Vasodilation to increases in flow was studied in isolated gracilis muscle arterioles of female endothelial nitric oxide synthase (eNOS)-knockout (KO) and female wild-type (WT) mice. Dilation to flow (0-10 microl/min) was similar in the two groups, yet calculated wall shear stress was significantly greater in arterioles of eNOS-KO than in arterioles of WT mice. Indomethacin, which inhibited flow-induced dilation in vessels of WT mice by approximately 40%, did not affect the responses of eNOS-KO mice, whereas miconazole and 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) abolished the responses. Basal release of epoxyeicosatrienonic acids from arterioles was inhibited by PPOH. Iberiotoxin eliminated flow-induced dilation in arterioles of eNOS-KO mice but had no effect on arterioles of WT mice. In WT mice, neither N(omega)-nitro-L-arginine methyl ester nor miconazole alone affected flow-induced dilation. Combination of both inhibitors inhibited the responses by approximately 50%. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) alone inhibited flow-induced dilation by approximately 49%. ODQ + indomethacin eliminated the responses. Thus, in arterioles of female WT mice, nitric oxide and prostaglandins mediate flow-induced dilation. When eNOS is inhibited, endothelium-derived hyperpolarizing factor substitutes for nitric oxide. In female eNOS-KO mice, metabolites of cytochrome P-450, via activation of large-conductance Ca2+-activated K+ channels of smooth muscle, mediate entirely the arteriolar dilation to flow.

The function of the nephron, the anatomical unit of the kidney, is segmented; at least 12 segments have been identified that differ in their morphology, transport properties and hormonal responsiveness. The medullary portion of the thick ascending limb of the loop of Henle (mTALH) has one of the highest concentrations of (Na+ + K+)ATPase found in mammalian tissues, reflecting the importance of this nephron segment in the regulation of extracellular fluid volume, as active sodium transport is driven by (Na+ + K+)ATPase. Here, in cells derived primarily from the mTALH of the outer medulla of rabbit kidney, we have identified a cytochrome P450-dependent monooxygenase system which metabolizes arachidonic acid to two biologically active oxygenated products; one of the products inhibits (Na+ + K+)ATPase and the other relaxes blood vessels. We report that formation of these oxygenated arachidonate metabolites is stimulated by arginine vasopressin (AVP) and salmon calcitonin (SCT).

We studied hydroxyeicosatetraenoic acid (HETE) release in response to ANG II from preglomerular microvessels (PGMVs), the vascular segment governing changes in renal vascular resistance. PGMVs were isolated from Sprague-Dawley rats and incubated with NADPH and hormones at 37 degrees C. Eicosanoids were extracted, and cytochrome P-450 (CYP)-derived HETEs were purified and quantitated by negative chemical ionization gas chromatography-mass spectroscopy. PGMVs produced primarily 20- and 19-HETEs, namely, 7.9 +/- 1.7 and 2.2 +/- 0.5 ng/mg protein, respectively. ANG II (5 nM) increased CYP-HETE release by two- to threefold; bradykinin, phenylephrine, and Ca(2+) ionophore were without effect. [Sar(1)]ANG II (0.1-100 microM) dose dependently stimulated 19- and 20-HETEs, an effect blocked by the AT(2)-receptor antagonist PD-123319 as well as by U-73122, a phospholipase C inhibitor. Microvascular 20-HETE release was increased more than twofold by the third day in response to ANG II (120 ng. kg(-1). min(-1)) infused subcutaneously for 2 wk; it was not further enhanced after 14 days, although blood pressure continued to rise. Thus an AT(2)-phospholipse C effector unit is associated with synthesis of a vasoconstrictor product, 20-HETE, in a key renovascular segment.

Abstract-We hypothesized that shear stress stimulates the release of epoxyeicosatrienoic acids (EETs) from arteriolar endothelium, which directly hyperpolarize smooth muscle. To test this hypothesis, a perfusion system, consisting of two separate, serially connected chambers (A and B), was used. A donor vessel, isolated from gracilis muscle of female NO-deficient mice and rats, was cannulated in chamber A. In chamber B, an endothelium-denuded detector vessel isolated from mesentery of these animals was cannulated. In the presence of indomethacin, 5, 10, and 20 dyne/cm 2 shear stress elicited dilation of donor vessels, followed by dilation of detector vessels. Changes in membrane potential of the detector vessel smooth muscle cells in response to the perfusate from 5 and 10 dyne/cm 2 shear stress-stimulated donor vessels was also recorded (by ϷϪ12 to Ϫ15 and Ϫ20 to Ϫ30 mV, respectively). Exposing detector vessels to 30 mmol/L KCl or pretreating them with iberiotoxin abolished their hyperpolarization and dilation to the flow of perfusate. Pretreatment of donor vessels with PPOH, an inhibitor of cytochrome P-450/epoxygenase, eliminated dilator responses in both donor and detector vessels, as well as the hyperpolarization of detector vessels. GC-MS analysis showed increasing release of EETs into the perfusate collected from 1, 5, and 10 dyne/cm 2 shear stress-stimulated arterioles, which was abolished by PPOH. Thus, EETs, released from endothelial cells of donor vessels stimulated with shear stress, hyperpolarize smooth muscle of downstream detector vessels, confirming their identity as endotheliumderived hyperpolarizing factors and suggesting that gap junctional communication may not be necessary for shear stress-stimulated EDHF-mediated vasodilation. Key Words: NO deficiency Ⅲ shear stress Ⅲ EET Ⅲ hyperpolarization Ⅲ arterioles A lthough the chemical identity of endothelium-derived hyperpolarizing factor (EDHF) remains controversial, epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acids by cytochrome P450 (CYP)/epoxygenase, have been identified as a potential EDHF in a variety of vascular beds, including coronary, cerebral, renal, skeletal muscle vasculature, and human forearm and subcutaneous microvessels. 1 However, some studies that question the existence of EDHF suggest that electrical coupling of endothelial and smooth muscle cells through gap junctions mediates the activity. The idea that EDHF/EETs activate potassium channels in endothelial cells, leading to either the release of K ϩ , 2-4 or spread of current from endothelial cells, [5][6][7][8][9] to hyperpolarize smooth muscle, has attracted attention, because histological evidence of the existence of gap junctions between endothelial and smooth muscle cells and between endothelial cells was provided. 10,11 It was also reported that hyperpolarization of endothelial cells by injection of current or administration of ACh was conducted downstream to cause dilation of arterioles through endothelial gap junctions, 12,13 a response that was inhibit...