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

Abstract

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...

Contribution of prostaglandin EP2receptors to renal microvascular reactivity in mice

“…Along these lines, Oyekan and McGiff (28) demonstrated that the endothelin-1-evoked decreases in renal blood flow and glomerular filtration were enhanced by COX inhibition. In contrast, indomethacin attenuated the increase in renal vascular resistance, the afferent arteriolar decrease in diameter, and renal microvascular smooth muscle cell calcium response to endothelin-1 (18,27). The results of the present study also suggest involvement of COXderived vasodilator and vasoconstrictor metabolites in the afferent arteriolar response to endothelin-1.…”

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid ω-hydroxylase activity

“…However, these results are confounded because heme oxygenase inducers also stimulate the formation of the vasodilatory gas carbon monoxide and may influence nitric oxide synthase activity and produce vasodilatory factors via the cGMP pathway (35). Dibromo-olefinic fatty acids are relatively specific inhibitors of arachidonic acid -/( -1)-hydroxylation and have been extensively used to characterize 20-HETE biological effects in vitro and in situ (17,21,46,54). The most potent inhibitor of arachidonic acid -hydroxylation to date is N-hydroxy-NЈ-(4-butyl-2-methylphenyl)-formamidine (HET-0016) (33).…”

Increased Excretion of Urinary 20-HETE in Rats With Cyclosporine-Induced Nephrotoxicity

“…(23). Endothelin-1 (ET-1) also stimulates the formation of 20-HETE and 20-HETE contributes to the renal vasoconstrictor response to this agonist as well (21,22). Thus, the present study examined that the changes in urinary excretion of 20-HETE and immunohistochemistry of CYP4A protein in kidneys from rats with CsA-induced nephrotoxicity because elevations in renal ANG II (29 -31) and ET-1 levels (24, 31) have been implicated in mediating the decline in renal function in this model.…”

“…20-HETE is also a potent constrictor of renal arterioles in rats (17,18) and dogs (19) and the vasoconstrictor response to 20-HETE is mediated by the blockade of large conductance Ca 2+ -activated K + -channels (19,20). Recent studies have also indicated that the production of 20-HETE is stimulated by ET-1 (21,22) and ANG II (23 -25) and that the elevation in the levels of 20-HETE in vascular smooth muscle cells potentiates the response to these vaso-constrictors in the renal vasculature. There is one report that CsA increases the expression of CYP4A enzymes and the omega-hydroxylation of fatty acids in the kidney of rats (26).…”