Introduction

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Angiotensin receptors are expressed in a wide variety of cell types and regulate important physiological activities such as blood pressure, electrolyte balance, hormone secretion, tissue growth and neuronal activity by interacting with specific receptors on target organs. Two angiotensin receptor subtypes, named AT₁ and AT₂, have been identified by expression cloning from various species, including humans (de Gasparo et al., 2000). The AT₁ receptors are defined by their sensitivity to losartan, a surmountable and selective AT₁ receptor antagonist, whereas AT₂ receptors are sensitive to the selective antagonist PD123319 (Bumpus et al., 1991).

The AT₁ subtype is expressed ubiquitously and is involved in all the well known biological functions of angiotensin II (Ang II). The intracellular signal transduction events triggered by AT₁ receptors are well characterized and include G-protein coupling, as well as activation of several tyrosine kinases. In contrast to the AT₁ subtype, the physiological role of the AT₂ receptor is still unclear (Nahmias and Strosberg, 1995).

Early studies suggested that the signaling pathways activated by the AT₂ receptor involve three major cascades of intracellular events: activation of protein phosphatases and protein dephosphorylation, regulation of the nitric oxide (NO)-cGMP system, and stimulation of phospholipase A₂ and release of arachidonic acid (Huang et al., 1996; Horiuchi et al., 1997, 1999; Cote et al., 1998; Gohlke et al., 1998; Zhu et al., 1998). One important emerging function of the AT₂ receptor is the attenuation of the physiological effects mediated by the AT₁ subtype. In numerous studies, including knockout experiments, the AT₂ receptor has been shown to counteract the effects of Ang II mediated by the AT₁ receptor, suggesting that AT₂ might provide a brake for the peptide signal. Since Ang II binds to the two receptor subtypes, AT₁ and AT₂, with similar affinity, the cellular response is highly dependent on the relative expression level and/or responsiveness of both receptors (Nouet and Nahmias, 2000).

In the rat anococcygeus smooth muscle, Ang II induces contractions in a concentration-dependent fashion by activating prejunctional receptors located at the sympathetic nerve endings. Activation of the prejunctional receptor results in enhancement of norepinephrine (NE) release, which induces contractile response by activation of postjunctional ₁-adrenoceptors (Doggrell and Woodruff, 1978; James and Leighton, 1987; Li et al., 1988).

The ultrastructure of nerve terminals in the rat anococcygeus muscle also offers support for a nonadrenergic, noncholinergic enervation of approximately 40% of the nerve terminals (Gibbins and Haller, 1979). Since NO was identified as the inhibitory transmitter utilized by the nonadrenergic, noncholinergic nerve terminals, these nerves have been termed nitrergic (Rand and Li, 1993).

In the present study, we tested the hypothesis that contractile responses to Ang II, in rat isolated anococcygeus smooth muscle preparations, are due to the activation of heterogeneous angiotensin receptor populations. To this end, we assessed the effects of losartan and PD123319, in the organ bath, in the presence and absence of the selective ₁-adrenoceptor antagonist, prazosin. The analysis of drug effects in this system and the prediction of angiotensin receptor population heterogeneity were performed with the Schild plot since the Schild regression has a slope that is different from unity (Kenakin, 1992) in a heterogeneous receptor system.

	Materials and Methods

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Tissue Preparation. Male Wistar rats (200-250 g) were decapitated and the anococcygeus muscle was removed as previously described by Gillespie (1972). Tissues were placed in 5 ml of physiological salt solution (PSS) as follows: 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO₃, 0.45 mM MgSO₄, 1.03 mM KH₂PO₄, 2.5 mM CaCl₂, 11.1 mM D-(+)-glucose, 0.067 mM disodium edetate, and 0.14 mM ascorbic acid. The PSS was gassed with 5% CO₂ and 95% O₂ and maintained at 37°C, pH 7.4, with periodic checking. Isotonic transducers were used to measure changes in isotonic contraction of the tissues, which were displayed on a Harvard Universal Student Oscillograph (Harvard Apparatus, Inc., Holliston, MA) at a resting tension of 1 g as previously analyzed (data not shown). Isolated muscles were allowed to equilibrate for 45 min before making experimental observations. The organ bath PSS was repeatedly replaced with fresh PSS every 15 min. After the equilibration period, tissues were stimulated with KCl (90 mM) to check their responsiveness.

