Molecular Cloning of Ovine Endothelial Nitric Oxide Synthase and Expression in COS-7 Cells

Abstract

While studies of human and bovine endothelial nitric oxide synthase (eNOS) demonstrate activation by Ca(2+)/calmodulin, recent progress demonstrates that eNOS phosphorylation can alter sensitivity to intracellular free calcium ([Ca(2+)](i)). 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 Ca(2+) 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 specific 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 [Ca(2+)](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 [Ca(2+)](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 Ca(2+)-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 COS-7 and P-UAEC, do so in association with changes in eNOS phosphorylation. Given this information we can now begin to dissect the relationship between Ca(2+) elevation and specific phosphorylation events in eNOS activation in the ovine model, and thereby gain insight into the possible basis for pregnancy-related dysfunction.

Direct Effects of Nicotine on Contractility of the Uterine Artery in Pregnancy

“…Indeed, previous studies have demonstrated that nicotine and its metabolic product cotinine significantly decreased eNOS mRNA and protein levels, resulting in impaired endothelial-dependent relaxations in the carotid artery rings after a 24-h treatment (Conklin et al, 2001). In addition to decreased eNOS protein levels, chronic nicotine treatment significantly attenuated eNOS phosphorylation at the site of Ser1179 that is involved in eNOS activation (Cale et al, 2005;Mount et al, 2007), suggesting a decrease in the function of the eNOS enzyme. In summary, the present study demonstrates for the first time that chronic, but not acute, nicotine treatment has direct effects on contractility of the uterine artery in pregnancy and inhibits endothelium-dependent relaxations, resulting in an enhanced vasoconstriction of the pregnant uterine artery.…”

“…eNOS protein and phosphorylation levels were determined with Western blot analysis, as described previously (Cale et al, 2005;Xiao et al, 2007). Arteries were homogenized in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 10 mM EDTA, 0.1% Tween 20, 0.1% ␤-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, and 5 g/ml aprotinin, pH 7.4.…”

Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells

“…QuikChange Site-Directed Mutagenesis kit (Stratagene; La Jolla, CA) was used to mutate ovine eNOS cDNA (pBK-CMV) (8) Thr497 to Asp (T497D: sense oligo 5Ј-GCG CAG GCA TCA CCA GGA AGA AGG ACT TTA AGG AAG T-3Ј), Thr 497 to Ala (T497A: sense oligo 5Ј-GCG CAG GCA TCA CCA GGA AGA AGG CCT TTA AGG AAG T-3Ј), or Ser1179 to Ala (S1179A: sense oligo 5Ј-GCC GTA TAC GTA CCC AGG CCT TTT CCC TGC AGG AG-3Ј). In addition, the double mutant (T497D/ S1179A) was generated by subjecting T497D to a second round of mutagenesis with the S1179A primers.…”

“…In addition, we previously isolated the ovine eNOS cDNA and described a COS-7 ovine eNOS cell model (8) to which we now compare UAEC phosphorylation and activation of eNOS. Use of the COS-7 eNOS model also allowed us to further determine how a phosphomimetic mutation of T497 affects phosphorylation of the other known eNOS sites and to compare its activity to eNOS with two mutations predicted to be detrimental to activity (T497D/S1179A).…”

Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

“…PCR primer pairs were designed from different exons of NOS3, c-Jun, or L-19 to discriminate PCR products that might arise from possible chromosomal DNA contaminants. Specifically, they were derived from the cDNA clones at the following nucleotides positioned at: 2029-2049 and 2359-2379 for rat c-jun (Sakai et al, 1989), 409-430 and 685-704 for sheep NOS3 (Cale et al, 2005) and 38-56 and 370-388 for sheep L19 (GenBank A# AY158223). The internal controls for NOS3 and L-19 were 171-190 and 561-580 of the pGL3-basic vector from Promega (Madison, WI).…”