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.
Patel
J M
JM
Research Service, Malcom Randall Department of Veterans Affairs Medical Center, University of Florida College of Medicine, Gainesville, Florida 32608, USA. Pateljm@medicine.ufl.edu
Li
Y D
YD
Zhang
J
J
Gelband
C H
CH
Raizada
M K
MK
Block
E R
ER
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism / Q000502:physiology; D037282:Calreticulin; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009994:Osmolar Concentration; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology*; D012261:Ribonucleoproteins / Q000378:metabolism*; D013552:Swine
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D037282:Calreticulin; D012261:Ribonucleoproteins; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium