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Evidence against the Hypothesis that Endothelial Endothelin B Receptor Expression Is Regulated by Relaxin and Pregnancy

Departments of Obstetrics, Gynecology, and Reproductive Sciences (L.J.K., J.N., K.H.-Y., K.D.D., K.P.C.) and Cell Biology and Physiology (K.P.C.), University of Pittsburgh School of Medicine and Magee Womens Research Institute, Pittsburgh, Pennsylvania 15213; and Department of Pathology (L.A.D.), University of New Mexico School of Medicine, Albuquerque, New Mexico 87131

Address all correspondence and requests for reprints to: Kirk P. Conrad, M.D., Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213. E-mail: rsikpc{at}mwri.magee.edu.

The endothelial endothelin B (ET_B) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ET_B receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 µg/h or vehicle for 5 d or 4–6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ET_B expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ET_B protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET_B expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET_B expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ET_B receptor protein has little or no role in relaxin stimulation of the endothelial ET_B/nitric oxide vasodilatory pathway.

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