Selectivity of [125I]-PD151242 for human, rat and porcine endothelin ETA receptors in the heart.

1. Endothelin-1 binds with high affinity to heart where it acts as a potent positive inotropic agent. Our aim was to characterize the labelled and unlabelled ETA-selective antagonist PD151242 in heart tissues derived from man, rat and pigs by use of radioligand binding techniques. 2. Binding of [125I]-PD151242 to sections of human left ventricle was time-dependent and reached equilibrium after 120 min at 23 degrees C with an association rate constant of 0.0235 min-1 nM-1. The binding was reversible at 23 degrees C with a dissociation rate constant of 0.00144 min-1. 3. Saturation binding assays with [125I]-PD151242 revealed a single population of high affinity ET receptors in human left ventricle (KD = 1.07 +/- 0.08 nM; Bmax = 29.8 +/- 4.2 fmol mg-1 protein), porcine left ventricle (KD = 1.92 +/- 0.27 nM; Bmax = 493 +/- 248 fmol mg-1 protein), and rat heart (KD = 0.64 +/- 0.08 nM; Bmax = 82.34.7 fmol mg-1 protein). 4. Unlabelled PD151242 competed with specific [125I]-ET-1 binding to human left ventricle tissue in a biphasic manner with high affinity binding to the ETA-site (KD = 7.21 +/- 2.83 nM) and lower affinity for the ETB-subtype (KD = 104 +/- 23 microM), indicating a greater than 10000 fold selectivity to the high affinity site. 5. The ETA-selective ligand FR139317 competed for [125I]-PD151242 binding in human left ventricle with nanomolar affinity (KD = 0.37 +/- 0.10 nM), whereas the ETB-selective compound, BQ3020, competed with only micromolar affinity (KD = 1.5 +/- 0.26 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of endothelin receptors on a human neuroblastoma cell line: evidence for the ETA subtype.

1. Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK-N-MC cell line, derived from a human neuroblastoma. 2. [125I]-ET-1 (2.5 x 10(-11) M) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 +/- 0.04, n = 3) with an apparent Kd of 1.4 +/- 0.3 x 10(-9) M and a Bmax of 3.1 +/- 1.0 pmol mg-1 protein. [125I]-ET-3 (2.5 x 10(-11) M), did not specifically bind to SK-N-MC cells. 3. The binding of [125I]-ET-1 was competitively inhibited by other ET isoforms, the order of potency being ET-1 greater than sarafotoxin S6b greater than ET-3. 4. Association of 1 nM [125I]-ET-1 at 37 degrees C reached apparent equilibrium at 60-80 min, with half-maximal binding being achieved at 12 min. 5. Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]-ET-1 dissociating. The actual amounts of [125I]-ET-1 dissociated were similar in both cases. 6. Incubation of [125I]-ET-3 with SK-N-MC cells at 37 degrees C for 60 min did not result in significant degradation of this peptide. However, [125I]-ET-1 was broken down by incubation with SK-N-MC cells, the pattern of degradation of dissociable [125I]-ET-1 (and that found in the supernatant) being different from that of non-dissociable [125I]-ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of endothelin ETA receptors masks the constrictor role of endothelin ETB receptors in rat isolated small mesenteric arteries

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ET_A and ET_B receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ET_A receptor antagonist, BQ-123; the ET_B receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ET_B receptor selective antagonist, BQ-788; and the ET_A/ET_B antagonist, TAK-044.
2. In 3rd generation mesenteric arteries, ET-1 (10⁻¹³10⁻⁷ M) produced concentration-dependent contractions (pD₂ 9.86). SRTX S6c (10⁻¹²10⁻⁷ M) also induced concentration-dependent contractions in 53% of arteries studied, although the E_max was much less than that obtained with ET-1 (10.7±2.9% vs 101.9±2.6% of the 60 mM KCl-induced contraction).
3. Neither ET_B receptor desensitization, by a supra-maximal concentration of SRTX S6c (10⁻⁷ M), nor incubation with BQ-788 (3×10⁻⁸ M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1.
4. In the presence of BQ-123 (10⁻⁶ M), responses to low concentrations of ET-1 (up to 10⁻¹⁰ M) were unaffected but responses to concentrations of ET-1 above 10⁻¹⁰ M were significantly inhibited.
5. SRTX S6c desensitization followed by incubation with BQ-123 (10⁻⁶ M) or co-incubation with BQ-788 (3×10⁻⁸ M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10⁻⁸ M and 3×10⁻⁷ M).
6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ET_A and ET_B receptors. The relative role of ET_B receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ET_B receptor desensitization or BQ-788. The effect of ET_B receptor desensitization or blockade is only revealed in the presence of ET_A receptor blockade, suggesting the presence of a ‘crosstalk'' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.

     

人脐静脉血管内皮细胞构建组织工程心脏瓣膜及其生理功能

目的 探讨人脐静脉血管内皮细胞(HUVECs)构建组织工程心脏瓣膜(TEHV)及其生理功能.方法 以脱细胞猪主动脉瓣作支架;将扩增的HUVECs种植在瓣膜上,体外静态构建TEHV,观察内皮细胞的形态和生长状况.收集瓣膜培养液,检测瓣膜内皮细胞分泌组织型纤溶酶原激活物(t-PA)、组织型纤溶酶原激活物抑制物(PAI-1)、前列环素(PGI2)、一氧化氮(NO)、内皮素(ET-1)的功能.结果 猪主动脉瓣膜中的细胞成分能完全去除,脱细胞瓣叶的生物力学特性同新鲜瓣叶相比无明显变化以HUVECs做种子细胞,成功构建TEHV;瓣膜表面的内皮细胞生长状态良好,长成连续的细胞层.细胞能够分泌t-PA、PAI-1、PGI2、NO、ET-1等.结论 以脱细胞猪主动脉瓣膜为支架,种植HUVECs成功构建TEHV,其表面HUVECs具有正常内皮细胞的生理功能.     

Role of endothelin ETA and ETB receptors in the guinea-pig urinary bladder contraction

The distribution and function of endothelin receptors in the guinea-pig urinary bladder were examined. Specific [125I]endothelin-1 binding sites with both the endothelin ET(A) and ET(B) receptor subtypes were distributed in the muscle layer. Endothelin-1 elicited a tonic contraction which was inhibited by cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ123) but not by N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (BQ788) and which was inhibited more strongly by a combination of BQ123 and BQ788. Sarafotoxin S6c elicited a contraction which was abolished by BQ788. The concentration of endothelin-1 in the muscle layer was 707.0+/-67.5 pg/g wet weight. Thus, endothelin-1 may regulate muscle tone via both subtypes of endothelin receptors in an autocrine manner in the guinea-pig urinary bladder.     

Evidence for a differential location of vasoconstrictor endothelin receptors in the vasculature.

1. There are at least two subtypes of vascular endothelin (ET) receptors. Stimulation of the ETA receptors on vascular smooth muscle cells leads to vasoconstriction, whereas activation of the ETB receptors on endothelial cells elicits vasodilatation. Several reports in the literature have suggested the presence of a vasoconstrictor non-ETA receptor on vascular smooth muscle which has pharmacological similarities to the ETB receptor. The present study was undertaken to determine the location of this ETB-like receptor within the vascular system. 2. Fourteen vascular smooth muscle preparations from six species were used to determine the effect of the ETA receptor antagonist, BQ-123, on concentration-response curves elicited by ET-1 and the ability of the ETB receptor agonist, sarafotoxin S6c, to cause contraction. The vessels fell into two categories. One group was sensitive to BQ-123 and insensitive to sarafotoxin S6c and, thus, probably contained ETA receptors. The other group, with vasoconstrictor ETB-like receptors, was insensitive to BQ-123 and sensitive to sarafotoxin S6c. 3. Vessels from cynomolgus monkeys, when studied in vitro, appeared to contain primarily ETA receptors, although the potency of BQ-123 was quite variable, suggesting the possibility of ETA receptor subtypes. In contrast, both ET-1 and sarafotoxin S6c, given as intravenous injections in conscious monkeys, produced transient, equipotent, and dose-related increases in blood pressure. The highest dose of sarafotoxin S6c (1 nmol kg-1, i.v.) also caused a marked secondary depressor response (-80 +/- 6 mmHg) that lasted approximately 10 min. The pressor responses suggest that the vasoconstrictor ETB-like receptors are present in cynomolgus monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of endothelin ETA and ETB receptors in the pathogenesis of monocrotaline-induced pulmonary hypertension

The functional roles of endothelin ETA and ETB receptors in the development of monocrotaline (MCT)-induced pulmonary hypertension were investigated using MCT-treated rats in the absence or presence of a daily administration of A-192621, a selective ETB receptor antagonist, ABT-627, a selective ETA receptor antagonist, or a combination of both drugs. Four weeks after the injection of saline or MCT (60 mg/kg, s.c.), cardiac hypertrophy, right ventricular systolic pressure and morphologic changes of pulmonary arteries were evaluated. Compared with the control animals, MCT produced marked pulmonary hypertension associated with increases in right ventricular pressure and hypertrophy, and pulmonary arterial medial thickening. These MCT-induced alterations were markedly suppressed by daily treatment with ABT-627 for 4 weeks (10 mg/kg/d, twice daily), whereas treatment with A-192621 significantly aggravated the above MCT-induced pathologic changes. The blockade of both receptor subtypes by a combination of A-192621 and ABT-627 also significantly improved the MCT-induced pathologic changes, to the same extent as with ABT-627 administration. Thus, an exaggerated response to MCT during ETB receptor blockade also seems to be mediated by ETA receptor activation, thereby suggesting that ETA receptor-mediated action is exclusively contributive to the pathogenesis of MCT-induced pulmonary hypertension, although we cannot rule out a protective role of ETB receptor-mediated action.     

Propofol stimulates nitric oxide release from cultured porcine aortic endothelial cells.

Propofol, an intravenous anaesthetic agent, causes marked vasodilatation in vivo. In the present study the effects of propofol on the release of nitric oxide (NO) from vascular endothelial cells was determined in vitro. Application of propofol to co-cultures of porcine aortic endothelial and smooth muscle cells resulted in a rapid increase in cyclic GMP formation. This increase was significantly inhibited following pretreatment of the cells with either NG-nitro-L-arginine (L-NOARG) or in the presence of haemoglobin. When applied to smooth muscle cells alone, propofol did not result in an increase in cyclic GMP levels. These results demonstrate that propofol stimulates the production and release of NO from cultured endothelial cells and suggest that the vasodilatation and hypotension observed when propofol is given in vivo may be due to NO release.     

Evidence for multiple endothelin receptors in the guinea-pig pulmonary artery and trachea.

1. The responses of the three peptides, endothelin 1 (ET-1), endothelin 2 (ET-2) and endothelin 3 (ET-3) were analysed on isolated circular segments of pulmonary arteries and trachea from the guinea-pig. 2. In the pulmonary artery, the vasomotor responses to the endothelins, expressed as the maximum contraction (Emax%), had the order ET-1 greater than ET-2 greater than ET-3 while the order of potency (pD2) was ET-1 = ET-2 greater than ET-3. ET-1 and ET-2 caused cross-desensitization, but did not affect the responses to ET-3. ET-3 did not cause cross-desensitization to ET-1 or ET-2 although it induced homologous desensitization. Finally, the effects of ET-1 and ET-2 were additive to those of ET-3. The additive effect of ET-3 to those of ET-1 or ET-2 was more difficult to demonstrate, given the profound contraction produced by ET-1 and to a lesser extent by ET-2. 3. In the trachea, the rank order of potency, additivity and desensitization were different from the pulmonary artery. Basically, all three peptides were equipotent but less potent than ET-1 in the artery. There was no evidence for additivity and only a slight tendency to tachyphylaxis was seen. 4. The guinea-pig pulmonary artery appears to be endowed with one receptor type which is sensitive to ET-1/ET-2 and with another receptor type which responds preferentially to ET-3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different role of endothelin ETA and ETB receptors and endothelial modulators in diabetes-induced hyperreactivity of the rabbit carotid artery to endothelin-1

The influence of diabetes on regulatory mechanisms and specific receptors implicated in the contractile response of isolated rabbit carotid arteries to endothelin-1 was examined. Endothelin-1 induced a concentration-dependent contraction that was greater in arteries from diabetic rabbits than in arteries from control rabbits. Endothelium removal or N(G)-nitro-L-arginine enhanced contractions in response to endothelin-1 only in control arteries, without modifying the endothelin-1 response in diabetic arteries. Indomethacin, furegrelate (thromboxane A(2) inhibitor), or cyclo-(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123; endothelin ET(A) receptor antagonist) inhibited the contractions in response to endothelin-1, the inhibition being greater in diabetic arteries than in control arteries. 2,6-Dimethylpiperidinecarbonyl-gamma-methyl-Leu-N(in)-(methoxycarbonyl)-D-Trp-D-Nle (BQ-788; endothelin ET(B) receptor antagonist) enhanced the contraction elicited by endothelin-1 in control arteries and displaced to the right the contractile curve for endothelin-1 in diabetic arteries. In summary, diabetes induces hyperreactivity of the rabbit carotid artery to endothelin-1 by a mechanism that at least includes: (1) enhanced activity of muscular endothelin ET(A) receptors; (2) impairment of endothelin ET(B) receptor-mediated nitric oxide (NO) release; and (3) enhancement of the production of thromboxane A(2).     

Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.     

Subtype-specific sorting of the ETA endothelin receptor by a novel endocytic recycling signal for G protein-coupled receptors

We have previously reported that endocytic sorting of ET(A) endothelin receptors to the recycling pathway is dependent on a signal residing in the cytoplasmic carboxyl-terminal region. The aim of the present work was to characterize the carboxyl-terminal recycling motif of the ET(A) receptor. Assay of truncation mutants of the ET(A) receptor with increasing deletions of the carboxyl-terminal tail revealed that amino acids 390 to 406 contained information critical for the ability of the receptor to recycle. This peptide sequence displayed significant sequence similarity to several protein segments confirmed by X-ray crystallography to adopt antiparallel beta-strand structures (beta-finger). One of these segments was the beta-finger motif of neuronal nitric-oxide synthase reported to function as an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domain-binding ligand. Based on these findings, the three-dimensional structure of the recycling motif of ET(A) receptor was predicted to attain a beta-finger conformation acting as an internal PDZ ligand. Site-directed mutagenesis at residues that would be crucial to the structural integrity of the putative beta-finger conformation or PDZ ligand function prevented recycling of the ET(A) receptor. Analysis of more than 300 G protein-coupled receptors (GPCRs) identified 35 different human GPCRs with carboxylterminal sequence patterns that fulfilled the structural criteria of an internal PDZ ligand. Among these are several receptors reported to follow a recycling pathway. In conclusion, recycling of ET(A) receptor is mediated by a motif with the structural characteristics of an internal PDZ ligand. This structural motif may represent a more general principle of endocytic sorting of GPCRs.     

Radioligand binding to muscarinic receptors of bovine aortic endothelial cells

1. Muscarinic receptors on endothelial cells of bovine thoracic aorta were characterized by binding assays in which (-)-[3H]-N-methyl quinuclidinyl benzilate ([3H]-NMeQNB) was used as radioligand. 2. Binding of [3H]-NMeQNB to crude membranes of freshly isolated endothelial cells was atropine-displaceable and of high affinity (KD = 0.48 nM) to a single class of sites (maximum binding capacity: 14 +/- 3 fmol mg-1 protein). Stereospecificity of the binding sites was demonstrated in experiments in which [3H]-NMeQNB binding was inhibited by dexetimide in the nanomolar range (KI = 0.63 nM) and by levetimide, its stereoisomer in the micromolar range (KI = 3.2 microM) (selectivity factor: approximately 5000). 3. Drug competition curves indicated a single class of binding sites for antagonists and the following apparent affinities (KI, nM): methyl atropine: 1.1: 4-diphenylacetoxy N-methyl piperidine methyl bromide (4-DAMP): 3.4; pirenzepine: 16; 11-[2-diethylamino-methyl)-1-piperidinyl- acetyl]-5,11-dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one (AF-DX 116); 2.500. Competition of acetylcholine with [3H]-NMeQNB was best described by two affinity sites (or states) (KH = 0.82 microM, KL = 1.6 microM). In the presence of guanylimido diphosphate [Gpp(NH)p] (100 microM), acetylcholine affinity (IC50) was slightly, but significantly reduced (factor approximately 4). 4. Binding of [3H]-NMeQNB to freshly harvested intact cells was also atropine-displaceable, stereospecific (selectivity factor: approximately 3500) and of high affinity (KD = 0.35 nM). The maximum binding capacity (9 +/- 2 fmol mg-1 total cell protein) was comparable to that of membranes and corresponded to approximately 900 binding sites per endothelial cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of the microtubular system in the endothelin-1 secretion from porcine aortic endothelial cells

The effects of certain microtubule-disrupting agents on endothelin-1 (ET-1) secretion from porcine aortic endothelial cells were studied. When endothelial cells were treated with thrombin (1 unit/mL), a significant increase in ET-1 secretion was detected in the incubation medium, while ET-1 secretion in the medium was diminished when the cells were treated simultaneously with either colchicine or vinblastine (10(-8)-10(-6) M). In such cases, however, the ET-1 content detected in the cells increased dose-dependently in accordance with the concentrations of the microtubule-disrupting agents. The intracellular accumulation of ET-1 was observed both in mitochondrial and microsomal fractions. On the other hand, thrombin produced a significant increase in polymerized tubulin content without affecting the total tubulin content. A thrombin-induced increase in the intracellular Ca2+ concentration of endothelial cells was inhibited by treatment with either colchicine or vinblastine. These results seem to indicate that the microtubular system may play an important role in ET-1 secretion from endothelial cells.     

Protamine induces elevation of cytosolic free Ca2+ in cultured porcine aortic endothelial cells.

