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 Ca(2+) concentration and by increased expression and release of the Ca(2+) binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In Ca(2+)-free medium and in the presence of the ANG II AT(1) and AT(2) receptor antagonists losartan and PD-123319 (1 microM each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular Ca(2+) release in PAEC (P < 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. ANG IV 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 IV 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 Ca(2+) mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC.