Introduction

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Vascular smooth muscle cells can be stimulated or inhibited by a variety of vasoactive agents that activate a network of signal transduction pathways. The identification of the receptors, ion channels and effectors associated with these pathways has revealed an increasing complexity in the modulatory mechanisms that may contribute to the regulation of vascular tone. The contraction of vascular smooth muscle by several agonists including NE and vasoactive peptides such as ANG II and vasopressin is determined by the activation of receptors associated with guanine nucleotide binding proteins that activate phospholipase C. This promotes the formation of Ins(1,4,5)P₃ and diacylglycerol (Altura and Altura, 1977; Legan et al., 1985; Nabika et al., 1985; Somlyo and Somlyo, 1994; Villalobos-Molina et al., 1982). Ins(1,4,5)P₃ then causes the mobilization of intracellular Ca⁺⁺ and vascular contraction. In comparison, relaxation of vascular smooth muscle can result from activation of receptors associated with the stimulation of adenylyl cyclase or guanylyl cyclase. Stimulation of adenylyl cyclase by agonists such as isoproterenol and PGI₂ increases cAMP which activates cAMP-dependent protein kinase (Bolton, 1979; Somlyo and Somlyo, 1994) or, under certain conditions, cGMP-dependent kinase (Jiang et al., 1992; Lincoln et al., 1990; Murthy and Makhlouf, 1995) to evoke vasodilation. Consequently, receptors linked to phospholipase C or adenylyl cyclase have opposing effects on the vascular smooth muscle cell, and interactions between these pathways could be important in the modulation of vascular tone.

Cross-talk between signaling pathways that activate either phospholipase C or adenylyl cyclase has been demonstrated in a variety of preparations. For example, activation of adenylyl cyclase with isoproterenol or forskolin to elevate cAMP in bovine iris sphincter muscle has been shown to inhibit carbachol-induced Ins(1,4,5)P₃ formation and smooth muscle contraction (Tachado et al., 1989, 1992). Similar results have been found in the rat aorta in which increases in cAMP inhibited the accumulation of Ins(1,4,5)P₃ and the contractile response to NE (Lincoln and Fisher-Simpson, 1983; Manolopoulos et al., 1991; Rapoport, 1991). In comparison, receptor activation of second messenger pathways which modulate Ins(1,4,5)P₃ turnover has been shown to enhance agonist-stimulated cAMP accumulation in rat pinealocytes and bovine adrenal cortical and glomerulosa cells (Baukal et al., 1994; Brami et al., 1987; Vanecek et al., 1985). Furthermore, we previously demonstrated an effect of ANG II to potentiate agonist-induced cAMP formation in cultured vascular smooth muscle cells (Kubalak and Webb, 1993). Such cross-talk in the vasculature may be a process by which ANG II sensitizes blood vessels to the counter-regulatory effects of dilator hormones that act through cAMP stimulation.

In the present study, we demonstrate a role for ANG II as a modulator of vasodilation in the intact blood vessel. We show that ANG II enhances the relaxant response of rat isolated aortic ring preparations to vasodilator agents that act specifically through adenylyl cyclase activation. The data indicate that the action of ANG II to stimulate contraction and concomitantly facilitate adenylyl cyclase activation may be an important mechanism in the regulation of vascular tone.

	Methods

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Tissue preparation. Male Sprague-Dawley rats (200-300 g) were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and exsanguinated. The thoracic aorta was removed and cleaned of excess connective tissue and fat and cut into ring segments (4 mm in length). The endothelium was removed in some experiments by gently abrading the intimal surface of the aortic rings with a wooden applicator rod. The rings were mounted horizontally under isometric conditions in a 10-ml organ bath by inserting a tungsten wire through the lumen of the vessel. The preparation was then anchored to a stationary support and another wire, similarly inserted, was connected to a Grass FT03C force-displacement transducer. The responses were recorded on a Grass ink-writing polygraph. The preparations were placed at a resting tension of 1.5 g and allowed to equilibrate for at least 1 h in Bülbring-modified Krebs' solution with the following composition (mM): NaCl, 133; KCl, 4.7; NaH₂PO₄, 1.35; NaHCO₃, 16.3; MgSO₄, 0.61; glucose, 7.8; and CaCl₂, 2.52, pH 7.2 (Bülbring, 1953). The solution was maintained at 37°C and aerated with 95% O₂ and 5% CO₂.

