Inhibition of renal nitric oxide synthesis with NG-monomethyl-L-arginine and NG-nitro-L-arginine PiiL A. NAESS, KNUT Institute for Experimental N-0407 Oslo 4, Norway
A. KIRKEBOEN, GEIR CHRISTENSEN, AND FREDRIK Medical Research, University of Oslo, Ullevaal Hospital,
Naess, P&l A., Knut A. Kirkebgen, Geir Christensen, and Fredrik Kiil. Inhibition of renal nitric oxide synthesis with NC;-monomethyl-L-arginine and N’=-nitro-L-arginine. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F939-F942, 1992.-In barbiturate-anesthetized dogs, the effects of intrarenal infusion of the two selective inhibitors of nitric oxide synthesis, N’;-monomethyl-L-arginine (L-NMMA) and NG-nitroL-arginine (NO-Arg), were compared. Basal renal blood flow (RBF) was reduced by 15 k 2% after L-NMMA at 0.28 pmol/ml, by 20 & 3% after NO-Arg at 0.07 pmol/ml, and by 31 t 5% after NO-Arg at 0.56 pmol/ml. Endothelium-dependent vasodilation induced by intrarenal infusion of acetylcholine was unaltered after L-NMMA, reduced by 24 ? 3% after NO-Arg at 0.07 pmol/ml, and reduced by 59 t 13% after NO-Arg at 0.56 ,umol/ml. Endothelium-independent vasodilation induced by intrarenal infusion of atria1 natriuretic factor was not reduced after L-NMMA and NO-Arg. This study shows that NO-Arg is more potent than L-NMMA in inhibiting basal renal nitric oxide synthesis. In contrast to L-NMMA, NO-Arg exerted an inhibitory effect on acetylcholine-induced renal vasodilation. Our findings indicate that one-third of the basal RBF and more than one-half of the increase in RBF during acetylcholine infusion are dependent on nitric oxide synthesis. acetylcholine; atria1 natriuretic factor; dog; endothelium-dependent vasodilation; endothelium-derived relaxing factor; endothelium-independent vasodilation; renal hemodynamics RELAXATION induced by acetylcholine is dependent on the presence of a functionally intact endothelium that releases an endothelium-derived relaxing factor (EDRF) (6). Recent studies have shown that nitric oxide constitutes, at least in part, the biological activity of EDRF (12, 17). Demonstration of the biosynthesis of nitric oxide from the amino acid L-arginine (16, 18) has led to the introduction of arginine analogues, such as N”-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (NO-Arg) as selective inhibitors of nitric oxide synthesis (7, 18, 20). The importance of nitric oxide to renal circulation in vivo is not known. Studies on isolated perfused rat kidneys in which the vessels were preconstricted with norepinephrine or methoxamine have indicated that acetylcholine induces renal vasodilation through an endothelium-dependent mechanism (2, 19). A reduction in renal blood flow (RBF) after intrarenal infusion of a selective inhibitor of nitric oxide synthesis has been demonstrated in only one in vivo study. In that study, Salom et al. (21) reported that, although RBF was reduced, acetylcholine-induced vasodilation was unaltered after intrarenal infusion of L-NMMA. The purpose of the present study was to investigate the role of nitric oxide in renal circulation in vivo. In vitro studies have demonstrated that NO-Arg is 4-70 times more potent than L-NMMA as a specific inhibitor of EDRF (7, 13, 15). In anesthetized dogs with denervated kidneys the effects of intrarenal infusion of
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L-NMMA and NO-Arg were compared with regard to basal RBF and endothelium-dependent (with acetylcholine) and endothelium-independent (with atria1 natriuretic factor) vasodilation. METHODS Animal preparation. Mongrel dogs (16-34 kg body wt) of either sex were anesthetized with pentobarbital sodium (25 mg/kg body wt iv) with maintenance doses of l-3 mg/kg. The dogs were intubated with an endotracheal tube and ventilated by a volume-regulated respirator (model 101, Princeton Medical Instruments, Natick, MA) to keep plasma pH between 7.37 and 7.42. Body temperature was kept constant by a heating pad. The urinary bladder was drained through a urethral catheter. The femoral artery was cannulated for arterial blood sampling and for blood pressure recording by a Statham P23 Gb pressure transducer (Gould Instruments). One kidney was exposed through a flank incision. All visible nerves were cut, and a curved 25gauge needle connected to a polyethylene tube was inserted in retrograde direction into the renal artery for drug infusion. An electromagnetic flow probe (Nycotron) was placed on the renal artery for measuring RBF. Experimental procedure. The effects of intrarenal infusion of L-NMMA and NO-Arg were examined with regard to basal RBF as well as to endothelium-dependent and -independent vasodilation in one group of six dogs. Before administration of the two nitric oxide inhibitors, acetylcholine and atria1 natriuretic factor were infused into the renal artery in each dog to select doses that increased RBF similarly. Each drug was infused until a stable RBF was reached (l-2 min). The selected doses of acetylcholine and atria1 natriuretic factor increased RBF by 24 t 2 and 28 * 6%, respectively. Because basal RBF varied from 105 to 270 ml/min between dogs, the arginine analogues were administered to give a fixed renal arterial blood concentration. The intrarenal infusion rate was kept constant at 1 ml/min. Before administration of L-NMMA, D-arginine and then L-arginine were infused into the renal artery for 5 min to yield a renal arterial concentration of 1.0 pmol/ml to exclude a direct vasoactive effect of these arginine enantiomers. L-NMMA was infused into the renal artery for 5 min to yield a renal arterial concentration of 0.28 pmol/ml. The selected doses of acetylcholine (1.0-2.0 pg/min) and atria1 natriuretic factor (50- 100 ng min -lo kg body wt-‘) were then infused in random order within 5 min after discontinuing L-NMMA infusion. Thereafter, renal arterial concentrations of D-arginine at 1.0 I.cmol/ml and then L-arginine at 1.0 pmol/ml were infused. Finally the selected doses of acetylcholine and atria1 natriuretic factor were infused for the third time. An identical experimental procedure was thereafter followed for NO-Arg at a low dose, to yield a renal arterial concentration of 0.07 ,umol/ml, and then at a high dose, to yield a renal arterial concentration of 0.56 pmol/ml. To determine which doses of NO-Arg produced maximal and minimal effects on RBF and the flow response to acetylcholine, experiments were performed on a second group of three dogs. NO-Arg at renal arterial concentrations (in pmol/ml) of 0.01, 0.035, 0.07, 0.56, and 1.12 were each infused for 5 min. The effects on RBF and acetylcholine-induced (1.0 pg/ml) vasodila-
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tion were examined for each dose of NO-Arg. The ability of L-arginine to reverse the effects of NO-Arg was examined for each dose of NO-Arg that reduced RBF or the vasodilatory response to acetylcholine. Drugs. Atria1 natriuretic factor, D-arginine hydrochloride, L-arginine hydrochloride, and NO-Arg were purchased from Sigma Chemical (St. Louis, MO). Acetylcholine was purchased from Dispersa. L-NMMA was a kind gift from Dr. H. Hodson, Wellcome Research Laboratories (Beckenham, UK). All drugs were dissolved in saline. Atria1 natriuretic factor was dissolved in 1% bovine serum albumin in saline. To dissolve the high dose of NO-Arg, sodium hydroxide was added to the solution. After NO-Arg was dissolved, pH was adjusted to 8.0. StatisticaL anaZysis. The data are means & SE. Each dog served as its own control, and the Friedman test (4) was used to calculate the probability values for multiple comparisons. The Wilcoxon two-tailed signed-rank test was used for paired statistical analysis. A probability value of P < 0.05 was regarded as statistically significant. RESULTS
Effect of L-NMMA and NO-Arg on basal RBF. Infusion of L-NMMA at a renal arterial concentration of 0.28 pmol/ml significantly reduced RBF by 15 rt 2% (Fig. 1) to a new steady state after 4-5 min and increased mean arterial blood pressure by 7 t 1%. When L-NMMA was discontinued, RBF was completely normalized in two dogs 10 min after stopping infusion of L-NMMA. In the other four dogs RBF remained low. Infusion of L-arginine at a renal arterial concentration of 1.0 pmol/ml did not influence RBF before L-NMMA infusion. After L-NMMA infusion L-arginine completely restored RBF. Infusion of the low dose of NO-Arg at a renal arterial concentration of 0.07 ,umol/ml reduced RBF by 20 t 3%, which is significantly more than the reduction seen after L-NMMA (Fig. 1). L-Arginine did not influence RBF before the low dose of NO-Arg. After infusion of the low dose of NO-Arg, L-arginine restored RBF only partially (by 48 t 13%). Infusion of the high dose of NO-Arg at a renal arterial concentration of 0.56 pmol/ml reduced RBF by 31 t 5%, which is significantly more than the reduction seen after the low dose of NO-Arg (Fig. 1). L-Arginine did not influence RBF before the high dose of NO-Arg. After infusion of the high dose of NO-Arg, L-arginine did not restore RBF significantly (by 17 t 10%). Mean arterial blood
HEMODYNAMICS
pressure was not affected by the two doses of NO-Arg. Infusion of D-arginine at a renal arterial concentration of 1 pmol/ml did not influence RBF either before or after the infusions of L-NMMA or NO-Arg. In the second group of three dogs, infusion of NO-Arg at renal arterial concentrations lower than 0.07 pmol/ml did not reduce RBF. Infusion of NO-Arg at a renal arterial concentration of 0.07 pmol/ml reduced RBF by on average 11%. Subsequent infusion of L-arginine at a renal arterial concentration of 7.0 pmol/ml reversed RBF by on average 50%. Infusion of NO-Arg at a renal arterial concentration of 0.56 ,umol/ml reduced RBF by on average 23%. Doubling the dose of NO-Arg to 1.12 pmol/ml did not reduce RBF further. Subsequent infusion of L-arginine at a renal arterial concentration of 20.0 pmol/ml did not reverse RBF after these high doses of NO-Arg. Effects of L-NMMA and NO-Arg on RBF responses to acetylcholine and atria1 natriuretic factor. After intrarenal infusion of L-NMMA, the increments in RBF caused by acetylcholine (1 .O-2.0 pg/min) and by atria1 natriuretic factor (50-100 ngemin -la kg body wt-l) were not significantly altered (Fig. 2). After intrarenal infusion of the low dose of NO-Arg, the flow response to acetylcholine was reduced by 24 t 3%. In contrast, the flow response to atria1 natriuretic factor was increased by 104 t 19% (Fig. 2). After infusion of L-arginine, the flow response to acetylcholine was not significantly different from the flow response to acetylcholine before infusion of the low dose of NO-Arg. After intrarenal infusion of the high dose of NO-Arg, the flow response to acetylcholine was reduced by 59 t 13%) whereas the flow response to atria1 natriuretic factor was increased by 41 t 9% (Fig. 2). After infusion of L-arginine, the flow response to acetylcholine was not significantly different from the flow response to acetylcholine before infusion of the high dose of NO-Arg. In the second group of three dogs, infusion of NO-Arg at renal arterial concentrations lower than 0.07 pmol/ml did not reduce the flow response to acetylcholine. NO-Arg at renal arterial concentrations (in hmol/ml) of 0.07,0.56, and 1.12 reduced the vasodilatory response to acetylcholine by an average of 22, 49, and 52%, respectively. L-Arginine completely restored the flow response to acetylcholine after each dose of NO-Arg. DISCUSSION
-s -5 -u g
L NMMA (0.28
~mol/ml)
NOARG (0.07
umol/ml)
NOARG (0.56
umol/ml)
O 10
0 z 20 al .-C 1;7
30
This study on anesthetized dogs shows that the two nitric oxide inhibitors, L-NMMA and NO-Arg, both reduce basal RBF. NO-Arg reduced basal RBF more than L-NMMA, and only NO-Arg inhibited the flow response to acetylcholine. Effect of L-NMMA and NO-Arg on basal RBF. L-NMMA reduced RBF by 15%. Doubling of infusion rates in two additional experiments had no further effect. Salom et al. (21) found a 13% reduction in RBF after 30 min intrarenal infusion of L-NMMA (50 pg. mine1 kg body wt-I) in anesthetized dogs. This response is similar to that found in our study although we infused L-NMMA in a concentration -10 times higher than in the study of Salom et al. (21). In both studies drugs were infused into the renal artery to minimize systemic effects. Systemic infusions of L-NMMA and NO-Arg in rats have indicated l
Fig. 1. Effect of intrarenal infusion of NG-monomethyl-L-arginine (L-NMMA) and NG -nitro-L-arginine (NO-Arg) on renal blood flow. * Significantly different from reduction induced by L-NMMA. t Significantly different from reduction induced by the low dose of NO-Arg. Values are means k SE in 6 dogs.