Experimental Procedures. The effects of losartan and PD123319 (0.01-10 nM) were studied after initial control cumulative concentration-effect curves to Ang II (0.1 nM-1 µM) had been obtained, and the antagonists then remained in the PSS for the remainder of the experiment. The antagonist incubation period was chosen as the required time to reach the equilibrium between drug and drug-receptor. This was performed by the administration of the Ang II EC₅₀ before and after (5-45 min) the antagonist (1 nM) had been added to the PSS. Required time was the minimum time required to induce the maximum inhibitory effect. The reversibility of the antagonist and antagonist-receptor interaction was analyzed by administration of the Ang II EC₅₀ before and after (0-20 min) antagonist (1 nM) had been washed away. A period of 45 min between the first and second Ang II administration was always sought. The interaction of AT₁ and AT₂ receptors with the NE-dependent response was studied via addition of losartan or PD123319 in the presence of the selective ₁-adrenoceptor antagonist, prazosin (1 µM), in the PSS. The prazosin concentration of 10 times higher than its pA₂ value in the rat anococcygeus muscle was chosen. For this, NE was administered cumulatively until a maximum response was obtained. Tissues were washed three times with PSS, and prazosin (10 nM-10 µM) remained in the organ bath (20 min) until a second cumulative curve for NE was constructed. Each tissue was used to test only one concentration of each antagonist.

Effect of N^G-Nitro-L-Arginine Methyl Ester (L-NAME). Cumulative concentration-effect curves for Ang II were obtained after tissue incubation with the NO-synthase inhibitor, L-NAME (100 µM, 30 min; Rand and Li, 1993), losartan, and PD123319 (3 nM) in the presence of prazosin (1 µM).

Effect of Ang II on the Precontraction Induced by Bethanechol. Tissue was equilibrated with prazosin (1 µM) associated, or not, with L-NAME (100 µM), losartan, and PD123319 (3 nM). After a 30-min incubation, the muscle was precontracted with bethanechol (EC₅₀ = 30 µM), a nonselective muscarinic agonist that does not undergo cholinesterase action. Bethanechol was used as the agonist, since some muscarinic cholinoceptors evoke contraction in the rat anococcygeus (Rand and Li, 1993). After a stable tone level was attained, cumulative concentration-effect curves for Ang II were obtained. The stability of the precontracted level tone was tested using the parallel bundle of muscle.

Drugs. Angiotensin II, L-NAME, and PD123319 were obtained from Sigma/RBI (Natick MA). Bethanechol, norepinephrine, and prazosin were obtained from Sigma-Aldrich (St. Louis, MO). Losartan potassium was kindly given by Dr. Ronald D. Smith (DuPont Merck Pharmaceutical Co. (Wilmington, DE).

