Simvastatin increases excitability in the hippocampus via a PI3 kinase-dependent mechanism.

Simvastatin is an HMG-CoA reductase inhibitor commonly used in the clinic to treat hypercholesterolemia. In addition, simvastatin has been shown to cross the blood-brain barrier and pleiotropic effects of simvastatin have been reported including anti-inflammatory properties, enhancement of neurite outgrowth, and memory enhancement properties. However, little has been reported on the effects of simvastatin on basal synaptic transmission and neuronal excitability. Here we report that simvastatin increases the fEPSP, the N-methyl-D-aspartate (NMDA) receptor-mediated fEPSP using extracellular recordings in the dendritic region of the CA1 of hippocampal slices taken from 8-week-old C57Black6J mice. In addition, we found that simvastatin perfusion causes a change in the input/output curve and a decrease of the paired-pulse facilitation ratio, indicating respectively an increase of the neuronal excitability and neurotransmitter release. We have also observed that acute application of simvastatin increased the amplitude of the compound action potential in the CA1 region. Notably, using LY294002, we have demonstrated that this effect was PI3K dependent and was occluded if the animals had previously received a diet supplemented with simvastatin. We have finally shown that the simvastatin-mediated increase of the compound action potential amplitude was also present in hippocampal slices from aged mice.

Microvascular endothelial dysfunction and its mechanism in a rat model of subarachnoid hemorrhage.

UNLABELLED: After subarachnoid hemorrhage (SAH), large cerebral arteries are prone to vasospasm. Using a rat model of SAH, we examined whether cortical microvessels demonstrate vasomotor changes that may make them prone to spasm and whether endothelial dysfunction may account for any observed changes. Two days after percutaneous catheterization into the cisterna magna, 0.3 mL of autologous blood was injected into the subarachnoid space. The brain tissue was harvested 20 min later, and microvessels were dissected from the parietal cortex. Vasomotor responses to the thromboxane analog U46619, the protein kinase C agonist phorbol acetate, endothelin-1, adenosine diphosphate, nitroprusside, and isoproterenol were examined in vitroin cerebral arterioles from the control, sham-operated, and SAH animals. Endothelial nitric oxide synthase (NOS3) messenger RNA and protein concentration was measured by northern and western blotting, respectively. Arterioles from the SAH animals demonstrated attenuated dilation to the endothelium-dependent dilator adenosine diphosphate and accentuated constriction to endothelin-1, while responses to the other agents tested were unchanged. NOS3 protein concentration was decreased, but NOS3 messenger RNA was increased after SAH. After SAH, cortical arterioles demonstrate endothelial dysfunction, which may be the basis for microvascular spasm. This is in part related to decreased NOS3, which occurs despite an increase in its transcription. IMPLICATIONS: Acute microvascular endothelial dysfunction may occur after subarachnoid hemorrhage and contribute to microvascular spasm.

Serotonin-induced human coronary microvascular contraction during acute myocardial ischemia is blocked by COX-2 inhibition.

Since serotonin (5-HT) is implicated in exacerbating acute coronary syndromes, we studied the reactivity of atrial coronary arterioles (70-140 microm) of atherosclerotic patients undergoing cardiac surgery to 5-HT, substance P (Sub P), and sodium nitroprusside by video-microscopy. Before ischemia, 5-HT-induced relaxation was not affected by NS398 (cyclooxygenase inhibitor), H2O2 or U63557A (thromboxane A2 synthase inhibitor), but was reduced by L-NNA. 5-HT elicited a potent contractile response after ischemia that was inhibited by NS398, Indo, and U63557A. While Sub P relaxation was decreased after ischemia, SNP relaxation was unchanged. The mRNA steady-state levels of NOS-3, NOS-2, prostacyclin synthase, and COX- 1 were not altered by ischemia. COX-2 mRNA and protein levels (Westernblotting), however, were increased (mean +/- SEM) 2.4 +/- 0.4 and 3.2 +/- 0.7 fold, respectively, in ischemic atrium corroborating with the immunohistochemistry of atrial tissue. It is concluded that myocardial ischemia enhanced contractile response of coronary arterioles to 5-HT maybe due to the stimulated prostaglandin release (likely thromboxane A2) secondary to induction of COX-2 expression. These findings may have implications regarding the cause of coronary spasm during acute myocardial ischemia.

Basic FGF reduces stunning via a NOS2-dependent pathway in coronary-perfused mouse hearts.

Basic fibroblast growth factor (FGF-2) may protect the heart from ischemia-reperfusion injury (stunning) by stimulating nitric oxide (NO) production. To test this hypothesis, we pretreated coronary-perfused mouse hearts with 1 microg/ml FGF-2 or vehicle control before the onset of ischemia. Intracellular calcium (Ca(i)(2+)) was estimated by aequorin, and NO release was measured with an NO-selective electrode. Hearts perfused with FGF-2 maintained significantly better left ventricular (LV) function during ischemia than hearts perfused with vehicle. FGF-2 significantly delayed the onset of ischemic contracture and improved LV recovery during reperfusion. Ca(i)(2+) was similar in both groups at baseline during ischemia and reperfusion. L-N(6)-(1-iminoethyl)lysine, a selective inhibitor of inducible NO synthase (NOS2), obliterated the protective effects of FGF-2. In transgenic hearts deficient in the expression of NOS2 (NOS2-/-), FGF-2 did not attenuate ischemia-induced LV dysfunction. Measurements of NO release demonstrated that FGF-2 perfusion significantly increased NO in wild-type but not in NOS2-/- hearts. We conclude that basic FGF attenuates myocardial stunning independent of alterations in Ca(i)(2+) by stimulating NO production via an NOS2-dependent pathway.

Increased pulmonary vascular contraction to serotonin after cardiopulmonary bypass: role of cyclooxygenase.

BACKGROUND: Pulmonary vascular resistance is frequently elevated after cardiopulmonary bypass (CPB). We examined if altered pulmonary microvascular reactivity to serotonin (5-HT) is due to altered expression of isoforms of nitric oxide synthase (NOS) or cyclooxygenase (COX). MATERIALS AND METHODS: Pigs (n = 8) were heparinized and placed on total CPB for 90 min and then perfused off CPB for 90 min. Noninstrumented pigs (n = 6) served as controls for vascular studies. Relaxation responses (% of precontraction) of microvessels (60-150 microm in diameter) were examined in vitro in a pressurized (20 mm Hg) no-flow state with video microscopic imaging. Expression of eNOS, iNOS, and inducible (COX-2) and constitutive (COX-1) cyclooxygenase was examined with Western blotting and reverse transcription polymerase chain reaction. RESULTS: Pulmonary vascular resistance (PVR) increased from 316 +/- 39 mm Hg x s/cm(5) at baseline to 495 +/- 53 at 60 min and 565 +/- 62 at 90 min after termination of CPB. 5-HT elicited a relaxation response (46.8 +/- 11. 8%) in precontracted control microvessels. This response was not affected by the NOS inhibitor N(G)-nitro-l-arginine. After CPB, pulmonary microvessels contracted significantly to 5-HT (-29 +/- 27%, P < 0.05 vs control). This response was partially inhibited (7 +/- 20%, P = 0.06) in the presence of the COX-2 inhibitor NS398, but was unaffected by the thromboxane synthase inhibitor U63557A (-20 +/- 19%). Expression of iNOS or COX-1 was not changed after CPB. Protein and mRNA expressions of COX-2 both increased significantly after CPB, while that of eNOS decreased by approximately 50%. CONCLUSIONS: PVR increased after CPB. This was associated with a hypercontractile response of isolated pulmonary microvessels to 5-HT that was in part mediated by the release of prostaglandins (but not thromboxane) and associated with increased expression of COX-2 and with decreased expression of eNOS.

Mesenteric dysfunction after cardiopulmonary bypass: role of complement C5a.

BACKGROUND: We investigated the effects of cardiopulmonary bypass (CPB) on ileal homeostasis, and the influence of functional inhibition of complement C5a on CPB-induced mesenteric injury. METHODS: Pigs were perfused on CPB for 1 hour and then perfused off CPB for an additional 2 hours. Antiporcine C5a monoclonal antibody (C5a MAb) was administered 20 minutes before onset of CPB to 6 pigs; 6 controls received saline vehicle. Total complement activity, ileal myeloperoxidase, and indices of ileal integrity were examined. RESULTS: Treatment with C5a MAb ameliorated CPB-induced abnormalities in endothelium-dependent relaxation to ADP and substance P, and the hypercontractile response to phenylephrine of ileal microvessels (88 to 168 microm). Ileal myeloperoxidase activity [units/g protein] was 41 +/- 11 in the C5a MAb group, compared to 83 +/- 13 in the saline group (19 +/- 10 base line). Total hemolytic complement activity was similar in the C5a MAb and saline groups (0.6 +/- 0.2 and 0.7 +/- 0.2 CH50 units). During CPB, ileal mucosal blood flow and mucosal pH, edema formation, and epithelial permeability deteriorated similarly in saline and C5a MAb groups. Inducible nitric oxide synthase (iNOS) mRNA expression was similar before and after CPB. CONCLUSIONS: CPB is associated with significant physiologic alterations in mucosal perfusion, epithelial permeability, edema formation, and blood flow regulation. Inhibition of C5a limits neutrophil-mediated impairment of ileal microvascular regulation after bypass, but does not improve extravascular mesenteric dysfunction after CPB.

PR39, a peptide regulator of angiogenesis.

Although tissue injury and inflammation are considered essential for the induction of angiogenesis, the molecular controls of this cascade are mostly unknown. Here we show that a macrophage-derived peptide, PR39, inhibited the ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1alpha protein, resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice. For the latter, coronary flow studies demonstrated that PR39-induced angiogenesis resulted in the production of functional blood vessels. These findings show that PR39 and related compounds can be used as potent inductors of angiogenesis, and that selective inhibition of hypoxia-inducible factor-1alpha degradation may underlie the mechanism of inflammation-induced angiogenesis.

Serotonin-induced coronary contraction increases after blood cardioplegia-reperfusion: role of COX-2 expression.

BACKGROUND: Coronary contraction has been implicated in causing suboptimal myocardial function after coronary bypass surgery. Addition of blood to cardioplegic solutions has been shown to improve endothelial function after cardioplegia. In this study, the effects of blood cardioplegia and brief reperfusion on vascular reactivity in patients with coronary artery disease and the expression (mRNA and protein) of enzymes involved in vasomotor regulation were examined. METHODS AND RESULTS: The atrial appendages of patients undergoing coronary artery surgery were harvested before cardiopulmonary bypass (control, n=8) and after bypass from a nonischemic tissue atrial segment exposed to cold, hyperkalemic blood cardioplegia (mean, 60 minutes) and a brief period (10 minutes) of reperfusion (CP-Rep, n=8). Responses of atrial arterioles were studied in vitro with video-microscopy. Reverse-transcriptase polymerase chain reaction and Western blotting were used to examine the expressions and protein content, respectively, of enzymes involved in vasomotor regulation. Serotonin caused a minimal dilation under baseline conditions but after CP-Rep elicited a potent contractile response that was inhibited in the presence of the selective inducible cyclooxygenase (COX-2) inhibitor NS398. Substance P caused an endothelium-dependent relaxation of atrial arterioles through release of nitric oxide, and ADP caused relaxation mediated through release of prostaglandins. After CP-Rep, relaxation to substance P was impaired, whereas endothelium-independent relaxation to nitroprusside and response to ADP were unchanged. Expression and protein level of COX-2 were significantly increased after CP-Rep. In contrast, expression of inducible (nitric oxide synthase-2) or constitutive endothelial (nitric oxide synthase-3) nitric oxide synthase, prostacyclin synthase, and constitutive cyclooxygenase (COX-1) were not altered after CP-Rep. CONCLUSIONS: CP-Rep increases serotonin-induced contraction of human microvessels caused by the release of products of COX-2 and the impaired release of nitric oxide. These findings have implications regarding altered coronary microvascular regulation and the cause of coronary spasm after cardiac surgery.

Attenuation of endothelium-dependent dilation of pig pulmonary arterioles after cardiopulmonary bypass is prevented by monoclonal antibody to complement C5a.

UNLABELLED: We examined whether pulmonary endothelial dysfunction associated with cardiopulmonary bypass (CPB) may be mediated by complement C5a in pigs. Pigs were placed on normothermic CPB for 1 h with or without a previous administration of 1.6 mg/kg anti-C5a monoclonal antibody (MAb), then reperfused for 2 h. Pulmonary tissue myeloperoxidase activity was measured. Expression of nitric oxide synthase (NOS) was measured by reverse transcriptase polymerase chain reaction and Western blotting. Pulmonary arterioles approximately 100 microm in diameter were preconstricted with the thromboxane analog U46619 1 microM, and relaxation responses to adenosine diphosphate 10(-9)-10(-4) M, substance P 10(-12)-10(-6) M, and sodium nitroprusside 10(-9)-10(-4) M were examined in vitro by videomicroscopy. Relaxation to the endothelium-dependent dilators adenosine diphosphate and substance P was attenuated after CPB; this attenuation was prevented by the previous administration of MAb. Relaxation to sodium nitroprusside was not affected by CPB. Neutrophil sequestration, as measured by MPO activity, increased after CPB, either with or without MAb. Transcription of NOS was unchanged by CPB, but translation of constitutive NOS was decreased after CPB, and this decrease was prevented by a previous administration of MAb. We conclude that pig pulmonary endothelial dysfunction associated with CPB may be mediated by C5a. The mechanism may involve changes in NOS translation. IMPLICATIONS: In pigs, pulmonary endothelial dysfunction may occur after cardiopulmonary bypass due to product(s) of complement activation.

Effect of sialyl Lewis(x) oligosaccharide on myocardial and cerebral injury in the pig.

BACKGROUND: Although administration of the sialyl Lewis(x) oligosaccharide may reduce myocardial injury after ischemia-reperfusion, its effect on coronary and cerebral microvascular regulation and its clinical application during cardiac operation have not been examined. METHODS: Pigs were placed on normothermic cardiopulmonary bypass after 30 minutes of left anterior descending coronary artery occlusion. The hearts were then arrested with cold high potassium cardioplegia. After 1 hour the cross-clamp was removed and the pigs were weaned from cardiopulmonary bypass and perfused for an additional 1 hour. CY-1503 (a sodium salt of the sialyl Lewis(x) oligosaccharide, n = 6) was administered before reperfusion. Six other pigs received saline vehicle. Endothelium-dependent relaxation of precontracted coronary and brain arterioles (70 to 180 microm) to adenosine 5'-diphosphate and endothelium-independent relaxation to sodium nitroprusside were studied in vitro with videomicroscopy. Control values were obtained from uninstrumented pigs. Myeloperoxidase activity in the myocardium and brain was measured to quantify neutrophil infiltration. Cardiac function and perfusion were assessed by left ventricular systolic pressure, maximum rate of increase of left ventricular pressure, left anterior descending coronary artery blood flow and percent segmental shortening, and cerebral vascular resistance, internal carotid artery blood flow, and the constitutively expressed and inducible isoform of nitric oxide synthase mRNA were measured. RESULTS: The impaired myocardial contractile function after ischemia and cardioplegia was not improved by administration of CY-1503. The reduced endothelium-dependent relaxation responses of coronary and brain arterioles during ischemia followed by cardioplegia and cardiopulmonary bypass were improved with CY-1503, but the altered pattern of organ perfusion was not improved. Myeloperoxidase activity was increased in the heart after ischemia-cardioplegia and in the brain after cardiopulmonary bypass. CY-1503 reduced myeloperoxidase activity in both the myocardium and in the brain. Expressions of myocardial inducible isoform or constitutively expressed nitric oxide synthase were not altered in the heart. CONCLUSIONS: Although the sialyl Lewis(x) oligosaccharide does reduce neutrophil infiltration and endothelial injury in the coronary and cerebral microcirculation after cardiopulmonary bypass, it does not have significant beneficial acute effects on organ perfusion or function in the myocardium or brain.

