Contribution of epoxyeicosatrienoic acids to flow-induced dilation in arteries of male ERalpha knockout mice: role of aromatase.

We studied the roles of estrogen receptors (ER) and aromatase in the mediation of flow-induced dilation (FID) in isolated arteries of male ERalpha-knockout (ERalpha-KO) and wild-type (WT) mice. FID was comparable between gracilis arteries of WT and ERalpha-KO mice. In WT arteries, inhibition of NO and prostaglandins eliminated FID. In ERalpha-KO arteries, N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited FID by approximately 26%, whereas indomethacin inhibited dilations by approximately 50%. The remaining portion of the dilation was abolished by additional administration of 6-(2-proparglyoxyphenyl)hexanoic acid (PPOH) or iberiotoxin, inhibitors of epoxyeicosatrienoic acid (EET) synthesis and large-conductance potassium channels, respectively. By using an electrophysiological technique, we found that, in the presence of 10 dyne/cm(2) shear stress, perfusate passing through donor vessels isolated from gracilis muscle of ERalpha-KO mice subjected to L-NAME and indomethacin elicited smooth muscle hyperpolarization and a dilator response of endothelium-denuded detector vessels. These responses were prevented by the presence of iberiotoxin in detector or PPOH in donor vessels. Gas chromatography-mass spectrometry (GC-MS) analysis indicated a significant increase in arterial production of EETs in ERalpha-KO compared with WT mice. Western blot analysis showed a significantly reduced endothelial nitric oxide synthase expression but enhanced expressions of aromatase and ERbeta in ERalpha-KO arteries. Treatment of ERalpha-KO arteries with specific aromatase short-interfering RNA for 72 h, knocked down the aromatase mRNA and protein associated with elimination of EET-mediation of FID. Thus, FID in male ERalpha-KO arteries is maintained via an endothelium-derived hyperpolarizing factor/EET-mediated mechanism compensating for reduced NO mediation due, at least in part, to estrogen aromatized from testosterone.

Aging enhances pressure-induced arterial superoxide formation.

The purpose of this study was to investigate the mechanisms that regulate superoxide (O(2)(*-)) production as a function of an acute elevation of intravascular pressure and age. Mesenteric arteries isolated from young (6 mo) and aged (24 mo) male Fischer 344 rats were used. O(2)(*-) production in vessels in response to 80 (normal pressure, NP) and 180 (high pressure, HP) mmHg was determined by the superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction assay. In vessels exposed to NP, O(2)(*-) production was significantly higher in aged than in young vessels (32.7 +/- 7.0 vs. 15.4 +/- 2.4 nmol.mg(-1).30 min(-1)). HP enhanced O(2)(*-) production in vessels of both groups, but the enhancement was significantly greater in aged than in young vessels (63.4 +/- 6.7 vs. 32.7 +/- 4.3 nmol.mg(-1).30 min(-1)). Apocynin (100 micromol/l) attenuated HP-induced increases in O(2)(*-) production in both groups, whereas allopurinol (100 micromol/l) and N(omega)-nitro-L-arginine methyl ester (100 mumol/l) inhibited the response only in aged vessels. Confocal microscopy showed increases in O(2)(*-) in response to HP in endothelial and smooth muscle layers of both groups, with much greater fluorescent staining in aged than in young rats and in the endothelium than in smooth muscle cells. No significant changes in NAD(P)H oxidase gene and protein expressions were observed in vessels of the two groups. Upregulation of protein expression of xanthine oxidase was detected in aged vessels. We conclude that NAD(P)H oxidase contributes importantly to HP-induced enhanced O(2)(*-) production in vessels of both young and aged rats, whereas xanthine oxidase and nitric oxide synthase-dependent O(2)(*-) production also contribute to the enhancement in mesenteric arteries of aged rats.

COX-2 contributes to the maintenance of flow-induced dilation in arterioles of eNOS-knockout mice.

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 microM), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by approximately 51% and approximately 58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by approximately 57%. The residual portion of the response was abolished by additional administration of Nomega-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice.

Epoxyeicosatrienoic acids are released to mediate shear stress-dependent hyperpolarization of arteriolar smooth muscle.

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/cm2 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/cm2 shear stress-stimulated donor vessels was also recorded (by approximately -12 to -15 and -20 to -30 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/cm2 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 endothelium-derived hyperpolarizing factors and suggesting that gap junctional communication may not be necessary for shear stress-stimulated EDHF-mediated vasodilation.