Clinical and Experimental Pharmacology and Physiology (1992) 19,376-379
SHORT COMMUNICATION
RENAL VASOCONSTRICTION IS MODULATED BY NITRIC OXIDE Julianne J. Reid and Michael J. Rand
Department of Pharmacology, University of Melbourne, Victoria, Australia (Received 13 December 1991)
SUMMARY 1. The effect of the nitric oxide synthesis inhibitor, NG-nitro-L-arginine (NOLA),has been examined on vascular reactivity in the rat isolated perfused kidney. 2. NOLA (10 pmol/L) had no effect on basal perfusion pressure, but significantly enhanced the vasoconstrictor responses to sympathetic nerve stimulation (1-16 Hz, 10 s) and noradrenaline (10-300 pmol). The enhancements were greater with the lower frequencies of stimulation and lower doses of noradrenaline. 3. The enhancing effect of 10 pmol/L NOLA on vasoconstrictor responses to nerve stimulation was partially prevented by 100 pmol/L L-arginine while 100 prnol/L D-arginine had no effect. 4. The results suggest that nitric oxide attenuates vasoconstrictor responses in the rat kidney, and provide evidence that nitric oxide has a physiological role in the modulation of vascular reactivity. Key words: kidney (rat), nitric oxide, NO-nitro-L-arginine, noradrenaline, sympathetic nerve stimulation.
INTRODUCTION Nitric oxide is an important endogenous vasodilator released from vascular endothelid cells (Palmer et al. 1987). The synthesis of nitric oxide from L-arginine is catalysed by the enzyme, nitric oxide synthase, which can be inhibited by a variety of L-arginine analogues, including NG-monomethyl-L-arginine(NMMA) and NG-nitro-L-arginine (NOLA; Moncada et al. 1991). Nitric oxide synthase inhibitors have been instrumental in identifying the physiological roles of endotheliumderived nitric oxide in the regulation of blood vessel
function. Systemic infusion of NMMA increases mean arterial blood pressure, suggestingthat a basal release of nitric oxide contributes to resting vascular tone (Rees et al. 1989; Vargas et al. 1990). A second physiological role for nitric oxide in the vasculature has been proposed, namely the modulation of vascular reactivity to other vasoactive agents (Vo et al. 1991). We have previously demonstrated that inhibition of nitric oxide synthesisby NOLA enhances the vasoconstrictor responses to sympathetic nerve stimulation
Correspondence: Dr J. J. Reid, Department of Pharmacology,University of Melbourne, Vic. 3052, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
377
Nitric oxide modulates renal vasoconstriction
and noradrenaline in the rat isolated caudal artery (Vo et al. 1991) and mesenteric arterial bed (Way et al. 1991). In this study, these observations have been extended to include the effect of NOLA on vasoconstrictor responses in the microvascular bed of the rat isolated perfused kidney.
METHODS Sprague-Dawley rats (250-400 g) of either sex were pretreated with heparin (1000 units/kp i.p. 30 rnin before the experiment) and anaesthetized with pentobarbitone sodium (80-100 mg/kg i.p.). The left kidney was isolated and perfused through the renal artery at a flow rate of 4 mL/min, as described by Boke & Malik (1983). The perfusion fluid was physiological salt solution (PSS), continuously bubbled with 95% 02/5% COz and maintained at 37"C, of the following composition (in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgCh 1.2, NaHC03 25.9, KH2P041.0, D( +) - glucose 10, Na pyruvate 2, NazEDTA 0.067, ascorbic acid 0.14. The perivascular nerves of the renal artery were stimulated with square wave pulses of 1 ms duration at supramaximal voltage (40 V), using a bipolar platinum electrode. Perfusion pressure was measured with a pressure transducer (Statham or Cobe CDX-111) and recorded on a Grass polygraph. Increases in perfusion pressure were taken as indices of vasoconstriction. After a stabilization period of 20-30 min, vasoconstrictor responses either to nerve stimulation (1 -16 Hz, 10 s) or to bolus injections of noradrenaline (10-300 pmol) were elicited at 3 and 5 rnin intervals, respectively. After 30-45 min, NOLA (10 pmol/L) was added to the perfusate. Where indicated, Larginine or D-arginine was included 20-30 rnin before the addition of NOLA. In time control experiments, NOLA was absent from the perfusate. In all experiments, the presence of functional endothelium was confirmed by the vasodilator action of acetylcholine (1 -30 pmol) in tissues precontracted with phenylephrine (0.3-1 pmol/L). It was also confirmed that NOLA (10 pmol/ L) reduced acetylcholine-induced vasodilatation by 50-100%. Results are expressed as mean f s.e.m., and differences between means were assessed by paired twotailed Student's t-test or one-way multivariate analysis of variance (MANOVA). A probability level of less than 0.05 was taken to indicate statistical significance.
RESULTS The increases in perfusion pressure produced by nerve stimulation (1-16 Hz,10 s; Fig. la) were abolished by 0.1 pmol/L tetrodotoxin or 1 pmol/L prazosin, indicating their neuronal origin and their mediation by activation of a-adrenoceptors. The vasoconstrictor responses to sympathetic nerve stimulation remained constant for up to 3 h (n = 7, data not shown). NOLA (10 pmol/L) did not significantly affect the basal perfusion pressure of 39.4 k 2.1 paired t-test, n = 15), but enhanced mmHg (P>0.05, the responses to stimulation (Fig. la). The enhancements had reached a maximum after 45-60 min, and were greater with lower, rather than higher, frequencies of stirnulation. After a 60 rnin exposure to 10 pmol/ L NOLA, the responses to nerve stimulation at 2, 4, 8 and 16 Hz for 10 s were significantly enhanced to 525 f67%, 442 f 87% 203 f23% and 131 f4%, respectively, of the initial responses before NOLA (P
SHORT COMMUNICATION
RENAL VASOCONSTRICTION IS MODULATED BY NITRIC OXIDE Julianne J. Reid and Michael J. Rand
Department of Pharmacology, University of Melbourne, Victoria, Australia (Received 13 December 1991)
SUMMARY 1. The effect of the nitric oxide synthesis inhibitor, NG-nitro-L-arginine (NOLA),has been examined on vascular reactivity in the rat isolated perfused kidney. 2. NOLA (10 pmol/L) had no effect on basal perfusion pressure, but significantly enhanced the vasoconstrictor responses to sympathetic nerve stimulation (1-16 Hz, 10 s) and noradrenaline (10-300 pmol). The enhancements were greater with the lower frequencies of stimulation and lower doses of noradrenaline. 3. The enhancing effect of 10 pmol/L NOLA on vasoconstrictor responses to nerve stimulation was partially prevented by 100 pmol/L L-arginine while 100 prnol/L D-arginine had no effect. 4. The results suggest that nitric oxide attenuates vasoconstrictor responses in the rat kidney, and provide evidence that nitric oxide has a physiological role in the modulation of vascular reactivity. Key words: kidney (rat), nitric oxide, NO-nitro-L-arginine, noradrenaline, sympathetic nerve stimulation.
INTRODUCTION Nitric oxide is an important endogenous vasodilator released from vascular endothelid cells (Palmer et al. 1987). The synthesis of nitric oxide from L-arginine is catalysed by the enzyme, nitric oxide synthase, which can be inhibited by a variety of L-arginine analogues, including NG-monomethyl-L-arginine(NMMA) and NG-nitro-L-arginine (NOLA; Moncada et al. 1991). Nitric oxide synthase inhibitors have been instrumental in identifying the physiological roles of endotheliumderived nitric oxide in the regulation of blood vessel
function. Systemic infusion of NMMA increases mean arterial blood pressure, suggestingthat a basal release of nitric oxide contributes to resting vascular tone (Rees et al. 1989; Vargas et al. 1990). A second physiological role for nitric oxide in the vasculature has been proposed, namely the modulation of vascular reactivity to other vasoactive agents (Vo et al. 1991). We have previously demonstrated that inhibition of nitric oxide synthesisby NOLA enhances the vasoconstrictor responses to sympathetic nerve stimulation
Correspondence: Dr J. J. Reid, Department of Pharmacology,University of Melbourne, Vic. 3052, Australia. Presented at the High Blood Pressure Research Council of Australia meeting on 12-13 December 1991, Adelaide, Australia.
