Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS

This study demonstrates that ANG IV-induced activation of lung endothelial cell nitric oxide synthase (ecNOS) is mediated through mobilization of Ca2+ concentration and by increased expression and release of the Ca2+ binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In Ca2+-free medium and in the presence of the ANG II AT(1) and AT(2) receptor antagonists losartan and PD-123319 (1 mu M each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular Ca2+ release in PAEC (P < 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. ANG TV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG TV stimulation (P < 0.05). Catalytic activity of purified ecNOS in the absence of calmodulin was increased in a concentration dependent fashion by calreticulin. Immunocoprecipitation studies revealed that ecNOS and calreticulin were coprecipitated in ANG IV-stimulated PAEC. These results demonstrate that ANG IV-mediated activation of ecNOS is regulated by intracellular Ca2+ mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC.