1 To assess the involvement of endothelial a 2 -adrenoceptors in the clonidine-induced vasodilatation, the mesenteric artery of Sprague Dawley rats was cannulated and perfused with Tyrode solution (2 ml min 71 ). We measured perfusion pressure, nitric oxide (NO) in the perfusate using chemiluminescence, and tissue cyclic GMP by RIA. 2 In phenylephrine-precontracted mesenteries, clonidine elicited concentration-dependent vasodilatations associated to a rise in luminal NO. One hundred nM rauwolscine or 100 mM L o -nitro-Larginine antagonized the clonidine-induced vasodilatation. Guanabenz, guanfacine, and oxymetazoline mimicked the clonidine-induced vasorelaxation. 3 In non-contracted mesenteries, 100 nM clonidine elicited a maximal rise of NO (123+13 pmol); associated to a peak in tissue cyclic GMP. Endothelium removal, L o -nitro-L-arginine, or rauwolscine ablated the rise in NO. One hundred nM aminoclonidine, guanfacine, guanabenz, UK14,304 and oxymetazoline mimicked the clonidine-induced surge of NO. Ten mM ODQ obliterated the clonidineinduced vasorelaxation and the associated tissue cyclic GMP accumulation; 10 ± 100 nM sildena®l increased tissue cyclic GMP accumulation without altering the clonidine-induced NO release. 4 a 2 -Adrenergic blockers antagonized the clonidine-induced rise in NO. Consistent with a preferential a 2D -adrenoceptor activation, the K B s for yohimbine, rauwolscine, phentolamine, WB-4101, and prazosin were: 6.8, 24, 19, 165, and 1489 nM, respectively. 5 Rat pretreatment with 100 mg kg 71 6-hydroxydopamine reduced 95% tissue noradrenaline and 60% neuropeptide Y. In these preparations, 100 nM clonidine elicited a rise of 91.9+15.5 pmol NO. Perfusion with 1 mM guanethidine or 1 mM guanethidine plus 1 mM atropine did not modify the NO surge evoked by 100 nM clonidine. 6 Clonidine and congeners activate endothelial a 2D -adrenoceptors coupled to the L-arginine pathway, suggesting that the antihypertensive action of clonidine involves an endothelial vasorelaxation mediated by NO release, in addition to presynaptic mechanisms. British Journal of Pharmacology (2001) 134, 957 ± 968

The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y 1 R and P2Y 2 R revealed a 6-to 8-fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y 2 R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y 1 R and P2Y 2 R were detected by RT-PCR and the mRNA coding for the P2Y 4 R, P2Y 6 R and P2Y 11 R was also identified. Application of 2-MeSADP and uridine triphosphate (UTP), preferential P2Y 1 R and P2Y 2 R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels.

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y 2 receptor (P2Y 2 R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-␤-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTPmediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1-10 M UTP, a selective P2Y 2 R agonist, displaced the P2Y 2 R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y 2 R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y 2 R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y 2 R in intact human blood vessels.

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.