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(Hypertension. 1997;30:585.)
© 1997 American Heart Association, Inc.
Articles |
Nitric Oxide Blunts Sympathetic Response of Pregnant Normotensive and Hypertensive Rat Arteries
From the Departments of Pharmacology and Internal Medicine (E.B.C.), School of Medicine of Ribeirão Preto, University of São Paulo, Brazil.
Correspondence to Dr Maria Cristina O. Salgado, Department of Pharmacology, School of Medicine, University of São Paulo, 14049-900 Ribeirão Preto, São Paulo, Brazil. E-mail mcdosalg{at}fmrp.usp.br
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Abstract |
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Abstract Rat pregnancy is associated with a blunted response to vasocontrictors both in vivo and in vitro as well as a decrease in arterial pressure. We examined the influence of pregnancy on neurally induced vasoconstrictor and vasodilator responses of the isolated mesenteric arterial bed from normotensive Wistar and spontaneously hypertensive nonpregnant and 20-day pregnant rats and determined the possible role of nitric oxide (NO) in modulating these responses. MAP (mm Hg) in pregnant normotensive (98±1, n=13) and hypertensive (136±5, n=13) rats was lower (P<.05) than in nonpregnant controls (114±2, n=14, and 174±3, n=12, respectively). In isolated mesenteric arterial beds, electrical field stimulation (EFS; 34 V, 3 ms, 10-64 Hz) of perivascular nerves at basal tone induced a frequency-dependent increase in perfusion pressure that was significantly (P<.001) greater in preparations from hypertensive compared with normotensive rats. Pregnancy was associated with a significant decrease in the maximal vasoconstrictor response elicited by EFS in both normotensive and hypertensive groups compared with their nonpregnant controls. In phenylephrine-preconstricted mesenteric beds, EFS (60 V, 1 ms, 1-8 Hz) elicited a similar frequency-dependent decrease in perfusion pressure in normotensive and hypertensive groups, but pregnancy did not influence these responses. In the presence of the NO synthase inhibitor N
-nitro-L-arginine (200
µmol/L), the maximal vasoconstrictor response induced by EFS was
significantly (P<.001) augmented in both normotensive and
hypertensive groups, and the differences observed between pregnant and
nonpregnant groups were abolished. Responses to sodium nitroprusside
were not affected by pregnancy, although they were greater in
preparations from hypertensive rats. These results indicate that NO
contributes to pregnancy-associated diminished vasoconstrictor response
to sympathetic stimulation in the mesenteric arterial bed
of both normotensive and hypertensive rats.
Key Words: nitric oxide • vasoconstriction • rats, inbred SHR • vasodilation • mesenteric arteries
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Introduction |
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Late pregnancy in normotensive rats as well as in SHR is characterized by a decrease in MAP.1 2 3 4 In normotensive rats this phenomenon is associated with decreased pressor responses to the administration of angiotensin II, NE, and vasopressin.5 6 7 Since it has been shown that pregnancy is associated with changes in the perivascular innervation of the uterine artery, it is possible that neural control of systemic blood vessels is also influenced by pregnancy.8 Relatively little attention has been directed toward the examination of whether pregnancy affects the responsiveness of blood vessels to vasoconstrictor or vasodilator nerves. A decrease in vasoconstrictor responses to sympathetic nerve activation has been reported in human uterine arteries9 and rabbit hindlimb vascular beds.10 Also, the vasoconstrictor response of the mesenteric bed in situ or in vitro to perivascular nerve stimulation has been shown to be reduced during late pregnancy in normotensive Sprague- Dawley and Wistar-Kyoto rats and SHR.11 12 13 A decrease in sympathetic neurotransmission in pregnancy has also been shown in rat mesenteric veins, whereas no changes were observed in isolated mesenteric arteries.14
Although endothelium-derived NO has been proposed to participate in the decrease in responsiveness to vasoconstrictor agents and systemic blood pressure, the mechanisms involved in these changes remain to be fully elucidated.11 12 13 Thus, the aim of this study was to determine the influence of pregnancy on neurally induced vasoconstrictor and vasodilator responses of the isolated mesenteric bed from Wistar normotensive rats and SHR and to examine the effect of NO synthase inhibition on the eventual modifications of these responses.
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Methods |
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The experiments were conducted on age-matched (body weight, 180 to 220 g) virgin (estrous day) or pregnant (20th day) Wistar normotensive rats and SHR. The stage of the estrous cycle and pregnancy was determined by examination of vaginal smears; the day when spermatozoa were present in the vaginal smears was labeled day 0 of pregnancy.
The day before the experiments, rats were anesthetized with ether and a polyethylene catheter (PE-50) was inserted into the thoracic aorta via the left carotid artery; the distal end was exteriorized through the animal’s back. On the day of the experiment, MAP of unanesthetized rat was recorded on a four-channel recorder (Hewlett Packard 7754 A). After MAP was measured, the rats were anesthetized with ether and the mesenteric bed was removed and prepared for perfusion in a water-jacket organ bath maintained at 37°C as previously described.15 In brief, the mesenteric arteries were perfused with a modified Krebs’ solution (in mmol/L: NaCl 120.0, KCl 4.7, CaCl2 3.0, MgCl2 1.4, NaHCO3 25.0, KH2PO4 1.2, glucose 11.0, and EDTA 0.03) equilibrated with a 95% O2/5% CO2 mixture at 37°C at a constant rate of 4 mL/min (LKB-2115 multiperpex pump). The mesenteric perfusion pressure was monitored with a pressure transducer (Hewlett-Packard 1280) connected to a sidearm of the mesenteric artery cannula. EFS of periarterial nerves was achieved through two bipolar platinum ring electrodes placed around the superior mesenteric artery. EFS at basal tone consisted of rectangular pulses (3 ms, 34 V) and variable frequency (10 to 64 Hz) applied for 20 seconds at 3-minute intervals. After the perfused mesenteric artery bed was allowed to equilibrate for 15 minutes, EFS (10 to 64 Hz) was applied and the increases in perfusion pressure were recorded. These vasoconstrictor responses were abolished by adding guanethidine (5 µmol/L, Sigma) or tetrodotoxin (1 µmol/L, Sigma) to the perfusion solution at the beginning of the perfusion period. In some preparations, L-NOArg (200 µmol/L, Sigma) was added to the perfusion solution, and its effect on the vasoconstrictor responses to EFS (10 to 64 Hz) was determined.
To investigate the vasodilator responses induced by EFS of perivascular nerves (1 ms, 60 V, 1 to 8 Hz, applied for 30 seconds, with the intervals between each stimulation being dependent on the time it took for perfusion pressure to return to baseline), both guanethidine (5 µmol/L) and atropine (1 µmol/L, Sigma) were added to the perfusion solution and, after the equilibration period, the mesenteric artery bed was preconstricted with PE (Sigma) to induce an increase of 60 mm Hg in perfusion pressure, after which the vessels were subjected to EFS (1 to 8 Hz). These vasodilator responses were abolished in the presence of tetrodotoxin (1 µmol/L), indicating their neurogenic origin.
In a separate series of experiments, the effect of the NO donor SNP (Sigma) was investigated in PE-precontracted mesenteric artery beds. Dose-response curves for SNP (10 to 1200 ng) were obtained by bolus injection of 10 to 50 µL of the solution into the perfusion stream before the pump.
All experiments were conducted in accordance with institutional guidelines on the use of animals in research. Results are expressed as mean±SE; however, frequencies that elicited 50% of the maximal response (F50) are reported as geometric means with their respective 95% confidence limits. The F50 values were calculated from linear regression analysis from the complete frequency-response curves by means of a computer program. Data were analyzed by one-way ANOVA followed by Student’s t test with Bonferroni correction, and P<.05 was taken as significant.
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Results |
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MAP of nonpregnant SHR was significantly higher than that of nonpregnant Wistar rats (174±3 mm Hg, n=12, versus 114±2 mm Hg, n=14; P<.05). Pregnancy was associated with a significant decrease in MAP in both normotensive rats (98±1 mm Hg, n=13) and SHR (136±5 mm Hg, n=13). The basal perfusion pressure of isolated mesenteric beds from nonpregnant animals was significantly higher in hypertensive rats (26.8±1.5 versus 21.6±0.7 mm Hg in normotensive, P<.05). In preparations from both normotensive and hypertensive rats, pregnancy was associated with significantly (P<.05) lower basal perfusion pressure (15.0±1.3 and 21.0±0.7 mm Hg, respectively).
EFS (10 to 64 Hz) of perivascular nerves at basal tone elicited frequency-dependent vasoconstrictor responses of the mesenteric beds isolated from all pregnant and nonpregnant rats. In mesenteric beds from nonpregnant animals, the frequency of the stimulus required for eliciting F50 was significantly (P<.001) lower in preparations from hypertensive (mean F50 [95% confidence limits], 19.4 [17.8 to 20.9] Hz; n=6) than normotensive (22.4 [21.2 to 23.6] Hz; n=6) rats, while the maximal vasoconstrictor response was significantly higher in the former group (286±9 versus 215±7 mm Hg, P<.05) (Fig 1, top).
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Pregnancy was associated with a significant decrease in the maximal vasoconstrictor response elicited by EFS when compared with nonpregnant rats in both normotensive (168±14 mm Hg, n=7, P<.05) and hypertensive (171±30 mm Hg, n=8, P<.001) groups. In addition, pregnancy induced a significant rightward shift of the frequency-response curve in the hypertensive group only(F50=27 [25 to 30] Hz). In contrast with the nonpregnant state, the frequency-response curves of preparations from pregnant normotensive and hypertensive animals were indistinguishable (Fig 1, top).
PE infusion induced an increase in mesenteric perfusion pressure from both normotensive and hypertensive rats. However, the concentration required to raise perfusion pressure by 60 mm Hg was significantly higher in preparations from both pregnant normotensive (17.5±1.0 versus 8.5±1.0 µmol/L) and hypertensive (44.0±8.0 versus 11.0±1.0 µmol/L) rats compared with their nonpregnant controls. In PE-preconstricted mesenteric beds and in the presence of guanethidine (5 µmol/L) and atropine (1 µmol/L), EFS (1 to 8 Hz) of perivascular nerves elicited frequency-dependent vasodilator responses in normotensive and hypertensive groups in both the pregnant and the nonpregnant states. No differences were observed in the responses between nonpregnant normotensive and hypertensive groups (n=7 each), and pregnancy did not influence these responses (Fig 1, middle).
L-NOArg (200 µmol/L) did not affect basal perfusion pressure of mesenteric beds from any of the groups. In mesenteric beds from nonpregnant animals, L-NOArg caused a significant shift to the left (F50=18 [17.0 to 19.5] Hz) of the vasoconstrictor frequency-response curve in the normotensive group only. In contrast, the maximal vasoconstrictor response induced by perivascular nerve EFS was significantly (P<.001) increased by L-NOArg in both normotensive (n=8) and hypertensive (n=6) groups (208±9 and 285±9 mm Hg, respectively). Interestingly, in the presence of L-NOArg the differences observed between either normotensive or hypertensive pregnant (n=6 each) and nonpregnant groups were abolished (Fig 1, bottom).
In preconstricted mesenteric beds from pregnant and nonpregnant normotensive or hypertensive rats, SNP caused a dose-dependent decrease in perfusion pressure (n=7 each). Vasodilation induced by SNP was significantly greater in preparations from hypertensive rats compared with those from normotensive rats. Nevertheless, pregnancy did not significantly influence mesenteric artery bed responses to SNP in either the normotensive or the hypertensive group (Fig 2).
