Endothelium-derived hyperpolarizing factor (EDHF) is released in response to agonists such as ACh and bradykinin and regulates vascular smooth muscle tone. Several studies have indicated that ouabain blocks agonist-induced, endothelium-dependent hyperpolarization of smooth muscle. We have demonstrated that epoxyeicosatrienoic acids (EETs), cytochrome P-450 metabolites of arachidonic acid, function as EDHFs. To further test the hypothesis that EETs represent EDHFs, we have examined the effects of ouabain on the electrical and mechanical effects of 14,15- and 11,12-EET in bovine coronary arteries. These arteries are relaxed in a concentration-dependent manner to 14,15- and 11,12-EET (EC(50) = 6 x 10(-7) M), bradykinin (EC(50) = 1 x 10(-9) M), sodium nitroprusside (SNP; EC(50) = 2 x 10(-7) M), and bimakalim (BMK; EC(50) = 1 x 10(-7) M). 11,12-EET-induced relaxations were identical in vessels with and without an endothelium. Potassium chloride (1-15 x 10(-3) M) inhibited [(3)H]ouabain binding to smooth muscle cells but failed to relax the arteries. Ouabain (10(-5) to 10(-4) M) increased basal tone and inhibited the relaxations to bradykinin, 11,12-EET, and 14,15-EET, but not to SNP or BMK. Barium (3 x 10(-5) M) did not alter EET-induced relaxations and ouabain plus barium was similar to ouabain alone. Resting membrane potential (E(m)) of isolated smooth muscle cells was -50.2 +/- 0.5 mV. Ouabain (3 x 10(-5) and 1 x 10(-4) M) decreased E(m) (-48.4 +/- 0.2 mV), whereas 11,12-EET (10(-7) M) increased E(m) (-59.2 +/- 2.2 mV). Ouabain inhibited the 11,12-EET-induced increase in E(m). In cell-attached patch clamp studies, 11,12-EET significantly increased the open-state probability (NP(o)) of a calcium-activated potassium channel compared with control cells (0.26 +/- 0.06 vs. 0.02 +/- 0.01). Ouabain did not change NP(o) but blocked the 14,15-EET-induced increase in NP(o). These results indicate that: 1) EETs relax coronary arteries in an endothelium-independent manner, 2) unlike EETs, potassium chloride does not relax the coronary artery, and 3) ouabain inhibits bradykinin- and EET-induced relaxations as has been reported for EDHF. These findings provide further evidence that EETs are EDHFs.

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To explore the hypothesis that altered vascular muscle signal transduction may underlie some of the vascular changes observed in hypertensive models, we measured expression of GTP-binding protein (G protein) o-subunits, G,, G i? and G q , in aortic muscle of reduced renal mass and sham-operated rats and proximal and distal aortic segments from rats with interrenal aortic coarctation (IR-AC). G protein expression was measured by immunoblot analysis. When we probed aortic muscle membrane with G, and G q a-subunit antibodies, we identified 41-and 42-kD immunoreactive proteins, respectively. Three immunoreactive bands specific to G, a-subunit antibody were resolved. Immunoreactive blot densities were compared. In aortic muscle membrane of reduced renal mass rats (blood pressure, 148 ±7 mm Hg), we found significantly reduced G, and G, blot densities compared with sham-operated controls (blood pressure, 99±12 mm Hg). There were no differences in G q blot densities between re-G TP-binding proteins (G proteins) are a family of heterotrimeric proteins composed of a-, /3-, and -y-subunits.1 -2 These regulatory proteins link cell surface receptors to intracellular intermediates and second messengers and thus are vital links and initiators of signal transduction in response to neurotransmitters and endogenous vasoactive agents in vascular smooth muscle. Given the massive literature regarding altered arterial reactivity to a variety of vasoactive substances in animals with experimental hypertension, we conducted the present studies to explore the hypothesis that arterial muscle membranes from rats with surgically induced hypertension show altered G protein expression compared with their normotensive counterparts. Methods Experimental Animal ModelsAdult male Sprague-Dawley rats (weight, 250±8 g; purchased from Sasco) were subjected to 75% reduction in renal mass by a two-stage operation to produce reduced renal mass (RRM) rats.3 -4 Normotensive control rats were subjected to the same surgical procedure except for removal of renal tissue (sham rats). After recovery for 1 week, both RRM and sham rats received a 4% NaCl diet for 2 weeks. Then rats were anesthetized with 40 mg/kg ketamine-HCI (Ketaset, Aveco Co) followed by 20 to 30 mg/kg sodium pentobarbital (Veterinary Labs, Inc). Catheters were placed into the femoral artery duced renal mass and control rats. G, and G, blot densities were significantly lower in IR-AC proximal aortic segments (carotid pressure, 165±5 mm Hg) and distal aortic segments (femoral pressure, 121 ±4 mm Hg) than in aortas of shamoperated controls. In contrast, G q expression was significantly increased in the high-pressure proximal aortic segments compared with low-pressure distal aortic segments from IR-AC rats. Thus, altered G protein expression occurs in aortic muscle from nongenetic rat models of hypertension. Given the differences between reduced renal mass and IR-AC models, it is clear that pressure is not the only variable regulating G protein expression and that hormonal and/or metabol...