ET(B) receptor-deficient rats exhibit reduced contraction to ET-1 despite an increase in ET(A) receptors.

Several disease states, including hypertension, are associated with elevations in plasma endothelin-1 (ET-1) and variable changes in vascular contraction to ET-1. The spotting lethal (sl) rat carries a deletion of the endothelin-B (ET(B)) receptor gene that prevents expression of functional ET(B) receptors, resulting in elevated plasma ET-1. On a normal diet, these rats are normotensive and thus provide an opportunity to study the vascular effects of chronically elevated ET-1 in the absence of hypertension. Studies were performed in rats homozygous for the ET(B) deficiency (sl/sl; n = 8) and in transgenic rats heterozygous for the ET(B) deficiency (sl/+; n = 8). Plasma ET-1 was elevated in sl/sl rats (3.85 +/- 0.55 pg/ml) compared with sl/+ rats (0.31 +/- 0.11 pg/ml). Mean arterial blood pressure in conscious unrestrained sl/sl and sl/+ rats was 101 +/- 5 and 107 +/- 6 mmHg, respectively. Concentration-dependent contractions to ET-1 (10(-11)-10(-8) M) were reduced in mesenteric small arteries (150-250 microm) from sl/sl rats, as indicated by an approximately 10-fold increase in EC(50). A selective ET(A) antagonist, A-127722 (30 nM), abolished contraction to ET-1 in both groups, whereas a selective ET(B) antagonist had no effect. Also, ET(B) agonists (IRL-1620 and sarafatoxin 6c) produced neither contraction nor relaxation in either group, indicating that contraction to ET-1 in this vascular segment was exclusively ET(A) dependent. Despite increased plasma ET-1, protein expression of ET(A) receptors in membrane protein isolated from mesenteric small arteries was increased in sl/sl compared with sl/+ rats, as shown by Western blotting. These results indicate that, in ET(B)-deficient rats, ET(A)-induced contraction is reduced in vessels normally lacking ET(B)-mediated effects. Reduced contraction may be related to elevated plasma ET-1 and occurs in the presence of increased ET(A) receptor protein expression, suggesting an uncoupling of ET(A) receptor expression from functional activity.

Dihydroxyeicosatrienoic acids are potent activators of Ca(2+)-activated K(+) channels in isolated rat coronary arterial myocytes.

1. Dihydroxyeicosatrienoic acids (DHETs), which are metabolites of arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs), have been identified as highly potent endogenous vasodilators, but the mechanisms by which DHETs induce relaxation of vascular smooth muscle are unknown. Using inside-out patch clamp techniques, we examined the effects of DHETs on the large conductance Ca(2+)-activated K(+) (BK) channels in smooth muscle cells from rat small coronary arteries (150-300 microM diameter). 2. 11,12-DHET potently activated BK channels with an EC(50) of 1.87 +/- 0.57 nM (n = 5). Moreover, the three other regioisomers 5,6-, 8,9- and 14,15-DHET were equipotent with 11,12-DHET in activating BK channels. The efficacy of 11,12-DHET in opening BK channels was much greater than that of its immediate precursor 11,12-EET. In contrast, AA did not significantly affect BK channel activity. 3. The voltage dependence of BK channels was dramatically modulated by 11,12-DHET. With physiological concentrations of cytoplasmic Ca(2+) (200 nM), the voltage at which the channel open probability was half-maximal (V(1/2)) was shifted from a baseline of 115.6 +/- 6.5 mV to 95.0 +/- 10.1 mV with 5 nM 11,12-DHET, and to 60.0 +/- 8.4 mV with 50 nM 11,12-DHET. 4. 11,12-DHET also enhanced the sensitivity of BK channels to Ca(2+) but did not activate the channels in the absence of Ca(2+). 11,12-DHET (50 nM) reduced the Ca(2+) EC(50) of BK channels from a baseline of 1.02 +/- 0.07 microM to 0.42 +/- 0.11 microM. 5. Single channel kinetic analysis indicated that 11,12-DHET did not alter BK channel conductance but did reduce the first latency of BK channel openings in response to a voltage step. 11,12-DHET dose-dependently increased the open dwell times, abbreviated the closed dwell times, and decreased the transition rates from open to closed states. 6. We conclude that DHETs hyperpolarize vascular smooth muscle cells through modulation of the BK channel gating behaviour, and by enhancing the channel sensitivities to Ca(2+) and voltage. Hence, like EETs, DHETs may function as endothelium-derived hyperpolarizing factors.