Data Analysis. Contractions were recorded as changes in the displacement (millimeters) from baseline and expressed as a percentage of the maximum response induced by KCl (90 mM) obtained at the beginning of each experiment. However, in the precontracting assays, data were expressed as percentages of the tone induced by bethanechol. Agonist concentration-response curves were fitted using a nonlinear interactive fitting program (GraphPad Prism 2.00; GraphPad Software Inc., San Diego, CA). Agonist potencies and maximum response are expressed as pD₂ (negative logarithm of the molar concentration of agonist producing 50% of the maximum response) and E_max (maximum effect elicited by the agonist), respectively. Concentration ratios (CRs) were determined from EC₅₀ values in the presence and absence of the antagonist. The concentration-response curves to agonists in the presence or absence of the antagonists were analyzed by plotting the negative logarithm of the ratio of concentrations of the agonist that produced the same effect (50% contraction) in the presence and absence of the antagonist minus 1 [log (CR  1)] against the negative logarithm of the concentration of antagonist (i.e., Schild plot analysis; Arunlakshana and Schild, 1959). The intercept on the abscissa yields the pA₂ value (negative logarithm of the concentration of antagonist that induces a 2-fold rightward shift of the concentration-response to the agonist), which is an indicator of the type of antagonism, i.e., a slope similar to 1 is considered to be competitive antagonism and the pA₂ value is similar to the pK_B value (the antagonist affinity estimate). Statistically significant differences were calculated by one-way analysis of variance (ANOVA) or by Student's t test analysis. P < 0.05 was considered as statistically significant.

	Results

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Effect of Losartan and PD123319. Ang II induced concentration-dependent contractions in the rat anococcygeus muscle (pD₂ = 8.68 ± 0.07; E_max = 76.92 ± 3.40%, n = 8). Time control experiments showed that two consecutive concentration-effect curves for this peptide could be obtained in the same tissue without any significant temporal change in agonist potency and maximum response (pD₂ = 8.79 ± 0.12; E_max = 73.39 ± 3.81%, n = 8; paired Student's t test, P > 0.05). Cumulative concentration-effect curves for Ang II were antagonized by losartan in a concentration-dependent fashion, with nonparallel rightward displacements (Fig. 1A). The Schild plot resulted in a linear regression with a slope of 0.51 (0.43-0.58), different from unity (two-tailed Student's t test, P < 0.05). The x-intercept indicated a pA₂ value of 10.89 (Fig. 2). PD123319, however, did not antagonize the agonist concentration-effect curves (Fig. 1B). The required time for the angiotensin receptor antagonists to reach the equilibrium state was tested; 30 min was required for the losartan effect to become stable. PD123319 did not produce any significant effect in the Ang II-induced contractions at all incubation times (Fig. 3A). Thus, 30 min was extrapolated as the required time for PD123319. The reversibility studies showed that losartan interacts with the AT₁ receptor in a reversible manner (Fig. 3B).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Inhibition by selective angiotensin receptor antagonists, losartan and PD123319, and by the selective 1-adrenoceptor antagonist, prazosin, of the contractile response to Ang II and NE. Concentration-response curves to agonists were carried out in isolated rat anococcygeus muscle in the absence and presence of the antagonists. Each symbol represents the mean ± S.E.M. of 8 to 40 experiments. Results are expressed as a percentage of the maximal contraction induced by 90 mM KCl at the beginning of the experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Schild plots of the antagonism of losartan (), losartan + PD123319 (0.1 nM) (), and prazosin (). The results for angiotensin antagonists are plotted from the data in Fig. 1.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Inhibitory effects of selective angiotensin receptor antagonists on the Ang II-induced contractions of the rat anococcygeus muscle. A demonstrates the effects of different incubation times on the inhibitory effects of 1 nM losartan () and PD123319 (). B shows the effects of different losartan washout times on the recovery of Ang II-induced contractions. One-way ANOVA: *, P < 0.05; ***, P < 0.001 compared with control group (0 min). Each symbol represents the mean ± S.E.M. of six experiments. Results are expressed as a percentage of the maximal contraction induced by 90 mM KCl at the beginning of the experiments.

Effect of Losartan and PD123319 Association. Incubation of several concentrations of losartan with PD123319 (0.1 nM) antagonized the cumulative concentration-effect curves for Ang II in a concentration-dependent fashion with parallel rightward displacements (Fig. 1C). The Schild plot resulted in a linear regression with a slope of 1.05 (0.84-1.25), which was not different from unity (two-tailed Student's t test). The x-intercept indicated a pK_B value for losartan of 9.32 (Fig. 2).