Materials. ()-Norepinephrine bitartrate (NE), ()-isoproterenol bitartrate, endothelin 1, ANG II, phenylephrine hydrochloride, phentolamine methanesulfonate, indomethacin, sodium nitroprusside, N⁶,2-O-dibutyryladenosine 3-5cyclic monophosphate (sodium salt; dibutyryl cAMP) and acetylcholine chloride were obtained from Sigma (St. Louis, MO). Iloprost was a gift from Schering AG (Berlin, Germany). Sodium pentobarbital was supplied by Abbott Laboratories (North Chicago, IL).

Statistical analysis. Contractions to KCl, phenylephrine and endothelin 1 are expressed in grams. Relaxant responses to isoproterenol, iloprost, dibutyryl cAMP and sodium nitroprusside are expressed as the percentage relaxation of the induced contraction. Data are expressed as the mean ± S.E. and were analyzed by two-way ANOVA for repeated measures or paired Student's t test where appropriate. A probability of less than .05 was considered significant.

	Results

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Effect of ANG II on isoproterenol-induced relaxation. We examined the influence of ANG II on isoproterenol-induced relaxation of isolated rat aorta using three different vasoconstrictors to test the selectivity of ANG II for a specific contractile agonist: 1) KCl, a vasoconstrictor that mediates contraction independently of receptor activation and Ins(1,4,5)P₃ formation, 2) phenylephrine, an alpha-1 adrenoceptor agonist and 3) endothelin 1, a peptide vasoconstrictor agonist (Yanagisawa et al., 1988). A representative trace illustrating the standard protocol is presented in figure 1. Potassium chloride (30 mM) produced a sustained contraction of the aortic ring preparations. Subsequent exposure of preparations to 0.1 µM ANG II evoked an additional contraction, but this effect was transient and the vessels completely returned to the initial level of induced tone after approximately 20 min. During this time, the contraction of paired, time-matched control preparations was sustained. Isoproterenol (0.01-1.0 µM) caused a concentration-dependent vasodilation of the KCl-precontracted preparations that was significantly enhanced after treatment with ANG II (P < .01) (fig. 2). The maximal relaxant response was increased from 81 ± 4% to 108 ± 5% (P < .001) but the EC₅₀ value for isoproterenol was unaffected by ANG II pretreatment (table 1). Both the initial contractile response to ANG II and the effect of ANG II to enhance isoproterenol-induced relaxation were blocked in aortae pretreated with the AT₁ receptor antagonist, losartan (data not shown).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Isolated ring preparation of rat aorta precontracted with KCl (30 mM) in the presence of indomethacin (1 µM) and phentolamine (1 µM). After the KCl-induced contraction had plateaued, ANG II (0.1 µM) was applied to the tissue. The peptide evoked a transient contraction that returned to the initial level of induced tone after about 20 min. Subsequently, a cumulative concentration-response curve to isoproterenol (0.01-1 µM) was established.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   ANG II enhancement of isoproterenol-induced vasodilation of rat aortic ring preparations precontracted with KCl. Vessels were precontracted to equivalent levels of tone with KCl (30 mM). Cumulative concentration-response curves to isoproterenol were constructed in the absence and presence of ANG II (0.1 µM), as described under "Methods." Data are expressed as percentage relaxation of the contraction to KCl. Each point represents the mean response ± S.E.; n = 12. Relaxant responses were significantly increased in preparations treated with ANG II compared with controls (P < .01; two-way ANOVA for repeated measures).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   ANG II enhancement of isoproterenol-induced vasodilation of rat aortic ring preparations precontracted with either (a) phenylephrine or (b) endothelin 1. Vessels were precontracted to equivalent levels of tone with either phenylephrine (1 µM) or endothelin 1 (3 nM). Cumulative concentration-response curves to isoproterenol were constructed in the absence and presence of ANG II (0.1 µM), as described under "Methods." Data are expressed as percentage relaxation of induced tone. Each point represents the mean response ± S.E.; n = 6 and 9 for phenylephrine- and endothelin 1-precontracted vessels, respectively. Relaxant responses were significantly increased in preparations treated with ANG II compared with controls (P < .05; two-way ANOVA for repeated measures).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   ANG II enhancement of isoproterenol-induced vasodilation of endothelium-denuded aortic ring preparations precontracted with phenylephrine. Vessels were precontracted to equivalent levels of tone with phenylephrine (30 nM). Cumulative concentration-response curves to isoproterenol were constructed in the absence and presence of ANG II (0.1 µM), as described under "Methods." Data are expressed as percentage relaxation of the contraction to phenylephrine. Each point represents the mean response ± S.E.; n = 9. Relaxant responses were significantly increased in preparations treated with ANG II compared with controls (P < .05; two-way ANOVA for repeated measures).

Selectivity of ANG II on receptor-stimulated cAMP-mediated vasodilation. To determine whether the enhancement of cAMP-mediated relaxation by ANG II was selective for beta adrenoceptors, we examined the influence of ANG II on the relaxant response of rat aortic ring preparations to iloprost, a stable PGI₂ analog. Iloprost (0.01-3 µM) caused concentration-dependent relaxations of KCl-precontracted ring preparations (fig. 5) though the dilation produced was less than that observed with isoproterenol (fig. 2). In control vessels, iloprost produced a maximal relaxation of 44 ± 6% compared with a maximal response of 81 ± 4% relaxation in vessels treated with isoproterenol. Nonetheless, exposure of aortae to ANG II significantly enhanced the concentration-response curve for iloprost-induced vasodilation (P < .05) (fig. 5). When vessels were pretreated with ANG II, the maximal relaxant effect of iloprost was increased to 68 ± 6%. The sensitivity of the aortae to iloprost was not altered by the peptide, however. The EC₅₀ for iloprost in control tissues was 3.1 ± 0.8 nM and 2.1 ± 0.3 nM in ANG II-treated preparations. Such findings indicate that ANG II enhancement of cAMP-mediated vasodilation in the intact vessel is not specific for beta adrenoceptors but is also observed when receptors for PGI₂ are activated. In each instance the principal effect is an increased effectiveness of the dilator agonist with no change in vascular sensitivity.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   ANG II enhancement of iloprost-induced vasodilation of rat aortic ring preparations precontracted with KCl. Vessels were precontracted to equivalent levels of tone with KCl (30 mM). Cumulative concentration-response curves to iloprost were constructed in the absence and presence of ANG II (0.1 µM), as described under "Methods." Data are expressed as percentage relaxation of the contraction to KCl. Each point represents the mean response ± S.E.; n = 9. Relaxant responses were significantly increased in preparations treated with ANG II compared with controls (P < .05; two-way ANOVA for repeated measures).

Effect of ANG II on dibutyryl cAMP-mediated vasodilation. Because the modulatory effect of ANG II on cAMP-mediated vasodilation was not selective for a specific receptor, we tested the possibility that ANG II may modify the dilator action of cAMP. A single concentration of dibutyryl cAMP (30 µM) was selected for use because this concentration evoked a consistent, time-dependent response. Vessels precontracted with KCl (30 mM) were maximally relaxed within 30 min after application of dibutyryl cAMP. Treatment of tissues with ANG II (0.1 µM) did not affect the time course or the magnitude of the relaxant response. The relaxant response of control and ANG II-treated tissues after 15 min incubation with dibutyryl cAMP was 54 ± 5% and 53 ± 5%, respectively. The maximal relaxation achieved in controls was 86 ± 5% and 90 ± 9% in preparations treated with ANG II (n = 6).

