receptors) to stimulate cAMP accumulation in cultured vascular smooth muscle cells. Certainly, the vascular reactivity results of Brizzolara-Gourdie and Webb (2) suggest that G_s could be the target of ANG II in that Iso as well as iloprost (both of which activate G_s-linked receptors) yield enhanced vasorelaxation with ANG II pretreatment. G_s has been a target protein of interest in the field of aging in that it has been shown that cholera toxin treatment of the aortas from aged rats produced significantly less cAMP accumulation compared with younger rats (21). Mader et al. (25) have shown that cholera toxin-induced labeling of G_s decreases with age in rat aortic membranes, whereas Western blotting showed no difference in G_s protein levels. Chapman et al. (5) have also shown that cholera toxin-induced vasorelaxation likewise declines with age in rat aortas.

Another hypothesis put forth in the literature that may explain the effect of ANG II on -AR signaling is through phosphatase activity (Fig. 6, pathway 3). Calcineurin (protein phosphatase 2B) is a calcium/calmodulin-regulated phosphatase that has been recently implicated in cardiovascular pathology (28). Baukal et al. (1) found that the effect of ANG II on agonist-induced cAMP production could be blocked by FK-506 and cyclosporin A, both of which are inhibitors of calcineurin. Furthermore, Shih and Malbon (36) showed that calcineurin was directly involved in -AR resensitization. In their model, calcineurin dephosphorylated and, therefore, resensitized -AR to agonist signaling. Therefore, the ANG II effect on -AR may be via a calcineurin action on the -AR itself. Age-related changes in calcineurin itself or in ANG II-mediated activation of calcineurin are, to our knowledge, unexplored.

A final hypothesis in understanding the effect of ANG II on -AR signaling is through ANG II-mediated effects on GRKs. -AR-phosphorylation causes profound decreases in the ability of the receptor to transduce signals in response to agonist binding (14). GRKs are a superfamily of kinases that phosphorylate and desensitize G protein-linked coupled receptors (32). Three GRKs, GRK-2, GRK-3 (also known as -AR kinase 1 and 2), and GRK-5, rapidly phosphorylate and desensitize not only -ARs but also many other G_s-linked receptors upon agonist binding in numerous tissues, including the cardiovascular system (7). GRKs have been shown to be regulated by agents that elevate PKC activity, such as ANG II. Taken together, the available data suggest that activation of GRKs by ANG II would decrease receptor-mediated responses. Thus a GRK hypothesis could explain the deficiency in the stimulatory effect of ANG II on -AR-mediated vasorelaxation in old animals, because they have upregulated GRK expression and activity (33). However, GRK activation would not be involved in the stimulatory mechanism of ANG II on Iso-mediated signaling observed in the aortas from young animals.

In summary, ANG II enhances -AR-mediated cAMP production and vasorelaxation in young but not old animals. It is well established that there is an age-related decline in -AR signaling that involves a mechanism associated with -AR function and/or coupling to adenylyl cyclase. The mechanisms involved with ANG II enhanced, -AR-mediated signaling are unknown but may involve adenylyl cyclase, G_s, or calcineurin. We suggest that aging may affect a factor common to both ANG II receptors and -AR signaling pathways, or aging may impair cross-talk between these two receptor pathways.