Rabbit Aortic Ring Preparation
Experiments were performed on isolated rabbit thoracic aorta from male Flanders hybrid rabbits (1.5–2.5 kg) obtained from a slaughterhouse. The thoracic aorta was carefully dissected and cleaned of adherent fat and connective tissue. Five-millimeter-wide rings were cut and mounted in a 10-mL organ bath containing Krebs solution of the following composition (mM): NaCl 128, KCl 4.7, NaHCO3 14.4, NaH2PO4 1.2, Na2-EDTA 0.1, CaCl2 2.5, glucose 11.1, pH 7.2. Krebs solution was kept at 37°C and aerated with 95% O2 and 5% CO2.
Isometric contractions were measured by using force-displacement transducers and were recorded under an initial tension of 2 g, which had been found to be the optimal tension for KCl-induced contraction (100 mM). All preparations were allowed to equilibrate for 90 minutes and were washed with Krebs solution at 15-minute intervals. The endothelium was kept intact in some rings, but in other groups the endothelium was removed by rubbing the luminal surface. Acetylcholine was used to test whether the endothelium had been removed. The rings were stimulated with NA 5.10−6 M and when the maximal contraction was achieved, acetylcholine 10−6 M was added to establish its relaxing effect.
Aortic rings (rubbed or unrubbed) were exposed to increasing doses of Ang II (10−10 to 2.5 10−6 M) at 90-minute intervals to construct 2 cumulative dose-response curves (CDRC I and CDRC II). Because many pharmacologists have indicated that the contractile response to Ang II is impaired as compared CDRC to multiple single doses, the maximal contractile response of the tissue was carefully reached every time. This means that intrinsic activity would not be blunted.
To evaluate Ang II-NA cross talk and the endothelium influence in this phenomenon, arteries with or without endothelium were treated with one CDRC to NA following the first CDRC to Ang II. Rings were rinsed and a 30-minute recovery period was allowed before the next exposure to Ang II (Fig. 1). To study the role of α1-ARs in the heterologous desensitization to Ang II the α1-ARs antagonist, prazosin 10−6 M was added to the bath 30 minutes before CDRC I in arteries with and without endothelium. Prazosin was removed by washing before the CDRC to NA. Furthermore, to establish the influence of agonists-exposition time in the cross talk, 2 stimulations with single doses of NA 5.10−7 M were allowed in arteries with and without endothelium. In matched experiments 1 CDRC to Ang II between the 2 stimulations with NA 5.10−7 M was performed.
In a similar protocol, 2 CDRC to NA (10−8 to 2.5 10−5 M) at 90-minute intervals were constructed both in rubbed and unrubbed arteries. The other group was treated with 1 CDRC to Ang II following the first CDRC to NA. Rings were rinsed and a 30-minute recovery period was allowed before the next exposure to NA. In matched experiments single CDRC to Ang II was constructed after a 60-minute rinsed period and then 1 CDRC to NA was performed. To study the role of AT1 receptors in the cross talk, losartan 10−7 M was added to the bath 30 minutes before the CDRC I to NA in arteries with endothelium. The AT1 receptor antagonist was removed by washing before the CDRC to Ang II.
Results are expressed as mg of isometric contraction or a percentage of the maximal contractile force obtained during the CDRC I for Ang II or NA.
Data are presented as mean values ± SEM and were analyzed by ANOVA with replications and Duncan test to evaluate CDRC. The pD2 (negative log of molar concentration of Ang II inducing 50% of the maximal contraction) and the maximal contractile response were calculated using a curve-fitting analysis program. Student t test paired or unpaired were used to compare pD2 values or maximal response. P less than 0.05 was considered statistically significant (two-tail test).
Effects of Ang II on Contractile Response of Arteries with and without Endothelium
The contractile response to Ang II (10−9 to 2.5.10−6 M) was dose dependent. The pD2 values of CDRC I were similar in arteries with and without endothelium. However, a significant shift to the right of second exposure to Ang II was observed in unrubbed (Fig. 2A) and rubbed (Fig. 2B) arteries. The quantity of the rightward shift was similar (n.s., ANOVA and Duncan test). No significant differences were found in maximal response from arteries with and without endothelium both in CDRC II and I (Table 1).
NA treatment between the first and the second exposure to Ang II increased Ang II desensitization in unrubbed (Fig. 2C) but not in rubbed aortic rings (Fig. 2D). Nevertheless, the maximal contractile response to Ang II-CDRC II was not modified by NA-treatment in arteries with endothelium but was enhanced in endothelium-denuded preparations (Table 1).
A single dose of NA 5.10−7 M was not able to modify either the contractile response or the pD2 in Ang II-CDRC both in arteries with and without endothelium (Table 2).
Effect of Prazosin Treatment on Contractile Response to Ang II in Arteries with and without Endothelium
In the presence of prazosin, a significant shift to the right of Ang II-CDRC I was observed in rubbed (pD2 = 8.02 ± 0.16, control; pD2 = 7.58 ± 0.09, prazosin, P < 0.01, n = 6) and unrubbed arteries (pD2 = 8.28 ± 0.08, control; pD2 = 7.35 ± 0.17 prazosin, P < 0.01, n = 6). Maximal contractile response was not modified. However, prazosin treatment was able to avoid the rightward shift of the second CDRC that has been observed in aortic rings with intact endothelium without any effect on the maximal response (Fig. 3). Furthermore, prazosin inhibited the maximal contractile response increase in endothelium-denuded arteries (Table 3).
Effects of NA on Contractile Response of Arteries with and without Endothelium
The contractile response to NA (10−8 to 2.5 10−5 M) was dose dependent. The pD2 values of CDRC I were similar in arteries with (6.37 ± 0.10) and without endothelium (5.93 ± 0.10). In addition, no significant differences in pD2 were observed in the second exposure to Ang II both in unrubbed (6.37 ± 0.08, n.s, n = 8;Fig. 4A) and rubbed arteries (5.97 ± 0.12, n.s, n = 8). However, the maximal contractile response was increased both in aortic rings with and without endothelium (Table 1).
Ang II treatment between the first and the second exposure to NA induced a significant rightward shift of the second CDRC to NA in unrubbed arteries (pD2= 1st: 6.37 ± 0.08; 2nd: 6.15 ± 0.06, P < 0.001, n = 14;Fig. 4B). However, in rubbed aortic rings no differences were found in pD2 values (1st: 6.21 ± 0.14; 2nd: 6.22 ± 0.06, n.s., n = 8). Furthermore, a maximal contractile response increase was also observed in rubbed and unrubbed aortic rings (Table 1).
Increase in contractile response to NA 5.10−7 M during the second stimulation was observed in aortic rings with endothelium (1st: 2226 ± 572 mg; 2nd: 3520 ± 715 mg, P < 0.001, n = 8) and without endothelium (1st: 2204 ± 640 mg; 2nd: 3287 ± 666 mg, P < 0.001, n = 8). This effect was not modified by treatment with Ang II between the 2 stimulations in arteries with endothelium (1st: 1780 ± 432 mg; 2nd: 2965 ± 540 mg, P < 0.001, n = 8) and denuded-endothelium (1st: 1962 ± 690 mg; 2nd: 3219 ± 864 mg, P < 0.001, n = 8).
Effect of Single CDRC to Ang II or NA on the Affinity and Maximal Response of Subsequent CDRC to NA or Ang II, Respectively
One CDRC to NA before single CDRC to Ang II caused a shift to the right in aortic rings with endothelium. pD2 values either in aortic rings without (control) and with previous exposure to NA were 8.48 ± 0.07 and 8.22 ± 0.08, respectively (P < 0.05, n = 14). In rubbed aortic rings no differences were found in pD2 (8.20 ± 0.16 vs 8.03 ± 0.08, n.s, n = 14). There were no significant differences in the maximal contractile response.
Nevertheless, 1 CDRC to Ang II before single CDRC to NA caused no differences in either affinity or the maximal contractile response in arteries with endothelium (pD2 = 6.26 ± 0.07 vs 6.37 ± 0.08, n.s., n = 8) and without endothelium (pD2 = 6.35 ± 0.12 vs 6.21 ± 0.06, n.s., n = 8).
Effect of Losartan Treatment on Contractile Response to NA in Arteries with and without Endothelium
Losartan did not modify either the pD2 (6.00 ± 0.27, control; 6.11 ± 0.25; losartan, n.s, n = 8) or the maximal contractile response (6034 ± 978 mg, control; 6228 ± 839, losartan, n.s, n = 8) of the first CDRC to NA. However, losartan treatment was able to avoid the rightward shift that has been observed in the second exposure to NA after Ang II treatment (Fig. 5). Increase in the maximal contractile response was not modified (1st: 6228 ± 839; 2nd: 7633 ± 998, P < 0.01, n = 8).
