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DOI: 10.1016/j.jsgi.2004.11.006 Molecular Cloning of Ovine Endothelial Nitric Oxide Synthase and Expression in COS-7 Cells
Department of Obstetncs and Gynecology and the Department of Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin
Department of Obstetncs and Gynecology and the Department of Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin; 202 S. Park St., 7E Meriter Hospital, Madison, WI 53715; imbird{at}facstaff.wisc.edu While studies of human and bovine endothelial nitric oxide synthase (eNOS) demonstrate activation by Ca2+ /calmodulin, recent progress demonstrates that eNOS phosphorylation can alter sensitivity to intracellular free calcium ([Ca2+]). The sheep, however, is widely used as a model for cardiovascular adaptation to pregnancy and ovine uterine artery endothelial cell (UAEC) eNOS undergoes pregnancy-specific (P) enhancement of activity associated with increased Ca2+ and protein kinase signaling in response to a number of agonists, including adenosine triphosphate (ATP). The degree of homology between the ovine and human full-length cDNAs was not previously known and yet is necessary to determine the validity in using an ovine model to study human physiology. The objectives of this study were to isolate and validate the clone of ovine eNOS cDNA and investigate ovine eNOS activation when expressed in COS-7 cells. The ovine eNOS cDNA has high homology to published human and bovine sequences and shares identity with the bovine amino acid sequence. When ovine eNOS was transiently expressed in COS-7 cells (COS-7/oeNOS), A23187 increased specfic catalytic activity in a dose- and time-dependent manner. A23187-stimulated activation of eNOS was, however, also accompanied by phosphorylation of eNOS S1179 and dephosphorylation of T497, demonstrating that an increase in [Ca2+]i may not be the sole mechanism of activation. The physiologic relevance of this was further underscored by the finding that ATP dose dependently increased peak [Ca2+]i and eNOS activity in COS-7/oeNOS, but also increased eNOS p-S1179 and decreased p-T497. This finding was similar to those in ovine P-UAEC treated with the Ca2+-mobilizing agonist ATP, wherein activation of eNOS was again concomitant with a rise p-S1179 as well as a slight decrease in p-T497. In conclusion, we describe the full-length ovine eNOS cDNA sequence and show that both physiologic and nonphysiologic calcium-mobilizing agents, which activate ovine eNOS in COS7 and PUAEC, do so in association with changes in eNOS phosphorylation. Given this information we can now begin to dissect the relationship between Ca2+ elevation and specflc phosphorylation events in eNOS activation in the ovine model, and thereby gain insight into the possible basis for pregnancy-related dysfunction.
Key Words: ERK1/2 • extracellular-signal regulated protein kinase • [Ca2+]i • intracellular free calcium concentration • calcium-activated calmodulin • uterine artery endothelial cells
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