Effect of ANG II on cGMP-mediated vasodilation. To determine the specificity of ANG II for signal transduction pathways coupled to adenylyl cyclase stimulation, we evaluated the effect of ANG II on the vasodilation of aortic ring preparations by sodium nitroprusside, a direct activator of soluble guanylyl cyclase. Sodium nitroprusside (0.01-3 µM) evoked a concentration-dependent relaxation of preparations precontracted with KCl (30 mM) that was not significantly affected by exposure to ANG II (0.1 µM) (fig. 6). The EC₅₀ values for sodium nitroprusside in control and ANG II-treated preparations were 34.8 ± 1.1 nM and 24.7 ± 6.7 nM, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The effect of ANG II on the relaxation of rat isolated aortic ring preparations by sodium nitroprusside. Vessels were precontracted to equivalent levels of tone with KCl (30 mM). Cumulative concentration-response curves to sodium nitroprusside were constructed in the absence and presence of ANG II (0.1 µM), as described under "Methods." Data are expressed as percentage relaxation of KCl-induced contraction. Each point represents the mean response ± S.E.; n = 6. Relaxant responses were not significantly affected by ANG II.

	Discussion

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We previously observed an effect of the vasoconstrictor peptide, ANG II, to synergistically enhance agonist-induced cAMP formation by isoproterenol, PGI₂ or adenosine in cultured vascular smooth muscle cells from rat aorta (Kubalak and Webb, 1993). This phenomenon was independent of any change in cell surface receptors or EC₅₀ values for the agonists but reflected an action of ANG II to increase maximal stimulation of cAMP production in the smooth muscle cell. In the present study, the significance of these cellular findings to the regulation of vascular tone in intact blood vessels was examined with isolated ring preparations of rat aorta. The data show that treatment of aortae with ANG II enhanced relaxation of these vessels by both the beta adrenoceptor agonist isoproterenol and the PGI₂ analog, iloprost. The maximal relaxant response to both vasodilators was significantly augmented after exposure of the tissues to ANG II, whereas the EC₅₀ values for relaxation were unaltered by peptide treatment. This potentiating action of ANG II was not influenced by removal of the endothelium but was blocked by the AT₁ receptor antagonist losartan. These results are consistent with our findings in cultured cells and point to an action of ANG II which serves to facilitate the relaxant effect of dilator agonists that act through cAMP formation.

The degree of vasodilation of isolated blood vessels can be influenced by the initial level of vascular tension (Eckly et al., 1994). In this study, application of ANG II to the aortic ring preparations caused an acute increase in the level of precontraction tone, but this effect was transient in nature and tone was always allowed to return to the initial level before the application of any vasodilator agonist. In addition, for each vasoconstrictor used, the effect of ANG II treatment was examined only in paired tissues that were contracted to equivalent levels of tension. Moreover, treatment of vessels with ANG II did not enhance the effect of all vasodilator agents tested. Relaxant responses to sodium nitroprusside, a vasodilator that stimulates cGMP, and to dibutyryl cAMP were unchanged by ANG II. Thus, the effect of ANG II to potentiate vascular relaxation by dilator agonists that stimulate cAMP formation is not a generalized phenomenon and cannot be attributed to the protocol used. The capacity of blood vessels to relax in response to vasodilators can also be influenced by the contractile agonist used to induce tone (Urquhart and Broadley, 1991). Consequently, three different contractile agents, KCl, phenylephrine and endothelin 1, were used to evaluate the specificity of ANG II effects. Potassium chloride evokes a contraction independently of second messenger formation, whereas the major mechanism of excitation-contraction coupling for phenylephrine and endothelin 1 is receptor-stimulated Ca⁺⁺ mobilization through Ins(1,4,5)P₃ generation (Masaki et al., 1994; Miller et al., 1993; Somlyo and Somlyo, 1994; Somlyo et al., 1985). The data clearly demonstrate that ANG II significantly enhanced the concentration-response curve for isoproterenol-induced vasodilation irrespective of the vasoconstrictor used for the initial induction of tone.

