Spreading dilatation to luminal perfusion of ATP and UTP in rat isolated small mesenteric arteries.

Levels of ATP achieved within the lumen of vessels suggest a key autacoid role. P2Y receptors on the endothelium may represent the target for ATP, leading to hyperpolarization and associated relaxation of vascular smooth muscle through the endothelium-dependent hyperpolarizing factor (EDHF) pathway. EDHF signals radially from the endothelium to cause dilatation, and appears mechanistically distinct from the axial spread of dilatation, which we showed occurs independently of a change in endothelial cell Ca2+ in rat mesenteric arteries. Here we have investigated the potential of P2Y receptor stimulation to evoke spreading dilatation in rat resistance small arteries under physiological pressure and flow. Triple cannulation of isolated arteries enables focal application of purine and pyrimidine nucleotides to the endothelium, avoiding potential complicating actions of these agents on the smooth muscle. Nucleotides were locally infused through one branch of a bifurcation, causing near maximal local dilatation attributable to EDHF. Dilatation then spread rapidly into the adjacent feed artery and upstream against the direction of luminal flow, sufficient to increase flow into the feed artery. The rate of decay of this spreading dilatation was identical between nucleotides, and matched that to ACh, which acts only on the endothelium. In contrast, focal abluminal application of either ATP or UTP at the downstream end of cannulated arteries evoked constriction, which only in the case of ATP was also associated with modest spread of dilatation. The non-hydrolysable ADP analogue, ADPbetaS, acting at P2Y1 receptors, caused robust local and spreading dilatation responses whether applied to the luminal or abluminal surface of pressurized arteries. Dilatation to nucleotides was sensitive to inhibition with apamin and TRAM-34, selective blockers of small- and intermediate-conductance Ca2+-activated K+ channels, respectively. These data demonstrate that direct luminal stimulation of P2Y receptor on the endothelium of rat mesenteric arteries leads to marked spreading dilatation and thus suggests that circulating purines and pyrimidines may act as important regulators of blood flow.

Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries.

In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (approximately 15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs (43Gap 26, 40Gap 27, and (37,43)Gap 27; 300 micromol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.