Endothelial nitric oxide modulatory role on vascular sympathetic reactivity

JB Sousa, P Fresco, C Diniz. REQUIMTE/FARMA, Faculty of Pharmacy, Department of Drug Sciences, Laboratory of Pharmacology; 4050-313, Portugal

Blood vessels endothelium is an important regulator of vascular tonus via release of various endothelium-derived substances (Furchgott and Zawadzki, 1980) such as nitric oxide (NO) and prostaglandins (Moncada et al., 1991). However, the modulatory role of these molecules has only been studied upon smooth muscle responsiveness and, no data has been gathered concerning their influence on vascular sympathetic reactivity at the neuroeffector junction. We aim at investigating the role of vascular endothelium in the modulation of noradrenaline release by evaluating the influence exerted by NO per se or on other, well established, prejunctionalmodulators such as adenosine.

Methods: Male Wistar rats (12 weeks-old, 270-360g) artery preparations with and without endothelium (mechanically removed), preincubated with 0.1 μM [3H]-noradrenaline (40 min, at 37ºC), were submitted to three-identical periods of stimulation (5 Hz, 100 pulses, 1 ms, 50 mA) every 20 min (S0-S2). Effects of drugs, such as NO donors (SNP and NONOate), a selective A2A adenosine receptor antagonist (SCH 58261), enzyme substrate/inhibitor (L-arginine, L-NAME), on NA release (estimated as tritium overflow) were studied. Results are expressed as mean±s.e.m; statistically significant differences were analysed by ANOVA followed by Holm-Sidak or Student’s t test (*p<0.05 or **p<0.001, from the respective control and #p<0.05 or ##p<0.001, from the endothelium intact artery preparations).

Results: L-arginine (1 mM) and sodium nitroprusside (SNP, 10µM, a NO donor) facilitated NA release (1.15±0.241%, n=6, **p<0.001 and 1.22±0.04%, n=6, **p<0.001, respectively) while L-NAME (100 µM; a NO synthesize inhibitor) inhibited noradrenaline release in endothelium-intact artery preparations (0.74±0.045%, n=20). SCH 58261 did not modify noradrenaline release in endothelium-intact artery preparations (0.97±0.08%, n=25, **p<0.001). However, in endothelium denuded artery preparations, L-NAME and L-arginine failed to influence noradrenaline release (1.03±0.082%, n=17, ##p<0.001 and 0.94±0.167%, n=7, respectively). Moreover, NO donors, SNP (0.76±0.061%, n=8, **p<0.001; ##p<0.001) and NONOate (0.63±0.036%, n=3, **p<0.001), as well as NBTI (5 µM, a nucleoside transporter inhibitor) and SCH 58261(20 nM,) had inhibited transmitter release up to 30% (0.48±0.058%, n=6, **p<0.001 and 0.66±0.05%, n=6, **p<0.001; ##p<0.001, respectively).

Conclusions: Our results support the occurrence of transsynaptic modulatory effect mediated by endothelial NO: in endothelium intact and denuded arterial preparations NO influences differently noradrenaline release and seem to modify the extracellular availability of adenosine and, subsequently, adenosine mediated effects.

1. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980 Nov 27;288(5789):373-6.

2. Berrazueta JR, López-Jaramillo P, Moncada S. Nitric oxide: from endogenous vasodilator to biologic mediator. Rev Esp Cardiol. 1990 Aug-Sep;43(7):421-31.

Joana Sousa thanks FCT for PhD grant: SFRH/BD/64911/2009