Intercellular Ca2+ waves propagate by regenerative IP3‐induced IP3 release

Abstract

The endothelium is a sheet of highly‐specialised cells that line every blood vessel. Endothelial cells detect, integrate and respond to information from numerous signals, passing this information to neighbouring endothelial cells via intercellular communication to coordinate vascular contractility, inflammation and exchange of products between the blood and surrounding tissues. A significant form of intercellular communication occurs by travelling spatial gradients of Ca2+ (‘Ca2+ waves’) that move between cells. While the underlying mechanisms are not understood, Ca2+ and IP3 diffusion through gap junctions are each proposed to underlie Ca2+ wave propagation in the endothelium. Our results show that IP3, but not Ca2+ or gap junctions, is required for Ca2+ wave propagation.To investigate the mechanisms underlying endothelial signal propagation, second order mesenteric arteries were dissected from rats, pinned out in an en face configuration and Ca2+ signals measured using Cal‐520 (5μM). Localised photorelease of caged‐IP3 (5μM) generated rapid rises in endothelial Ca2+ at the site of photo‐activation. The Ca2+ rise subsequently propagated across cells outside the photolysis site. The propagated Ca2+ rise reflects cell‐cell communication since no uncaging occurred outside the photolysis site.To investigate the contribution of Ca2+ to signal propagation, Ca2+ buffering was spatiotemporally controlled using local photoactivation of the membrane permeant Ca2+ buffer, Diazo‐2. Release of the Ca2+ buffer prevented signal propagation within the uncaging region alone; signal propagation still occurred outside the uncaging area. This result suggests that IP3, but not Ca2+, underlies wave propagation. Supportive evidence for this conclusion was provided by photolysis of caged Ca2+(NP‐EGTA AM, 5μM). Photorelease of caged Ca2+ generated a Ca2+ rise that remained localised to the uncaging region and did not propagate outside the photolysis site, even with progressively increased Ca2+ loads. To verify these results, ACh (1μM) or ionomycin (10μM) were applied to restricted populations of cells by pressure ejection from a puffer pipette. A comparable Ca2+ rise was generated in each case, however the ACh generated a substantial Ca2+ wave that propagated upstream of the activated cells while the Ca2+ rise from ionomycin did not. These results suggest that Ca2+ is not responsible for propagating Ca2+ waves in the endothelium. The data also questions the involvement of gap junctions in wave transmission. If gap junctions were present and open, the Ca2+ rise generated by ionomycin would have been expected to diffuse to neighboring cells. This was not observed. Furthermore, the gap junction blockers gap27 and 18αGA, while successful in blocking FRAP, did not inhibit wave propagation. Together, these results suggest that IP3 is essential for Ca2+ wave propagation, but Ca2+ or gap junctions are not.