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Discussion |
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The present study shows that constrictor responses of mesenteric resistance vessels induced by perivascular nerve stimulation are significantly reduced in late-pregnant normotensive rats and SHR and that the NO synthase inhibitor L-NOArg reversed this reduction. Since perivascular nerves were activated by EFS, the observed reduction in vasoconstrictor responses could have resulted from a decreased response to sympathetic nerves or from an increase in response to vasodilator nerve activation. The latter possibility seems unlikely, since no differences were observed in the vasodilator responses induced by perivascular nerve activation in the presence of guanethidine or atropine. In our study, we have confirmed that the vasoconstrictor response induced by perivascular nerve activation is mediated by sympathetic nerves, since guanethidine abolished these responses. Thus, the decrease in the vasoconstrictor response appears to be related to a decrease in the response to sympathetic nerve activation, which could have resulted from prejunctional and/or postjunctional changes. Previous reports have shown that sympathetic nerve–induced vasoconstrictor responses are diminished in pregnancy.10 11 12 13 16 It is possible that the density of sympathetic innervation in the mesenteric bed decreases during pregnancy, as has been reported for the guinea pig uterine artery.8 On the other hand, NE-induced vasoconstriction of isolated vessels has been shown to be decreased in late pregnancy,17 which would suggest that the diminished response to sympathetic nerve activation observed in the present study resulted from postjunctional changes. Consistent with this interpretation, the concentration of PE required to increase perfusion pressure by 60 mm Hg was higher in mesenteric beds from pregnant animals. It is noteworthy, however, that no changes in responsiveness to NE in isolated mesenteric bed from pregnant Sprague-Dawley rats11 12 13 or in sympathetic responses of isolated mesenteric arteries have been reported.18 19
Interestingly, the vasoconstrictor responses of mesenteric resistance vessels to sympathetic nerve activation in nonpregnant SHR observed in the present study, while significantly higher than normotensive rats, became similar to the normotensive rats in late pregnancy, indicating that pregnancy-associated factors exert a powerful influence on neurovascular function. It has been suggested that enhanced production of NO is involved in the decrease in vascular tone and responsiveness to vasoconstrictor agents associated with pregnancy.11 12 20 Therefore, the effect of the NO synthase inhibitor L-NOArg on vasoconstrictor responses induced by sympathetic nerve activation was also determined. L-NOArg enhanced the vasoconstrictor responses in both normotensive and hypertensive pregnant rats, making them indistinguishable from those of nonpregnant animals. These findings are consistent with those reported by Chu and Beilin11 for the SHR but contrast with those reported by the same authors12 for WKY animals, in which an NO-independent vasodilator mechanism was proposed to explain the diminished vascular reactivity. Our results suggest that NO is responsible for the blunted response of mesenteric resistance vessels to sympathetic nerve activation observed in late pregnancy in both normotensive animals and SHR. NO could be acting as a functional antagonist at the smooth muscle level and/or as a modulator of NE release by sympathetic nerves.21 It has been shown that in vessels with both noradrenergic and nitrergic innervation subjected to EFS, noradrenergic contractions are enhanced by NO synthase inhibitors.22 These observations indicate that electrical stimulation activated both noradrenergic and nitrergic transmission and that NO has a modulatory influence on the response to sympathetic nerve activation. This influence has been attributed to a postjunctional effect and not to prejunctional inhibition of NE transmission, since NO synthase inhibitors did not alter the amount of NE released by EFS.21 23 24 25 However, an endothelium-derived factor has been shown to inhibit NE release from sympathetic nerves in the rabbit carotid artery.26 The fact that L-NOArg did not alter the basal perfusion pressure indicates that under our conditions, there was no substantial basal production of NO and that perivascular nerve stimulation was associated with activation of NO synthesis. Whether NO synthesis stimulated by perivascular nerve activation occurs in endothelial or smooth muscle cells or in perivascular nerves was not investigated in the present study. Endothelial NO synthesis could have been activated by NE released during sympathetic nerve activation or by an increase in shear stress secondary to vasoconstriction. Alternatively, NO synthesis could have occurred in perivascular nitrergic nerves present in this preparation. This explanation seems unlikely, since NO synthase inhibitors failed to affect, or even enhance, the relaxations of the mesenteric bed induced by perivascular nerve activation, which would suggest that the vasodilation induced by these nerves was not mediated by NO.27 Finally, it cannot be excluded that NO synthesis occurred in smooth muscle cells. Although vascular smooth muscle cells do not express any isoform of NO synthase under normal conditions, it is possible that pregnancy induces the expression of this enzyme in these cells.
The fact that in both normotensive and hypertensive rats pregnancy was not associated with any significant change in the vasodilator affect of the NO donor SNP indicates that enhanced production of NO seems to be entirely responsible for the decreased vasoconstrictor response to sympathetic nerve activation observed in late pregnancy.
In conclusion, our study provides evidence indicating that NO contributes to pregnancy-associated diminished vasoconstrictor responses of mesenteric resistance vessels to sympathetic nerve activation in both normotensive and hypertensive rats. Furthermore, these data suggest that pregnancy is associated with an increase in NO production in response to sympathetic nerve activation in both normotensive and hypertensive rats.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants to Gustavo Ballejo and Maria Cristina O. Salgado from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The authors acknowledge Osmar Vettori and Tadeu F. Vieira for expert technical assistance.
Received March 17, 1997; first decision April 17, 1997; accepted May 1, 1997.
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