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NITRIC F
120-
2
m
: Acetylcholine
m
: Atrial
natriuretic
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F941
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factor
-2 loo3 2 80-0 : E 600 5L 40caI ; 200, ;s -c O-
Fig. 2. Effect of intrarenal infusion of L-NMMA and NO-Arg on renal blood flow responses to acetylcholine and atria1 natriuretic factor. * Significantly different from flow response to acetylcholine before infusion of NO-Arg. t Significantly different from flow response to atria1 natriuretic factor before infusion of NO-Arg. Values are means t SE in 6 dogs. Before (0.28
LNMMA pmol/ml)
After (0.28
LNMMA
Before
pmol/ml)
(0.07
NOARG pmol/ml)
After (0.07
NOARG
Before
pmol/ml)
(0.56
that tonic release of EDRF may influence renal vascular resistance (1,22,23). Large, acute increments in systemic arterial blood pressure in those studies probably raised renal vascular resistance by activating the autoregulatory mechanism (1). The importance of direct renal vasoconstriction caused by L-NMMA and NO-Arg is, therefore, difficult to assess from those studies. The effect of intrarenal infusion of NO-Arg has not previously been studied. In our study both doses of NOArg induced greater reductions in basal RBF than did L-NMMA. Infusion of the high dose of NO-Arg reduced RBF by as much as 31%, indicating that basal renal vascular tone is substantially dependent on nitric oxide synthesis. Doubling the dose of NO-Arg to a renal arterial concentration of 1.12 pmol/ml in three additional experiments had no further effect on RBF. Radermacher et al. (19) infused equimolar doses of L-NMMA and NO-Arg in two groups of isolated perfused rat kidneys preconstricted with norepinephrine. In both their and our studies the increase in renal vascular resistance was approximately twice as large after NO-Arg as after L-NMMA administration. In our study L-arginine restored RBF after administration of L-NMMA. This is in agreement with the hypothesis that L-NMMA competes with L-arginine for the nitric oxide-forming enzyme (18, 20). On the other hand, L-arginine did not completely reestablish RBF after infusion of NO-Arg. L-Arginine restored ~50% of the reduction in RBF caused by the small dose of NO-Arg, but L-arginine had no effect on RBF after administration of the large dose of NO-Arg. In three additional experiments higher concentrations of L-arginine did not fully reestablish RBF after infusion of NO-Arg. Previous studies concerning the reversibility of the effects of NO-Arg have given equivocal results. In a study on aortic ring preparations Moore et al. (13) reported complete reversal of the effects of NO-Arg after L-arginine administration. In contrast, Kobayashi and Hattori (9) found almost irreversible inhibition of the vasorelaxation induced by acetylcholine after treatment with NOArg in the same preparation. Mugge et al. (14) demonstrated that a 30-fold higher concentration of L-arginine only partially reversed the effects of NO-Arg on blood flow to heart and hindlimb in rabbits. Effects of L-NMMA and NO-Arg on RBF responses to acetylcholine and atria1 natriuretic factor. In studies on isolated rat kidneys inhibitors such as hemoglobin (2),
NOARG ymol/ml)
After (0.56
NOARG pmol/ml)
gossypol (2, 19), and methylene blue (19) reduced the vasodilator effect of acetylcholine. Tolins et al. (22) found in clearance studies on anesthetized rats that intravenous injection of L-NMMA prevented both the fall in systemic blood pressure and the increase in para-aminohippurate clearance during intravenous infusion of acetylcholine. In our study L-NMMA did not reduce acetylcholine-induced vasodilation. Salom et al. (21) found that intrarenal infusion of L-NMMA did not reduce acetylcholine-induced vasodilation in anesthetized dogs. But L-NMMA completely blocked the vasodilator effect of acetylcholine after pretreatment with an inhibitor of prostaglandin synthesis, meclofenamate, a finding in agreement with results obtained by Lahera et al. (10). Salom et al. (21) proposed that the acetylcholine-induced increase in RBF was mediated by both prostaglandins and EDRF and that the hemodynamic effect of EDRF could be fully compensated for by vasodilating prostaglandins during EDRF blockade with L-NMMA. On the other hand, Luscher et al. (II) found in a study on rat renal arterial ring preparations that inhibition of prostaglandin synthesis with indomethacin augmented the vasorelaxation induced by acetylcholine. They concluded that acetylcholine appeared to release both EDRF and a vasoconstrictor prostanoid from the renal artery. In our study the RBF response to acetylcholine was reduced after infusion of both doses of NO-Arg in a dose-dependent manner. After infusion of the high dose of NO-Arg, the vasodilatory response to acetylcholine was reduced by as much as 59%. Hence we have shown that NO-Arg reduced a large fraction of both basal RBF and acetylcholine-induced renal vasodilation without pretreatment with a prostaglandin synthesis inhibitor. Since NO-Arg exerted no inhibitory effect on the endothelium-independent vasodilation caused by infusion of atria1 natriuretic factor, the responses to NO-Arg probably reflect inhibition of nitric oxide synthesis in vivo. Doubling the dose of NO-Arg from a renal arterial concentration of 0.56 to 1.12 pmol/ml in three additional experiments did not reduce the vasodilatory response to acetylcholine further. That inhibition of renal nitric oxide synthesis failed to completely prevent acetylcholine-induced vasodilation is in accordance with findings in other studies (18, 20). The remaining vasodilation might signify incomplete blocking of nitric oxide synthesis. The role of other relaxing factors such as the
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F942
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endothelium-dependent hyperpolarizing factor (3, 5) is not yet clear. After infusion of NO-Arg, intrarenal infusion of L-Arg failed to normalize RBF but restored the vasodilatory response to acetylcholine. This may indicate different mechanisms for nitric oxide synthesis during basal release and during receptor stimulation with acetylcholine. Atria1 natriuretic factor has been shown to be an endothelium- independent vasodilator (24). In our study the vasodilatory response to atria1 natriuretic factor was not reduced after intrarenal infusion of L-NMMA or NO-Arg. In contrast, the vasodilatory response to atria1 natriuretic factor was increased after infusion of both doses of NOArg (Fig. 2). Because infusion of norepinephrine also increases the vasodilatory effect of atria1 natriuretic factor on the renal circulation (8), elevated vascular tone may increase preglomerular responsiveness to atria1 natriuretic factor. In conclusion, NO-Arg was more potent than L-NMMA in inhibiting basal renal nitric oxide synthesis. In contrast to L-NMMA, NO-Arg exerted an inhibitory effect on acetylcholine-induced renal vasodilation. Our findings indicate that one-third of the basal RBF and more than one-half of the increase in RBF during acetylcholine infusion are dependent on nitric oxide synthesis. We thank B. Amundsen, B. Austbe, H. B&en, K. Eriksen, M. R. Holthe, G. S. Jensen, S. Leraand, 0. Moen, A. C. Nilsen, G. Torgersen, and T. Verpe for skilled assistance. This work was supported by Anders Jahre’s Fund for the Promotion of Science and Professor Carl Semb’s Research Fund. Address reprint requests to P. A. Naess. Received
17 July
1991; accepted
in final
form
4 December
1991.
REFERENCES 1. Baylis, C., P. Harton, and K. Engels. Endothelial derived relaxing factor controls renal hemodynamics in the normal rat kidney. J. Am. Sot. Nephrol. 1: 875-881, 1990. 2. Burton, G. A., S. MacNeil, A. de Jonge, and J. Haylor. Cyclic GMP release and vasodilation induced by EDRF and atria1 natriuretic factor in the isolated perfused kidney of the rat. Br. J. Pharmacol. 99: 264-268, 1990. 3. Chen, G., H. Suzuki, and A. H. Weston. Acetylcholine releases an endothelium-derived relaxing factor and EDRF from rat blood vessels. Br. J. PharmacoZ. 95: 1165-1174, 1988. 4. Conover, W. J. Practical Nonparametric Statistics. New York: Wiley, 1980, p. 299-300. 5. Feletou, M., and P. M. Vanhoutte. Endothelium-dependent hyperpolarization of the canine coronary artery smooth muscle. Br. J. Pharmacol. 93: 515-524, 1988. 6. Furchgott, R. F., and J. V. Zawadski. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature Lond. 288: 373- 376, 1980. 7. Ishii, K., B. Chang, J. F. Kerwin, Z. J. Hiang, and F. Murad. NW-nitro-L-arginine: a potent inhibtor of endotheliumderived relaxing factor formation. Eur. J. Pharmacol. 176: 219223, 1990.
HEMODYNAMICS
8. Kimura, K., Y. Hirata, S. Nanba, A. Tojo, H. Matsuoka, and T. Sugimoto. Effects of atria1 natriuretic peptide on renal arterioles: morphometric analysis using microvascular casts. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F936-F944, 1990. 9. Kobayashi, Y., and K. Hattori. Nitroarginine inhibits endothelium-derived relaxation. Jpn. J. PharmacoZ. 52: 167-169, 1990. IO. Lahera, V., M. G. Salom, M. J. Fiksen-Olsen, L. Raij, and J. C. Romero. Effects of NG-monomethyl-L-arginine and L-arginine on acetylcholine renal response. Hypertension DaZlas 15: 659663, 1990. 11. Luscher, T. F., D. Diederich, E. Weber, P. M. Vanhoutte, and F. R. Buhler. Endothelium-dependent responses in carotid and renal arteries of normotensive and hypertensive rats. Hypertension Dallas ItI: 573-578, 1988. 12. Moncada, S., R. M. J. Palmer, and E. A. Higgs. The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension Dallas 12: 365-372, 1988. 13. Moore, P. K., 0. A. Al-Swayeh, N. W. S. Chong, R. A. Evans, and A. Gibson. L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilation in vitro. Br. J. Pharmacol. 99: 408-412, 1990. 14. Mugge, A., J. A. G. Lopez, D. J. Piegors, K. R. Breese, and D. D. Heistad. Acetylcholine-induced vasodilation in rabbit hindlimb in vivo is not inhibited by analogues of L-arginine. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H242-H247, 1991. 15. Mulsch, A., and R. Busse. NG-nitro-L-arginine (N5-(imino(nitroamino)methyl)-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. Naunyn Schmiedeberg’s Arch. Pharmacol. 341: 143147, 1990. 16. Palmer, R. M. J., D. S. Ashton, and S. Moncada. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature Lond. 333: 664-666, 1988. 17. Palmer, R. M. J., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature Lond. 327: 524-526, 1987. 18. Palmer, R. M. J., D. D. Rees, D. S. Ashton, and S. Moncada. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem. Biophys. Res. Commun. 153: 1251-1256, 1988. 19. Radermacher, J., U. Forstermann, and J. C. Frolich. Endothelium-derived relaxing factor influences renal vascular resistance. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F9-F17, 1990. 20. Rees, D. D., R. M. J., Palmer, H. F. Hodson, and S. Moncada. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br. J. Pharmacol. 96: 418-424, 1989. 21. Salom, M. G., V. Lahera, and J. C. Romero. Role of prostaglandins and endothelium-derived relaxing factor on the renal response to acetylcholine. Am. J. Physiol. 260 (Renal FZuid EZectrolyte Physiol. 29): F145-F149, 1991. 22. Tolins, J. P., R. M. J. Palmer, S. Moncada, and L. Raij. Role of endothelium-derived relaxing factor in regulation of renal hemodynamic responses. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H655-H662, 1990. 23. Welch, W. J., C. S. Wilcox, K. Aisaka, S. S. Gross, 0. W. Griffith, B. M. A. Fontoura, T. Maack, and R. Levi. Nitric oxide synthesis from L-arginine modulates renal vascular resistance in isolated perfused and intact rat kidneys. J. Cardiouusc. Pharmacol. 17, Suppl. 3: S165-S168, 1991. 24. Winquist, R. J., E. P. Faison, S. A. Waldman, K. Schwartz, F. Murad, and R. M. Rapoport. Atria1 natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle. Proc. NutZ. Acad. Sci. USA 81: 7661-7664, 1984.
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A. KIRKEBOEN, GEIR CHRISTENSEN, AND FREDRIK Medical Research, University of Oslo, Ullevaal Hospital,
Naess, P&l A., Knut A. Kirkebgen, Geir Christensen, and Fredrik Kiil. Inhibition of renal nitric oxide synthesis with NC;-monomethyl-L-arginine and N’=-nitro-L-arginine. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F939-F942, 1992.-In barbiturate-anesthetized dogs, the effects of intrarenal infusion of the two selective inhibitors of nitric oxide synthesis, N’;-monomethyl-L-arginine (L-NMMA) and NG-nitroL-arginine (NO-Arg), were compared. Basal renal blood flow (RBF) was reduced by 15 k 2% after L-NMMA at 0.28 pmol/ml, by 20 & 3% after NO-Arg at 0.07 pmol/ml, and by 31 t 5% after NO-Arg at 0.56 pmol/ml. Endothelium-dependent vasodilation induced by intrarenal infusion of acetylcholine was unaltered after L-NMMA, reduced by 24 ? 3% after NO-Arg at 0.07 pmol/ml, and reduced by 59 t 13% after NO-Arg at 0.56 ,umol/ml. Endothelium-independent vasodilation induced by intrarenal infusion of atria1 natriuretic factor was not reduced after L-NMMA and NO-Arg. This study shows that NO-Arg is more potent than L-NMMA in inhibiting basal renal nitric oxide synthesis. In contrast to L-NMMA, NO-Arg exerted an inhibitory effect on acetylcholine-induced renal vasodilation. Our findings indicate that one-third of the basal RBF and more than one-half of the increase in RBF during acetylcholine infusion are dependent on nitric oxide synthesis. acetylcholine; atria1 natriuretic factor; dog; endothelium-dependent vasodilation; endothelium-derived relaxing factor; endothelium-independent vasodilation; renal hemodynamics RELAXATION induced by acetylcholine is dependent on the presence of a functionally intact endothelium that releases an endothelium-derived relaxing factor (EDRF) (6). Recent studies have shown that nitric oxide constitutes, at least in part, the biological activity of EDRF (12, 17). Demonstration of the biosynthesis of nitric oxide from the amino acid L-arginine (16, 18) has led to the introduction of arginine analogues, such as N”-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (NO-Arg) as selective inhibitors of nitric oxide synthesis (7, 18, 20). The importance of nitric oxide to renal circulation in vivo is not known. Studies on isolated perfused rat kidneys in which the vessels were preconstricted with norepinephrine or methoxamine have indicated that acetylcholine induces renal vasodilation through an endothelium-dependent mechanism (2, 19). A reduction in renal blood flow (RBF) after intrarenal infusion of a selective inhibitor of nitric oxide synthesis has been demonstrated in only one in vivo study. In that study, Salom et al. (21) reported that, although RBF was reduced, acetylcholine-induced vasodilation was unaltered after intrarenal infusion of L-NMMA. The purpose of the present study was to investigate the role of nitric oxide in renal circulation in vivo. In vitro studies have demonstrated that NO-Arg is 4-70 times more potent than L-NMMA as a specific inhibitor of EDRF (7, 13, 15). In anesthetized dogs with denervated kidneys the effects of intrarenal infusion of
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Copyright
KIIL
L-NMMA and NO-Arg were compared with regard to basal RBF and endothelium-dependent (with acetylcholine) and endothelium-independent (with atria1 natriuretic factor) vasodilation. METHODS Animal preparation. Mongrel dogs (16-34 kg body wt) of either sex were anesthetized with pentobarbital sodium (25 mg/kg body wt iv) with maintenance doses of l-3 mg/kg. The dogs were intubated with an endotracheal tube and ventilated by a volume-regulated respirator (model 101, Princeton Medical Instruments, Natick, MA) to keep plasma pH between 7.37 and 7.42. Body temperature was kept constant by a heating pad. The urinary bladder was drained through a urethral catheter. The femoral artery was cannulated for arterial blood sampling and for blood pressure recording by a Statham P23 Gb pressure transducer (Gould Instruments). One kidney was exposed through a flank incision. All visible nerves were cut, and a curved 25gauge needle connected to a polyethylene tube was inserted in retrograde direction into the renal artery for drug infusion. An electromagnetic flow probe (Nycotron) was placed on the renal artery for measuring RBF. Experimental procedure. The effects of intrarenal infusion of L-NMMA and NO-Arg were examined with regard to basal RBF as well as to endothelium-dependent and -independent vasodilation in one group of six dogs. Before administration of the two nitric oxide inhibitors, acetylcholine and atria1 natriuretic factor were infused into the renal artery in each dog to select doses that increased RBF similarly. Each drug was infused until a stable RBF was reached (l-2 min). The selected doses of acetylcholine and atria1 natriuretic factor increased RBF by 24 t 2 and 28 * 6%, respectively. Because basal RBF varied from 105 to 270 ml/min between dogs, the arginine analogues were administered to give a fixed renal arterial blood concentration. The intrarenal infusion rate was kept constant at 1 ml/min. Before administration of L-NMMA, D-arginine and then L-arginine were infused into the renal artery for 5 min to yield a renal arterial concentration of 1.0 pmol/ml to exclude a direct vasoactive effect of these arginine enantiomers. L-NMMA was infused into the renal artery for 5 min to yield a renal arterial concentration of 0.28 pmol/ml. The selected doses of acetylcholine (1.0-2.0 pg/min) and atria1 natriuretic factor (50- 100 ng min -lo kg body wt-‘) were then infused in random order within 5 min after discontinuing L-NMMA infusion. Thereafter, renal arterial concentrations of D-arginine at 1.0 I.cmol/ml and then L-arginine at 1.0 pmol/ml were infused. Finally the selected doses of acetylcholine and atria1 natriuretic factor were infused for the third time. An identical experimental procedure was thereafter followed for NO-Arg at a low dose, to yield a renal arterial concentration of 0.07 ,umol/ml, and then at a high dose, to yield a renal arterial concentration of 0.56 pmol/ml. To determine which doses of NO-Arg produced maximal and minimal effects on RBF and the flow response to acetylcholine, experiments were performed on a second group of three dogs. NO-Arg at renal arterial concentrations (in pmol/ml) of 0.01, 0.035, 0.07, 0.56, and 1.12 were each infused for 5 min. The effects on RBF and acetylcholine-induced (1.0 pg/ml) vasodila-
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tion were examined for each dose of NO-Arg. The ability of L-arginine to reverse the effects of NO-Arg was examined for each dose of NO-Arg that reduced RBF or the vasodilatory response to acetylcholine. Drugs. Atria1 natriuretic factor, D-arginine hydrochloride, L-arginine hydrochloride, and NO-Arg were purchased from Sigma Chemical (St. Louis, MO). Acetylcholine was purchased from Dispersa. L-NMMA was a kind gift from Dr. H. Hodson, Wellcome Research Laboratories (Beckenham, UK). All drugs were dissolved in saline. Atria1 natriuretic factor was dissolved in 1% bovine serum albumin in saline. To dissolve the high dose of NO-Arg, sodium hydroxide was added to the solution. After NO-Arg was dissolved, pH was adjusted to 8.0. StatisticaL anaZysis. The data are means & SE. Each dog served as its own control, and the Friedman test (4) was used to calculate the probability values for multiple comparisons. The Wilcoxon two-tailed signed-rank test was used for paired statistical analysis. A probability value of P < 0.05 was regarded as statistically significant. RESULTS
Effect of L-NMMA and NO-Arg on basal RBF. Infusion of L-NMMA at a renal arterial concentration of 0.28 pmol/ml significantly reduced RBF by 15 rt 2% (Fig. 1) to a new steady state after 4-5 min and increased mean arterial blood pressure by 7 t 1%. When L-NMMA was discontinued, RBF was completely normalized in two dogs 10 min after stopping infusion of L-NMMA. In the other four dogs RBF remained low. Infusion of L-arginine at a renal arterial concentration of 1.0 pmol/ml did not influence RBF before L-NMMA infusion. After L-NMMA infusion L-arginine completely restored RBF. Infusion of the low dose of NO-Arg at a renal arterial concentration of 0.07 ,umol/ml reduced RBF by 20 t 3%, which is significantly more than the reduction seen after L-NMMA (Fig. 1). L-Arginine did not influence RBF before the low dose of NO-Arg. After infusion of the low dose of NO-Arg, L-arginine restored RBF only partially (by 48 t 13%). Infusion of the high dose of NO-Arg at a renal arterial concentration of 0.56 pmol/ml reduced RBF by 31 t 5%, which is significantly more than the reduction seen after the low dose of NO-Arg (Fig. 1). L-Arginine did not influence RBF before the high dose of NO-Arg. After infusion of the high dose of NO-Arg, L-arginine did not restore RBF significantly (by 17 t 10%). Mean arterial blood
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pressure was not affected by the two doses of NO-Arg. Infusion of D-arginine at a renal arterial concentration of 1 pmol/ml did not influence RBF either before or after the infusions of L-NMMA or NO-Arg. In the second group of three dogs, infusion of NO-Arg at renal arterial concentrations lower than 0.07 pmol/ml did not reduce RBF. Infusion of NO-Arg at a renal arterial concentration of 0.07 pmol/ml reduced RBF by on average 11%. Subsequent infusion of L-arginine at a renal arterial concentration of 7.0 pmol/ml reversed RBF by on average 50%. Infusion of NO-Arg at a renal arterial concentration of 0.56 ,umol/ml reduced RBF by on average 23%. Doubling the dose of NO-Arg to 1.12 pmol/ml did not reduce RBF further. Subsequent infusion of L-arginine at a renal arterial concentration of 20.0 pmol/ml did not reverse RBF after these high doses of NO-Arg. Effects of L-NMMA and NO-Arg on RBF responses to acetylcholine and atria1 natriuretic factor. After intrarenal infusion of L-NMMA, the increments in RBF caused by acetylcholine (1 .O-2.0 pg/min) and by atria1 natriuretic factor (50-100 ngemin -la kg body wt-l) were not significantly altered (Fig. 2). After intrarenal infusion of the low dose of NO-Arg, the flow response to acetylcholine was reduced by 24 t 3%. In contrast, the flow response to atria1 natriuretic factor was increased by 104 t 19% (Fig. 2). After infusion of L-arginine, the flow response to acetylcholine was not significantly different from the flow response to acetylcholine before infusion of the low dose of NO-Arg. After intrarenal infusion of the high dose of NO-Arg, the flow response to acetylcholine was reduced by 59 t 13%) whereas the flow response to atria1 natriuretic factor was increased by 41 t 9% (Fig. 2). After infusion of L-arginine, the flow response to acetylcholine was not significantly different from the flow response to acetylcholine before infusion of the high dose of NO-Arg. In the second group of three dogs, infusion of NO-Arg at renal arterial concentrations lower than 0.07 pmol/ml did not reduce the flow response to acetylcholine. NO-Arg at renal arterial concentrations (in hmol/ml) of 0.07,0.56, and 1.12 reduced the vasodilatory response to acetylcholine by an average of 22, 49, and 52%, respectively. L-Arginine completely restored the flow response to acetylcholine after each dose of NO-Arg. DISCUSSION
-s -5 -u g
L NMMA (0.28
~mol/ml)
NOARG (0.07
umol/ml)
NOARG (0.56
umol/ml)
O 10
0 z 20 al .-C 1;7
30
This study on anesthetized dogs shows that the two nitric oxide inhibitors, L-NMMA and NO-Arg, both reduce basal RBF. NO-Arg reduced basal RBF more than L-NMMA, and only NO-Arg inhibited the flow response to acetylcholine. Effect of L-NMMA and NO-Arg on basal RBF. L-NMMA reduced RBF by 15%. Doubling of infusion rates in two additional experiments had no further effect. Salom et al. (21) found a 13% reduction in RBF after 30 min intrarenal infusion of L-NMMA (50 pg. mine1 kg body wt-I) in anesthetized dogs. This response is similar to that found in our study although we infused L-NMMA in a concentration -10 times higher than in the study of Salom et al. (21). In both studies drugs were infused into the renal artery to minimize systemic effects. Systemic infusions of L-NMMA and NO-Arg in rats have indicated l
Fig. 1. Effect of intrarenal infusion of NG-monomethyl-L-arginine (L-NMMA) and NG -nitro-L-arginine (NO-Arg) on renal blood flow. * Significantly different from reduction induced by L-NMMA. t Significantly different from reduction induced by the low dose of NO-Arg. Values are means k SE in 6 dogs.
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NITRIC F
120-
2
m
: Acetylcholine
m
: Atrial
natriuretic
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factor
-2 loo3 2 80-0 : E 600 5L 40caI ; 200, ;s -c O-
Fig. 2. Effect of intrarenal infusion of L-NMMA and NO-Arg on renal blood flow responses to acetylcholine and atria1 natriuretic factor. * Significantly different from flow response to acetylcholine before infusion of NO-Arg. t Significantly different from flow response to atria1 natriuretic factor before infusion of NO-Arg. Values are means t SE in 6 dogs. Before (0.28
LNMMA pmol/ml)
After (0.28
LNMMA
Before
pmol/ml)
(0.07
NOARG pmol/ml)
After (0.07
NOARG
Before
pmol/ml)
(0.56
that tonic release of EDRF may influence renal vascular resistance (1,22,23). Large, acute increments in systemic arterial blood pressure in those studies probably raised renal vascular resistance by activating the autoregulatory mechanism (1). The importance of direct renal vasoconstriction caused by L-NMMA and NO-Arg is, therefore, difficult to assess from those studies. The effect of intrarenal infusion of NO-Arg has not previously been studied. In our study both doses of NOArg induced greater reductions in basal RBF than did L-NMMA. Infusion of the high dose of NO-Arg reduced RBF by as much as 31%, indicating that basal renal vascular tone is substantially dependent on nitric oxide synthesis. Doubling the dose of NO-Arg to a renal arterial concentration of 1.12 pmol/ml in three additional experiments had no further effect on RBF. Radermacher et al. (19) infused equimolar doses of L-NMMA and NO-Arg in two groups of isolated perfused rat kidneys preconstricted with norepinephrine. In both their and our studies the increase in renal vascular resistance was approximately twice as large after NO-Arg as after L-NMMA administration. In our study L-arginine restored RBF after administration of L-NMMA. This is in agreement with the hypothesis that L-NMMA competes with L-arginine for the nitric oxide-forming enzyme (18, 20). On the other hand, L-arginine did not completely reestablish RBF after infusion of NO-Arg. L-Arginine restored ~50% of the reduction in RBF caused by the small dose of NO-Arg, but L-arginine had no effect on RBF after administration of the large dose of NO-Arg. In three additional experiments higher concentrations of L-arginine did not fully reestablish RBF after infusion of NO-Arg. Previous studies concerning the reversibility of the effects of NO-Arg have given equivocal results. In a study on aortic ring preparations Moore et al. (13) reported complete reversal of the effects of NO-Arg after L-arginine administration. In contrast, Kobayashi and Hattori (9) found almost irreversible inhibition of the vasorelaxation induced by acetylcholine after treatment with NOArg in the same preparation. Mugge et al. (14) demonstrated that a 30-fold higher concentration of L-arginine only partially reversed the effects of NO-Arg on blood flow to heart and hindlimb in rabbits. Effects of L-NMMA and NO-Arg on RBF responses to acetylcholine and atria1 natriuretic factor. In studies on isolated rat kidneys inhibitors such as hemoglobin (2),
NOARG ymol/ml)
After (0.56
NOARG pmol/ml)
gossypol (2, 19), and methylene blue (19) reduced the vasodilator effect of acetylcholine. Tolins et al. (22) found in clearance studies on anesthetized rats that intravenous injection of L-NMMA prevented both the fall in systemic blood pressure and the increase in para-aminohippurate clearance during intravenous infusion of acetylcholine. In our study L-NMMA did not reduce acetylcholine-induced vasodilation. Salom et al. (21) found that intrarenal infusion of L-NMMA did not reduce acetylcholine-induced vasodilation in anesthetized dogs. But L-NMMA completely blocked the vasodilator effect of acetylcholine after pretreatment with an inhibitor of prostaglandin synthesis, meclofenamate, a finding in agreement with results obtained by Lahera et al. (10). Salom et al. (21) proposed that the acetylcholine-induced increase in RBF was mediated by both prostaglandins and EDRF and that the hemodynamic effect of EDRF could be fully compensated for by vasodilating prostaglandins during EDRF blockade with L-NMMA. On the other hand, Luscher et al. (II) found in a study on rat renal arterial ring preparations that inhibition of prostaglandin synthesis with indomethacin augmented the vasorelaxation induced by acetylcholine. They concluded that acetylcholine appeared to release both EDRF and a vasoconstrictor prostanoid from the renal artery. In our study the RBF response to acetylcholine was reduced after infusion of both doses of NO-Arg in a dose-dependent manner. After infusion of the high dose of NO-Arg, the vasodilatory response to acetylcholine was reduced by as much as 59%. Hence we have shown that NO-Arg reduced a large fraction of both basal RBF and acetylcholine-induced renal vasodilation without pretreatment with a prostaglandin synthesis inhibitor. Since NO-Arg exerted no inhibitory effect on the endothelium-independent vasodilation caused by infusion of atria1 natriuretic factor, the responses to NO-Arg probably reflect inhibition of nitric oxide synthesis in vivo. Doubling the dose of NO-Arg from a renal arterial concentration of 0.56 to 1.12 pmol/ml in three additional experiments did not reduce the vasodilatory response to acetylcholine further. That inhibition of renal nitric oxide synthesis failed to completely prevent acetylcholine-induced vasodilation is in accordance with findings in other studies (18, 20). The remaining vasodilation might signify incomplete blocking of nitric oxide synthesis. The role of other relaxing factors such as the