Effects of ischemic preconditioning on myocardial perfusion, function, and microvascular regulation.

BACKGROUND: Ischemic preconditioning (PC) has been advocated as a method to preserve myocardial function and perfusion during minimally invasive direct coronary bypass (MIDCAB). We examined the effects of PC on indexes of myocardial function, perfusion, and endothelial and beta-adrenergic coronary regulation after 30 minutes of ischemia and 60 minutes of reperfusion (IR). METHODS AND RESULTS: Five groups of pigs were studied: (1) PC-IR: PC by 3 cycles of 5-minute left anterior descending coronary artery occlusion (CO) and 5-minute reperfusion (Rep) + 30 minutes of CO + 60 minutes of Rep; (2) IR alone: 30 minutes of CO + 60 minutes of Rep; (3) PC alone; (4) PC-IR-glibenclamide (GLIB): PC-IR + infusion of GLIB; (5) control: noninstrumented. Reactivity (in vitro) of coronary arterioles (70 to 150 microns) from the myocardial area at risk was examined with video microscopy. beta-Adrenergic microvascular relaxations to isoproterenol, forskolin, and 8-bromo-cAMP were significantly reduced after IR alone (P < 0.05 versus control, 2-way ANOVA). PC before IR restored these responses to normal (P < 0.05 PC-IR versus IR alone), and GLIB abolished this effect of PC. Subepicardial endothelium-dependent microvascular relaxation to ADP was significantly reduced after IR alone (P < 0.01 versus control) but was preserved in both the PC-IR and PC-IR-GLIB groups (P < 0.05 versus IR alone). The response of vessels to ADP from the subendocardium was significantly reduced in all groups compared with the control response (all P < 0.05 versus control). Nitroprusside elicited a similar response in vessels from all groups. PC before IR did not affect the reduced myocardial percent segmental shortening or left ventricular maximal dP/dt, did not affect myocardial perfusion in the subepicardium or subendocardium, and did not change expression of the inducible or the constitutively expressed isoforms of nitric oxide synthase. CONCLUSIONS: PC before IR preserves beta-adrenergic signal transduction in coronary smooth muscle through a KATP channel mechanism, whereas PC preserves endothelium-dependent relaxation in the subepicardium through a mechanism not related to KATP channels or the enhanced expression of nitric oxide synthase. Nevertheless, PC does not improve short-term myocardial function or baseline myocardial perfusion after IR. Thus, the short-term beneficial role of PC in myocardial protection during MIDCAB may be limited.

