Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells

Abstract

Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and alpha-2 adrenoceptors (alpha-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and alpha-2 adrenoceptors, partly inhibited L-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of alpha-2 AR, at very low concentrations completely inhibited NO formation. Like L-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of alpha-2 AR was very potent in activating cellular NO, thus indicating a possible role for alpha-2 AR in L-arginine-mediated NO synthesis. D-arginine also activated NO production and could be inhibited by imidazoline and alpha-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated L-arginine-mediated NO synthesis, thus indicating mediation via G proteins. L-type Ca(2+) channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the L-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of L-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development.

Fig. 1.

Structures of guanidinium-containing compounds. Idazoxan and moxonidine are the ligands for I-receptor and α-2 AR. The guanidinium groups are marked with a dotted box.

Fig. 2.

Arginine dose–response and the effect of NOS inhibitor and receptor antagonist. (A) Dose-dependent activation of cellular NO synthesis by l-arginine (∗, P < 0.05 vs. 0; n = 3). (B) Effect of eNOS inhibition with 5 mM l-N^G-nitroarginine methyl ester (l-NAME) on l-arginine (5 mM) -initiated NO₂⁻/NO₃⁻ formation. (∗, P = 0.08 vs. 5 mM l-arginine; n = 3). (C) Effect of idazoxan (10 μM) on l-arginine (5 mM) -mediated NO₂⁻/NO₃⁻ formation in HUVEC cultures (#, P = 0.033 vs. 5 mM l-arginine; n = 3). The experiments were carried out in 12-well plates at 120,000 cells per well in 95% air/5% CO₂ incubator at 37°C for 30 min. The NO₂⁻/NO₃⁻ levels were measured by a modified Griess reaction that can detect NO₂⁻/NO₃⁻ levels in the lower nanomolar range.

Fig. 3.

Effect of α-2 AR antagonist on the l-arginine and A23187-mediated NO formation. (A and B) The cell cultures (120,000 cells per well) were treated with either 5 mM l-arginine (A) or 1 μM A23187 (B) in the presence and absence of 0.2 nM rauwolscine for 30 min at 37°C in a 5% CO₂/95% air incubator. The supernatants were assayed for NO₂⁻/NO₃⁻ by modified a Griess reaction. (A) n = 4; ∗, P < 0.05 vs. 5 mM l-arginine. (B) n = 4; ∗, P < 0.05 vs. none. (C) Dose–response of α-2 AR agonist dexmedetomidine. The cells (180,000 cells per well) were treated with increasing concentrations of dexmedetomidine for 60 min. The culture supernatants were used for a NO₂⁻/NO₃⁻ assay by a Griess reaction (n = 4; ∗, P < 0.05 vs. 0.001 nM dexmedetomidine). (D) The cultures (120,000 cells per well) were treated with 5 mM d-arginine in the presence and absence of 0.2 nM rauwolscine and 50 μM idazoxan for 30 min in Krebs solution at 37°C in 5% CO₂/95% air incubator. The cell supernatants were assayed for NO₂⁻/NO₃⁻ by a modified Griess reaction (n = 4; ∗, P = 0.009 vs. 5 mM d-arginine; #, P = 0.007 vs. 5 mM d-arginine; $, P = 0.031 vs. 5 mM d-arginine).

Fig. 4.

Effect of pertussis toxin (PTx) on NO synthesis. The cell cultures (120,000 cells per well) were pretreated with pertussis toxin at 200 and 400 ng/ml for 2 h, followed by l-arginine (5 mM) in the presence and absence of pertussis toxin for 30 min at 37°C in a 5% CO₂/95% air incubator. The culture supernatants were assayed for NO₂⁻/NO₃⁻ by modified Griess reaction (n = 3; ∗, P < 0.05 vs. + 400 ng/ml PTx; #, P = 0.077 vs. none).

Fig. 5.

Ca²⁺ dependence of l-arginine activity and agmatine-mediated cellular NO synthesis. (A) Effect of Ca²⁺ channel blocker and phospholipase C inhibitor on NO formation. The cell cultures were treated with 5 mM l-arginine in the presence and absence of 5 μM nifedipine and 3 μM U73122. The experiments were conducted at 37°C for 30 min in a 5% CO₂/95% air incubator. The culture supernatants were assayed for NO₂⁻/NO₃⁻ by using a modified Griess reaction (n = 3; ∗, P < 0.05 vs. none). (B) Fluorescence recording of fluo-4-loaded cells (1 μM in a six-well plate; 150,000 cells per well) with excitation at 488 nm and emission at 526 nm. A 10 mM aliquot of l-arginine was added (at arrow), and a time course of Ca²⁺ fluorescence was recorded using a Leica deconvolution microscope with a Xenon light source. Digitized images were captured with Slidebook software, and this software is used for time lapse and excitation of the fluorophore probe. The data are representative of three similar recordings. (C) Effect of α-2 AR antagonist on l-arginine-mediated dilation. The gracilis anticus muscle segments of the first-order gracilis muscle arterioles were isolated as described in Materials and Methods. The individual arteriolar segments were cannulated at both ends in a water-jacketed vessel chamber. The distal micropipette was connected to a stopcock, and the proximal micropipette was connected to a reservoir, the height of which was adjusted to achieve 80-mmHg intraluminal pressure. Test compounds (l-arginine and rauwolscine) were included in the superfusion buffer (∗, P < 0.05 vs. + 0.2 nM rauwolscine; n = 3). (D) Effect of α-2 AR antagonist on the agmatine-mediated NO formation. The 90% confluent cells preloaded with 5 μM DAF-FM diacetate were treated with 10 μM agmatine in the presence and absence of 0.2 nM rauwolscine for 10 min as described in Materials and Methods. The fluorescence as a measure of NO synthesis was measured using a UV confocal microscope (n = 3; ∗, P < 0.05 vs. untreated).