Barber, Dustan A.

Experiments were designed to characterize endothelin receptors in arteries after chronic increases in blood flow. A fistula was created between the femoral artery and vein in one hindlimb of dogs; contralateral blood vessels were sham operated. Sham- and fistula-operated arteries were removed 6 wk postoperatively. Some arteries were prepared for measurement of isometric force or for isolation of membrane proteins. Other arteries were used for histological staining with an endothelin-B (ETB) receptor antibody. In arteries suspended for the measurement of isometric force, endothelin-1 produced concentration-dependent increases in tension that were significantly greater in fistula- than in sham-operated arteries without endothelium. The ETB-receptor-selective peptide sarafotoxin S6c produced concentration-dependent increases in tension only in fistula-operated arteries. In receptor-binding studies of membrane proteins, Scatchard analysis of saturation binding with 125I-labeled endothelin-1 (125I-endothelin-1) indicated that the total number of receptors was greater in fistula-operated arteries; affinity was threefold less in fistula- than in sham-operated arteries. Competitive displacement of 125I-endothelin-1 by endothelin-3 was significant for a two-site model in membranes prepared from sham-and fistula-operated arteries. Competitive inhibition of 125I-endothelin-1 binding by sarafotoxin S6c was significant for a one-site binding model in all arteries. Sarafotoxin S6c binding sites were elevated significantly in fistula-operated arteries. Immunohistochemical staining for the ETB receptor was significantly greater in both the endothelium and smooth muscle of fistula- than in sham-operated arteries. These results suggest that chronic increases in blood flow upregulate endothelin receptors, including ETB receptors in arterial smooth muscle.

Experiments were designed to determine whether endogenous physiological fluctuations in sex steroid hormones affect expression or functional responses of endothelin receptors. Coronary arteries from sexually mature male, female, and ovariectomized pigs were prepared either for receptor binding or measurement of isometric force in organ chambers. Competitive binding of 125I-labeled endothelin-1 was significant for a one-site model with unlabeled endothelin-1 and a two-site model with unlabeled endothelin-3 and sarafotoxin S6c in all pigs. The total number of binding sites for all endothelin ligands was not different between male and female pigs. Binding affinities for the high-affinity binding site for both endothelin-3 and sarafotoxin S6c were significantly greater (lower inhibition constant) in membranes prepared from female pigs with high endogenous estrogen. In organ chamber experiments, contractions to endothelin-1 but not endothelin-3 or sarafotoxin S6c were significantly greater in coronary arterial rings from female compared with male pigs and were not affected significantly by removal of the endothelium or by treatment of the rings with either indomethacin (10(-5) mol/l) or the combination of indomethacin and NG-monomethyl-L-arginine (10(-4) mol/l). These results suggest endogenous fluctuations in estrogen are associated with an increase in affinity of a high-affinity endothelin receptor in coronary arterial smooth muscle of female pigs. In addition, independent of endogenous estrogen status, coronary arteries from female pigs generate significantly greater contractions to endothelin-1 compared with male pigs. This phenomenon occurs at the level of smooth muscle and is not dependent on the endothelium or synthesis of nitric oxide or prostaglandins.

Experiments were designed to determine whether normal fluctuations in endogenous sex steroid hormones and/or gender affect endothelium-dependent relaxations in coronary arteries, and, if so, to identify endothelium-derived factors contributing to these differences. Coronary arteries from sexually mature, gonadally intact male and female pigs or ovariectomized pigs were prepared either for study of isometric force in organ chambers or for measurement of prostanoids and activity of nitric oxide (NO) synthase. In organ chamber studies, neither the magnitude nor the sensitivity of endothelium-dependent relaxations correlated with endogenous estrogen or progesterone in female pigs. However, relaxations to bradykinin and UK-14304 were significantly greater and/or shifted leftward in arterial rings from female compared with male pigs. Indomethacin (10−5mol/l) increased endothelium-dependent relaxations only in arteries from male pigs. NG-monomethyl-l-arginine reduced endothelium-dependent relaxations to a similar extent in coronary arteries from either sex. Neither production nor response to thromboxane A2or prostacyclin differed in coronary arteries from male compared with female pigs. Activity for calcium-dependent or -independent NO synthase was similar in both sexes. These results suggest that normal fluctuations in endogenous sex steroid hormones do not affect endothelium-dependent relaxations in coronary arteries from female pigs. There are, however, gender differences in endothelium-dependent relaxations that are indomethacin sensitive and may be due to cyclooxygenase products other than thromboxane A2or prostacyclin.

Experiments were designed to determine whether nitric oxide was the mediator of increased endothelium-dependent relaxations in veins proximal to an arteriovenous fistula. A fistula was prepared between femoral arteries and veins in dogs. After 6 wk, veins proximal to the fistula were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers. In some rings the endothelium was removed deliberately. NG-monomethyl-L-arginine (L-NMMA) caused contraction in three of six fistula-operated veins with and without endothelium. In rings contracted submaximally with prostaglandin F2 alpha, acetylcholine and the alpha 2-adrenergic agonist UK-14,304 cause e tylcholine and the alpha 2-adrenergic agonist UK-14,304 caused endothelium-dependent, concentration-dependent relaxations that were greater in fistula compared with sham-operated veins. These relaxations were reduced by L-NMMA. Calcium ionophore A23187 caused comparable endothelium-dependent relaxations in fistula- and sham-operated veins that were unaffected by L-NMMA. There were no differences in either calcium-dependent or -independent activity of nitric oxide synthase isolated from fistula- and sham-operated veins. Positive staining for nitric oxide synthase was present in both the endothelium and media of fistula-operated veins. These results indicate that nitric oxide mediates increased endothelium-dependent relaxations to acetylcholine and alpha 2-adrenergic agonists in fistula-operated veins. Therefore, chronic increases in blood flow and oxygen tension modify selectively receptor-coupled production of nitric oxide in endothelium and smooth muscle of veins.

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

Background Adventitial gene transfer may serve as a tool to study vascular biology and may have therapeutic potential. We investigated the hypothesis that adenovirus-mediated transfer of the gene for endothelial nitric oxide synthase (eNOS) to the adventitia would alter vascular reactivity. Methods and Results Rabbit carotid arteries were surgically isolated and adenoviral vectors encoding eNOS (AdeNOS) or β-galactosidase instilled into the periarterial sheath at a concentration of 1×10 10 pfu/mL. Arteries were harvested 4 days later for immunostaining, NOS enzymatic assay, measurement of cGMP, and vasomotor studies. Transgene expression in the adventitia was confirmed by histochemistry for β-galactosidase and immunostaining for eNOS with a monoclonal antibody. Calcium-dependent NOS enzymatic activity and cGMP levels were significantly greater in the AdeNOS-transduced arteries. Maximal contractions to phenylephrine (10 −5 mol/L) were diminished in the AdeNOS-transduced arteries (4.6±0.2 versus 5.6±0.2 g; P <.05), but in the presence of the eNOS inhibitor N G -monomethyl- l -arginine (3×10 −4 mol/L) there was no difference between the two groups (7.1±0.2 versus 7.5±0.3 g; P =NS). Relaxations to calcium ionophore obtained during submaximal contractions to phenylephrine were significantly enhanced in the AdeNOS-transduced arteries (−log EC 50 , 7.77±0.08 versus 7.45±0.07; P <.02). Conclusions We conclude that eNOS gene transfer to the adventitia alters vascular reactivity, as demonstrated by diminished contractile responses to phenylephrine and enhanced relaxations to calcium ionophore. This may represent a therapeutic strategy for vascular diseases characterized by decreased bioavailability of NO.