Cholesterol feeding impairs endothelium-dependent relaxation of rabbit aorta

Experiments were designed to assess the effect of cholesterol feeding on the endothelium-mediated relaxation of the rabbit aorta to acetylcholine. Age-matched male New Zealand white rabbits were fed either a 2% cholesterol diet or standard rabbit chow. The animals were anaesthetized with sodium pentobarbitone and sacrificed after 4 and 8 weeks on these diets. Rings were prepared from the proximal thoracic aorta and examined in tissue baths. These rings were contracted first with norepinephrine (-6 log mol/L) and acetylcholine was added to demonstrate the endothelium-mediated relaxation. The endothelium-dependent relaxation was significantly less in aortas from rabbits fed the 2% cholesterol diet than in aortas from animals fed the conventional diet. This impairment of relaxation was apparent after both 4 and 8 weeks of cholesterol feeding. In both groups of animals no relaxation was seen in rings from which the endothelium was removed. These results show that cholesterol feeding leads to an impairment of endothelium-mediated relaxation of the rabbit aorta to acetylcholine.     

Possible mechanism of captopril induced endothelium-dependent relaxation in isolated rabbit aorta

The mechanism of captopril, an angiotensin converting enzyme (ACE) inhibitor with sulfhydryl group (SH) in its structure, to produce an endothelium-dependent vasorelaxation was studied. In rabbit aorta with intact endothelium and precontracted with phenylephrine, captopril and superoxide dismutase (SOD) produced dose-dependent relaxation. Lisinopril, an ACE inhibitor without a -SH group in its structure, did not produce endothelium-dependent relaxation. It was observed that captopril, like SOD, produced the relaxation by protecting the EDRF from getting inactivated by superoxide anions as pyrogallol and methylene blue inhibited both the captopril and SOD-mediated relaxation. The free radical scavenging action of captopril is further substantiated by the observation that captopril, but not lisinopril, inhibited FeCl₃/ascorbic acid-induced lipid peroxidation in whole tissue homogenates of rabbit aorta to a level comparable to that of SOD. These results suggest that endothelium-dependent vasodilation produced by captopril may be due to its ability to scavenge superoxide anion and this property may be ascribed to the -SH group present in its structure.     

Thrombin induces endothelium-dependent relaxation of pig coronary arteries

At nanomolar concentrations, the proteolytic enzyme thrombin caused a reversible concentration-dependent relaxation of PGF2 alpha-precontracted pig coronary artery ring segments with intact endothelium. After mechanical removal of the endothelium both thrombin- and bradykinin-induced relaxation disappeared. The thrombin-induced relaxation was inhibited by the tightbinding inhibitor hirudin in a concentration-dependent manner.     

Role of intracellular Ca2+ in endothelium-dependent contraction and relaxation of rabbit intrapulmonary arteries

We examined whether Ca(2+) mobilizers induce endothelium-dependent contraction and relaxation (EDC and EDR) in isolated rabbit intrapulmonary arteries. Ionomycin (10(-7) M) and A-23187 (10(-7) M), both Ca(2+) ionophores, and thapsigargin (10(-6) M), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, caused a contraction in the non-contracted preparations, and a transient relaxation followed by a transient contraction and sustained relaxation in the precontracted preparations. Endothelium-removal abolished the contraction and transient relaxation (EDC and EDR) but not sustained relaxation (endothelium-independent relaxation, EIR). In the noncontracted preparations, ionomycin-induced EDC was significantly attenuated by quinacrine (10(-5) M), manoalide (10(-6) M), both phospholipase A(2) inhibitors, indomethacin (10(-5) M) and aspirin (10(-4) M), both COX inhibitors, and ozagrel (10(-5) M), a TXA(2) synthetase inhibitor. In the precontracted arteries, EDR was markedly reduced by L-NAME (10(-4) M), a NOS inhibitor, and methylene blue (10(-6) M), a guanylate cyclase inhibitor, and was enhanced by indomethacin, aspirin and ozagrel, probably due to inhibition of EDC. ZM230487, a 5-lipoxygenase inhibitor, had no effect on EDR. EIR was not affected by L-NAME, indomethacin or ZM230487. Arachidonic acid (10(-6) M) evoked EDC sensitive to indomethacin and ozagrel. L-Arginine (10(-3) M) caused EDR sensitive to L-NAME in the ionomycin-stimulated preparations. In conclusion, Ca(2+) mobilizers cause EDC and EDR via production of TXA(2) and NO, respectively.     

Interleukin-1 impairs both vascular contraction and relaxation in rabbit isolated aorta.

Incubation of rabbit aortic rings with interleukin-1 (100 U/ml) in vitro led to a depressed contractile response to norepinephrine, whether the endothelium was present or not. In both cases norepinephrine-induced contraction was restored in the presence of NG-methyl-L-arginine (300 microM), an inhibitor of nitric oxide synthesis. In interleukin-1-treated rings precontracted with norepinephrine (1 microM), the relaxing response to acetylcholine was totally suppressed independently on the presence of endothelium. High concentrations of acetylcholine (greater than 1 microM) induced a slight contraction which was of lower amplitude than that obtained in control endothelium-denuded rings and was increased in the presence of NG-methyl-L-arginine. These results show that interleukin-1 (i) affects not only vascular contraction but also relaxation and (ii) involves both endothelial and non-endothelial factors. These observations suggest an impairment of the whole vascular reactivity during septic shock.     

Visfatin causes endothelium-dependent relaxation in isolated blood vessels

Visfatin is a novel adipocyte-derived cytokine. We hypothesized that visfatin could directly affect vascular reactivity. To test the hypothesis, effects of visfatin on contraction of isolated blood vessels were examined. In endothelium-intact rat aorta, pretreatment with visfatin (100 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 μM)-induced contraction. In NA (100 nM)-pre-contracted aorta, visfatin (1-100 ng/ml) directly induced a relaxation. Although an N^G-Nitro-l-arginine methyl ester (300 μM, 15 min) inhibited the relaxation, an insulin receptor inhibitor, AGL2263 (10 μM, 20 min) was ineffective. Visfatin (100 ng/ml, 20 min) induced a phosphorylation of eNOS at serine 1177 and a de-phosphorylation of eNOS at threonine 495. Visfatin also induced a phosphorylation of Akt at serine 473 and a substrate of cGMP-dependent protein kinase, vasodilator stimulated phosphoprotein at serine 239. Present study revealed for the first time that visfatin has a vasodilating effect on isolated blood vessels, which is mediated via endothelium-derived NO.     

Exercise preserves endothelium-dependent relaxation in coronary arteries of hypercholesterolemic male pigs.

We tested the hypothesis that exercise training (Ex) attenuates hypercholesterolemia-induced impairment of endothelium-dependent relaxation (EDR) in male porcine coronary arteries [left anterior descending coronary arteries (LAD)] by increasing nitric oxide (NO) release [due to increased endothelial NO synthase (NOS) expression] and/or increased bioactivity of NO. Adult male pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20-24 wk. Pigs were Ex or remained sedentary (Sed) for 16-20 wk, beginning after 4 wk on diet. Four groups of pigs were used: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. HF enhanced LAD contractions induced by KCl, aggregating platelets (AP), and serotonin (5-HT). AP and 5-HT produced EDR after blockade of cyclooxygenase with indomethacin (Indo) and smooth-muscle 5-HT(2) receptors with ketanserin. HF impaired EDR induced by AP, 5-HT, and bradykinin. Results indicate a decreased contribution of NO to EDR in HF-Sed LADs, because the percentage of bradykinin-induced EDR inhibited by N(G)-nitro-L-arginine methyl ester was 27% in NF-Sed and 34% in NF-Ex but only 17% in HF-Sed. Also, N(G)-nitro-L-arginine methyl ester + Indo results indicate that release of an Indo-sensitive vasoconstrictor contributes to blunted EDR in HF-Sed LAD. Immunoblot and immunohistochemistry results indicate the following: 1) LAD endothelial NOS protein content was similar among groups; 2) HF decreased LAD superoxide dismutase (SOD) but increased caveolin-1 content; and 3) Ex increased SOD content of HF LADs. We conclude that HF impairs EDR by impairing the contribution of NO released from NOS (due to decreased SOD and increased caveolin-1 protein content) and by production of an Indo-sensitive vasoconstrictor. Ex preserves EDR in HF LADs by decreasing the production of the constrictor and increasing NO-release by NOS and/or NO bioactivity and bioavailability.     

Hyperlipoproteinemia impairs endothelium-dependent vasodilation

Atherogenic lipoproteins can cause endothelial dysfunction in the initial stage of atherogenesis. In our study we examined 134 patients with defined hyperlipoproteinemia (non-HDL cholesterol>4.1 mmol/l or triglycerides>2.5 mmol/l or taking any of lipid lowering drugs)--94 men and 40 women. The subgroup of controls of comparable age contained 54 normolipidemic individuals--30 men and 24 women. Patients with hyperlipoproteinemia revealed significantly lower ability of endothelium-dependent flow-mediated vasodilation (EDV) measured on brachial artery (4.13+/-3.07 vs. 5.41+/-3.82 %; p=0.032) and higher carotid intima media thickness than normolipidemic controls (0.68+/-0.22 vs. 0.58+/-0.15 mm; p=0.005). In regression analysis, EDV correlated significantly with plasma concentrations of oxLDL (p<0.05) HDL-cholesterol (p<0.05), Apo A1 (p<0.05), ATI (p<0.01) and non-HDL cholesterol (p<0.05). Patients with hyperlipoproteinemia showed higher plasma levels of oxLDL (65.77+/-9.54 vs. 56.49+/-7.80 U/l; p=0.015), malondialdehyde (0.89+/-0.09 vs. 0.73+/-0.08 micromol/l; p=0.010) and nitrites/nitrates (20.42+/-4.88 vs. 16.37+/-4.44 micromol/l; p=0.018) indicating possible higher long-term oxidative stress in these patients.     

Exercise training preserves endothelium-dependent relaxation in brachial arteries from hyperlipidemic pigs.

We tested the hypothesis that exercise training (Ex) attenuates the effects of hyperlipidemia on endothelial function by enhancing NO-mediated vasorelaxation in porcine brachial (Br) arteries. Adult female pigs were fed a normal-fat (NF) or high-fat (HF) diet for 20 wk. Four weeks after initiation of the diet, pigs underwent Ex or remained sedentary (Sed) for 16 wk. Relaxation to ACh was impaired by HF (P = 0.03). The combination of HF and Sed impaired ACh-induced relaxation more than HF or Sed alone (P = 0.0002). Relaxation to high doses of bradykinin (BK) was impaired by HF (P = 0.0002). Ex significantly improved ACh-induced relaxation (P = 0.01) and tended to improve relaxation to BK (P = 0.38). To determine the mechanism(s) by which HF and Ex affected relaxation to ACh and BK, relaxation was assessed in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. In the presence of l-NAME, Indo, or l-NAME + Indo, ACh-induced relaxation was no longer different between HF and NF arteries; however, relaxation remained greater in Ex than in Sed arteries. In the presence of l-NAME or Indo, BK-induced relaxation was no longer altered by HF but was enhanced by Ex. In the presence of l-NAME + Indo, BK-induced relaxation was enhanced by HF and Ex. These data indicate that hyperlipidemia impairs ACh- and BK-induced relaxation by impairing NO- and PGI(2)-mediated relaxation. Ex attenuates the effects of HF by enhancing a vasodilator mechanism independent of NO and PGI(2).     

Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF

Standard treatments for erectile dysfunction (ED) (i.e., PDE5 inhibitors) are less effective in diabetic patients for unknown reasons. Endothelium-dependent relaxation (EDR) of human corpus cavernosum (HCC) depends on nitric oxide (NO), while in human penile resistance arteries (HPRA) endothelium-derived hyperpolarizing factor (EDHF) and NO participate. Here we show that diabetes significantly reduced EDR induced by acetylcholine (ACh) in HCC and HPRA. Relaxation attributed to EDHF was also impaired in HPRA from diabetic patients. The PDE5 inhibitor, sildenafil (10nM), reversed diabetes-induced endothelial dysfunction in HCC, but not in HPRA. Calcium dobesilate (DOBE; 10 microM) fully reversed diabetes-induced endothelial dysfunction in HPRA by specifically potentiating the EDHF-mediated component of EDR. Impairment by diabetes of NO and EDHF-dependent responses precluded the complete recovery of endothelial function in HPRA by sildenafil. This could explain the poor clinical response to PDE5 inhibitors of diabetic men with ED and suggests that a pharmacological approach that combines enhancement of NO/cGMP and EDHF pathways could be necessary to treat ED in many diabetic men.     

Chronic intermittent hypoxia impairs endothelium-dependent dilation in rat cerebral and skeletal muscle resistance arteries

The goal of the present study was to evaluate the effects of relatively short-term chronic intermittent hypoxia (CIH) on endothelial function of resistance vessels in the skeletal muscle and cerebral circulations. Sprague-Dawley rats were exposed to 14 days of CIH (10% fraction of inspired oxygen for 1 min at 4-min intervals, 12 h/day, n = 6). Control rats (n = 6) were housed under normoxic conditions. After 14 days, resistance arteries of the gracilis muscle (GA) and middle cerebral arteries (MCA) were isolated and cannulated with micropipettes, perfused and superfused with physiological salt solution, and equilibrated with 21% O2-5% CO2 in a heated chamber. The arteries were pressurized to 90 mmHg, and vessel diameters were measured via a video micrometer before and after exposure to ACh (10-7-10-4 M), sodium nitroprusside (10-6 M), and acute reduction of Po2 in the perfusate/superfusate (from 140 to 40 mmHg). ACh-induced dilations of GA and MCA from animals exposed to CIH were greatly attenuated, whereas responses to nitroprusside were similar to controls. Dilations of both GA and MCA in response to acute reductions in Po2 were virtually abolished in animals exposed to CIH compared with controls. These findings suggest that exposure to CIH reduces the bioavailability of nitric oxide in the cerebral and skeletal muscle circulations and severely blunts vasodilator responsiveness to acute hypoxia.     

Visfatin impairs endothelium-dependent relaxation in rat and human mesenteric microvessels through nicotinamide phosphoribosyltransferase activity

Visfatin, also known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase (Nampt), is an adipocytokine whose circulating levels are enhanced in metabolic disorders, such as type 2 diabetes mellitus and obesity. Circulating visfatin levels have been positively associated with vascular damage and endothelial dysfunction. Here, we investigated the ability of visfatin to directly impair vascular reactivity in mesenteric microvessels from both male Sprague-Dawley rats and patients undergoing non-urgent, non-septic abdominal surgery. The pre-incubation of rat microvessels with visfatin (50 and 100 ng/mL) did not modify the contractile response to noradrenaline (1 pmol/L to 30 μmol/L), as determined using a small vessel myograph. However, visfatin (10 to 100 ng/mL) concentration-dependently impaired the relaxation to acetylcholine (ACh; 100 pmol/L to 3 μmol/L), without interfering with the endothelium-independent relaxation to sodium nitroprusside (1 nmol/L to 3 μmol/L). In both cultured human umbilical vein endothelial cells and rat microvascular preparations, visfatin (50 ng/mL) stimulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, as determined by lucigenin-derived chemiluminiscence. The relaxation to ACh impaired by visfatin was restored by the NADPH oxidase inhibitor apocynin (10 μmol/L). Additionally, the Nampt inhibitor APO866 (10 mmol/L to 10 μmol/L), but not an insulin receptor-blocking antibody, also prevented the stimulation of NADPH oxidase and the relaxation impairment elicited by visfatin. Accordingly, the product of Nampt activity nicotinamide mononucleotide (100 nmol/L to 1 mmol/L) stimulated endothelial NADPH oxidase activity and concentration-dependently impaired ACh-induced vasorelaxation. In human mesenteric microvessels pre-contracted with 35 mmol/L potassium chloride, the endothelium-dependent vasodilation to bradykinin (1 nmol/L to 3 μmol/L) was equally impaired by visfatin and restored upon co-incubation with APO866. In conclusion, visfatin impairs endothelium-dependent relaxation through a mechanism involving NADPH oxidase stimulation and relying on Nampt enzymatic activity, and therefore arises as a potential new player in the development of endothelial dysfunction.     

