Losartan Reduces Phenylephrine Constrictor Response in Aortic Rings From Spontaneously Hypertensive Rats
Role of Nitric Oxide and Angiotensin II Type 2 Receptors
Nitric oxide seems to be involved in the mechanisms underlying the antihypertensive and renal responses of losartan in spontaneously hypertensive rats (SHR). We investigated the contribution of nitric oxide to the effect of this angiotensin II (Ang II) type 1 (AT1) receptor antagonist on the constrictor response of phenylephrine in aortic rings from SHR. Furthermore, since it has been suggested that Ang II could bind to unblocked AT2 receptors, during administration of an AT1 receptor antagonist, we also studied the effect of the AT2 receptor antagonist PD 123319 on the contractile response to phenylephrine in aortic rings from SHR. To this end, we studied dose-response curves of phenylephrine (10−9 to 10−5 mol/L) in the presence and absence of losartan (10−9, 10−7, and 10−5 mol/L) in SHR aortic rings. Preincubation with losartan reduced the constrictor response to phenylephrine but not to KCl (10 to 120 mmol/L) in a dose-dependent manner. On the other hand, the presence of captopril (10−5 mol/L) in the incubation medium did not alter the response to phenylephrine, even at the dose of 10−3 mol/L. The reduced response to phenylephrine in the presence of losartan was abolished in both endothelium-denuded rings and rings treated with a nitric oxide synthesis inhibitor. A similar situation was observed in PD 123319–pretreated rings, in which the effect of losartan on the contractile response to phenylephrine was reversed. Losartan was not able to stimulate the production of aortic cGMP compared with the control group. Likewise, losartan did not modify the relaxing responses to either acetylcholine or sodium nitroprusside in phenylephrine-preconstricted aortic rings. Furthermore, losartan did not alter isometric tension in aortic rings in either basal or phenylephrine-preconstricted conditions. These data demonstrate that Ang II potentiates the vasoconstriction induced by phenylephrine through the stimulation of AT1 receptors. Moreover, AT2 receptors and nitric oxide appear to be involved in this effect.
Angiotensin II (Ang II), the primary effector of the renin-angiotensin system, produces vasoconstriction not only through the interaction with AT1 receptors in the vascular smooth muscle but also through its ability to modulate sympathetic neural function.1 In this respect, it is well established that Ang II can facilitate peripheral sympathetic function through multiple mechanisms, including an increase in catecholamine biosynthesis, stimulation of ganglionic cells, release of catecholamine from the adrenal medulla, and attenuation of prejunctional reuptake.2 3 4 5 In addition, it has been shown that subthreshold concentrations of Ang II increase the contractile response of vascular smooth muscle to sympathetic nerve stimulation and adrenoceptor activation in isolated vessels from normotensive rats.6 7 Likewise, Qiu et al8 have reported that perindoprilat reduced the decrease in diameter and blood flow induced by phenylephrine in mesenteric arteries from both SHR and Wistar-Kyoto rats. Moreover, since similar results have been reported with the AT1 receptor antagonist losartan, it might seem that AT1 receptors are involved in the modulation of sympathetic neural functions.8
Losartan reduces blood pressure in both essential and experimental hypertension.9 10 Although its hypotensive action is mainly attributed to its ability to antagonize the binding of Ang II to AT1 receptors,10 additional mechanisms appear to be involved in this effect. In this regard, we have previously reported that administration of NO synthesis inhibitors impairs the blood pressure–lowering action induced by losartan in rats in both short-term11 and longer9 treatments. In addition, we have also observed that pretreatment with L-NAME reduces the acute renal hemodynamic effects of a low dose of losartan in anesthetized SHR.12 These data suggest that NO might be involved in both the antihypertensive and renal actions of AT1 receptor antagonists. Therefore, since AT1 receptors seem to mediate the modulation of the sympathetic neural function exerted by Ang II,8 we designed the present study to investigate the effect of NO synthesis inhibition in the action of losartan on the vascular responses to phenylephrine in aortic rings from SHR. In addition, and as we have previously implied, some of the renal effects of losartan could depend on the actions of Ang II through AT2 receptors, since pretreatment with PD 123319, an AT2 receptor antagonist, reduces the increase in natriuresis and diuresis induced by losartan in anesthetized SHR.12 Therefore, we also investigated the role of AT2 receptors in the action of losartan on the constrictor response to phenylephrine.
Experiments were conducted in 18- to 21-week-old male SHR (Charles River, Barcelona, Spain) maintained under controlled conditions of light and temperature. Rats were fed a normal rat chow (A.04, Panlab) and had free access to tap water. All experimental procedures were approved by the Institutional Animal Care and Use Committee according to the guidelines for ethical care of experimental animals of the European Community.
Preparation of Vascular Rings
On the day of the experiment, the descending thoracic aorta was excised from anesthetized rats. The aortas were immediately transferred to ice-cold Krebs' bicarbonate buffer (composition in mmol/L: NaCl 118.4, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11), cleaned of periadventitial tissue, and cut transversely into ring segments (3.0 mm in length). In some experiments, the luminal surface of aortic rings was rubbed with a small piece of polyethylene tubing for removal of the endothelium.
Each ring was placed in a tissue bath filled with Krebs' bicarbonate buffer (37°C) bubbled with 95% O2/5% CO2 and was attached to a force-displacement transducer (model FT03, Grass Instrument Co) coupled to a polygraph (Grass model 7E) for measurement of isometric tension. Rings were allowed to equilibrate for 60 to 90 minutes at a resting tension of 2 g with changes of buffer every 15 minutes. In preliminary experiments, we found that 2 g of resting tension is optimal for the expression of KCl-induced contraction of SHR aortic rings. Experiments were performed when isometric tension was stable. At the end of the experiment, the presence or absence of functional endothelium was verified in all aortic rings by observing whether relaxation occurred on exposure to acetylcholine (10−5 mol/L). After the experiments were completed, aortic rings were allowed to dry at least 24 hours and then were weighed.
We conducted a first group of experiments to investigate the effect of losartan on the contractile response to phenylephrine. For this purpose, aortic rings from SHR were preincubated for 15 minutes with three different doses of losartan (10−9, 10−7, and 10−5 mol/L) before being exposed to phenylephrine (10−9 to 10−5 mol/L). In a related study, we assessed the effect of losartan (10−5 mol/L) on the contractile response of KCl (10 to 120 mmol/L). We evaluated the effectiveness of losartan in blocking AT1 receptors at the dose for which the maximal inhibition of the contractile response of phenylephrine was reached (10−5 mol/L) by ascertaining that the constrictor responsiveness to Ang II (50 nmol) was totally blunted after the addition of losartan (1.12±0.08 versus 0.01±0.001 g of tension) to the incubation medium. In a parallel study, we evaluated, as a control, the effect of captopril (10−5 mol/L) on the contractile response of phenylephrine (10−9 to 10−5 mol/L). We evaluated the effectiveness of captopril in inhibiting Ang II formation in preliminary experiments by ascertaining that the constrictor responsiveness to Ang I (50 nmol) was blunted after the addition of captopril (0.98±0.05 versus 0.12±0.01 g of tension) to the incubation medium.
To study the role of NO in the effect of losartan on the contractile response to phenylephrine, we assessed the effect of the NO synthesis inhibitor L-NAME (10−4 mol/L) on dose-response curves to phenylephrine (10−9 to 10−5 mol/L) in aortic rings preincubated or not with losartan (10−5 mol/L) for 15 minutes. In another series of experiments, we also evaluated the role of the endothelium. For this purpose, we compared the dose-response curves to phenylephrine in endothelium-denuded rings preincubated or not with losartan for 15 minutes. In a complementary study, we measured the ability of losartan to stimulate cGMP production in the descending thoracic aorta, according to a published procedure.13 Briefly, aortas were transversely cut in half and preincubated in oxygenated Krebs' bicarbonate buffer containing 3-isobutyl-1-methylxanthine (IBMX, 0.5 mmol/L) for 1 hour in a shaking water bath at 37°C. After an equilibration period, the buffer was replaced with fresh buffer containing IBMX and the following agents: losartan (10−5 mol/L) alone, L-NAME (10−4 mol/L) alone, and losartan (10−5 mol/L) plus L-NAME (10−4 mol/L). Aortas incubated only with vehicle (Krebs') were used as controls. After 5 minutes of incubation, the aortic segments were snap-frozen in liquid nitrogen and subsequently homogenized in ice-cold trichloroacetic acid (10 g/100 mL). Homogenates were extracted with diethyl ether, and the aqueous phase was evaporated under vacuum. After reconstitution in radioimmunoassay buffer, cGMP was measured with a commercial kit (Advanced Magnetics).
The main part of the relaxing effect of acetylcholine in aortic rings of rats depends on NO synthesis14 ; therefore, in another series of experiments, we determined the effect of losartan (10−5 mol/L) on the vasorelaxation induced by acetylcholine. To this end, we compared the separate effects of cumulative additions of acetylcholine (10−9 to 10−5 mol/L) on the isometric tone of aortic rings contracted with a submaximal dose of phenylephrine (10−6 mol/L) and exposed or not to losartan (10−5 mol/L). We used the same dose of phenylephrine in both conditions, although the absolute contractions reached were different in both groups. Our decision was based on preliminary experiments in which we observed that aortic rings exposed to losartan and contracted with phenylephrine at a dose 10 times higher than in controls, in order to reach the same degree of tension, showed a reduction in the vasorelaxation induced by acetylcholine compared with control rings (49±3% versus 61.5±3% of maximal relaxation, P<.05; n=4). In a parallel study, we also examined the effect of losartan (10−5 mol/L) on the endothelium-independent relaxation induced by sodium nitroprusside (10−10 to 10−6 mol/L). Likewise, to study whether losartan is able to induce by itself a vasorelaxing action, we investigated the effect of cumulative addition of losartan (10−9 to 10−5 mol/L) on the isometric tone of aortic rings contracted with a submaximal dose of phenylephrine (10−6 mol/L).
We evaluated the role of AT2 receptors in the effect of losartan on the contractile response to phenylephrine in another group of experiments. We assessed the effect of the AT2 receptor antagonist PD 123319 (10−5 mol/L) on dose-response curves to phenylephrine (10−9 to 10−5 mol/L) in aortic rings preincubated or not with losartan (10−5 mol/L). The dose of PD 123319 was based on a previous report.15
Losartan was obtained from Merck Sharp & Dohme SA, and PD 123319 was a gift from Parke-Davis. All other drugs were purchased from Sigma Chemical Co. IBMX was dissolved in dimethyl sulfoxide and further diluted in Krebs' buffer. Stock solutions of all other drugs were prepared in distilled water and diluted to the desired concentration with buffer immediately before the experiment. Concentrations are expressed as the final molar concentration in the organ chamber.
Calculations and Statistical Analysis
For agents that elicit contraction of aortic rings, results are expressed as the change in isometric tension induced by the agonist, normalized by the dry weight of the vascular rings. For agents that elicit relaxation of phenylephrine-preconstricted aortic rings, the response is expressed as percent reduction of tension in the preconstricted state. The negative logarithm of the concentration of phenylephrine and KCl producing both 50% of the maximal response (pD2) and the maximal contraction were determined from the individual dose-response curves by linear regression analysis (over the range of 20% to 80% of the maximal response) and by fitting a hyperbolic curve, respectively. Results are expressed as mean±SE of rings from seven rats unless otherwise specified. Single-variable comparisons were made with a one-way ANOVA; all other data were analyzed by two-way ANOVA for multiple comparisons followed by the Newman-Keuls test if differences were noted. The null hypothesis was rejected at a value of P<.05.
Fig 1⇓ depicts the dose-response curves induced by 10−9 to 10−5 mol/L phenylephrine in SHR aortic rings in the presence and absence of either losartan (10−9, 10−7, or 10−5 mol/L; top) or captopril (10−5 mol/L, bottom). Neither losartan nor captopril at the doses used was able to modify the basal tone of aortic rings. Rings exposed to 10−7 and 10−5 mol/L losartan elicited a lesser maximal contractile response to phenylephrine than control rings (Fig 1⇓, top; Table 1⇓), although the reduction in the maximal response induced by losartan was more evident at 10−5 mol/L (Table 1⇓). By contrast, rings pretreated with 10−9 mol/L losartan developed a phenylephrine-induced contraction similar to those observed in control rings (Fig 1⇓, top; Table 1⇓). Furthermore, no differences were observed in pD2 values between control rings and rings pretreated with any losartan dose (Table 1⇓). Moreover, the increases in isometric tension induced by 10 to 120 mol/L KCl were similar in rings incubated or not with 10−5 mol/L losartan (maximal response: 1.53±0.02 versus 1.48±0.03 g/mg of tissue).
Since both maximal response (1.53±0.07 versus 1.59±0.12 g/mg of tissue, respectively; Fig 1⇑, bottom) and pD2 (6.9±0.1 versus 6.7±0.1, respectively) were similar in control and captopril-treated rings, it would seem that the exposure of aortic rings to captopril did not modify the phenylephrine-induced contraction. Moreover, the addition of 10−3 mol/L captopril to the incubation medium did not modify either the maximal response (1.67±0.09 g/mg of tissue, n=5) or pD2 (6.8±0.09, n=5) compared with the control group (1.6±0.05 g/mg of tissue and 6.9±0.06, maximal response and pD2, respectively).The contraction induced by phenylephrine in aortic rings exposed to both 10−5 mol/L captopril and 10−5 mol/L losartan was similar (1.25±0.05 g/mg of tissue and 6.78±0.07, maximal response and pD2, respectively; n=4) to the contraction observed in rings exposed only to 10−5 mol/L losartan (Table 1⇑).
The addition of the NO synthesis inhibitor L-NAME significantly increased the contractile response to phenylephrine compared with that of control rings (Table 2⇓). Moreover, the presence of L-NAME in the medium reversed the minor contraction induced by phenylephrine in the presence of losartan (Fig 2⇓, top; Table 2⇓). Rubbing the lumen of aortic rings induced a leftward shift of the dose-response curves to phenylephrine similar to that observed in L-NAME–treated rings (Table 2⇓). Furthermore, removal of aortic endothelium also eliminated the reduction in the phenylephrine-induced contraction evoked by exposure of the rings to losartan (Fig 2⇓, bottom; Table 2⇓). Losartan was not able to stimulate aortic cGMP production compared with control aortas (8.4±2 versus 9±2 pmol/mg protein, n=4). The addition of L-NAME to the incubation medium reduced cGMP in a similar manner in all groups (control: 3.9±0.8 pmol/mg protein; losartan: 4.9±1; n=4).
Fig 3⇓ illustrates the effect of losartan preincubation on the vasorelaxation induced by either acetylcholine or sodium nitroprusside in aortic rings preconstricted with a submaximal dose of phenylephrine. Exposure of the rings to losartan did not modify the relaxing response to cumulative doses of either acetylcholine (top) or sodium nitroprusside (bottom). In addition, increasing doses of losartan (10−9 to 10−5 mol/L) did not alter the isometric tone of aortic rings precontracted with a submaximal dose of phenylephrine (1.23±0.1 versus 1.18±0.12 g, before versus after losartan; n=5).
The presence of PD 123319 in the incubation medium did not modify the contractile response to phenylephrine compared with the response of control rings (Table 2⇑). By contrast, preincubation of rings with this AT2 receptor antagonist reversed the effect of losartan on the contractile response to phenylephrine in aortic rings from SHR because it normalized the maximal response to this α1-agonist (Fig 4⇓, Table 2⇑).
The present study demonstrates that preincubation of SHR aortic rings with the AT1 receptor antagonist losartan reduces phenylephrine-induced contractions in a dose-dependent manner. This suggests a facilitatory role, through AT1 receptors, of endogenous Ang II in phenylephrine vasoconstriction. In addition, since the attenuation of the contractile response to phenylephrine induced by losartan disappears in both endothelium-denuded aortic rings and L-NAME–pretreated vascular rings, this effect appears to be mediated by endothelium-derived NO. Moreover, as the reduction in the phenylephrine-induced contraction evoked by the exposure of the rings to losartan is blunted in the presence of the AT2 receptor antagonist PD 123319, the participation of this AT2 receptor in the effect of the AT1 receptor antagonist losartan also could be proposed.
The facilitatory action of Ang II in neurogenic vasoconstriction has been demonstrated in isolated vessels, in which the administration of subpressor doses of Ang II potentiates the contractile responses of vascular smooth muscle to either sympathetic nerve stimulation or adrenoceptor activation.6 7 In accordance with these observations, we found in the present study that the AT1 receptor antagonist losartan attenuated the contractile response to phenylephrine in SHR aortic rings, suggesting that Ang II, through AT1 receptors, plays a mediatory role in neurogenic vasoconstriction. A similar result was observed by Qiu et al8 in mesenteric arteries of both SHR and Wistar-Kyoto rats, in which losartan reduced the decrease in diameter and blood flow induced by phenylephrine. All these data suggest that Ang II not only increases the levels of catecholamines2 3 4 5 but also plays a mediatory role in the vasoconstrictor action of phenylephrine. Therefore, it is possible to postulate that the hypotensive effect of losartan could be partially due to the reduction in the sympathetic neural function that is augmented in hypertension.16 17
As losartan did not modify the contractile response to KCl, the effect of this AT1 receptor antagonist on the contractile action of phenylephrine appears to be specific to this agent and not a general effect on the contractile mechanisms of smooth muscle cells. In addition, it has been shown that losartan by itself lacked effects on intracellular inositol triphosphate levels in different tissues,18 19 20 and losartan did not modify21 or even increase20 the intracellular concentration of free calcium in rat smooth muscle cells. Moreover, it has been shown that losartan inhibits the increase induced by Ang II, and not by other vasoconstrictors, in both the phosphoinositide signaling system and the elevation of intracellular free calcium.20
It should be noted that the mechanism underlying the reduction in the phenylephrine-induced tone exerted by losartan should not be through partial α1-adrenoceptor antagonism because it has been reported that losartan did not present α1-antagonist activity.22 This effect appears to involve an endothelium-derived factor, since the attenuated maximal contractile response to phenylephrine observed in aortic rings exposed to losartan disappears after endothelium removal. Furthermore, as pretreatment with the NO synthesis inhibitor L-NAME also reverses the altered vasoconstrictor response to phenylephrine induced by losartan, this effect seems to be related to NO. However, the mechanism by which NO can be involved in the attenuation of the phenylephrine-induced vasoconstriction exerted by losartan is not totally clear at this point. It seems that losartan was not able to stimulate NO synthesis because it was not able to augment the cGMP levels in aortic rings, nor was it able to increase the dilator action of acetylcholine. Furthermore, losartan did not modify the isometric tension of SHR aortic rings in either basal or phenylephrine-precontracted conditions. In view of the present data, it could be proposed that under the present experimental conditions, losartan does not stimulate NO production in both basal and stimulated conditions. Similarly, we have previously observed that the acute blood pressure–lowering effect of losartan in both SHR and rats with aortic coarctation–induced hypertension is not accompanied by elevation of cGMP levels in the thoracic aorta.23 24 It should be mentioned that the impairment of the vasodepressor response to acute losartan caused by pretreatment with an NO synthesis inhibitor in SHR is normalized in animals concurrently receiving sodium nitroprusside to correct for the loss of endogenous NO.23 These data suggest that the presence of NO is necessary for the full expression of the effect of losartan on the contractile response to phenylephrine in SHR aortic rings. Consequently, it could be proposed that during NO synthesis inhibition, an imbalance between endogenous vasodilator-vasoconstrictor factors could exist, resulting in impaired effects of losartan.