Myocardial VEGF expression after cardiopulmonary bypass and cardioplegia.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a heparin-binding glycoprotein that plays a critical role in angiogenesis, vascular remodeling, and regulation of vascular tone and permeability. Because myocardial and peripheral edema and systemic hypotension occur frequently after cardiac operations, we examined the effects of cardiopulmonary bypass (CPB) and cardioplegia on gene expressions of VEGF protein and the VEGF tyrosine kinase receptor flk-1 and coronary vascular responses to VEGF. METHODS AND RESULTS: Pigs (n = 6) were placed on normothermic CPB and hearts were arrested for 1 hour with a hyperkalemic, cold blood cardioplegic solution. Pigs were then separated from CPB and perfused off CPB for an additional 2 hours. Myocardial and skeletal muscle specimens were obtained for Northern analysis of VEGF protein, flk-1 receptor, and basic fibroblast growth factor (bFGF) mRNA before CPB and after 2 hours of reperfusion. Isolated, precontracted coronary arterioles in pre-CPB dilated potently to exogenous VEGF (dilation = 26 +/- 4% of precontraction at 10(-12) mol/L VEGF). Cardioplegia-reperfusion was associated with a 4 +/- 2-fold (P < 0.05 vs pre-CPB) increase in myocardial VEGF protein mRNA, whereas no similar increase was observed in the skeletal muscle. Flk-1 mRNA was increased 6 +/- 3-fold (P < 0.05 vs pre-CPB) after reperfusion, whereas it was unchanged in the skeletal muscle. Relaxations of precontracted coronary arterioles to VEGF were significantly increased (40 +/- 6% at 10(-12) mol/L, P < 0.05 vs pre-CPB) after 2 hours of reperfusion, but those to the endothelium-dependent vasodilator ADP and the endothelium-independent vasodilator nitroprusside were not changed, suggesting that the VEGF receptors remain intact and function is selectively upregulated. In contrast, relaxation responses of microvessels to bFGF were not altered after cardioplegia-reperfusion, and there was no increase in bFGF mRNA in either myocardium or skeletal muscle. CONCLUSIONS: This study shows that VEGF protein and its flk-1 receptor gene expressions are selectively increased and the potent VEGF-induced vascular responses are enhanced in the coronary microcirculation after blood cardioplegia. The respective parameters are unchanged in the skeletal muscle after normothermic CPB. These findings may have important implications regarding postoperative coronary blood flow regulation, increases in myocardial edema, and vascular remodeling after cardioplegia-reperfusion.

Anti-C5a monoclonal antibody reduces cardiopulmonary bypass and cardioplegia-induced coronary endothelial dysfunction.

OBJECTIVE: Because C5a induces tissue injury by activating polymorphonuclear leukocytes, the hypothesis was that inhibition of C5a activity would reduce cardioplegia-related injury. METHODS: Pigs were placed on cardiopulmonary bypass. The hearts were arrested for 1 hour with hyperkalemic cardioplegia. Pigs were then separated from bypass, and the hearts were reperfused for 2 hours. Anti-porcine C5a monoclonal antibody (1.6 mg/kg, intravenously; n = 6) was administered 20 minutes before the onset of cardiopulmonary bypass. Six pigs received saline solution vehicle. Reactivity of coronary arterioles was studied in vitro with videomicroscopy. Microvessels from uninstrumented pigs served as controls for vascular studies. RESULTS: Endothelium-dependent relaxation to adenosine diphosphate (percent relaxation of precontraction) was reduced after cardioplegic reperfusion (63% +/- 14% vs 77% +/- 10% in control at 10 micromol/L; P =.01). This impairment in endothelium-dependent relaxation was improved with anti-porcine C5a monoclonal antibody (80% +/- 22%; P =.01 vs saline solution), as was the impaired endothelium-dependent relaxation to clonidine (64% +/- 12% control; 26% +/- 17% saline solution; 55% +/- 24% anti-porcine C5a monoclonal antibody at 10 micromol/L; P =.01 saline solution vs control or anti-porcine C5a monoclonal antibody). Myeloperoxidase activity was significantly decreased (0.2 +/- 0.2 units/g protein; P =.04) in the anti-porcine C5a monoclonal antibody group compared with 5.2 +/- 2.7 in the saline solution group. CH50 2 hours after bypass was not statistically different (0.57 +/- 0.41 unit and 0.65 +/- 0.41 unit, respectively) between the anti-porcine C5a monoclonal antibody and saline solution groups. Despite less myocardial polymorphonuclear leukocyte infiltration after C5a inhibition, maximum rate of rise of left ventricular pressure, percent segmental shortening, and blood flow through the left anterior descending coronary artery were similar in the anti-porcine C5a monoclonal antibody and saline solution groups. CONCLUSIONS: Inhibition of C5a limits neutrophil-mediated impairment of endothelium-dependent relaxation after cardiopulmonary bypass and cardioplegic reperfusion, but it has no effect on short-term myocardial functional preservation.