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endothelium-dependent hyperpolarizing factor (3, 5) is not yet clear. After infusion of NO-Arg, intrarenal infusion of L-Arg failed to normalize RBF but restored the vasodilatory response to acetylcholine. This may indicate different mechanisms for nitric oxide synthesis during basal release and during receptor stimulation with acetylcholine. Atria1 natriuretic factor has been shown to be an endothelium- independent vasodilator (24). In our study the vasodilatory response to atria1 natriuretic factor was not reduced after intrarenal infusion of L-NMMA or NO-Arg. In contrast, the vasodilatory response to atria1 natriuretic factor was increased after infusion of both doses of NOArg (Fig. 2). Because infusion of norepinephrine also increases the vasodilatory effect of atria1 natriuretic factor on the renal circulation (8), elevated vascular tone may increase preglomerular responsiveness to atria1 natriuretic factor. In conclusion, NO-Arg was more potent than L-NMMA in inhibiting basal renal nitric oxide synthesis. In contrast to L-NMMA, NO-Arg exerted an inhibitory effect on acetylcholine-induced renal vasodilation. Our findings indicate that one-third of the basal RBF and more than one-half of the increase in RBF during acetylcholine infusion are dependent on nitric oxide synthesis. We thank B. Amundsen, B. Austbe, H. B&en, K. Eriksen, M. R. Holthe, G. S. Jensen, S. Leraand, 0. Moen, A. C. Nilsen, G. Torgersen, and T. Verpe for skilled assistance. This work was supported by Anders Jahre’s Fund for the Promotion of Science and Professor Carl Semb’s Research Fund. Address reprint requests to P. A. Naess. Received
17 July
1991; accepted
in final
form
4 December
1991.
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8. Kimura, K., Y. Hirata, S. Nanba, A. Tojo, H. Matsuoka, and T. Sugimoto. Effects of atria1 natriuretic peptide on renal arterioles: morphometric analysis using microvascular casts. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F936-F944, 1990. 9. Kobayashi, Y., and K. Hattori. Nitroarginine inhibits endothelium-derived relaxation. Jpn. J. PharmacoZ. 52: 167-169, 1990. IO. Lahera, V., M. G. Salom, M. J. Fiksen-Olsen, L. Raij, and J. C. Romero. Effects of NG-monomethyl-L-arginine and L-arginine on acetylcholine renal response. Hypertension DaZlas 15: 659663, 1990. 11. Luscher, T. F., D. Diederich, E. Weber, P. M. Vanhoutte, and F. R. Buhler. Endothelium-dependent responses in carotid and renal arteries of normotensive and hypertensive rats. Hypertension Dallas ItI: 573-578, 1988. 12. Moncada, S., R. M. J. Palmer, and E. A. Higgs. The discovery of nitric oxide as the endogenous nitrovasodilator. Hypertension Dallas 12: 365-372, 1988. 13. Moore, P. K., 0. A. Al-Swayeh, N. W. S. Chong, R. A. Evans, and A. Gibson. L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilation in vitro. Br. J. Pharmacol. 99: 408-412, 1990. 14. Mugge, A., J. A. G. Lopez, D. J. Piegors, K. R. Breese, and D. D. Heistad. Acetylcholine-induced vasodilation in rabbit hindlimb in vivo is not inhibited by analogues of L-arginine. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H242-H247, 1991. 15. Mulsch, A., and R. Busse. NG-nitro-L-arginine (N5-(imino(nitroamino)methyl)-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. Naunyn Schmiedeberg’s Arch. Pharmacol. 341: 143147, 1990. 16. Palmer, R. M. J., D. S. Ashton, and S. Moncada. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature Lond. 333: 664-666, 1988. 17. Palmer, R. M. J., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature Lond. 327: 524-526, 1987. 18. Palmer, R. M. J., D. D. Rees, D. S. Ashton, and S. Moncada. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem. Biophys. Res. Commun. 153: 1251-1256, 1988. 19. Radermacher, J., U. Forstermann, and J. C. Frolich. Endothelium-derived relaxing factor influences renal vascular resistance. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F9-F17, 1990. 20. Rees, D. D., R. M. J., Palmer, H. F. Hodson, and S. Moncada. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br. J. Pharmacol. 96: 418-424, 1989. 21. Salom, M. G., V. Lahera, and J. C. Romero. Role of prostaglandins and endothelium-derived relaxing factor on the renal response to acetylcholine. Am. J. Physiol. 260 (Renal FZuid EZectrolyte Physiol. 29): F145-F149, 1991. 22. Tolins, J. P., R. M. J. Palmer, S. Moncada, and L. Raij. Role of endothelium-derived relaxing factor in regulation of renal hemodynamic responses. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H655-H662, 1990. 23. Welch, W. J., C. S. Wilcox, K. Aisaka, S. S. Gross, 0. W. Griffith, B. M. A. Fontoura, T. Maack, and R. Levi. Nitric oxide synthesis from L-arginine modulates renal vascular resistance in isolated perfused and intact rat kidneys. J. Cardiouusc. Pharmacol. 17, Suppl. 3: S165-S168, 1991. 24. Winquist, R. J., E. P. Faison, S. A. Waldman, K. Schwartz, F. Murad, and R. M. Rapoport. Atria1 natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle. Proc. NutZ. Acad. Sci. USA 81: 7661-7664, 1984.
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