Diffusion of nitric oxide across cell membranes of the vascular wall requires specific connexin-based channels

NO is generated within cells and frequently must be transferred to responsive neighboring cells, as occurs in the endothelium-dependent relaxation of smooth muscle cells observed in blood vessels. It is thought that NO diffuses freely across cell membranes, but it may also permeate through low resistant membrane pathways. Here, we describe the participation of connexin (Cx)-formed channels in the NO transport across cell membranes and between endothelial and smooth muscle cells. We used a water-soluble NO donor of high molecular weight (S-nitrosylated albumin, BSA-NO) that does not permeate through cell membranes or Cx-based channels and the NO-sensitive dye 4,5-diaminofluorescein diacetate to detect changes of intracellular NO concentration. We found that NO generated in the extracellular space was not detected intracellularly in Cx-deficient HeLa cells, suggesting that cell membrane represents a significant diffusion barrier for NO transfer. However, Cx-based channels provide efficient pathways for NO signaling because NO opened and permeated hemichannels expressed in HeLa cells transfected with Cx43, Cx40, or Cx37. In contrast, NO closed hemichannels of HeLa-Cx32 cells, which otherwise are permeable to NO if are opened by a divalent cation-free extracellular solution. Consistent with this, blockade of Cx-based channels abolished the myoendothelial NO transfer and associated NO-dependent vasodilation induced by acethylcholine. These results indicate that Cx-based channels play a key role in the NO-dependent tonic control of vascular function and may direct the NO signal to specific targets, which provides a novel mechanistic basis for the critical role of Cxs in cell–cell communication in the vessel wall.This article is part of the Special Issue Section entitled ‘Current Pharmacology of Gap Junction Channels and Hemichannels’.

► The participation of connexin-formed membrane channels in NO transfer was evaluated.
► The cell membrane represents a significant barrier for rapid intercellular NO signaling.
► NO opens and permeates hemichannels formed by connexins 43, 40, or 37.
► NO transfer from endothelial to smooth muscle cells requires connexin-formed channels.
► The ACh-induced NO-mediated dilation requires functional connexin-formed channels.