Connexin hemichannels-mediated Ca 2+ entry results in nitric oxide production in in situ injured endothelial cells Everardo Avelino Secondo anno Ciclo XXII

Injury provokes a Ca 2+ wave characterized by two phases, peak and plateau PEAK ~ 60% > intracellular stores depletion (P 2 Y 1,2,12,13 ) ~ 40 > Ca2+ influx (P 2 X and Gap junctions) PLATEAU Ca2+ influx (Gap junctions) Introduction

Endothelium functions: Angiogenesis and vasculogenesis Preserve a surface unreactive toward circulating cells Mantain thromboresistence Inhibit vascular tone Inhibit cells growth in outer layer of the vesell wall. Introduction

Nitric oxide measurement DAF FM DA Fluorescent dye Indicator of NO production Emission: 510nm Excitation: 490nm

Injury augments nitric oxide production in rat aortic endothelium

Injury induced NOP* depend on extracellular Ca 2+ * Nitric oxide production

Capacitative Ca 2+ entry does not sustain injury-induced NOP

Putative blockers of CxHcs* reduce NO synthesis induced by Ca 2+ entry in injured endothelium *Connexin hemichannels

Putative blockers of CxHcs* reduce NO synthesis induced by Ca 2+ entry in injured endothelium *Connexin hemichannels

Connexin mimetic peptides reduce both injury-induced Ca 2+ elevation and NO production in ra aortic EC’s Pre-incubation

Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s Pre-incubation

Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s Accute effect

Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s

Summary

Nucleus Intracellular Ca2+ stores Ca 2+ ATPase Gap ATP TRPV 4 IP 3 R PG PLC IP3 Ca 2+ eNOS CaM Inhibit CCE Ca 2+ Activates NCCE ? Summary

Preeliminar results