Published online before print August 11, 2003, doi: 10.1161/01.HYP.0000088362.50484.4C
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(Hypertension. 2003;42:802.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
SR141716A-Sensitive Enhancement of ET-1 Hypotensive Effect by Chronic NOS Inhibition
From the Departments of Pharmacology and Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil.
Correspondence to Dr Maria Cristina O. Salgado, Department of Pharmacology, School of Medicine-USP, 14049-900 Ribeirão Preto, SP, Brazil. E-mail mcdosalg{at}fmrp.usp.br
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Abstract |
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The present study evaluated the potential mechanism involved in the hypotensive effect induced by ET-1 in rats treated with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) in the drinking water during 7 days. Hypertension developed in the L-NAME–treated rats (164±3 versus 112±1 mm Hg in untreated control rats), and the hypotensive effect of ET-1 (100 pmol/kg IV) was significantly enhanced compared with control rats (32±2% versus 20±1% fall in mean arterial pressure). The enhanced ET-1 hypotensive effect in L-NAME–treated rats was abolished by the ETB receptor antagonist BQ-788 but was unaltered by the cyclooxygenase inhibitor diclofenac, the cytochrome P450 inhibitor fluconazole, or the potassium channel blockers apamin, glibenclamide, tetraethylammonium, and 4-aminopyridine. Pretreatment with the cannabinoid CB1 receptor antagonist SR141716A significantly reduced the hypotensive response to ET-1 in L-NAME–treated rats (20±1%), although it did not modify the response in untreated control rats (17±1%). These findings indicate that in rats under chronic NOS inhibition, the hypotensive effect of ET-1 is unexpectedly enhanced and appears to be mediated by a non-NO/non-prostanoid mechanism and involves an SR141716A-sensitive mechanism triggered by ETB receptor activation.
Key Words: endothelin • L-NAME • nitric oxide • prostaglandins • potassium channels
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Introduction |
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Intravascular administration of endothelin-1 (ET-1) causes a transient decrease followed by prolonged increase in arterial blood pressure in rats and other species.1 The hypertensive effect has been attributed to the activation of ETA receptors, whereas the hypotensive effect has been attributed to the activation of ETB receptors,2 although ETB receptors have been also shown to contribute to the hypertensive response.3 Initial observations in isolated and perfused rat lungs or mesentery showed that the activation of ET receptors in endothelial cells also stimulates the production of NO and prostacyclin (PGI2), suggesting that they could mediate the vasodilator effect and/or counteract the vasoconstrictor effect of ET-1.4 Studies in vivo have shown that the acute administration of NO synthase (NOS) inhibitors do not inhibit or attenuate only partially the hypotensive effect of ET-1, suggesting that NO-independent mechanisms contribute to this effect.5–7 In the present study, we report that the hypotensive effect of ET-1 administration is paradoxically enhanced in rats under chronic NOS inhibition and discuss the results of experiments designed to examine the potential mechanisms involved in this apparently NO-independent, enhanced vasodilation.
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Methods |
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Male Wistar rats (250 to 300 g) were housed under conditions of constant temperature (22°C) and exposed to a 12-hour dark-light cycle and were offered either tap water (control group) or water containing L-NAME at a concentration adjusted daily such that rats received 60 mg/kg per day. On the 6th day, under tribromoethanol anesthesia (250 mg/kg IP), the animals were instrumented with femoral venous and arterial catheters (PE-50 soldered to PE-10, Clay Adams) filled with heparinized saline (500 IU/mL) and exteriorized through the animal’s back. After the surgical procedures, the rats were allowed to drink L-NAME or tap water until the beginning of the experiment on the next day. All experiments were conducted in accordance with institutional guidelines on the use of animals in research.
Twenty hours after the surgical procedures, arterial blood pressure recording was performed with a pressure transducer (Statham P23 Gb), and the amplified (Hewlett-Packard 8805-A) signal was fed to a computer acquisition board (analog-to-digital converter CAD-12/36, software Aqdados; Lynx Tecnologia Eletrônica). Mean arterial pressure (MAP) and heart rate (HR) were derived from the arterial pulse pressure. A 30-minute period of stabilization was allowed, after which a single dose of ET-1 (100 pmol/kg) was given intravenously to control or L-NAME–treated rats. For comparison, the hypotensive response to bradykinin (20 nmol/kg IV) was also studied in control and chronically L-NAME–treated rats. To investigate the mechanism involved in the L-NAME–resistant hypotensive response to ET-1, the following drugs were administered intravenously to L-NAME–treated rats before the injection of 100 pmol/kg of ET-1: the selective ETB-receptor antagonist BQ-788 (400 µg/kg, 10 minutes before ET-1); the cyclooxygenase (COX) inhibitor diclofenac (4 mg/kg, 30 minutes before ET-1); the cytochrome P450 inhibitor fluconazole (350 mg/kg, 10 minutes before ET-1); the ATP-sensitive potassium channel inhibitor glibenclamide (40 µmol/kg, 6 minutes before ET-1); the voltage-sensitive potassium channel blocker 4-aminopyridine (4-AP, 20 µmol/kg, 6 minutes before ET-1); the calcium-activated potassium channels blockers apamin (80 nmol/kg) and tetraethylammonium (TEA, 180 µmol/kg) 15 minutes before ET-1; or the cannabinoid CB1-receptor antagonist SR141716A (15 mg/kg, 10 minutes before ET-1). The effect of SR141716A on the ET-1 hypotensive effect was also investigated in control rats. The concentrations of potassium channel inhibitors were obtained from in vivo studies with the use of normotensive anesthetized rats.8
Drugs
ET-1, L-NAME, sodium diclofenac, glibenclamide, 4-AP, apamin, TEA, and bradykinin were purchased from Sigma Chemical Co, and fluconazole from Pfizer Inc. SR141716A was provided by Research Biochemicals International as part of the Chemical Synthesis Program of the National Institute of Mental Health, Contract N01MH30003. Stock solution of fluconazole was prepared in 0.1N HCl and of glibenclamide in 0.1N NaOH, with the pH adjusted to 7.4; all other drugs were dissolved in distilled water.
