Novel mechanism of nitric oxide-mediated regulation of resistance artery function by the control of connexin and pannexin-formed channels

Abstract

Gap junctions are intercellular channels formed by two hemichannels provided by adjacent cells. Hemichannels, in turn, are assembled by protein subunits known as connexins (Cxs). Functional hemichannels can also remain unpaired and allow the release of paracrine signals or Ca2+ entry. In addition to connexins, pannexin-1 (Panx-1), a member of another protein family that only forms hemichannels, has also been detected in the vessel wall of resistance arteries. Gap junctions connect endothelial and smooth muscle cells, and then, it is thought that these channels play a central role in the coordination of vascular wall function and in the conduction of vasomotor signals along the vessel length. Control of vasomotor tone in resistance arteries and arterioles is essential in the regulation of arterial blood pressure and blood flow distribution. Nitric oxide (NO) production by the enzyme endothelial NO synthase (eNOS) has been ascribed as the major vasodilator signal activated in endothelial cells. Although the endothelium-mediated vasodilation depends exclusively on NO in large conduit vessels, an additional vasodilator mechanism associated with the endothelium-dependent hyperpolarization of smooth muscle cells also plays a prominent role in the control of vasomotor tone in small resistance arteries and arteriole. It is thought that the smooth muscle hyperpolarization relies on electrotonic transmission of a hyperpolarizing current from endothelial cells to smooth muscle cells via direct cell-to-cell communication through gap junction channels located at discrete points of contact of these two cell types. Although it is thought that connexin-formed hemichannels are closed in physiological conditions with a normal extracellular Ca2+ concentration, it has been reported that NO-mediated S-nitrosylation induces opening of Cx43-formed channels. These data indicate that activity of Cx-formed gap junction channels or hemichannels in endothelial cells may be controlled by NO production. In contrast, the effect of NO on pannexin-formed channels is controversial, since NO has been found to activate or inhibit these channels. The contribution of NO-mediated S-nitrosylation to the control of gap junction connecting endothelial or smooth muscle cells, and then, to the coordination of vascular function and conduction of vasomotor responses along the length of resistance vessels has not been studied. Based on these antecedents, we now propose to analyze the mechanisms involved in the NO-mediated control of gap junction channels and connexin- or Panx-formed hemichannels of resistance vessels. In addition, we propose to evaluate the involvement of these regulation mechanisms in the conduction of vasomotor responses along the length of resistance vessels. The goals of this proposal will be developed in isolated mesenteric vessels of rat, cremaster muscle microcirculation in vivo and primary cultures of endothelial cells. Adult, male C57 Bl/6 and endothelial cell specific pannexin-1 knockout mice, and male Sprague-Dawley rats will be used for all experiments. To obtain the results will be used the analytical techniques of Western blot, Immunocytochemistry, Proximity Ligation Assay, Protein S-nitrosylation analysis by Biotin switch assay and Proximity Ligation Assay, Dye Coupling to assess intercellular gap junction communication and intracellular recordings of membrane potential. (AU)