Constitutive expression of arginase in microvascular endothelial cells counteracts nitric oxide‐mediated vasodilatory function

Abstract

SPECIFIC AIMS Arginase shares a common substrate, L-arginine, with nitric oxide synthase (NOS). It is speculated that argi-nase, if it is expressed in the endothelium, might play a pivotal role in the regulation of NO-mediated vasodila-tion by reducing the L-arginine availability to NOS. In the present study, we tried to determine whether arginase is expressed and active in coronary arterioles and to demonstrate whether endothelial arginase can influence NO production and play a functional role in regulating NO-mediated dilation of coronary microves-sels. We determined the expression and cellular local-ization of arginase in porcine coronary microvessels (50 –200 ␮m) by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and studied NO production and vasodilation of isolated coronary arterioles (60 –110 ␮m) with and without arginase inhibition. PRINCIPAL FINDINGS 1. Arginase mRNA and protein are constitutively expressed in coronary arterioles We determined whether arginase mRNA is present in the isolated coronary arterioles using RT-PCR. Argi-nase I (131 bp), but not arginase II (360 bp), transcript was detected in coronary arterioles after PCR (Fig. 1). Positive controls for the arginase I and II transcripts were revealed in liver and kidney tis-sues, respectively. To determine the cellular localization of arginase I in the coronary arteriole, vessels were subjected to immu-nohistochemical study. The background level of stain-ing was determined in an isolated vessel treated with the nonimmune mouse serum, secondary antibody, and fluorescein avidin D. In the presence of anti-arginase I monoclonal antibody, high levels of immu-nostaining, as represented by a pseudo-color spectral display, were detected in both the endothelial and smooth muscle cells. 2. Arginase inhibitor ␣-difluoromethylornithine (DFMO) specifically potentiates endothelium-dependent, NO-mediated coronary arteriolar dilation