Data Analysis
The data are presented as mean±SEM. Basal MAP and HR were analyzed by nonpaired Student t test, and the analysis of percentage changes in MAP and HR were performed by nonpaired Mann-Whitney followed by the post hoc Student-Newman-Keuls test. Significant differences were considered at a value of P<0.05.
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Results |
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Compared with normotensive control rats, L-NAME–treated awake rats exhibit a significantly higher MAP (164±3 versus 112±1 mm Hg, P<0.0001) and HR (404±7 versus 367±4 bpm, P<0.0001). As shown in Figure 1, in awake normotensive rats, ET-1 (100 pmol/kg IV) produced a rapid transient fall in mean arterial blood pressure followed by slowly developing and long-lasting pressor response. In L-NAME–treated rats, in contrast, ET-1 induced a significantly greater fall in MAP and a blunted pressor effect (Figure 1). The reflex tachycardia associated with the hypotensive effect induced by ET-1 was similar in both groups, whereas, as expected, the reflex bradycardia was of lesser magnitude in L-NAME–treated rats (Figure 1). The hypotensive effect induced by bradykinin (20 nmol/kg IV) was unaffected by chronic L-NAME treatment (28±2% versus 26±2% fall in MAP in control normotensive rats, n=8).
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The effects of COX and cytochrome P450 inhibitors, potassium channel blockers, as well as of the ETB receptor antagonist BQ-788 on L-NAME-resistant fall in MAP elicited by ET-1 are shown in Figure 2. Pretreatment with diclofenac decreased basal MAP (140±4 mm Hg, n=8, P<0.002) but did not affect the hypotensive response to ET-1. Fluconazole (MAP=164±8 mm Hg, n=8) and the potassium channel blockers glibenclamide (MAP= 160±5 mm Hg, n=8), 4-AP (MAP=164±4 mm Hg, n=8), apamin (MAP=162±7 mm Hg, n=8), and TEA (MAP=150±5 mm Hg, n=8, P<0.02 versus control L-NAME) pretreatment failed to modify the hypotensive effect induced by ET-1 in L-NAME–treated rats. The administration of the ETB receptor antagonist BQ-788 did not affect basal MAP (161±4 mm Hg, n=8) but practically abolished the ET-1 hypotensive effect (from 32±2% to 3±2% fall in MAP) in L-NAME–treated rats.
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Pretreatment with SR141716A did not affect basal MAP (158±4 mm Hg, n=8) but significantly (P<0.0002) reduced the hypotensive response to ET-1 in L-NAME–treated rats (Figure 3) to similar values of that in untreated control rats (MAP=107±3 mm Hg, n=3), although it did not modify the hypotensive response to ET-1 in untreated rats.
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Discussion |
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The main findings of the present study were (1) in rats made hypertensive by 1-week administration of L-NAME, the hypotensive effect of ET-1 administration was unexpectedly enhanced, whereas the hypertensive effect is blunted, (2) only the enhancement of the ET-1 hypotensive effect of L-NAME–treated rats was annulled by SR141716A, and (3) ETB blockade fully blocked the hypotensive effect of ET-1. The augmented hypotensive effect does not appear to result simply from the elevated blood pressure of these animals, since the hypotensive effect of bradykinin, which is also partially resistant to NOS inhibitors,9 was not enhanced. Although L-NAME hypertensive rats exhibit attenuated reflex tachycardia,10 this alteration seems unlikely to have contributed to the enhanced hypotensive effect of ET-1, since the hypotensive effect of bradykinin was unaltered in L-NAME rats. Therefore, it seems that chronic NO deficiency modifies selectively the pressor responses to ET-1.
