Endothelin-Mediated Calcium Signaling in Preglomerular Smooth Muscle Cells

Abstract

Abstract-This study was performed to test the hypothesis that endothelin peptides differentially influence intracellular calcium concentration ([Ca 2ϩ ] i ) in preglomerular microvascular smooth muscle cells (MVSMC), in part through activation of endothelin (ET) A receptors. Experiments were performed in vitro with the use of single MVSMC freshly isolated from rat preglomerular microvessels. The effect of ET-1, ET-2, and ET-3 on [Ca 2ϩ ] i was measured with the use of the calcium-sensitive dye, fura 2, and standard fluorescence microscopy techniques. Baseline [Ca 2ϩ ] i averaged 84Ϯ3 nmol/L (nϭ141 cells from 23 dispersions). ET-1 concentrations of 1, 10, and 100 nmol/L evoked peak increases in [Ca 2ϩ ] i of 48Ϯ16, 930Ϯ125, and 810Ϯ130 nmol/L, respectively. The time course of the [Ca 2ϩ ] i response was biphasic, beginning with a rapid initial increase followed by a sustained plateau phase or a period during which [Ca 2ϩ ] i oscillated sharply. Similar responses were observed after ET-2 administration. In contrast, ET-3 stimulated monophasic increases in [Ca 2ϩ ] i of only 14Ϯ5, 33Ϯ16, and 44Ϯ19 nmol/L at peptide concentrations of 1, 10, and 100 nmol/L, respectively. These responses are significantly smaller than responses to ET-1 or ET-2, respectively. The relative contributions of calcium mobilization and calcium influx in the response to ET-1 were also evaluated. Removal of calcium from the bathing medium did not significantly alter the peak response to 10 nmol/L ET- influx is postulated to increase smooth muscle contractility and may contribute to the prolonged vasoconstriction commonly associated with endothelin-mediated responses. Initially, the renal vasoconstrictor actions of ET-1 were thought to involve ET A receptors exclusively, with ET B receptors producing vasodilation by stimulating nitric oxide release from endothelial cells. However, the development of selective ET A receptor antagonists revealed that non-ET A receptors contribute some of the ET-1-mediated vasoconstriction. ET B receptor-mediated vasoconstriction has been demonstrated in vivo in the renal circulation of the rat. 5,6 Endothelin receptors have been identified in the kidney, and reports suggest that rat preglomerular microvessels express both ET A and ET B receptors 7,8 ; however, there are only limited data demonstrating the functional distribution of ET A and ET B receptors along preglomerular resistance vessels. 6 Our laboratory has recently established the methods needed for obtaining viable vascular smooth muscle cells from freshly isolated preglomerular microvascular tissue. 9 We used this preparation to determine the effect of receptor selective endothelin peptides on [Ca 2ϩ ] i in preglomerular vascular smooth muscle cells. Additional studies were performed to determine the relative contribution of calcium influx and calcium mobilization on the increase in [Ca 2ϩ ] i induced by endothelin.

Endothelin B receptor Ca2+ signaling in shark vascular smooth muscle: participation of inositol trisphosphate and ryanodine receptors

“…The [Ca 2+ ]i response of shark anterior mesenteric artery VSM to ETBR stimulation in Ca 2+ -free Ringer was characterized by a prolonged peak of approximately 100·s. This contrasts with the short duration spike (<25·s) typical of the responses seen in mammalian VSM (Schroeder et al, 2000). The current studies were performed intentionally at 20-21°C, because shark cells are accustomed to ambient temperatures of 13-17°C in the sea, and our experience is that they lose viability at temperatures above 25°C.…”

Cytochrome P450 and Cyclooxygenase Metabolites Contribute to the Endothelin-1 Afferent Arteriolar Vasoconstrictor and Calcium Responses

“…A major product of the CYP450 hydroxylase pathway is the endogenous renal microvascular vasoconstrictor metabolite 20-HETE. 26 30 In addition, the peak responses to ET-1 are generated primarily by mobilization of Ca 2ϩ from intracellular stores, and the sustained elevations in [Ca 2ϩ ] i are the result of calcium influx. 30 CYP450 hydroxylase inhibition significantly attenuated the peak response at the lower doses and at higher doses of ET-1 diminished the sustained [Ca 2ϩ ] i phase, which supports the concept that 20-HETE produced by the vascular smooth muscle cell contributes to ET-1-evoked preglomerular vasoconstriction.…”

The Vascular Endothelin System in Hypertension - Recent Patents and Discoveries

“…Also, ET-1, -2 and -3 could induce vascular contraction via activation of VSM ET B2 receptors [64]. For example, both ET A and ET B receptors are present and produce vasoconstriction in the renal circulation [65]. Also, studies using selective ET receptor agonists and antagonists have suggested the presence of the constrictor ET B receptors in human subcutaneous and rat mesenteric arteries, albeit in low numbers.…”

“…Vascular contraction to ET-1 is increased in rat hearts during ischemia/ reperfusion and in the rat pulmonary circulation in pulmonary hypertension [46]. Evidence indicates that a defect in VSM regulation of intracellular Ca 2+ may play a role in the augmented vascular reactivity to ET-1 in some forms of experimental hypertension [65,79]. However, vascular contraction to ET is unchanged in the aorta of SHR and is even decreased in the mesenteric arteries of DOCA-salt hypertensive rats [46,64].…”