Effects of coronary artery disease on expression and microvascular response to VEGF.

The effects of coronary artery disease (CAD) on human coronary microvascular responses to vascular endothelial growth factor (VEGF) and the alterations of the myocardial expressions of VEGF and its flk-1 and flt-1 receptors were examined in 48 patients. Microvascular studies were performed in vitro with video microscopy. The expressions of VEGF and its receptors were examined using Northern analysis of total mRNA, and the expressions of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were examined by RT-PCR. VEGF and hepatocyte growth factor (HGF) caused potent relaxations of microvessels. These responses were reduced in the presence of NG-nitro-L-arginine and the tyrosine kinase inhibitor genistein or in microvessels from patients with CAD. Relaxations to substance P and sodium nitroprusside were similar in both groups. The substance P response was abolished in the presence of NG-nitro-L-arginine. The expression of VEGF and its receptors and the expression of cNOS and iNOS were not altered in patients with CAD. In conclusion, VEGF and HGF elicit the release of nitric oxide through activation of tyrosine kinase receptors. CAD is associated with reduced vascular responses to both VEGF and HGF; this is not likely due to a reduced expression of VEGF or flt-1 or flk-1 receptors and not due to a generalized endothelium dysfunction despite the presence of mild hypercholesterolemia in these patients with CAD. These findings may have important implications regarding the efficacy of endogenous and exogenous VEGF in patients with risk factor for CAD.

Clotrimazole is a potent vasodilator of the rat coronary microcirculation.

Clotrimazole (CLT), used in the treatment of patients with sickle cell disease, directly blocks Ca2+-activated K+ (K+Ca) channels in red cells and in portal vein smooth muscle cells by a cytochrome P450(cyt P450)-independent mechanism. Therefore, we examined the effects of CLT on vasomotor tone of coronary arterioles. Rat coronary arterioles (80-180 micro(m) in diameter) were studied in vitro in a pressurized no-flow state with a video microscopy. CLT (0.1 micromol/L) elicited in nonprecontracted vessels a small contraction (<10% baseline diameter, P < 0.05 vs time control), consistent with blockade of a hyperpolarizing K+ channel. However, similar contraction was produced by the cyt P450 blocker 17-octadecynoic acid (17-ODYA, 100 micromol/L), suggesting possible involvement of arachidonate metabolites of cyt P450. In contrast, microvessels precontracted with the thromboxane A2 analog U46619 dilated in response to CLT [>90% relaxation of the U46619-induced precontraction at 100 micromol/L (P < 0.01 vs time control)] and its structural analogs flutrimazole (FLT), UR-4055, UR4057, UR-4058, and UR-4059. This relaxation was cyt P450-independent, since the in vivo CLT metabolite (CLT-carbinol) was equipotent with CLT, and 17-ODYA did not promote relaxation. CLT-induced dilation was not inhibited by the nitric oxide synthase inhibitor NGnitro-l-arginine (100 micromol/L, P > 0.5) or affected by endothelial denudation (P > 0.5). Thus, CLT at concentrations >1 micromol/L is a potent vasodilator of rat coronary arterioles. This dilation is likely mediated through a vascular smooth muscle mechanism independent of cyt P450 and is not modulated by nitric oxide or by the endothelium. This effect may arise from CLT's reported ability to inhibit voltage-gated Ca2+ channels or to inhibit, in some tissues, Ca2+ release from intracellular stores. The CLT- and FLT-induced relaxation may be a property common to this class of drugs and have clinical applicability.