Determination of pA₂ for Prazosin. NE induced concentration-dependent contractions in the rat anococcygeus muscle (pD₂ = 6.06 ± 0.04; E_max = 110.82 ± 1.52%, n = 40). Prazosin antagonized NE-induced contractions in a concentration-dependent fashion (Fig. 1D). Results showed a pA₂ value equal to 7.60 (Fig. 2), a value in line with that reported by Doggrell and Paton (1978) in the rat anococcygeus muscle. Therefore, the chosen prazosin concentration was 1 µM.

Effect of Losartan and PD123319 in the Presence of Prazosin. Prazosin completely changed the effects of losartan and PD123319. In the presence of prazosin, losartan did not antagonize the cumulative concentration-effect curves for Ang II (Fig. 1E). Conversely, PD123319 markedly enhanced the E_max values for Ang II in a concentration-dependent fashion (Fig. 1F).

Effect of L-NAME. Results showed that L-NAME significantly enhanced the E_max and the pD₂ of Ang II in the presence of prazosin (Fig. 4). However, the incubation of L-NAME with angiotensin receptor antagonists did not produce any synergetic effect, since there was no statistical difference between these groups (Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effects of prazosin () associated with L-NAME (), PD123319 (), L-NAME + PD123319 (), losartan (), and L-NAME + losartan () on the Ang II-induced contraction of the rat anococcygeus muscle. Each symbol represents the mean ± S.E.M. of six experiments. Results are expressed as a percentage of the maximal contraction induced by 90 mM KCl at the beginning of the experiments.

Effect of Ang II on the Precontraction Induced by Bethanechol. The precontraction induced by bethanechol remained stable for at least 30 min. Ang II induced a biphasic action (Fig. 5), from 0.1 nM to 0.03 µM, the peptide enhanced the tone in a concentration-dependent manner. This effect was not significantly altered by angiotensin receptor antagonists or by L-NAME (one-way ANOVA, P > 0.05). Conversely, from 0.03 µM to 10 µM, Ang II produced a concentration-dependent relaxant response that was not altered by losartan. However, PD123319 and L-NAME inhibited the Ang II-induced relaxation in a significant manner (Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of Ang II on the precontraction induced by bethanechol (30 µM) in the rat anococcygeus muscle. The assay was reproduced in the absence () and presence of losartan (), PD123319 () and L-NAME (). Each symbol represents the mean ± S.E.M. of four experiments. Results are expressed as a percentage of the tone induced by bethanechol before the addition of Ang II.

	Discussion

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The selective antagonism of receptors with simple competitive antagonists offers a method of determining whether receptor populations are heterogeneous, by comparison with a model that assumes receptor homogeneity. This approach is based on the assumption that the Schild regressions result in a seemingly linear regression with a slope of unity and an intercept of K_B for competitive antagonists (Kenakin, 1992). In this study, we evaluated the effects of losartan and PD123319 on Ang II-induced contractions.

PD123319 did not produce any significant effect. Following incubation with losartan, Ang II-induced curves shifted rightward of the control in a concentration-dependent manner. Interestingly, this effect was followed by a decrease in E_max, resulting in a Schild plot slope of less than 1. Several mechanisms have been postulated to explain this effect: 1) tachyphylaxis to the agonist, 2) equilibrium conditions between antagonist and antagonist-receptors have not been attained, (3) the interaction between antagonist and antagonist-receptor is not reversible, 4) heterogeneous receptor populations are observed, 5) the agonist is, in part, an indirect-acting agonist, and 6) the antagonist is not competitive (Kenakin, 1981, 1992).

Results demonstrated that two consecutive curves for Ang II could be obtained in the same tissue with no significant difference. The equilibrium conditions were analyzed and used in all the experimental procedures. Moreover, inhibitor washout experiments were consistent with earlier reports showing that radiolabeled losartan rapidly dissociates from Ang II binding sites (Chiu et al., 1990). Thus, the first, second, and third possibilities were discarded.

