Abstract-Arachidonic acid metabolites contribute to the endothelin-1 (ET-1)-induced decrease in renal blood flow, but the vascular sites of action are unknown. Experiments performed in vitro used the rat juxtamedullary nephron preparation combined with videomicroscopy. The response of afferent arterioles to ET-1 was determined before and after cytochrome P450 (CYP450) or cyclooxygenase (COX) inhibition. Afferent arteriolar diameter averaged 20Ϯ1 m (nϭ17) at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.001 to 10 nmol/L ET-1 caused a graded decrease in diameter of the afferent arteriole. Vessel diameter decreased by 30Ϯ2% and 41Ϯ2% in response to 1 and 10 nmol/L ET-1, respectively. The afferent arteriolar response to ET-1 was significantly attenuated during administration of the CYP450 hydroxylase inhibitor , such that afferent arteriolar diameter decreased by 19Ϯ3% and 22Ϯ3% in response to 1 and 10 nmol/L ET-1, respectively. COX inhibition also greatly attenuated the vasoconstriction elicited by ET-1, whereas the CYP450 epoxygenase inhibitor N-methylsulfonyl-6-(2-proparglyoxyphenyl) hexanamide enhanced the ET-1-mediated vascular response. Additional studies were performed using freshly isolated smooth muscle cells prepared from preglomerular microvessels. Renal microvascular smooth muscle cells were loaded with the calcium-sensitive dye fura 2 and studied by use of single-cell fluorescence microscopy. Basal renal microvascular smooth muscle cell [Ca 2ϩ ] i averaged 95Ϯ3 nmol/L (nϭ42). ET-1 (10 nmol/L) increased microvascular smooth muscle cell [Ca 2ϩ ] i to a peak value of 731Ϯ75 nmol/L before stabilizing at 136Ϯ8 nmol/L. Administration of DDMS or the COX inhibitor indomethacin significantly attenuated the renal microvascular smooth muscle cell calcium response to ET-1. These data demonstrate that CYP450 hydroxylase and COX arachidonic acid metabolites contribute importantly to the afferent arteriolar diameter and renal microvascular smooth muscle cell calcium responses elicited by ET-1. Key Words: endothelin-1 Ⅲ renal hemodynamics Ⅲ cytochrome P450 Ⅲ cyclooxygenase Ⅲ cytosolic calcium Ⅲ microcirculation E ndothelin-1 (ET-1) is a 21-amino-acid peptide synthesized by endothelial cells that acts as a potent vasoconstrictor on vascular smooth muscle cells. [1][2][3] Elevations in circulating and tissue ET-1 levels have been implicated in a number of pathological states, including acute renal failure, cyclosporine nephrotoxicity, and hypertension. 4 -6 The ET-1 effects on the renal circulation are consistent with the view that the peptide plays a crucial role in maintaining fluid and electrolyte homeostasis. Intrarenal infusion of ET-1 decreases renal blood flow and glomerular filtration rate while increasing sodium excretion. 3,7,8 Previous studies have demonstrated that ET-1 constricts the afferent arteriole 9 -13 ; however, the preglomerular vascular smooth muscle cellular signaling mechanisms responsible are not well understood. ET-1 activates G proteins that in turn stimulate phospholipase C...

Abstract-The current studies were performed to determine the contribution of calcium mobilization and voltage-dependent calcium influx to the increase in [Ca 2ϩ ] i elicited by ATP and UTP. Suspensions of freshly isolated smooth muscle cells were prepared from preglomerular microvessels by enzymatic digestion and loaded with the Ca 2ϩ -sensitive dye fura 2. ] i response to ATP and UTP was also assessed in the absence of extracellular calcium. In these studies, exposure to 100 mol/L ATP induced a transient peak increase in [Ca 2ϩ ] i , with the plateau phase being totally abolished. In contrast, exposure to 100 mol/L UTP under calcium-free conditions resulted in no detectable change in the UTP-mediated increase in [Ca 2ϩ ] i . The role of L-type calcium channels in the response was assessed with the calcium channel antagonist diltiazem. Incubation with diltiazem (10 mol/L) markedly reduced the response to ATP, whereas the response to UTP was only slightly reduced. These data demonstrate that both ATP and UTP directly stimulate a biphasic increase in [Ca 2ϩ ] i in renal microvascular smooth muscle cells. Furthermore, the data suggest that the elevation of [Ca 2ϩ ] i elicited by ATP is largely dependent on calcium influx through L-type calcium channels, whereas the response to UTP appears to derive primarily from mobilization of calcium from intracellular stores. The mechanism of this vasoconstriction is at least partially dependent on the activation of voltage-gated calcium channels. 1 Subsequently, it was shown that P2 receptor inactivation or blockade significantly attenuated pressure-mediated afferent arteriolar vasoconstrictor responses. 5 This observation led to the postulate that activation of P2 receptors plays an important role in mediating renal microvascular autoregulatory responses; however, the specific P2 receptor subtype involved remains to be determined.Recently, it was reported that P2 receptor-mediated renal vasoconstriction can be elicited by several different purine-or pyrimidine-based compounds that exhibit varying selectivity for different P2 receptor subtypes. 5,6 ATP and UTP evoke strikingly different afferent arteriolar vasoconstrictor responses; thus, suggesting the involvement of P2X (ATP) and P2Y (UTP) purinoceptor subtypes. 5 P2X and P2Y purinoceptors are structurally different receptor proteins that use different signal transduction pathways 7-13 ; however, the signal transduction cascade for each has not been thoroughly investigated in the renal microcirculation. Clarification of the calcium signaling pathways accessed by ATP and UTP is important to advancing our understanding of the mechanisms by which P2 receptors influence renal microvascular tone and may provide insight into the specific P2 receptor subtype or subtypes involved in the autoregulatory response.We have recently established the methods needed for obtaining viable vascular smooth muscle cells from freshly isolated preglomerular microvascular tissue. 14 We used this preparation to determine the effect of ATP a...

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.