High-salt diet impairs vascular relaxation mechanisms in rat middle cerebral arteries

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or a high-salt (HS) diet (4% NaCl) for 3 days or 4 wk. PO(2) reduction to 40-45 mmHg, the stable prostacyclin analog iloprost (10 pg/ml), and stimulatory G protein activation with cholera toxin (1 ng/ml) caused vascular smooth muscle (VSM) hyperpolarization, increased cAMP production, and dilation in cerebral arteries from rats on a LS diet. Arteries from rats on a HS diet exhibited VSM depolarization and constriction in response to hypoxia and iloprost, failed to dilate or hyperpolarize in response to cholera toxin, and cAMP production did not increase in response to hypoxia, iloprost, or cholera toxin. Low-dose angiotensin II infusion (5 ng x kg(-1) x min(-1) i.v.) restored normal responses to reduced PO(2) and iloprost in arteries from animals on a HS diet. These observations suggest that angiotensin II suppression with a HS diet leads to impaired relaxation of cerebral arteries in response to vasodilator stimuli acting at the cell membrane.     

Kinins mediate kallikrein-induced endothelium-dependent relaxations in isolated canine coronary arteries.

ACE inhibitors elicit the release of endothelium-derived relaxing factors in perfused isolated canine arteries (Mombouli and Vanhoutte, J. Cardiovasc. Pharmacol. 1991, 18: 926-927); this action is antagonized by bradykinin-receptor antagonists suggesting that it is mediated by local kinin generation. The effects of exogenous tissular kallikrein (porcine) were examined in vitro in the isolated canine coronary artery. Isometric tension was measured in blood vessel rings (with and without endothelium) contracted with prostaglandin F2 alpha. The kallikrein elicited relaxations in rings with, but not in those without, endothelium. This response was augmented by the angiotensin converting enzyme inhibitor perindoprilat, and it was antagonized by the selective B2-kinin receptor antagonist HOE 140 and aprotinin, an inhibitor of tissular kallikrein. These data suggest that in the canine coronary artery, kallikrein causes relaxations that may be mediated by kinins generated from endogenous kininogens present in the vascular wall.     

Developmental changes in endothelium-dependent relaxation of pulmonary arteries: role of EDNO and prostanoids