Results obtained in the present study showed that desensitization to Ang II contractile response in rabbit aortic rings (rubbed and unrubbed) is not reversible after a 90-minute recovery period after the first exposition. As early as 1980, Gunther et al 26 reported the first direct evidence, by radioligand binding assay, that Ang II regulates the number of its own receptors in resistance vasculature. Further investigations revealed that AT1 receptor activation is subject to a negative feedback, in that increased levels of Ang II diminish and decreased Ang II concentrations enhance AT1 receptor activation. 27,28
In a previous work we demonstrated in rabbit aorta that the presence of an intact endothelium increased Ang II–desensitization. 25 Furthermore, there are 2 mechanisms involved in the development of Ang II-tachyphylaxis; one involves endothelium influence and one occurs at the level of the smooth muscle and is endothelium-independent. The endothelium-dependent tachyphylaxis is related with the intrinsic contractile property and the endothelium-independent tachyphylaxis is related with the loss of affinity. In the present work the recovery time after the first CDRC to Ang II was larger and no differences were found in the maximal contractile response between the first and the second CDRC to Ang II. This result suggests the endothelium-dependent desensitization disappearance. However, a shift to the right of the CDRC II to Ang II was observed in rubbed and unrubbed arteries. This would mean that the endothelium-independent tachyphylaxis can not be reversed by increasing the recovery time. This result is in agreement with the previous finding of Kuttan and Sim 12 and Gruetter et al 29 in rat aorta. Tachyphylaxis is associated with changes at the receptor level (ie, change in affinity and coupling efficiency). The factors involved can be derived from the endothelium or the smooth muscle cell. Kai et al 15 found in intact vascular smooth muscle cells homologous desensitization of the IP3 response to the subsequent stimulation, but not downregulation of AT1AR or protein Gαq/Gα11 after short-term exposure (stimulation with single dose during 10 minutes) to Ang II.
Recently, it has been shown that multiple agonists other than Ang II modulate AT1 receptor function. 30 This phenomenon, referred to as heterologous AT1 receptor regulation, is induced by various factors (eg, glucocorticoids, aldosterone, forskolin, nitric oxide, etc.), including NA, all of which downregulate AT1 receptor expression. Yang et al 8 demonstrated that negative feedback regulation of Ang II receptors by NA is a result of a decrease in AT1 receptor gene expression in WKY brain neurons. In addition, neurons of SHR brain lack this downregulatory mechanism. The action of NA on AT1 receptors involves the α1A-adrenergic receptor and thus provides an example of the cross talk between these 2 receptors in the neurons.
Taking account of these data about the heterologous desensitization of the response to Ang II produced by NA prolonged infusion, we investigated the effects of interpolating one CDRC to NA after the first CDRC to Ang II. The result was an increase of the Ang II-desensitization in unrubbed arteries. This is in agreement with Seasholtz et al. 16 However, in aortic rings without endothelium the maximal contractile response was enhanced. A single dose of NA 5.10−7 M did not modify either the affinity or the maximal contractile response to Ang II both in rubbed and unrubbed arteries. That would mean this is a time- dependent phenomenon. Therefore, these results demonstrate endothelium-dependent enhancement of Ang II-contractile response desensitization induced by NA. The role of endothelium in this phenomenon will be discussed later. On the other hand, the increased intrinsic activity may be due to the fact that no counteracting action of endothelium-relaxing factors was observed and the equilibrium was displaced by release of vasoconstrictors; for that reason, the maximal response observed was greater.
A significant shift to the right in CDRC I to Ang II was observed in rubbed and unrubbed aortic rings treated with prazosin. This result is in agreement with Zimmerman et al 31 and Zimmerman, 2 who demonstrated that Ang II can facilitate peripheral sympathetic function through multiple mechanisms both at level of the central nervous system and peripherally at the level of the sympathetic nerve ending. In the latter case, it also may be the result either of an increased release of NA from sympathetic nerves, or the inhibition of NA uptake by sympathetic nerves, 32 or a direct effect on the excitation-coupling vascular smooth muscle mechanism. 33 In the present study, when the α1-AR was blocked with prazosin during the first stimulation with Ang II, NA released from nerve ending by Ang II could not interact with its receptor. Therefore, the results obtained mean that α1-AR stimulation would enhance Ang II, not only in contractile response but affinity. These data are in agreement with Majewsky et al. 4 However, prazosin was able to avoid the shift to the right of Ang II-CDRC II in arteries with endothelium. Furthermore, the increase in the maximal contractile response observed in rubbed aortic rings was blocked. These data prove a role of α1-AR in the desensitization to Ang II because α1-AR blocking during the first stimulation with Ang II eliminated completely the loss of affinity observed in the second stimulation. In addition, the inhibitory effect of prazosin in the improvement of the maximal response observed in rubbed arteries suggests the sensitization of the contractile mechanism caused by α1-AR stimulation during the first CDRC to Ang II and the subsequent CDRC to NA. This effect was not observed in unrubbed arteries on account of the endothelium-counteracting action. To check the heterologous desensitization induced by NA without previous stimulation of AT1 receptor, a single CDRC to NA was performed before 1 CDRC to Ang II. Results showed that NA stimulation shifted to the right the Ang II CDRC in unrubbed arteries. That means no previous AT1 receptor stimulation was necessary to the heterologous desensitization. Taken together, these findings would suggest on the one hand the presence of cross talk between α1-AR and Ang II receptors and on the other hand an endothelium influence in such phenomenon.
