In the isolated rat mesenteric bed, the 1 min perfusion with 100 nM anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 ± 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5 -iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB 1 receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

To evaluate whether sympathetic activity induces nitric oxide (NO) production, we perfused the rat arterial mesenteric bed and measured luminally accessible norepinephrine (NE), NO, and cGMP before, during, and after stimulation of perivascular nerves. Electrical stimulation (1 min, 30 Hz) raised perfusion pressure by 97 ± 7 mmHg, accompanied by peaks of 23 ± 3 pmol NE, 445 ± 48 pmol NO, and 1 pmol cGMP. Likewise, perfusion with 10 μM NE induced vasoconstriction coupled to increased NO and cGMP release. Electrically elicited NO release depended on stimulus frequency and duration. Endothelium denudation with saponin abolished the NO peak without changing NE release. Inhibition of NO synthase with 100 μM N ω-nitro-l-arginine reduced basal NO and cGMP release and blocked the electrically stimulated and exogenous NE-stimulated NO peak while enhancing vasoconstriction. Blocking either sympathetic exocytosis with 1 μM guanethidine or α1-adrenoceptors with 30 nM prazosin abolished the electrically evoked vasoconstriction and NO release. α2-Adrenoceptor blockade with 1 μM yohimbine reduced both vasoconstriction and NO peak while increasing NE release. In summary, sympathetically released NE induces vasoconstriction, which triggers a secondary release of endothelial NO coupled to cGMP production.

Epinephrine plays a key role in the control of vasomotor tone; however, the participation of the NO/cGMP pathway in response to beta-adrenoceptor activation remains controversial. To evaluate the involvement of the endothelium in the vascular response to epinephrine, we assessed NO production, endothelial NO synthase phosphorylation, and tissue accumulation of cGMP in the perfused arterial mesenteric bed of rat. Epinephrine elicited a concentration-dependent increase in NO (EC(50) of 45.7 pM), which was coupled to cGMP tissue accumulation. Both NO and cGMP production were blocked by either endothelium removal (saponin) or NO synthase inhibition (N(omega)-nitro-L-arginine). Blockade of beta(1)- and beta(2)-adrenoceptors with 1 microM propranolol or beta(3)-adrenoceptor with 10 nM SR 59230A displaced rightward the concentration-NO production curve evoked by epinephrine. Selective stimulation of beta(1)-, beta(2)-, or beta(3)-adrenoceptors also resulted in NO and cGMP production. Propranolol (1 microM) inhibited the rise in NO induced by isoproterenol or the beta(2)-adrenoceptor agonists salbutamol, terbutaline, or fenoterol. Likewise, 10 nM SR 59230A reduced the effects of the beta(3)-adrenoceptor agonists BRL 37344, CGP 12177, SR 595611A, or pindolol. The NO production induced by epinephrine and BRL 37344 was associated with the activation of the phosphatidylinositol 3-kinase/Akt pathway and phosphorylation of eNOS in serine 1177. In addition, in anaesthetized rats, bolus administration of isoproterenol, salbutamol, or BRL 37344 produced NO-dependent reductions in systolic blood pressure. These findings indicate that beta(1)-, beta(2)-, and beta(3)-adrenoceptors are coupled to the NO/cGMP pathway, highlighting the role of the endothelium in the vasomotor action elicited by epinephrine and related beta-adrenoceptor agonists.

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Na-Ca exchanger (NCX) contributes to control the intracellular free Ca concentration ([Ca]), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively. In addition, both blockers abolished the NO production activated by ACh in intact mesenteric arteries. In contrast, the inhibition of NCXrm did not affect the vasodilator responses induced by the Ca ionophore, ionomycin, and the NO donor, S-nitroso- N-acetylpenicillamine. Furthermore, SEA0400, KB-R7943, and a small interference RNA directed against NCX1 blunted the increase in [Ca] induced by ACh or ATP in cultured endothelial cells. The analysis by proximity ligation assay showed that the NO-synthesizing enzyme, eNOS, and NCX1 were associated in endothelial cell caveolae of intact mesenteric resistance arteries. These results indicate that the activation of NCXrm has a central role in Ca-mediated vasodilation initiated by ACh in endothelial cells of resistance arteries.-Lillo, M. A., Gaete, P. S., Puebla, M., Ardiles, N. M., Poblete, I., Becerra, A., Simon, F., Figueroa, X. F. Critical contribution of Na-Ca exchanger to the Ca-mediated vasodilation activated in endothelial cells of resistance arteries.