The enhancement of vasodilation by ANG II was not specific for beta adrenoceptors because the peptide also potentiated the relaxation produced by the PGI₂ analog, iloprost. The maximal iloprost-induced relaxant response was increased from 44 ± 6% in control aortae to 68 ± 6% in vessels treated with ANG II. When a vasoconstrictor peptide such as ANG II activates phospholipase C to initiate contraction of vascular smooth muscle, it simultaneously stimulates phospholipase A₂ to promote the local formation of PGI₂ and other dilator prostaglandins (Moncada et al., 1977; Webb, 1982). In the present study, all experiments were conducted in the presence of the cyclooxygenase inhibitor indomethacin to eliminate the influence of these eicosanoids. However, local production of vasodilator prostaglandins such as PGE₂ and PGI₂ which act through adenylyl cyclase activation is considered to be an important feedback mechanism for modulating the effects of constrictor agents and, in this context, the action of ANG II to facilitate the relaxant effects of such prostaglandins may be of particular relevance.

An alternative pathway by which relaxation of blood vessels may be elicited involves the stimulation of guanylyl cyclase in vascular smooth muscle cells. Vasodilators such as sodium nitroprusside produce vascular relaxation by direct activation of soluble guanylyl cyclase to increase cGMP levels (Kukovetz et al., 1979; Murad et al., 1978). In contrast to the observations for isoproterenol and iloprost, relaxation of KCl-contracted aortae by sodium nitroprusside was not affected by exposure of tissues to ANG II. The relaxation of preparations by direct application of dibutyryl cAMP was also unaltered by ANG II treatment. These results indicate that the action of ANG II to potentiate arterial vasodilation is selective for agonists that stimulate adenylyl cyclase and suggest that the principal effect of the peptide is to amplify cAMP production, as demonstrated in our cellular experiments (Kubalak and Webb, 1993), without changing the subsequent dilator action of the nucleotide once formed.

Enhancement of agonist-stimulated cAMP production by ANG II has been observed in bovine adrenal cortical and glomerulosa cells (Baukal et al., 1994; Brami et al., 1987) and myocytes from hypertrophied rat heart (Sunga and Rabkin, 1994), as well as in vascular smooth muscle cells (Kubalak and Webb, 1993). The mechanism for this effect appears complex and varies with cell type. For example, a role for the protein phosphatase, calcineurin, has been proposed for adrenal glomerulosa cells whereas a change in the inhibitory guanine nucleotide binding protein, G_i, was suggested in hypertrophied myocytes. In comparison, the data indicate that ANG II enhancement of cAMP stimulation in vascular smooth muscle cells results largely from the peptide's action to elevate intracellular Ca⁺⁺ which then combines with calmodulin to facilitate adenylyl cyclase activation, an idea supported by the expression in these cells of type III adenylyl cyclase (Webb et al., 1995), a Ca⁺⁺-calmodulin-sensitive isoform of the effector (Choi et al., 1992).

In summary, exposure of arterial blood vessels to the vasoconstrictor peptide ANG II was found to enhance the relaxant effect of agonists that act through the stimulation of cAMP formation. This action was manifest as an increase in maximal response whereas the sensitivity of vessels to the vasodilators was unaltered. The most pronounced effect of ANG II treatment was on the vascular relaxation produced by iloprost, an analog of PGI₂. This is of particular interest because when ANG II initiates vascular constriction, it simultaneously promotes the local formation of PGI₂ as well as other counter-regulatory prostaglandins, and the present data indicate that the peptide has yet an additional action to amplify the effectiveness of these dilator agents. When viewed collectively, such a sequence of events provides a tightly integrated mechanism for the modulation of vascular tone and for buffering the arterial blood vessel against overstimulation by vasoconstrictor peptides.