No homologous desensitization of the contractile response to NA was observed with the present protocol. However, an increase endothelium-independent of the maximal contractile response was observed. This phenomenon was not time-dependent because it was also observed with single doses to NA. Previous reports have shown that exposure to elevated catecholamines or other receptor agonists in vivo or in vitro result in desensitization of α1-AR–mediated vascular smooth muscle contraction in rat or rabbit aorta. 10,34 The disagreement with the present results may be due to the different NA incubation and recovery periods used in the experiments. Treatment with 1 CDRC to Ang II after the first CDRC to NA caused a rightward shift of the second CDRC in unrubbed arteries. Losartan added during the first stimulation with NA blocked this effect. However, 1 CDRC to Ang II performed before 1 CDRC to NA was not able to induce NA desensitization both in rubbed and unrubbed arteries. These findings may suggest that cross talk between α1-AR and AT1-receptor during the first CDRC to NA is necessary for inducing heterologous desensitization. These data, with the data mentioned previously, support the view that endothelium plays an important role in the cross talk between Ang II and adrenergic receptors. According to the literature, cardiovascular signaling cross talk mediates both short- and long-term events, and coordination of the individual contributory pathways is regulated at various signaling junctions, particularly the G protein, AC, PK, and MAPK levels. 35 It has been previously reported that endothelium influence vascular smooth muscle intrinsic activity. However, endothelium influence on the hormone-receptor affinity has not yet been established. Oriowo et al 36 proposed that factors in the microenvironment of the receptor could alter the affinity of the receptor. The possible involvement of such factors is also suggested from the observation that the endothelium may be a source of endothelium-derived modulating factors, which influence the affinity of the angiotensin receptor. 12
Griendling et al 37 reported that when Ang II binds to its AT1 receptor in vascular smooth muscle, it initiates a biphasic response activating phospholipase C (PLC) and later on phospholipase D (PLD). In contrast to the PLC response, PLD activation does not appear to desensitize significantly for as long as 1 hour. This pathway is the most important source of phosphatidic acid and diacylglycerol and probably represents the major pathway by which protein kinase C (PKC) remains activated. On the other hand, potassium channels that are inhibited by internal ATP (KATP channels) provide a critical link between metabolism and cellular excitability. Light et al 38 demonstrated that PKC acts on KATP channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac KATP channels, thereby increasing their open probability at physiological ATP concentrations. In a previous paper 25 we found that Glibenclamide (KATP. blocker) was the only one potassium channels blocker able to avoid the loss of affinity on the second CDRC to Ang II. Furthermore, the KATP channels opener cromakalin increased the desensitization to Ang II in aortas without endothelium. Taking into account these data from the literature, we hypothesize that endothelium-dependent hyperpolarization induced by KATP channels-activation might modify Ang II affinity. That means a role of KATP channels in the endothelium-dependent cross talk between α1-AR and Ang II receptor is possible. Nevertheless, further studies are necessary to establish the mechanisms involved in the endothelium-dependent cross talk.
In summary, we have provided the first direct evidence that there is an endothelium-dependent cross talk between α1-AR and Ang II receptors in smooth muscle of rabbit aortic rings. Although is well established that the renin-angiotensin system is implicated in the development and maintenance of blood pressure elevation, numerous aspects about the role of Ang II in pathogenesis of essential hypertension are unclear. Cross talk between the renin-angiotensin system and sympathetic nervous system has received some attention in the literature and a more thorough appraisal of recent articles in this area is required. An altered regulation of Ang II at the cellular and molecular level could be fundamental in the pathology of essential hypertension. Both in vitro studies in cultured cells and data from whole animal experiments indicate that Ang II signaling in hypertension is upregulated. Lack of cross talk between the renin-angiotensin system and sympathetic nervous system might account for this phenomenon. By extension, it seems reasonable to assume that endothelial dysfunction associated with hypertension could play an important role in this interaction.
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