Enhanced endothelin-1 response and receptor expression in small mesenteric arteries of insulin-resistant rats.

Hyperinsulinemia, a primary feature of insulin resistance, is associated with increased endothelin-1 (ET-1) activity. This study determined the vascular response to ET-1 and receptor binding characteristics in small mesenteric arteries of insulin-resistant (IR) rats. Rats were randomized to control (C) (n = 32) or IR (n = 32) groups. The response to ET-1 was assessed (in vitro) in arteries with (Endo+) and without (Endo-) endothelium. In addition, arteries (Endo+) were pretreated with the ET(B) antagonist A-192621 or the ET(A) antagonist A-127722. Finally, binding characteristics of [(125)I]ET-1 were determined. Results showed that in Endo+ arteries the maximal relaxation (E(max)) to ET-1 was similar between C and IR groups; however, the concentration at 50% of maximum relaxation (EC(50)) was decreased in IR arteries. In Endo- arteries, the E(max) to ET-1 was enhanced in both groups. Pretreatment with A-192621 enhanced the E(max) and EC(50) to ET-1 in both groups. In contrast, A-127722 inhibited the ET-1 response in all arteries in a concentration-dependent manner; however, a greater ET-1 response was seen at each concentration in IR arteries. Maximal binding of [(125)I]ET-1 was increased in IR versus C arteries although the dissociation constant values were similar. In conclusion, we found the vasoconstrictor response to ET-1 is enhanced in IR arteries due to an enhanced expression of ET receptors and underlying endothelial dysfunction.

12-lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation.

Noncyclooxygenase metabolites of arachidonic acid (AA) have been proposed to mediate endothelium-dependent vasodilation in the coronary microcirculation. Therefore, we examined the formation and bioactivity of AA metabolites in porcine coronary (PC) microvascular endothelial cells and microvessels, respectively. The major noncyclooxygenase metabolite produced by microvascular endothelial cells was 12(S)-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product. 12(S)-HETE release was markedly increased by pretreatment with 13(S)-hydroperoxyoctadecadienoic acid but not by the reduced congener 13(S)-hydroxyoctadecadienoic acid, suggesting oxidative upregulation of 12(S)-HETE output. 12(S)-HETE produced potent relaxation and hyperpolarization of PC microvessels (EC(50), expressed as -log[M] = 13.5 +/- 0.5). Moreover, 12(S)-HETE potently activated large-conductance Ca(2+)-activated K(+) currents in PC microvascular smooth muscle cells. In contrast, 12(S)-HETE was not a major product of conduit PC endothelial AA metabolism and did not exhibit potent bioactivity in conduit PC arteries. We suggest that, in the coronary microcirculation, 12(S)-HETE can function as a potent hyperpolarizing vasodilator that may contribute to endothelium-dependent relaxation, particularly in the setting of oxidative stress.

Cytochrome P450 activity and endothelial dysfunction in insulin resistance.

Impaired endothelium-dependent relaxation attributable to nitric oxide/prostacyclin-independent factor (endothelium-dependent hyperpolarizing factor; EDHF) has been demonstrated in the small mesenteric arteries of insulin-resistant rats. The purpose of this study was to determine if modulation of the cytochrome P450 enzyme system would restore EDHF-mediated relaxation in insulin-resistant rats. Sprague-Dawley rats were randomized to control (n = 32) or insulin-resistant (n = 32) groups. Each group was further randomized to treatment (n = 48) or placebo (n = 16). Miconazole (3 days) and phenobarbital (3 and 14 days) achieved cytochrome P450 inhibition and induction, respectively. Following drug treatment, mean arterial pressure was measured and vascular function was assessed in small mesenteric arteries in vitro. Specifically, acetylcholine-induced relaxation alone and in the presence of indomethacin plus N-nitro-L-arginine (LNNA) or KCl was determined. Miconazole reduced the maximal relaxation in response to acetylcholine in control rats. Similarly, in the presence of LNNA plus indomethacin, acetylcholine-induced relaxation was impaired in the miconazole-treated control group versus the placebo group, whereas relaxation in the presence of KCl was unchanged. Miconazole did not affect relaxation in insulin-resistant arteries. In contrast, 3- and 14-day treatment with phenobarbital significantly improved acetylcholine-induced relaxation in insulin-resistant arteries. Likewise, acetylcholine-mediated relaxation in the presence of LNNA plus indomethacin was also improved after phenobarbital treatment, while relaxation in the presence of KCl was unchanged. Phenobarbital treatment did not affect the control group. Miconazole treatment increased the mean arterial pressure in control rats, while 14-day phenobarbital treatment normalized the mean arterial pressure in insulin-resistant rats. Cytochrome P450 induction results in the restoration of EDHF-mediated relaxation in small mesenteric arteries and the normalization of mean arterial pressure in insulin-resistant rats. Thus, endothelial dysfunction secondary to insulin resistance can be reversed by the induction of cytochrome P450.