O'Donnell, Denise C., Mary L. Tod, and John B. Gordon.Developmental changes in endothelium-dependent relaxation of pulmonary arteries: role of EDNO and prostanoids. J. Appl. Physiol. 81(5): 2013-2019, 1996.Wehypothesized that maturational changes in both prostaglandin andendothelium-derived nitric oxide (EDNO) activity contribute todevelopmental changes in endothelium-dependent relaxation of newbornpulmonary arteries. Responses to endothelium-dependent vasodilatorsacetylcholine, bradykinin, and calcium ionophore A-23187 weredetermined in phenylephrine-constricted third- and fourth-generation(1- to 2-mm-diameter) pulmonary artery rings from 2-day (2d)- and 1-mo(1m)-old lambs under control conditions (Con), after inhibition of EDNOsynthesis withN-nitro-L-arginine(L-NNA), after inhibition ofprostanoid synthesis with meclofenamate (Mec), or both modulators withboth inhibitors. Endothelium-independent responses to sodiumnitroprusside (SNP) were also measured in Con rings.Endothelium-dependent relaxation was greater in 2d than 1m Con rings,particularly at high concentrations when an increase in tensionoccurred in 1m rings. L-NNAattenuated endothelium-dependent relaxation more in 2d rings, and SNPcaused greater relaxation in 2d rings. However, Mec abolished allage-related differences by attenuating relaxation in 2d rings andconstriction in 1m rings. These data suggest that developmental changesin endothelium-dependent responses of ovine pulmonary artery rings reflect both a decrease in EDNO activity and maturational differences in the relative influence of dilator and constrictor prostanoids.

     

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.     

L-Arginine does not improve endothelium-dependent relaxation inin vitro Watanabe rabbit thoracic aorta

Summary The findings that even a singlein vitro addition of L-arginine is able to normalize endothelium function in cerebral vessel from diet-induced hypercholesterolemic rabbits prompted us to investigate if similar results could be obtained on Watanabe rabbits thoracic aorta, in which we previously demonstrated low content of the amino acid.L-Arginine (1 mM) preincubated for 45 minutes before the addition of drugs for studing endothelium-dependent vascular relaxation, did not modify the effect of acetylcholine on aortic isolated preparations. The lack of any effect by L-arginine indicates that the amino acid deficiency is not main cause of the impairment of endothelium function. The muscarinic receptor functionality affected by atherosclerotic process and/or the increased synthesis of EDCFs could account for the reduced endothelium-dependent relaxation.     

Possible role of P-450-derived metabolites in endothelium-dependent relaxation of rat small mesenteric arteries

We reported previously that acetylcholine (ACh)-induced endothelium-dependent relaxation of rat mesenteric microvessels depended both on nitric oxide (NO) and on a charybdotoxin (CTX)-sensitive endothelium-derived hyperpolarizing vasodilator. Cytochrome P450 (CYP)-dependent arachidonic acid metabolites act in some systems as hyperpolarizing vasodilators. We sought to quantitate contributions of such metabolites to the CTX-sensitive component of ACh-induced vasodilation in isolated rat mesenteric resistance arteries. ACh relaxed these vessels nearly completely (93.3+/-1.2%, n = 71); cyclooxygenase inhibition with indomethacin did not diminish this response (94.3+/-11.4%, n = 9). NO synthase inhibition with Nitro-L-arginine (NNLA) reduced relaxation by 30% (n = 54, p<0.05). Pretreatment of vessels with CYP inhibitors, either clotrimazole (CTM) or 17-octadecynoic acid (17-ODYA), or with selective K+ channel inhibitors, either tetraethyammonium acetate (TEA) or CTX, each led to similar small reductions in maximal relaxation (17%, 22%, 16%, and 9% respectively, n = 3-6). Combined pretreatment with NNLA + either (CTM or 17-ODYA) or (TEA or CTX) each led to similar maximal relaxations (52.2+/-4.8%, 50.6+/-9.2, 37.6+/-8.6%, and 44.1+/-11.5%, respectively, n = 6-35; p<0.05 for NNLA+[CTM or TEA or CTX] vs NNLA alone). Combined pretreatment with NNLA+CTM+(CTX or TEA) led to similar maximal relaxations (43.0+/-7.3%, 43.7+/-15%, n = 6-11) that did not differ from values in vessels pretreated with either NNLA+CTM or NNLA+(CTX or TEA). We conclude that the ACh-induced vasodilation was insensitive to cyclooxygenase inhibition, partially sensitive to NO synthase inhibition, and that the K+ channel blockers TEA and CTX identified the same minor component of ACh relaxation as did the CYP inhibitor CTM.