IDENTIFICATION OF AN IP(3) RECEPTOR IN ENDOTHELIAL-CELLS

In this study we have used saponin to permeabilize bovine endothelial cell membranes in order to directly test the involvement of IP3 in regulating internal Ca2+ release. Our results indicate that the release of internal Ca2+ occurs as early as 1-3 seconds after IP3 addition. This IP3-induced internal Ca2+ release can be inhibited by heparin (an IP3 receptor antagonist). Further binding of [H-3]IP3 to saponin-permeabilized bovine endothelial cells reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (K(d)) of almost-equal-to 0.50 (+/- 0.03) nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the bovine endothelial cell IP3 receptor (almost-equal-to 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Immunofluorescence data indicates that the IP3 receptor is preferentially located at the perinuclear region of the cells. In addition, PCR analysis of first-strand cDNAs from both bovine endothelial cells and rat brain tissues reveals that the IP3 receptor transcript in bovine endothelial cells belongs to the short non-neuronal form and not the long neuronal form detected in rat brain tissue. These findings suggest that the IP3 receptor in endothelial cells is both structurally and functionally analogous to that reported in non-neuronal cell systems and probably plays an important role in agonist-induced endothelial cell activation. (C) 1994 Wiley-Liss, Inc.