Metformin improves vascular function in insulin-resistant rats.

This study assessed the effect of metformin treatment on insulin, mean arterial pressure (MAP), and endothelial function in insulin-resistant (IR) rats. In addition, we assessed the direct effect of metformin in vitro. Sprague-Dawley rats were randomized to control (n=28) or IR (n=28) groups. Rats were further randomized to receive metformin (300 mg/kg) or placebo for 2 weeks. MAP and insulin were measured. Subsequently, a third-order branch of the superior mesenteric artery was isolated, and endothelial function was assessed. Specifically, dose-response experiments of acetylcholine (ACh) with or without N-nitro-L-arginine (LNNA) were performed. For in vitro experiments, mesenteric arteries were removed from untreated control and IR rats and treated with metformin (100 micromol/L) before ACh+/-LNNA. MAP and insulin levels were improved in IR-metformin compared with IR-placebo rats. Maximal relaxation (E(max)) to ACh was enhanced in IR-metformin (92+/-2%) compared with IR-placebo rats (44+/-4%) (P<0.05). Relaxation in response to ACh+LNNA was greater in IR-metformin (33+/-4%) than in IR-placebo rats (12+/-4%) but remained depressed compared with control rats (E(max)=68+/-5%). The control group was not affected by metformin. In vitro treatment of arteries with metformin in response to ACh produced results similar to those in the experiments with metformin-treated rats. Although metformin improves metabolic abnormality in IR rats, this action does not appear to mediate its effect on vascular function. Both in vivo and in vitro metformin improved ACh-induced relaxation in IR rats to control levels, apparently through nitric oxide-dependent relaxation. These data suggest that metformin improves vascular function through a direct mechanism rather than by improving metabolic abnormalities.

Impaired endothelium-mediated relaxation in coronary arteries from insulin-resistant rats.

OBJECTIVE: The insulin resistance syndrome is associated with atherosclerosis and cardiovascular events; however, the underlying mechanism of vascular dysfunction is unknown. The purpose of the current study was to assess endothelium- and smooth-muscle-mediated vasodilation in isolated coronary arteries from insulin-resistant rats and to determine whether insulin resistance alters the activity of the specific endothelium-derived relaxing factors. METHODS: Male Sprague-Dawley rats were randomized to insulin resistance or control. Insulin resistance was induced by a fructose-rich diet. After 4 weeks of diet, coronary arteries were removed and vascular function was assessed in vitro using videomicroscopy. Acetylcholine (10(-9)-3 x 10(-5) M)- or sodium-nitroprusside (10(-9)-3 x 10(-4) M)-induced relaxations were determined. To evaluate the role of the specific endothelium-derived relaxing factors, several inhibitors were used, including N-nitro-L-arginine (LNNA), charybdotoxin/apamin (CTX/apamin), and indomethacin. RESULTS: Studies with nitroprusside showed that smooth-muscle-dependent relaxation did not differ between insulin resistance and control groups. In contrast, maximal relaxation (E(max)) to acetylcholine was decreased in the insulin resistance group (56 +/- 7%) versus control (93 +/- 3%). LNNA pretreatment further impaired E(max) in the IR group from 56 +/- 7 to 17 +/- 2% (p < 0.01). In control, E(max) was only slightly impaired by LNNA (93 +/- 3 to 63 +/- 6%; p < 0.05). The addition of CTX/apamin also decreased relaxation in the control group (93 +/- 3 to 47 +/- 7%; p < 0.05), whereas relaxation in insulin-resistant rats was not affected (45 +/- 5% with CTX/apamin vs. 56 +/- 7% with acetylcholine alone, NS). Pretreatment with indomethacin did not affect relaxation in either group, while pretreatment with the combination of LNNA and CTX/ apamin completely abolished relaxation in both groups. CONCLUSIONS: Endothelium-dependent relaxation is impaired in small coronary arteries from insulin-resistant rats. The mechanism of this defect is related to a decrease in an endothelium-dependent, nitric oxide/prostanoid-independent relaxing factor or endothelium-derived hyperpolarizing factor.