In the present study, we tested the possibility that, in losartan-incubated tissues, inhibition of Ang II-induced contractions would result not only from the blockade of the angiotensin AT₁ receptor, but also from the activation of the angiotensin AT₂ sites. Results showed that in the presence of PD123319, losartan antagonized the Ang II-induced contractions in a concentration-dependent fashion with no E_max decrease. In this situation, the Schild plot exhibited a slope of unity and produced a pK_B value of 9.32, suggesting that losartan binds to the AT₁ receptor with high affinity, as do the majority of nonpeptide antagonists (Schambye et al., 1994). Since PD123319 is a selective AT₂ receptor antagonist up to 500 nM (Whitebread et al., 1989; Brechler et al., 1993), a nonselective effect was not considered.

Since Ang II binds to its two receptor subtypes, AT₁ and AT₂, with a similar affinity, the tissue response is highly dependent on the relative responsiveness of both receptors (Nouet and Nahmias, 2000). Therefore, when the AT₁ subtype is inhibited by losartan and the AT₂ receptor is free to interact with Ang II, the AT₂-mediated effect becomes predominant, and losartan becomes a noncompetitive antagonist. In fact, some studies have demonstrated that the activation of AT₂ by Ang II can induce effects opposite to the AT₁-mediated effects. A few studies, in rats, have suggested that Ang II has relaxant effects that are mediated by AT₂ receptors, thereby counteracting the constrictor action of Ang II at the smooth muscle site (Scheuer and Perrone, 1993; Israel et al., 2000).

It has been shown that Ang II exerts profound physiological effects in catecholaminergic neurons that are associated with the stimulation of turnover, synthesis, and release of NE by increasing the vesicular trafficking in catecholaminergic neurons (Wang et al., 2001). Since Ang II activates prejunctional receptors located at sympathetic neurons of the rat anococcygeus (Li et al., 1988), we evaluated the nature of these receptors by incubating the tissue in the presence of prazosin, losartan, and PD123319. Prazosin inhibited the Ang II-induced contractions, confirming that Ang II is, in part, an indirect-acting agonist, which could contribute to the apparent noncompetitive antagonism of losartan. The residual contractile effect of Ang II was unaffected by losartan, indicating that the losartan-sensitive component of the contractile response to peptide is mediated by AT₁ receptors located on sympathetic nerves. These results are in agreement with those of Wang et al. (2001), who demonstrated that Ang II increases NE vesicular trafficking in rat catecholaminergic neurons via an AT₁ receptor-mediated mechanism.

Conversely, PD123319 surprisingly enhanced the Ang II-induced contractions in a concentration-dependent fashion in the presence of prazosin, suggesting the existence of AT₂ receptors with an inhibitory function in the rat anococcygeus muscle. These results are in agreement with early results that indicate the involvement of AT₂ receptors with inhibitory signals (Hein et al., 1995; Siragy et al., 1999; Nouet and Nahmias, 2000) and suggest that the AT₂-mediated effect can be seen only if the sympathetic component is inhibited, since the NE-mediated response is the predominant component of the muscular tonus.