377
Nitric oxide modulates renal vasoconstriction
and noradrenaline in the rat isolated caudal artery (Vo et al. 1991) and mesenteric arterial bed (Way et al. 1991). In this study, these observations have been extended to include the effect of NOLA on vasoconstrictor responses in the microvascular bed of the rat isolated perfused kidney.
METHODS Sprague-Dawley rats (250-400 g) of either sex were pretreated with heparin (1000 units/kp i.p. 30 rnin before the experiment) and anaesthetized with pentobarbitone sodium (80-100 mg/kg i.p.). The left kidney was isolated and perfused through the renal artery at a flow rate of 4 mL/min, as described by Boke & Malik (1983). The perfusion fluid was physiological salt solution (PSS), continuously bubbled with 95% 02/5% COz and maintained at 37"C, of the following composition (in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgCh 1.2, NaHC03 25.9, KH2P041.0, D( +) - glucose 10, Na pyruvate 2, NazEDTA 0.067, ascorbic acid 0.14. The perivascular nerves of the renal artery were stimulated with square wave pulses of 1 ms duration at supramaximal voltage (40 V), using a bipolar platinum electrode. Perfusion pressure was measured with a pressure transducer (Statham or Cobe CDX-111) and recorded on a Grass polygraph. Increases in perfusion pressure were taken as indices of vasoconstriction. After a stabilization period of 20-30 min, vasoconstrictor responses either to nerve stimulation (1 -16 Hz, 10 s) or to bolus injections of noradrenaline (10-300 pmol) were elicited at 3 and 5 rnin intervals, respectively. After 30-45 min, NOLA (10 pmol/L) was added to the perfusate. Where indicated, Larginine or D-arginine was included 20-30 rnin before the addition of NOLA. In time control experiments, NOLA was absent from the perfusate. In all experiments, the presence of functional endothelium was confirmed by the vasodilator action of acetylcholine (1 -30 pmol) in tissues precontracted with phenylephrine (0.3-1 pmol/L). It was also confirmed that NOLA (10 pmol/ L) reduced acetylcholine-induced vasodilatation by 50-100%. Results are expressed as mean f s.e.m., and differences between means were assessed by paired twotailed Student's t-test or one-way multivariate analysis of variance (MANOVA). A probability level of less than 0.05 was taken to indicate statistical significance.
RESULTS The increases in perfusion pressure produced by nerve stimulation (1-16 Hz,10 s; Fig. la) were abolished by 0.1 pmol/L tetrodotoxin or 1 pmol/L prazosin, indicating their neuronal origin and their mediation by activation of a-adrenoceptors. The vasoconstrictor responses to sympathetic nerve stimulation remained constant for up to 3 h (n = 7, data not shown). NOLA (10 pmol/L) did not significantly affect the basal perfusion pressure of 39.4 k 2.1 paired t-test, n = 15), but enhanced mmHg (P>0.05, the responses to stimulation (Fig. la). The enhancements had reached a maximum after 45-60 min, and were greater with lower, rather than higher, frequencies of stirnulation. After a 60 rnin exposure to 10 pmol/ L NOLA, the responses to nerve stimulation at 2, 4, 8 and 16 Hz for 10 s were significantly enhanced to 525 f67%, 442 f 87% 203 f23% and 131 f4%, respectively, of the initial responses before NOLA (P
