Role of L-Arginine in Angiotensin II Induced Hypertension in Rats

It is widely accepted that nitric oxide (NO) regulates arterial blood pressure by influencing systemic vascular resistance and fluid and electrolyte homeostasis. Despite the large amount of research on this subject, the factors important in the control of NO production in the kidney have not been clearly defined. One potential site of regulation of NO production is by controlling the cellular availability of L-Arginine (L-Arg), the substrate for NO synthase. Recent studies from our laboratory have indicated that the cellular mechanisms of L-Arg transport in endothelial cells and renal epithelial cells play an important role in NO production in the mammalian kidney with resulting effects on kidney function and arterial blood pressure. Moreover, previous studies demonstrated that supplementation of exogenous L-Arg is beneficial in preventing salt sensitive hypertension. The present studies were performed to assess the mechanisms by which the supplementation of exogenous L-Arg blunts angiotensin II-dependent hypertension in rats. Based on these previous studies, work in this dissertation tested the hypothesis that cellular L-Arginine (L-Arg) uptake attenuates angiotensin II (Ang II) mediated hypertension in rats by increasing nitric oxide, blunting renal vasoconstriction and enhancing the pressure natriuresis mechanism..;Aim 1 of this dissertation determined if administration of exogenous L-Arg was able to attenuate the Ang II induced hypertension and associated renal damage in male Sprague Dawley rats. For this study, male Sprague Dawley rats were infused intravenously with either Ang II (20 ng/Kg/min) or Ang II plus L-Arg (L-Arg:300 mug/ Kg/min) for a period of 9 days with continuous monitoring of mean arterial blood pressure. A control group of rats were infused with saline (vehicle; 1 ml/hr). Results of this chronic study demonstrated that intravenous co-infusion of L-Arg (300 mug/ Kg/min) blunted the increase in mean arterial blood pressure by 45% from the elevated levels in the Ang II infused rats. Along with this the glomerular damage (represented by the urinary albumin excretion and kidney histological sections) observed in the Ang II infused rats was significantly reduced with L-Arg administration.;Plasma nitrate levels, measured as an index of NO production, were significantly elevated in L-Arg co-infused rats, demonstrating that the effects of L-Arg were mediated by an increased production of NO. Further studies were performed to examine if L-Arg administration was effective in reversing the hypertension that was already established with chronic infusion of Ang II. Commencement of L-Arg administration following establishment of Ang II mediated hypertension, had a minor effect in ameliorating the Ang II hypertension.;In the second aim of this dissertation, the influence of exogenous L-Arg to modulate the effects of Ang II on the pressure natriuresis and diuresis relationship and intra renal blood flow was examined. Co-administration of L-Arg (300 mug/Kg/min) prevented the Ang II-mediated suppression of the pressure natriuresis and diuresis relationship. The beneficial effects of exogenous L-Arg appeared to be mediated via the vasculature since the effects of acute infusion of Ang II (20 ng/Kg/min) to reduce total renal blood flow (RBF) and glomerular filtration rate (GFR) were reversed by L-Arginine infusion. L-Arg administration was also effective in restoring the blunted superficial cortical blood flow and outer medullary blood flow that was caused by acute infusion of Ang II in these rats. Moreover, urinary nitrate levels were significantly elevated in the L-Arg infused rats. From these studies, it is concluded that the beneficial effects of L-Arg to reverse the anti-natriuretic and anti-diuretic effects of Ang II in the kidney are mediated by stimulation of NO in the vasculature.;The objective of the third aim was to determine if cellular L-Arg uptake mechanisms in the vasculature were responsible for the observed antihypertensive effects of L-Arg. In vitro experiments were performed in Ang II stimulated isolated aortic rings from Sprague Dawley rats. The contractile force developed in these rings in response to incubation with various pharmacological agents, was assessed with a force transducer. Incubation of aortic rings with physiological doses (10-4M) of L-Arg attenuated the dose dependent increase in contractile force when the rings were incubated with successively higher doses of Ang II (10-10M to 3x10-7M). As a measure of NO production, L-Arg incubated vessels had a greater diaminofluorescein (DAF) dye fluorescence intensity when compared to vehicle-treated vessels. The influence of L-Arg to blunt Ang IImediated contraction was eliminated by endothelial denudation or incubation with the NOS inhibitors L-NAME or LNMMA. These experiments indicated that the effects of L-Arg to blunt Ang II-mediated vasoconstriction in the aortic rings were endothelium-dependent, NOS-dependent, and associated with increased NO production.;Endothelial cells possess two distinct transport systems, a y + and y+L system, important for L-Arg uptake. Cellular L-Arg uptake by the y+ and y+L systems are competitively inhibited by cationic amino acids. The y+L system is competitively inhibited by neutral amino acids; but anionic amino acids do not alter L-Arg uptake by either system. Further experiments were performed to examine the mechanisms of cellular L-Arg uptake in the aortic rings. The influence of L-Arg to blunt Ang II-dependent vasoconstriction was attenuated by the addition of cationic amino acids (L-Ornithine, L-Lysine, L-HomoArginine) or neutral amino acids (L-Serine, L-Threonine) in the presence of an excess dose (10 -3M) of L-Arg. The anionic amino acids, L-Aspartate or L-Glutamate did not influence L-Arg's effects on Ang II-mediated contraction. These studies demonstrated that L-Arg blunts Ang II-mediated vascular contraction by an endothelial- and NOS-dependent mechanism that involves cellular uptake of L-Arg via y+ or y+L transporters.;In conclusion, the studies performed in this dissertation demonstrate that the beneficial effects of exogenous L-Arg administration to attenuate Ang II-dependent hypertension and renal disease are mediated by increased cellular uptake of L-Arg in endothelial cells. The cellular uptake, which is mediated by y+ or y+L transporters, increases NO production and reduces the vasoconstrictor effects of Ang II in the renal vasculature. As a result, the vasoconstrictive effects of Ang II to reduce GFR and RBF and retain sodium and water are attenuated and the hypertension and accompanying renal damage are minimized.