It has been suggested that in the presence of a specific AT1 receptor antagonist, Ang II might bind to unblocked AT2 receptors and partially mediate the actions of the AT1 receptor antagonist. Indeed, we have observed that preincubation with the AT2 receptor antagonist PD 123319 abolishes the effect of losartan, suggesting that AT2 receptors are required for the full expression of the action of losartan on the vasoconstriction induced by phenylephrine. Similarly, we have previously reported that the excretory effects induced by acute administration of losartan in anesthetized SHR12 depend on the actions of Ang II through AT2 receptors. Since it has been reported that Ang II is able to stimulate NO synthesis via AT2 receptors in endothelial cells25 and isolated coronary microvessels,15 it is plausible to suggest a link between the mechanism underlying the participation of AT2 receptors and NO. However, a stimulation of NO mediated by AT2 receptors in the presence of losartan cannot be proposed.
The present study also showed that preincubation with captopril did not modify the dose-response curves of phenylephrine in SHR aortic rings alone or in the presence of losartan. Similarly, Atkinson et al26 have observed that captopril did not alter the sympathetically mediated vasoconstriction in SHR tail arteries. The fact that losartan but not captopril reduced the phenylephrine-induced tone in SHR aortic rings suggests different explanations. One possibility is that the captopril dose (10−5 mol/L) used was not high enough to be effective in inhibiting Ang II formation. However, as this dose did blunt the constrictor response to Ang I, this seems not to be the case. Furthermore, even a dose 100 times higher was not able to modify the constrictor effect of phenylephrine. Since several studies have shown that ACE inhibitors reduce catecholamine-induced vasoconstriction in pithed SHR26 27 or in blood-perfused vessels,8 another possible explanation for the lack of effect of captopril in isolated SHR vessels could be that there is not good tissue penetration of this drug. However, if this is the case, it appears to be a feature of captopril expressed only in vessels from SHR because it has been reported that ACE inhibitors reduce the vasoconstrictor action of adrenergic agonists in isolated vessels from normotensive animals.28 Another explanation for the differences observed between captopril and losartan may be a prior occupancy of the Ang II receptors before tissue collection. However, although we do not have any evidence to clarify this point, it is possible to expect that Ang II would be washed out during ring preparation. Finally, the differences between the AT1 receptor antagonist and the ACE inhibitor observed in this study could be due to the fact that the Ang II involved in the potentiation of the phenylephrine-induced vasoconstriction could be locally formed through a non-ACE enzymatic pathway. Similarly, it has been shown that Ang II generated through a non-renin, non-ACE mechanism potentiates the sympathetic contractile responses in isolated vessels from normotensive animals, since the administration of either renin or ACE inhibitors did not alter the facilitatory role of the neurogenic vasoconstriction induced by either Ang I or the renin substrates.6 7 Indeed, in addition to the renin-ACE enzymatic axis, there may exist multiple pathways of angiotensin production in the blood vessel wall, such as the chimase pathway, as has been suggested for the heart.29
In view of the present results, it can be concluded that in SHR aortic rings, Ang II potentiates the vasoconstriction induced by phenylephrine through AT1 receptors, because losartan inhibited this effect. Moreover, this action of losartan appears to depend on the interaction of Ang II and AT2 receptors. In addition, the presence of NO seems to be necessary for the full expression of the effect of losartan on the contractile response to phenylephrine in SHR aortic rings.
Selected Abbreviations and Acronyms
|Ang I, II||=||angiotensin I, II|
|AT1, AT2||=||angiotensin type 1, type 2 (receptor)|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by a grant from Comisión Interministerial de Ciencia y Tecnología, Spain (SAF-1549-C02-01). The authors thank Lucila Krauss and Antonio Carmona for their excellent technical assistance, Merck Sharp & Dohme for kindly providing losartan, and Anthony DeMarco for his editorial assistance.
- Received March 16, 1996.
- Revision received April 10, 1996.
- Revision received July 8, 1996.
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