Effect of clenbuterol on non-endothelial nitric oxide release in rat mesenteric arteries and the involvement of β-adrenoceptors

Abstract

1. The aim of the present study was to explore the contribution of adrenergic, sensory and nitrergic innervations to the inhibitory effects of the β₂-adrenoceptor agonist clenbuterol on responses to electrical field stimulation (EFS, 200 mA, 0.3 ms, 1–16 Hz, for 30 s, at 1 min interval) in rat mesenteric artery segments without endothelium and the possible involvement of adrenergic, sensory and nitrergic innervations.

2. Clenbuterol (1 μM) reduced EFS-induced contractile responses, and this effect was reversed by the β-antagonist propranolol (1 μM) (contraction at 16 Hz expressed as % of 75 mM K⁺-induced contraction was: control, 69±9, clenbuterol, 31±6, n=13, P<0.001; control, 83±5, clenbuterol+propranolol 70±7, n=11, P>0.05).

3. In arteries preincubated with [³H]-noradrenaline (NA), clenbuterol did not modify the tritium overflow evoked by EFS (200 mA, 0.3 ms, 4 Hz, for 60 s; ratio between tritium release in the second and first stimuli was: control, 0.80±0.05 and clenbuterol added before second stimulus, 0.91±0.11, n=5, P>0.05).

4. The nitric oxide (NO) synthase inhibitors N^G-monomethyl-L-arginine (L-NMMA) and N^G-nitro-L-arginine methyl ester (L-NAME) (10 and 100 μM), and the guanylate cyclase inhibitor methylene blue (10 μM) increased the contractions caused by EFS (% contraction at 16 Hz, control, 81±7, n=26; 10 μM L-NMMA, 109±12, n=8, P<0.05; methylene blue, 119±6, n=6, P<0.05). However, these contractions were decreased by the NO synthase substrate L-arginine 10 μM (14±6%, n=6, P<0.001), but not modified by either the sensory neurones toxin capsaicin (0.5 μM, 75±6%, n=6, P>0.05) or the protein synthesis inhibitor cycloheximide (10 μM, 83±6%, n=8, P>0.05). None of these drugs altered the concentration-response curves to exogenous NA (n=7).

5. Pretreatment with capsaicin or cycloheximide did not modify the reduction of the EFS-evoked contraction provoked by clenbuterol. However the presence of L-NMMA (or L-NAME) or methylene blue did decrease the effect of clenbuterol (% contraction at 16 Hz, clenbuterol, 31±6, n=13; clenbuterol+10 μM L-NMMA, 93±11, n=8, P<0.05; clenbuterol+methylene blue, 90±7, n=6, P<0.05).

6. These results suggest that the reduction caused by clenbuterol in the contraction induced by EFS in rat mesenteric arteries seems to be mediated by NO release, through the activation of β₂-adrenoceptors probably present on nitrergic nerves.

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