NO, via its target Cx37, modulates calcium signal propagation selectively at myoendothelial gap junctions.

Background: Gap junctional calcium signal propagation (transfer of calcium or a calcium releasing messenger via gap junctions) between vascular cells has been shown to be involved in the control of vascular tone. We have shown before that nitric oxide (NO) inhibits gap junctional communication in HeLa cells exclusively expressing connexin 37 (HeLa-Cx37) but not in HeLa-Cx40 or HeLa-Cx43. Here we studied the effect of NO on the gap junctional calcium signal propagation in endothelial cells which, in addition to Cx37, also express Cx40 and Cx43. Furthermore, we analyzed the impact of NO on intermuscle and on myoendothelial gap junction-dependent calcium signal propagation. Since specific effects of NO at one of these three junctional areas (interendothelial/ myoendothelial/ intermuscle) may depend on a differential membrane localization of the connexins, we also studied the distribution of the vascular connexins in small resistance arteries.

Results: In endothelial (HUVEC) or smooth muscle cells (HUVSMC) alone, NO did not affect gap junctional Ca2+ signal propagation as assessed by analyzing the spread of Ca2+ signals after mechanical stimulation of a single cell. In contrast, at myoendothelial junctions, it decreased Ca2+ signal propagation in both directions by about 60% (co-cultures of HUVEC and HUVSMC). This resulted in a longer maintenance of calcium elevation at the endothelial side and a faster calcium signal propagation at the smooth muscle side, respectively. Immunohistochemical stainings (confocal and two-photon-microscopy) of cells in co-cultures or of small arteries revealed that Cx37 expression was relatively higher in endothelial cells adjoining smooth muscle (culture) or in potential areas of myoendothelial junctions (arteries). Accordingly, Cx37 - in contrast to Cx40 - was not only expressed on the endothelial surface of small arteries but also in deeper layers (corresponding to the internal elastic lamina IEL). Holes of the IEL where myoendothelial contacts can only occur, stained significantly more frequently for Cx37 and Cx43 than for Cx40 (endothelium) or Cx45 (smooth muscle).

Conclusion: NO modulates the calcium signal propagation specifically between endothelial and smooth muscle cells. The effect is due to an augmented distribution of Cx37 towards myoendothelial contact areas and potentially counteracts endothelial Ca2+ signal loss from endothelial to smooth muscle cells. This targeted effect of NO may optimize calcium dependent endothelial vasomotor function.