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Agmatine Induced NO Dependent Rat Mesenteric Artery Relaxation and Its Impairment in Salt-Sensitive Hypertension

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Agmatine Induced NO Dependent Rat Mesenteric Artery Relaxation and Its Impairment in Salt-Sensitive Hypertension

Tushar V Gadkari et al. Nitric Oxide.

Abstract

l-Arginine and its decarboxylated product, agmatine are important mediators of NO production and vascular relaxation. However, the underlying mechanisms of their action are not understood. We have investigated the role of arginine and agmatine in resistance vessel relaxation of Sprague-Dawley (SD) and Dahl salt-sensitive hypertensive rats. Second or 3rd-order mesenteric arterioles were cannulated in an organ chamber, pressurized and equilibrated before perfusing intraluminally with agonists. The vessel diameters were measured after mounting on the stage of a microscope fitted with a video camera. The gene expression in Dahl rat vessel homogenates was ascertained by real-time PCR. l-Arginine initiated relaxations (EC50, 5.8±0.7mM; n=9) were inhibited by arginine decarboxylase (ADC) inhibitor, difluoromethylarginine (DFMA) (EC50, 18.3±1.3mM; n=5) suggesting that arginine-induced vessel relaxation was mediated by agmatine formation. Agmatine relaxed the SD rat vessels at significantly lower concentrations (EC50, 138.7±12.1μM; n=22), which was compromised by l-NAME (l-N(G)-nitroarginine methyl ester, an eNOS inhibitor), RX821002 (α-2 AR antagonist) and pertussis toxin (G-protein inhibitor). The agmatine-mediated vessel relaxation from high salt Dahl rats was abolished as compared to that from normal salt rats (EC50, 143.9±23.4μM; n=5). The α-2A AR, α-2B AR and eNOS mRNA expression was downregulated in mesenteric arterioles of high-salt treated Dahl hypertensive rats. These findings demonstrate that agmatine facilitated the relaxation via activation of α-2 adrenergic G-protein coupled receptor and NO synthesis, and this pathway is compromised in salt-sensitive hypertension.

Keywords: ADC; AR; Agmatine; Arginine decarboxylase; DFMA; Hypertension; Nitric oxide; PTx; adrenergic receptor; arginine decarboxylase; difluoromethylarginine; eNOS; endothelial nitric oxide synthases; l-Arginine; l-NAME; l-NG-nitroarginine methyl ester; pertussis toxin; α-2 Adreno receptor.

Figures

Figure 1
Figure 1. Concentration dependant relaxation responses to L-arginine and agmatine
(a). Dose-response to intraluminal perfusion of L-arginine (n = 9) in SD rat mesenteric arterioles and after pre-treatment with ADC blocker, DFMA (0.5 mM)(n = 5); #P < 0.05 vs. L-arginine. (b). Concentration dependent dose response curve to intraluminal perfusion of agmatine in rat mesenteric arterioles in the presence and absence of an eNOS blocker, L-NAME (0.5 mM)(n = 4) and ADC blocker, DFMA (0.5 mM)(n = 3). Values are mean ± SE with; *P < 0.05 vs. agmatine; **P > 0.05 vs. agmatine. (c). Dose response to agmatine in SD rat vessels was obtained in the presence and absence of an α-2 AR antagonist, RX821002 (50 nM) (n = 6); *P < 0.05 vs. agmatine. (d). Agmatine relaxation response in the absence and presence of G-protein inhibitor, PTx (100 nM). Values are mean ± SE (n = 4); *P < 0.05 vs. agmatine.
Figure 2
Figure 2. Relaxation response to agmatine and L-arginine in Dahl salt-sensitive hypertension
(a) Agmatine and (b) L-arginine-mediated relaxation observed in salt-sensitive Dahl rats maintained on high salt, HS (4% NaCl) and normal salt, NS (0.49% NaCl) for 5 weeks, (a: n = 5, b: n = 3). Values are mean ± SE, #P < 0.05 vs. L-arginine, *P < 0.05 vs. agmatine.
Figure 3
Figure 3. Compromised nitric oxide activity in Dahl salt-sensitive hypertension
(a) Plasma nitrite concentrations in HS and NS fed Dahl rats (n = 3). (b) Real Time PCR data for α-2A AR, α-2B AR and eNOS mRNA expression. Mesenteric artery tissue was harvested and total RNA isolated from NS and HS rats. The semi-quantitative RT-PCR was carried out using SYBR Green technology in a StepOne RT-PCR system. Values are mean ± SE and expressed as fold change in mRNA expression in HS rats as compared to that in NS rats (n = 4).
Figure 4
Figure 4
Schematic representation of the proposed receptor-mediated hypothesis for arginine/agmatine stimulation of vascular NO synthesis.

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