EDHF-mediated relaxation is impaired in fructose-fed rats.

Insulin resistance (IR) is associated with endothelial dysfunction. A defect in endothelium-dependent relaxation via outward potassium conductance has been observed in mesenteric arteries from IR rats. The purpose of this study was to assess whether this defect in endothelium-dependent relaxation was due to impaired endothelium-derived hyperpolarizing factor (EDHF) and to determine which specific potassium channel(s) are involved in relaxation. This was accomplished by using specific potassium channel inhibitors in the presence of nitric oxide synthase and cyclooxygenase inhibition. In addition, we sought to assess the function of smooth muscle cell adenosine triphosphate (ATP)-dependent potassium (K(ATP)) channels. Sprague-Dawley rats were randomized to control or IR. To determine EDHF-mediated relaxation, acetylcholine (ACh)-induced (10(-9)-10(-5) M) relaxation was measured (in vitro) in mesenteric arteries in the presence of indomethacin (10(-5) M) and N-nitro-L-arginine (L-NNA) (10(-4) M). Subsequently the combination of charybdotoxin (CTX) (0.1 microM) and apamin (0.5 microM) or glibenclamide (Glib) (10 microM) was added to the bath to inhibit KCa or K(ATP), respectively. In separate experiments, relaxation to pinacidil (10(-13)-10(-5) M), a K(ATP) activator, was assessed in vessels with intact endothelium, endothelium denuded, or with L-NNA. Maximal relaxation to ACh in the presence of L-NNA and indomethacin was 68+/-6% for control and 12+/-3% for IR (p<0.01). The addition of CTX + apamin almost abolished EDHF-mediated relaxation in control (Emax, 8+/-5% vs. 68+/-6%; p<0.01), whereas Glib had little affect. Neither CTX + apamin nor Glib had any affect on IR. Additionally, IR arteries were less sensitive to pinacidil than were controls (EC50, 1.5+/-0.9 microM vs. 5x10(-4)+/-3x10(-4) microM, respectively; p<0.01). Endothelial removal or L-NNA pretreatment of control arteries decreased the response to pinacidil similar to IR, whereas IR vessels were unaffected. EDHF-mediated relaxation is impaired in IR arteries. In addition, the K(Ca) channel appears to be imperative for activity of EDHF in rat small mesenteric arteries. Moreover, activation of K(ATP) channels by pinacidil is impaired in IR, and this appears to be a result of endothelial dysfunction.

Endothelial dysfunction precedes hypertension in diet-induced insulin resistance.

The insulin-resistant (IR) syndrome may be an impetus for the development of hypertension (HTN). Unfortunately, the mechanism by which this could occur is unclear. Our laboratory and others have described impaired endothelium-mediated relaxation in IR, mildly hypertensive rats. The purpose of the current study is to determine if HTN is most likely a cause or result of impaired endothelial function. Sprague-Dawley rats were randomized to receive a fructose-rich diet for 3, 7, 10, 14, 18, or 28 days or were placed in a control group. The control group received rat chow. After diet treatment, animals were instrumented with arterial cannulas, and while awake and unrestrained, their blood pressure (BP) was measured. Subsequently, endothelium-mediated relaxation to acetylcholine was determined (in vitro) by measuring intraluminal diameter of phenylephrine-preconstricted mesenteric arteries ( approximately 250 microM). Serum insulin levels were significantly elevated in all groups receiving fructose feeding compared with control, whereas there were no differences in serum glucose levels between groups. Impairment of endothelium-mediated relaxation starts by day 14 [mean percent maximal relaxation (Emax): 69 +/- 10% of baseline] and becomes significant by day 18 (Emax: 52 +/- 11% of baseline; P < 0.01). However, the mean BP (mmHg) does not become significantly elevated until day 28 [BP: 132 +/- 1 (day 28) vs. 116 +/- 3 (control); P < 0.05]. These findings demonstrate that both IR and endothelial dysfunction occur before HTN in this model and suggest that endothelial dysfunction may be a mechanism linking insulin resistance and essential HTN.