Endothelin A and B receptors of preglomerular vascular smooth muscle cells

Background. The endothelin (ET) receptors are subclassified into ETA. which are purely vasoconstrictive, and ETB. The ETB receptors may cause either vasodilation by stimulating the release of nitric oxide from endothelial cells, or vasoconstriction of vascular smooth muscle cells (VSMC). The relative contribution of ETA and ETB receptors to calcium signaling and vasoconstriction in the renal microcirculation is not clear. Our goal was to study the cytosolic calcium concentration ([Ca2+](i)) responses of fresh rat preglomerular VSMC and afferent arterioles to agonists and antagonists of ETA and ETB receptors in rats. Methods. Fresh VSMC and afferent arterioles were isolated using the magnetized microsphere/sieving technique, followed by gentle collagenase digestion. [Ca2+](i) was measured with fura-2 ratiometric fluorescence. Results. Afferent arterioles and VSMC responded to ET-1 stimulation with a rapid peak increase in [Ca2+](i) (Delta = 287 +/- 81 and 342 +/- 55 nmol/L, respectively). The ETB receptor agonist IRL 1620 stimulated a rise in [Ca2+](i) in afferent arterioles (106 +/- 35 nmol/L) subsequent addition of ET-1 at the IRL 1620 to stimulate ETA receptors caused a second peak that was twice as large (213 44 nmol/L). In VSMC, the ETB agonist peak increase was 99 12 nmol/L; addition of ET-1 then increased [Ca2+](i) by 294 +/- 23 nmol/L. The ETB inhibitor BQ-788 prevented stimulation of [Ca2+](i) by IRL 1620 in afferent arterioles and VSMC: subsequent stimulation of ETA receptors with ET-1 caused an increase in [Ca2+](i) (239 +/- 17 and 248 +/- 22 nmol/L). Pretreatment with the selective ETA inhibitor PD 156707 attenuated but did not abolish the responses to ET-1. suggesting that the residual [Ca2+](i) response was caused by ETB stimulation. Conclusion. These results indicate that fresh preglomerular VSMC as well as afferent arterioles have both ETA and ETB receptors, and that the rapid peak [Ca2+](i) responses to the ETB agonist IRL 1620 are less than half that of subsequent stimulation of ETA receptors with ET-1. The similarity of findings in isolated VSMC and afferent arterioles Suggests that responses in VSMC in our arteriolar preparation overshadow any potential contribution of endothelial cells when reagents are administered abluminally.