Less efficient prejunctional adenosine receptor-mediated inhibitory effects in mesenteric vessels of spontaneously hypertensive rats (SHR)

C Rocha-Pereira1, JB Sousa1, P Fresco1, J Gonçalves1, SM Arribas2, MC González2, F Ferreirinha3, P Correia-de-Sá3, C Diniz1. 1REQUIMTE/FARMA, Laboratório de Farmacologia, Faculdade de Farmácia, Universidade do Porto, Departamento de Ciências do Medicamento, 4050-313, Portugal, 2Facultad de Medicina, Universidad Autónoma de Madrid, Departamento de Fisiología, 28029, Spain, 3Instituto de Ciências Biomédicas Abel Salazar-Universidade do Porto-ICBAS-UP, Laboratório de Farmacologia, 4050-313, Portugal

The mesenteric circulation plays an important role in the regulation and maintenance of systemic blood pressure (1). Adenosine is a potent regulator of vascular tone and exerting its effects through prejunctional modulation of perivascular sympathetic neurotransmission by activating A1, A2A, A2B and A3 receptors (2). In this study, we aimed at investigating the role of presynaptic adenosine receptors in mesenteric vessels, both veins (MV) and arteries (MA) and if possible, establish a relationship between adenosine receptor-mediated effects in the mesenteric circulation and hypertension.

Functional study: MA and MV segments from control (WKY) and spontaneously hypertensive rats (SHR), pre-incubated with 0.1μM [3H]-NA, were submitted to four-identical periods of stimulation (2Hz, 200pulses, 1ms, 80mA) every 20min (S1-S4). The effects of selective adenosine receptor (AR) agonists and/or antagonists on NA release (estimated as tritium overflow) were studied. Morphological study: MA/MV-segments were longitudinally opened, paraformaldehyde-fixed, incubated (overnight, 4ºC) with primary individual anti-AR antibodies (anti-A1/A2A/A2B/A3) and anti-tyrosine hydroxylase (TH) and followed by fluorescent secondary antibodies; tissues were mounted on a slide exposing the adventitia and visualized by confocal microscopy. Results are expressed as mean±s.e.m; p<0.05 was taken to reflect statistically significant differences from the appropriate control (ANOVA followed by Holm-Sidak or Student’s t test).

CPA (100 nM) inhibited NA release in MV and MA but inhibition was less evident in SHR vessels (MV-SHR: 65.90±2.86% vs WKY: 45.55±3.80%; n=7-9; p<0.001; and MA-SHR: 74.36±2.85% vs WKY: 61.68±2.74%; n=14; p<0.001). IB-MECA (100 nM) inhibited NA release in WKY MV (84.89±4.30%; n=5; p<0.05), an effect not observed in WKY MA or in SHR MA/MV. CGS 21680 (up to 100 nM) failed to facilitate NA release in MV from both strains but induced a slight facilitation in MA from WKY and SHR (1.24±5.18% and 1.21±7.73%; n=5; p<0.05). NECA (1, 3 and 10 μM), in the presence of A1 (DPCPX: 20 nM) and A3 (MRS 1523: 1 μM) antagonists failed to change tritium overflow in MA/MV from both SHR and WKY. Confocal microscopy showed that all AR-subtypes are located on the same structure as TH-staining sympathetic nerves both in MA and MV.

This study shows for the first time that sympathetic nerves of MA and MV present all AR-subtypes and characterized the modulatory effects mediated by A1-, A2A-, A2B- and A3-adenosine receptors in the mesenteric vessels. Data revealed a major contribution of A1 receptors in both vessels. A3 receptors reduced NA release only in MV, whereas A2A receptors facilitated NA release in MA. Our findings also showed that in hypertensive vessels the reduced A1 receptor-mediated inhibition as well as the absence of A3 receptor-mediated inhibition of NA release can increase NA levels in the synaptic cleft, both in arteries and veins, which might conduce to an increase of vascular reactivity.

References: (1) Takenaga and Kawasaki, 1999. Eur J Pharmacol.367:239-245; (2) Diniz et al., 2008. Curr Pharm Des.14:1698-1722.

Acknowledgements: Ações Integradas Luso-Espanholas E-73/10; C. Rocha-Pereira thanks FCT for PhD grant (SFRH/BD/32161/2006).