Functional and molecular characterization of nitric oxide synthase in the endometrium and myometrium of pregnant sheep during the last third of gestation

Abstract

Objective: This study was undertaken to characterize the biochemical and expression profiles of the nitric oxide synthase isoforms present in the sheep uterus during late gestation. Study Design: Myometrium and endometrium were obtained from 28 time-mated pregnant sheep that were under halothane general anesthesia. Tissues were kept frozen at –80°C until they were homogenized for the measurement of (1) nitric oxide synthase activity according to the carbon 14–labeled arginine-citrulline conversion assay, (2) nitric oxide synthase protein mass according to Western blot analysis, and (3) nitric oxide synthase messenger ribonucleic acid according to the ribonuclease protection assay. The nitric oxide synthase activity assay included 8 parallel treatments for biochemical characterization, in particular with the arginase inhibitors ornithine and (+)-S-2-amino-5-iodoacetamidopentanoic acid. Results: The biochemical characterization of nitric oxide synthase indicated that the predominant form of nitric oxide synthase in endometrium and myometrium (80%-90%) was calcium-calmodulin dependent. In endometrium 50% of reduced nicotinamide adenine dinucleotide–dependent arginine metabolism was accounted for by the presence of alternative arginine metabolic pathways. Expressions of type 1 and type 3 nitric oxide synthase were demonstrated in endometrium and myometrium by Western blot and ribonuclease protection assay. Although no significant decrease in nitric oxide synthase activity or protein mass was observed, a significant decrease in myometrial type 1 nitric oxide synthase messenger ribonucleic acid occurred in sheep not in labor at 140 days’ gestation ( P < .05 by analysis of variance; term is 144 ± 5 days). Conclusion: In the gravid sheep uterus the predominant nitric oxide synthase isoforms are type 1 in myometrium and type 3 in endometrium. Despite a decrease in type 1 nitric oxide synthase messenger ribonucleic acid, enzymatic activity and type 1 nitric oxide synthase protein mass do not decrease before parturition. (Am J Obstet Gynecol 1999;181:116-25.)