By Hussam A.S. Murad Department of Pharmacology and Therapeutics Faculty of Medicine, Ain Shams University By Hussam A.S. Murad Department of Pharmacology and Therapeutics Faculty of Medicine, Ain Shams University

L-arginine is the precursor of nitric oxide. It is transformed by nitric oxide synthase (NOS) to nitric oxide, an important endothelium- derived vasoactive mediator involved in CVS regulation. L-arginine, constitutes about 5% of the amino acid content of the adult diet. Supplementation with L-arginine is the only effective way to deliver more L-arginine to the individual. Supplementation with exogenous L-arginine improves NO- mediated vascular functions in vivo, although its baseline plasma concentration is about 25-fold higher than the Michaelis-Menten constant ( Km ) of the isolated purified endothelial NO synthase in vitro. This "L-arginine paradox" occurs in many cardiovascular diseases, as hypertension and ischemia. ACE inhibitors with a sulfhydryl (SH) group improve endothelial dysfunction through increased protection and stability of NO by scavenging superoxide anions. On the other hand, SH group causes multiple immune-based adverse effects. Lisinopril is an ACEI without an SH group. L-arginine is the precursor of nitric oxide. It is transformed by nitric oxide synthase (NOS) to nitric oxide, an important endothelium- derived vasoactive mediator involved in CVS regulation. L-arginine, constitutes about 5% of the amino acid content of the adult diet. Supplementation with L-arginine is the only effective way to deliver more L-arginine to the individual. Supplementation with exogenous L-arginine improves NO- mediated vascular functions in vivo, although its baseline plasma concentration is about 25-fold higher than the Michaelis-Menten constant ( Km ) of the isolated purified endothelial NO synthase in vitro. This "L-arginine paradox" occurs in many cardiovascular diseases, as hypertension and ischemia. ACE inhibitors with a sulfhydryl (SH) group improve endothelial dysfunction through increased protection and stability of NO by scavenging superoxide anions. On the other hand, SH group causes multiple immune-based adverse effects. Lisinopril is an ACEI without an SH group. Introduction

 The Research Question: Would addition of the nitric oxide precursor; L-arginine to lisinopril, substitute for the lack of lisinopril’s protective free radicle scavenging effect on nitric oxide resulting in an improvement of lisinopril’s effects on endothelial dysfunction ?  Thus, the present work was designed to study whether L-arginine can modify effects of lisinopril on systolic blood pressure and isolated aortic rings in hypertensive rats.  The Research Question: Would addition of the nitric oxide precursor; L-arginine to lisinopril, substitute for the lack of lisinopril’s protective free radicle scavenging effect on nitric oxide resulting in an improvement of lisinopril’s effects on endothelial dysfunction ?  Thus, the present work was designed to study whether L-arginine can modify effects of lisinopril on systolic blood pressure and isolated aortic rings in hypertensive rats. Aim of the Study

(I) Induction of DOCA-salt hypertension in rats: Methods mmHg The blood pressure was measured by Harvard indirect tail-cuff method before and 4 weeks after DOCA-salt administration. Rats with systolic blood pressure of 140 mmHg or more were considered hypertensive. The blood pressure was measured by Harvard indirect tail-cuff method before and 4 weeks after DOCA-salt administration. Rats with systolic blood pressure of 140 mmHg or more were considered hypertensive.

(II) The experimental design: The DOCA-salt hypertensive rats were divided into 4 groups (6 rats per group) and treated as follows :  Group I (Control group).  Group II (L-arginine group): received L-arginine ( 20 mg/ml drinking water ) for 4 weeks.  Group III (Lisinpril group): received lisinopril (2 mg/kg/day) by gastric gavage for 4 weeks.  Group IV (L-arginine and lisinopril group): received L-arginine (20 mg/ml drinking water) and lisinopril (2 mg/kg/day by gastric gavage) for 4 weeks. (II) The experimental design: The DOCA-salt hypertensive rats were divided into 4 groups (6 rats per group) and treated as follows :  Group I (Control group).  Group II (L-arginine group): received L-arginine ( 20 mg/ml drinking water ) for 4 weeks.  Group III (Lisinpril group): received lisinopril (2 mg/kg/day) by gastric gavage for 4 weeks.  Group IV (L-arginine and lisinopril group): received L-arginine (20 mg/ml drinking water) and lisinopril (2 mg/kg/day by gastric gavage) for 4 weeks. Methods

 After 4 weeks the effects of L-arginine, lisinopril and a combination of both were estimated on: A.Systolic blood pressure. B.Norepinephrine-induced contractions (g) of the isolated perfused aortic ring. C.Acetylcholine-induced relaxations (% of the maximal relaxation) of the isolated perfused aortic ring.  After 4 weeks the effects of L-arginine, lisinopril and a combination of both were estimated on: A.Systolic blood pressure. B.Norepinephrine-induced contractions (g) of the isolated perfused aortic ring. C.Acetylcholine-induced relaxations (% of the maximal relaxation) of the isolated perfused aortic ring. Methods

(A)Effects of L-arginine, lisinopril and a combination of both on systolic blood pressure in DOCA-salt hypertensive rats : Results L-arginine, lisinopril and the combination of both produced statistically significant reductions in systolic blood pressure. Lisinpril produced a statistically significant reduction compared with L-arginine and the combination of both produced a statistically significant reduction compared with each agent alone.

Results (A): Control group. (B): L-arginine group. (C): Lisinopril group. (D): L-arginine + Lisinopril group. N 1 -N 5 : – M Norepinephrine. (A): Control group. (B): L-arginine group. (C): Lisinopril group. (D): L-arginine + Lisinopril group. N 1 -N 5 : – M Norepinephrine. (B) Effects of L-arginine, lisinopril and a combination of both on norepinephrine-induced contractions (g) of isolated perfused aortic ring preparation from pretreated DOCA-salt hypertensive rats :

Results (C)Effects of L-arginine, lisinopril and a combination of both on acetylcholine-induced relaxation (% of the maximal relaxation) of isolated perfused aortic ring preparation from pretreated DOCA- salt hypertensive rats : (A): Control group. (B): L-arginine group. (C): Lisinopril group. (D): L-arginine + Lisinopril group. A 1 -A 5 : – M Acetylcholine. (A): Control group. (B): L-arginine group. (C): Lisinopril group. (D): L-arginine + Lisinopril group. A 1 -A 5 : – M Acetylcholine.

 Supplementation with L-arginine has been shown to improve endothelial functions augmenting effects of lisinopril in hypertensive rats due to increasing NO synthesis despite saturating intracellular L-arginine concentrations.  This "L-arginine paradox“ may be explained by an elevated level of asymmetric dimethylarginine (ADMA, an endogenous competitive inhibitor of NO synthase) because ADMA, in concentrations found in many patho-physiological conditions; inhibits vascular NO production.  Also independent of substrate effects of L-arginine to NO synthase, L-arginine may regulate enzyme reaction kinetics. In addition, sequestration of arginine to regions in the cell that are poorly accessible to NO synthase may account for situations in which increasing arginine drives enzyme activity, even when arginine is available in apparent excess.  Supplementation with L-arginine has been shown to improve endothelial functions augmenting effects of lisinopril in hypertensive rats due to increasing NO synthesis despite saturating intracellular L-arginine concentrations.  This "L-arginine paradox“ may be explained by an elevated level of asymmetric dimethylarginine (ADMA, an endogenous competitive inhibitor of NO synthase) because ADMA, in concentrations found in many patho-physiological conditions; inhibits vascular NO production.  Also independent of substrate effects of L-arginine to NO synthase, L-arginine may regulate enzyme reaction kinetics. In addition, sequestration of arginine to regions in the cell that are poorly accessible to NO synthase may account for situations in which increasing arginine drives enzyme activity, even when arginine is available in apparent excess. Discussion

 L-arginine limits the increase in systolic blood pressure in rats with nephropathy and lisinopril alone and the combination of both agents are more effective. Lisinopril limits formation of renal endothelin-1 (ET-1). Also NO counteracts formation and effects of ET-1. Consequently, L- arginine with lisinopril would represent a novel strategy for patients with severe nephropathy not completely responsive to ACE inhibition due to restoring the nitric oxide/ET-1 balance.  Supplementation of L-arginine, in patients with pulmonary hypertension increases NO production, decreases pulmonary vascular resistance and mean pulmonary arterial pressure. Consequently, oral supplementation of L- arginine may have beneficial effects on hemodynamics and exercise capacity in these hypertensive patients.  L-arginine limits the increase in systolic blood pressure in rats with nephropathy and lisinopril alone and the combination of both agents are more effective. Lisinopril limits formation of renal endothelin-1 (ET-1). Also NO counteracts formation and effects of ET-1. Consequently, L- arginine with lisinopril would represent a novel strategy for patients with severe nephropathy not completely responsive to ACE inhibition due to restoring the nitric oxide/ET-1 balance.  Supplementation of L-arginine, in patients with pulmonary hypertension increases NO production, decreases pulmonary vascular resistance and mean pulmonary arterial pressure. Consequently, oral supplementation of L- arginine may have beneficial effects on hemodynamics and exercise capacity in these hypertensive patients. Discussion

 In conclusion, L-arginine augments effects of lisinopril in hypertensive rats and it seems to provide a hopeful prospect for the treatment of cardiovascular diseases. However, more studies evaluating the effects of long-term treatment with L- arginine are needed. Conclusion