Interestingly, neither prazosin nor angiotensin receptor antagonists abolished the Ang II-induced contractions. These results suggest the existence of non-AT₁, non-AT₂ postjunctional angiotensin receptors in the rat anococcygeus muscle. Thus, Ang II may act on two types of sites, AT₁ and non-AT₁, non-AT₂ sites, in the presence of PD123319. By evaluating the Hill coefficient (n_H) derived from the cumulative concentration-effect curves for Ang II, we analyzed this prediction. Given that n_H different from 1 indicates negative (n_H < 1) or positive (n_H > 1) cooperativity (Moore and Scanlon, 1989), control curves for Ang II produced n_H < 1 (n_H = 0.85 ± 0.07), suggesting the presence of at least two different binding sites with negative cooperativity. Conversely, inhibition of the AT₂ site with PD123319 (0.1 nM) produced n_H > 1 (n_H = 1.20 ± 0.10), suggesting the presence of two binding sites following a positive cooperativity relationship. Thus, these results account for the presence of three binding sites, which combine to make a single signal. In fact, inhibition of both the PD123319-sensitive site and the losartan-sensitive site by association of PD123319 with losartan (0.1 nM) produced a n_H of unity (n_H = 1.07 ± 0.12). Thus, it may be concluded that, despite no significant change in the pD₂ and E_max parameters of the Ang II-induced curves, PD123319 may alter the slope of the contractile responses and Ang II may act on AT₁ and non-AT₁, non-AT₂ sites, in the presence of PD123319.

It has been shown that the inhibitory AT₂-mediated effect of Ang II may be mediated either directly or via the release of inhibitory autacoids such as NO (Siragy and Carey, 1997; Cote et al., 1998; Siragy et al., 1999; Israel et al., 2000). Since the rat anococcygeus smooth muscle has a rich nitrergic enervation, it may be postulated that the inhibitory AT₂-mediated effect is mediated by NO production. We tested this hypothesis by incubating the rat anococcygeus preparations with the NO-synthase inhibitor, L-NAME, in the presence of prazosin, losartan, and PD123319. Results showed that L-NAME markedly counteracted the inhibitory effect of prazosin on the Ang II-induced contractions. In contrast, association of L-NAME with prazosin and the selective angiotensin receptor antagonists, particularly PD123319, did not produce any synergistic or additional effect. However, the fact that PD123319 potentiates Ang II-induced contraction to the same degree as L-NAME is not unequivocal evidence that AT₂ receptors couple to NO.

To verify the presence or absence of an AT₂ coupled to NO relaxation or inhibition of contraction, Ang II was added to precontracted tissues previously incubated with prazosin, losartan, and PD123319. Results demonstrated that Ang II produced a biphasic effect, which consisted of a contractile phase followed by a relaxant phase. The contractile phase may be due to activation of non-AT₁, non-AT₂ receptors since none of the compounds altered this response. Conversely, PD123319 and L-NAME markedly inhibited the relaxant phase, demonstrating that the mechanism underlying the inhibitory AT₂-mediated effect of Ang II is highly dependent upon NO generation.

These results are not surprising in view of the fact that the effects of selective stimulation of either angiotensin AT₁ or AT₂ receptors have been shown to oppose each other in various biological systems. Most of the known effects of Ang II, such as increase in blood pressure, stimulation of myocyte hypertrophy, vasopressin release, and drinking are attributed to the stimulation of the angiotensin AT₁ receptor (Ganz and Perfetto, 1990; Everet et al., 1994). There is evidence that some of these actions are under inhibitory control by angiotensin AT₂ receptors. Indeed, AT₂ receptor subtypes antagonize both pressor and growth effects of the AT₁ receptor subtypes (Hein et al., 1995; Ichiki et al., 1995; Nakajima et al., 1995, Israel et al., 2000) and the centrally mediated Ang II-induced release of vasopressin and drinking (Höhle et al., 1995). Thus, the existence of an equilibrium between constrictor AT₁ and inhibitory AT₂ receptor could be proposed. This balance could play an important role in the mechanism of action of angiotensin AT₁ receptor antagonists. Consequently, when Ang II action is inhibited by losartan, the effects of angiotensin AT₂ receptor stimulation may be overexposed, inducing errors in the antagonist profile analysis or in receptor characterization.

In conclusion, the present study demonstrated that there is a negative cross-talk between angiotensin AT₁ and AT₂ receptor subtypes in the rat anococcygeus smooth muscle. We speculate that the AT₁ subtype mediates contractile response via NE release and that the AT₂ subtype mediates inhibition of the contractile response via NO generation, probably by stimulation of nitrergic nerve endings.