The fact that the hypotensive effect of ET-1 was abolished by ETB antagonist, together with the blunted hypertensive effect, which is mediated mainly by ETA receptors, would suggest that either a downregulation of the latter or an upregulation of the former or a combination of both alterations could have contributed to the observed phenomenon. It has been shown that NOS inhibition leads to an increase in ET release,11,12 which could in turn modulate the expression of its receptors.12,13 This is unlikely to explain the present observations, since it implies that ETA but not ETB receptors are more susceptible to downregulate when exposed to continuous high levels of the peptide, which is not consistent with reports showing that ETB receptors are downregulated by continuous exposure to ET.14 Alternatively, the lack of hypertensive response in the absence of NO production would suggest that ET-1–induced hypertensive response in control rats involves the inhibition of NO action. On the other hand, the diminished NO production could also explain the enhanced hypotensive effect of ET-1, since it has been reported that NO inhibits the production of endothelium-derived hyperpolarizing factor (EDHF),15 potential mediators of the NOS inhibitors resistant to the hypotensive effect of ETB receptor activation.
It is unlikely that vasodilator prostanoids, especially PGI2, which has been shown to be produced in response to ET-1 and to diminish the hypertensive effect of ET-1 in normotensive rats,4 are involved in the enhanced hypotensive effect of ET-1, since diclofenac did not affect the ET-1 hypotensive effect in animals under chronic NOS inhibition. Similarly, the fact that fluconazole, an inhibitor of the cytochrome P4502C enzyme,16 which has been postulated to be involved in the production of vasodilator arachidonic acid metabolites,17 did not affect the hypotensive effect of ET-1 would suggest that these metabolites are also unlikely to mediate the enhanced hypotensive effect of ET-1.
The most intriguing finding of the present study was that the enhanced ET-1 hypotensive effect in L-NAME–treated rats is reduced by SR141716A, whereas this compound failed to influence the hypotensive effect of ET-1 in control normotensive rats. SR141716A was originally described as a selective CB1 receptor antagonist18 and has been shown to antagonize the vasodilator effect of the endocannabinoid anandamide,19 which would suggest that the enhancement of the ET-1 hypotensive effect in L-NAME–treated rats is mediated by anandamide or other endocannabinoid, such as 2-arachidonoglycerol,20 capable of activating SR-sensitive receptors. Noteworthy, it has recently been shown that chronic NOS inhibition enhances the relaxant effect of anandamide against noradrenaline-induced vasoconstriction.21 Interestingly, NO has been shown to stimulate anandamide uptake in endothelial cells22; it is not inconceivable that the lack of NO resulted in less uptake of anandamide produced in response to ET-1 and consequently enhanced the vasodilator effect of anandamide. More recent studies, however, indicate that mechanisms other than CB1 receptor activation participate in the vasodilation induced by anandamide, including the activation of vanilloid VR1 receptor and release of CGRP23,24 or a SR141716A-sensitive mechanism that is unrelated to CB-1 receptor activation.25 In addition, we cannot discard that an action of SR141716A unrelated to the blockade of cannabinoid receptors, such as inhibition of gap junction26 or of potassium channels,27 could have also contributed to the observed annulment of the enhanced hypotensive effect of ET-1 in L-NAME hypertensive animals.
The results of the experiments performed to detect the potential involvement of potassium channels in the hypotensive effect of ET-1 suggest that if this effect involves an EDHF-like mechanism, this is unlikely to involve apamin, 4-AP, glibenclamide, or TEA-sensitive potassium channels. It is unlikely that the lack of effect of the potassium channel blockers on the ET-1 hypotensive effect resulted from insufficient doses of these inhibitors, since TEA and 4-AP reduced basal heart frequency (data nor shown) and in preliminary experiments the dose of glibenclamide used abolished the hypoglycemic effect of cromakalim (data not shown). Interestingly, 4-AP at a lower dose than the one used in the present study has been shown to reduce the hypotensive effect of bradykinin in normotensive anesthetized rats.8 The fact that the hypotensive effect of bradykinin, which is also partially resistant to NOS inhibitors, was not modified by chronic L-NAME administration, would suggest that different mechanisms mediate the hypotensive effect of these peptides.
In conclusion, this study shows that in rats under chronic inhibition of NOS, the hypotensive effect of ET-1 is enhanced and appears to be mediated by a non-NO/non–prostanoid-dependent, and SR 141716A–sensitive mechanism triggered by ETB receptor activation. Whether the latter mechanism involves the enhanced production of an endocannabinoid remains to be elucidated.
Perspectives
Chronic inhibition of NO production resulted in an enhanced hypotensive effect of ET-1 mediated by an SR 141716A–sensitive mechanism triggered by ETB receptor activation. Noteworthy, this mechanism does not contribute to the hypotensive effect of ETB receptor activation when NO is being produced. These findings revealed a novel biological effect of NO, which is the modulation of signaling pathways involved in ETB receptor activation in endothelial cells. Whether these pathways implicate the enhanced production and/or bioavailability of an endocannabinoid remains to be elucidated.
Received May 5, 2003; first decision June 16, 2003; accepted July 16, 2003.
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