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Pressor Effect of NG-Monomethyl-L-Arginine in SHRSP
Pressor
Effect
of NG-Monomethyl-L-Arginine
in SHRSP
Kazuo AISAKA, Aki MITANI, Yasuo KITAJIMA' and Takafumi ISHIHARA Laboratoryof ExperimentalPharmacolgyand ' Gene Technology, SuntoryInstitutefor BiomedicalResearch,Mishima-gun,Osaka618, Japan AcceptedSeptember10, 1990 Abstract-Pressor effects of NG-monomethyl-L-arginine (L-NMMA), a selective inhibitor of nitric oxide production from L-arginine, on mean blood pressure (MBP) were investigated in conscious Wistar Kyoto rats (WKY) and stroke-prone spon taneously hypertensive rats (SHRSP). L-NMMA (0.1-10 mg/kg, i.v.) elicited a dose-dependent increase in the MBP of WKY and SHRSP. The pressor response to L-NMMA was more marked in SHRSP than in WKY. These results suggest that nitric oxide may play an important role in the blood pressure regulation in the conscious SHRSP. Endothelial cells can modurate vascular smooth muscle tone via the synthesis and re lease of one or more relaxing factors (1). The vasodilatation in response to many endo genous substances including acetylcholine is mediated by endothelium-derived relaxing factors (EDRFs), which were first described by Furchgott and Zawadzki (2). One of the EDRFs has been identified as nitric oxide (NO) or a related nitroso-compound (3-5). NO is derived from a novel biosynthetic pathway involving the oxidation of a gua nidino nitrogen of L-arginine (6-8). Recently, it was reported that NG-monomethyl-L arginine (L-NMMA) competitively inhibits the generation of NO from L-arginine (8, 9). Thus, L-NMMA can be utilized as a tool to selectively inhibit NO production. One of us and others have proposed that NO may not only mediate the action of numerous vaso dilating substances, but may also participate in arterial pressure homeostasis (10, 11). In the anesthetized guinea pig (10, 12) and rabbit (11), L-NMMA, but not D-NMMA, elicits a large and sustained increase in ar terial blood pressure; this response is re versed by L-arginine. These findings prompted us to test the hypothesis that the production of arginine-derived nitric oxide in the vascula ture is modified by long term severe hyper tension. In order to test this, we investigated the effect of L-NMMA on MBP in normal
Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Twenty-five to 38 week-old male SHRSP and WKYwere used. Under sodium thiopental (40 mg/kg, i.p.) anesthesia, indwelling polyethylene catheters (PE 10) were inserted into the distal aorta via the femoral artery for blood pressure measurement and into the jugular vein for drug administration. The catheters were filled up with heparin sodium solution (500 U/ml) to prevent blood coagu lation. Blood pressure (BP) was measured with the aid of a pressure transducer (MPU 0.5A, Nihon Kohden, Tokyo), and heart rate (HR) was determined with a heart rate counter (AT-600T, Nihon Kohden) triggered by the BP pulse. More than 3 days after the surgical opera tion described above, each animal was transferred into an individual translucent cylindrical box made of vinyl chloride polymer (25 cm in diameter and 30 cm in height). After connecting the previously implanted catheter with a pressure transducer, BP and HR were allowed to stabilize for 1 to 2 hours, during which the animal became accustomed to the box. First, the effect of physiological saline solution (vehicle) given intravenously (i.v.) in a volume of 0.1 ml/100 g was studied for 20 min. Immediately thereafter, 0.1, 1 and 10 mg/kg of L-NMMA were administered to the same animal one dose after the other at
20 min-intervals. Changes in BP and HR were continuously recorded on a polygraph recorder (RM-6200, Nihon Kohden). Animal behavior was observed concomitantly. L NMMA was synthesized according to the method of Corbin and Reporter (13). The crude L-NMMA was purified on a column of Dowex 50x8 (NH4+ form) using 0.15 M NH40H as an eluent. The solution was con centrated and neutralized with hydrochloric acid to yield L-NMMA HCI; the concentration of pure compounds was determined by amino acid analysis. The results obtained were ex pressed as the mean±S.E. After the stabilization of BP in animals, the
mean BP (MBP) values in WKY and SHRSP were 113±4 mmHg (N=6) and 173±8 mmHg (N=8), respectively. The administration of L-NMMA (0.1-10 mg/kg, i.v. bolus injec tion) into the WKY and SHRSP elicited a pressor response that increased in magnitude with increasing doses of L-NMMA without causing any change in gross behavior. Be cause both diastolic and systolic levels were increased to the same extent, the pulse pres sue remained constant. The time course of the L-NMMA-induced increase in MBP is shown in Fig. 1. The onset of action occurred within the first minute of administration; a 10 mg/kg, i.v.-bolus dose of L-NMMA elicited
Fig. 1. Effect of NG-monomethyl-L-arginine on mean blood pressure and heart rate in conscious WKY (0, N=6) and SHRSP (0, N=8). The changes of mean blood pressure (JMBP) and heart rate (4HR) from the values before administration of saline are shown. Control MBP and HR in WKY and SHRSP were 113±4 and 173±8 mmHg and 295±11 and 307±10 beats/min, respectively.
the most marked increase in MBP, which did not recover to the initial level within 60 min. L-NMMA tended to elicit a moderate brady cardia at a dose of 10 mg/kg in both groups (Fig. 1). The pressor response to L-NMMA is due to an inhibition of the synthesis of endothe lium derived NO from L-arginine because the response could be abolished by treatment with L-arginine (10, 11). Accordingly, our findings imply that in the conscious rat, endothelium-derived NO is spontaneously released and plays a significant role in the regulation of peripheral vascular resistance and hence of arterial blood pressure. The finding that the pressor response to L-NMMA was more marked in SH RSP suggests that NO synthesis in endothelial cells contributes to the regulation of blood pressure, especially in the hypertensive rat. L-NMMA elicited a moderate bradycardia in conscious rats, and it had a similar effect in anesthetized guinea pigs and rabbits (10, 11). It has been already reported that heart rate decrease was mediated by the baroreceptor reflex because the decrease was abolished by pretreatment with atropine (10). Accordingly, we speculate that the syn thesis/release or vasodilating effect of nitric oxide in hypertension is changed, and the vasculature itself has a local regulatory sys tem that can modulate blood pressure. Acknowledgments: We express our gratitude to Drs. T. Ogawa and M. Kimoto of Tokushima Uni versity School of Medicine and Dr. T. Kasai of Hokkaido University for the useful advice on the synthesis of L-NMMA. The authors also thank Dr. T. Noguchi, the director of our institute, for his support and encouragement throughout this study.
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References 1 Furchgott, R.F. and Vanhoutte, P.M.: Endo thelium-derived relaxing and contracting factors. FASEBJ. 3, 2007-2018 (1989) 2 Furchgott, R.F. and Zawadzki, J.V.: The obligatory role of endothelial cells in the relax ation of arterial smooth muscle by acetylcholine. Nature 288, 373-376 (1980) 3 Ignarro, L.J., Byrns, R.E., Buga, G.M. and Wood, K.S.: Endothelium-derived relaxing factor from pulmonary artery and vein possesses phar
13
macologic and chemical properties identical to those of nitric oxide radical. Circ. Res. 61, 866 879 (1987) Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E. and Chaudhuri, G.: Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. NatI. Acad. Sci. U.S.A. 84, 9265-9269 (1987) Palmer, R.M.J., Ferrige, A.G. and Moncada, S.: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526 (1987) Palmer, R.M.J., Ashton, D.S. and Moncada, S.: Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664-666 (1988) Schmidt, H.H.H.W., Nau, H., Xittfoht, W., Gerlach, J., Prescher, K.E., Klein, M. M., Niroomand, F. and Bohme, E.: Arginine is a physiological precursor of endothelium-derived nitric oxide. Eur. J. Pharmacol. 154, 213-216 (1988) Sakuma, I., Stuehr, D.J., Gross, S.S., Nathan, C. and Levi, R.: Identification of arginine as a precursor of endothelium-derived relaxing factor. Proc. NatI. Acal. Sci. U.S.A. 85, 8664-8667 (1988) Aisaka, K., Gross, S.S., Griffith, O.W. and Levi, R.: L-Arginine availability determines the duration of acetylcholine-induced systemic vasodilation in vivo. Biochem. Biophys. Res. Commun. 163, 710-717 (1989) Aisaka, K., Gross, S.S., Griffith, OW. and Levi, R.: NG-Methylarginine, an inhibitor of endothelium derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo ? Biochem. Biophys. Res. Commun. 160, 881-886 (1989) Rees, D.D., Palmer, R.M.J. and Moncada, S.: Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc. NatI. Acad. Sci. U.S.A. 86, 3375-3378 (1989) Aisaka, K., Gross, S.S., Steinberg, C., Griffith, O.W. and Levi, R.: NG-Monomethyl-L-arginine, an inhibitor of endothelium-derived nitric oxide synthesis, abbreviates acetylcholine-induced vasodilatation in the guinea-pig. In Nitric Oxide from L-Arginine: A Bioregulatory System, Edited by Moncada, S., Chapter 40, Elsevier Science Publishers, Amsterdam (1990) Corbin, J.L. and Reporter, M.: NG-Methylated arginine; A convenient preparation of NG methylarginine. Anal. Biochem. 57, 310-312 (1978)
Effect
of NG-Monomethyl-L-Arginine
in SHRSP
Kazuo AISAKA, Aki MITANI, Yasuo KITAJIMA' and Takafumi ISHIHARA Laboratoryof ExperimentalPharmacolgyand ' Gene Technology, SuntoryInstitutefor BiomedicalResearch,Mishima-gun,Osaka618, Japan AcceptedSeptember10, 1990 Abstract-Pressor effects of NG-monomethyl-L-arginine (L-NMMA), a selective inhibitor of nitric oxide production from L-arginine, on mean blood pressure (MBP) were investigated in conscious Wistar Kyoto rats (WKY) and stroke-prone spon taneously hypertensive rats (SHRSP). L-NMMA (0.1-10 mg/kg, i.v.) elicited a dose-dependent increase in the MBP of WKY and SHRSP. The pressor response to L-NMMA was more marked in SHRSP than in WKY. These results suggest that nitric oxide may play an important role in the blood pressure regulation in the conscious SHRSP. Endothelial cells can modurate vascular smooth muscle tone via the synthesis and re lease of one or more relaxing factors (1). The vasodilatation in response to many endo genous substances including acetylcholine is mediated by endothelium-derived relaxing factors (EDRFs), which were first described by Furchgott and Zawadzki (2). One of the EDRFs has been identified as nitric oxide (NO) or a related nitroso-compound (3-5). NO is derived from a novel biosynthetic pathway involving the oxidation of a gua nidino nitrogen of L-arginine (6-8). Recently, it was reported that NG-monomethyl-L arginine (L-NMMA) competitively inhibits the generation of NO from L-arginine (8, 9). Thus, L-NMMA can be utilized as a tool to selectively inhibit NO production. One of us and others have proposed that NO may not only mediate the action of numerous vaso dilating substances, but may also participate in arterial pressure homeostasis (10, 11). In the anesthetized guinea pig (10, 12) and rabbit (11), L-NMMA, but not D-NMMA, elicits a large and sustained increase in ar terial blood pressure; this response is re versed by L-arginine. These findings prompted us to test the hypothesis that the production of arginine-derived nitric oxide in the vascula ture is modified by long term severe hyper tension. In order to test this, we investigated the effect of L-NMMA on MBP in normal
Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Twenty-five to 38 week-old male SHRSP and WKYwere used. Under sodium thiopental (40 mg/kg, i.p.) anesthesia, indwelling polyethylene catheters (PE 10) were inserted into the distal aorta via the femoral artery for blood pressure measurement and into the jugular vein for drug administration. The catheters were filled up with heparin sodium solution (500 U/ml) to prevent blood coagu lation. Blood pressure (BP) was measured with the aid of a pressure transducer (MPU 0.5A, Nihon Kohden, Tokyo), and heart rate (HR) was determined with a heart rate counter (AT-600T, Nihon Kohden) triggered by the BP pulse. More than 3 days after the surgical opera tion described above, each animal was transferred into an individual translucent cylindrical box made of vinyl chloride polymer (25 cm in diameter and 30 cm in height). After connecting the previously implanted catheter with a pressure transducer, BP and HR were allowed to stabilize for 1 to 2 hours, during which the animal became accustomed to the box. First, the effect of physiological saline solution (vehicle) given intravenously (i.v.) in a volume of 0.1 ml/100 g was studied for 20 min. Immediately thereafter, 0.1, 1 and 10 mg/kg of L-NMMA were administered to the same animal one dose after the other at
20 min-intervals. Changes in BP and HR were continuously recorded on a polygraph recorder (RM-6200, Nihon Kohden). Animal behavior was observed concomitantly. L NMMA was synthesized according to the method of Corbin and Reporter (13). The crude L-NMMA was purified on a column of Dowex 50x8 (NH4+ form) using 0.15 M NH40H as an eluent. The solution was con centrated and neutralized with hydrochloric acid to yield L-NMMA HCI; the concentration of pure compounds was determined by amino acid analysis. The results obtained were ex pressed as the mean±S.E. After the stabilization of BP in animals, the
mean BP (MBP) values in WKY and SHRSP were 113±4 mmHg (N=6) and 173±8 mmHg (N=8), respectively. The administration of L-NMMA (0.1-10 mg/kg, i.v. bolus injec tion) into the WKY and SHRSP elicited a pressor response that increased in magnitude with increasing doses of L-NMMA without causing any change in gross behavior. Be cause both diastolic and systolic levels were increased to the same extent, the pulse pres sue remained constant. The time course of the L-NMMA-induced increase in MBP is shown in Fig. 1. The onset of action occurred within the first minute of administration; a 10 mg/kg, i.v.-bolus dose of L-NMMA elicited
Fig. 1. Effect of NG-monomethyl-L-arginine on mean blood pressure and heart rate in conscious WKY (0, N=6) and SHRSP (0, N=8). The changes of mean blood pressure (JMBP) and heart rate (4HR) from the values before administration of saline are shown. Control MBP and HR in WKY and SHRSP were 113±4 and 173±8 mmHg and 295±11 and 307±10 beats/min, respectively.
the most marked increase in MBP, which did not recover to the initial level within 60 min. L-NMMA tended to elicit a moderate brady cardia at a dose of 10 mg/kg in both groups (Fig. 1). The pressor response to L-NMMA is due to an inhibition of the synthesis of endothe lium derived NO from L-arginine because the response could be abolished by treatment with L-arginine (10, 11). Accordingly, our findings imply that in the conscious rat, endothelium-derived NO is spontaneously released and plays a significant role in the regulation of peripheral vascular resistance and hence of arterial blood pressure. The finding that the pressor response to L-NMMA was more marked in SH RSP suggests that NO synthesis in endothelial cells contributes to the regulation of blood pressure, especially in the hypertensive rat. L-NMMA elicited a moderate bradycardia in conscious rats, and it had a similar effect in anesthetized guinea pigs and rabbits (10, 11). It has been already reported that heart rate decrease was mediated by the baroreceptor reflex because the decrease was abolished by pretreatment with atropine (10). Accordingly, we speculate that the syn thesis/release or vasodilating effect of nitric oxide in hypertension is changed, and the vasculature itself has a local regulatory sys tem that can modulate blood pressure. Acknowledgments: We express our gratitude to Drs. T. Ogawa and M. Kimoto of Tokushima Uni versity School of Medicine and Dr. T. Kasai of Hokkaido University for the useful advice on the synthesis of L-NMMA. The authors also thank Dr. T. Noguchi, the director of our institute, for his support and encouragement throughout this study.
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References 1 Furchgott, R.F. and Vanhoutte, P.M.: Endo thelium-derived relaxing and contracting factors. FASEBJ. 3, 2007-2018 (1989) 2 Furchgott, R.F. and Zawadzki, J.V.: The obligatory role of endothelial cells in the relax ation of arterial smooth muscle by acetylcholine. Nature 288, 373-376 (1980) 3 Ignarro, L.J., Byrns, R.E., Buga, G.M. and Wood, K.S.: Endothelium-derived relaxing factor from pulmonary artery and vein possesses phar
13
macologic and chemical properties identical to those of nitric oxide radical. Circ. Res. 61, 866 879 (1987) Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E. and Chaudhuri, G.: Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. NatI. Acad. Sci. U.S.A. 84, 9265-9269 (1987) Palmer, R.M.J., Ferrige, A.G. and Moncada, S.: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526 (1987) Palmer, R.M.J., Ashton, D.S. and Moncada, S.: Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664-666 (1988) Schmidt, H.H.H.W., Nau, H., Xittfoht, W., Gerlach, J., Prescher, K.E., Klein, M. M., Niroomand, F. and Bohme, E.: Arginine is a physiological precursor of endothelium-derived nitric oxide. Eur. J. Pharmacol. 154, 213-216 (1988) Sakuma, I., Stuehr, D.J., Gross, S.S., Nathan, C. and Levi, R.: Identification of arginine as a precursor of endothelium-derived relaxing factor. Proc. NatI. Acal. Sci. U.S.A. 85, 8664-8667 (1988) Aisaka, K., Gross, S.S., Griffith, O.W. and Levi, R.: L-Arginine availability determines the duration of acetylcholine-induced systemic vasodilation in vivo. Biochem. Biophys. Res. Commun. 163, 710-717 (1989) Aisaka, K., Gross, S.S., Griffith, OW. and Levi, R.: NG-Methylarginine, an inhibitor of endothelium derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo ? Biochem. Biophys. Res. Commun. 160, 881-886 (1989) Rees, D.D., Palmer, R.M.J. and Moncada, S.: Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc. NatI. Acad. Sci. U.S.A. 86, 3375-3378 (1989) Aisaka, K., Gross, S.S., Steinberg, C., Griffith, O.W. and Levi, R.: NG-Monomethyl-L-arginine, an inhibitor of endothelium-derived nitric oxide synthesis, abbreviates acetylcholine-induced vasodilatation in the guinea-pig. In Nitric Oxide from L-Arginine: A Bioregulatory System, Edited by Moncada, S., Chapter 40, Elsevier Science Publishers, Amsterdam (1990) Corbin, J.L. and Reporter, M.: NG-Methylated arginine; A convenient preparation of NG methylarginine. Anal. Biochem. 57, 310-312 (1978)