To test the hypothesis that protamine influences calcium movement in endothelial cells, we measured the concentration of intracellular free calcium ([Ca2+]i) in cultured porcine aortic endothelial (PAE) cells in Krebs solution (2.5mM Ca2+, pH 7.4) at 37 degrees C, by fura-2 fluorimetry. The basal [Ca2+]i of PAE cells was 113+/-18 nM (n=6). Protamine increased [Ca2+]i in a concentration-dependent manner (EC50, the concentration having 50% of the maximum effect, 1.4+/-0.3 microg mL(-1), n=6). The response of PAE cells to 100 microg mL(-1) protamine (330+/-80 nM, n=6) was blocked by a Ca2+ chelator, 5 mM glycoletherdiaminetetraacetic acid (EGTA; 131+/-16 nM, n=6), and by a non-selective Ca2+ channel blocker, 3 mM Co2+ (134+/-14 nM, n=6). These results suggest that Ca2+ influx through cell-membrane Ca2+ channels is mainly responsible for the protamine-induced Ca2+ elevation.     

Evidence for an intracellular action of platelet-activating factor in bovine cultured aortic endothelial cells

Bovine culture endothelial cells (BAECs) generate platelet-activating factor (Paf) following activation by bradykinin (Bk 0.1 nM), the ionophore, A23187 (3 microM), and ATP (10 microM), but Paf is not released from the cells. These stimuli also elicit generation of prostacyclin (PGI2). The specific and competitive Paf receptor antagonists, WEB 2086 (0.1-1.0 microM) and CV 6209 (0.01-0.1 microM), inhibited Bk-, A23187- and, to a lesser extent, ATP-induced PGI2 generation but had no effect on basal PGI2 generation. These data suggest a role for intracellular Paf in signal transduction.     

Evidence for muscarinic receptors in endothelial cells from combined functional and binding studies

The aim of this study was to characterize muscarinic receptors of the bovine coronary artery by means of a combination of mechanical relaxation and contraction responses and radioligand binding data. Fresh helical strips of bovine coronary artery with intact endothelium relaxed in response to low concentrations (0.03-1 microM) of acetylcholine (ACh) and contracted at higher concentrations while endothelium-denuded strips only contracted. The ED50 for relaxation was 0.13 microM and that for contraction 1.8 microM (without endothelium); in the presence of endothelium, contraction dose-response curves were shifted to the right and the maximum contraction was reduced. In order to determine the location of the receptors mediating vasorelaxation, apparent affinity constants (KA) of ACh for relaxant and contractile effects were determined by irreversible blockade of a fraction of receptors with propyl benzilylcholine mustard (PBCM). The affinity constants (KA) were 0.22 microM for relaxation and 13 microM (with endothelium) and 20 microM (without endothelium) for contraction. In competition binding experiments against the muscarinic antagonist, [3H]N-methylscopolamine ([3H]NMS), the apparent affinity (KI) of ACh for binding sites in homogenates of endothelium-free coronary artery was 16 microM which was not different from the affinity constant determined in functional contraction experiments. Thus, the affinity constant of ACh determined for relaxation responses with endothelium-preserved vessels had no correlate in the binding affinity as determined with endothelium-free arteries. These findings indicate that bovine coronary arteries are relaxed by ACh through muscarinic receptors located on the endothelium whereas contractions are mediated by receptors on smooth muscle cells.     

Reduced hyperpolarization in endothelial cells of rabbit aortic valve following chronic nitroglycerine administration

This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the hyperpolarization induced by acetylcholine (ACh) in aortic valve endothelial cells (AVECs) of the rabbit and, if so, whether antioxidant agents can normalize this downregulated hyperpolarization. ACh (0.03-3 microM) induced a hyperpolarization through activations of both apamin- and charybdotoxin-sensitive Ca2+-activated K+ channels (K(Ca)) in rabbit AVECs. The intermediate-conductance K(Ca) channel (IK(Ca)) activator 1-ethyl-2-benzimidazolinone (1-EBIO, 0.3 mM) induced a hyperpolarization of the same magnitude as ACh (3 microM). The ACh-induced hyperpolarization was significantly weaker, although the ACh-induced [Ca2+]i increase was unchanged, in NTG-treated rabbits (versus NTG-untreated control rabbits). The hyperpolarization induced by 1-EBIO was also weaker in NTG-treated rabbits. The reduced ACh-induced hyperpolarization seen in NTG-treated rabbits was not modified by in vitro application of the superoxide scavengers Mn-TBAP, tiron or ascorbate, but it was normalized when ascorbate was coadministered with NTG in vivo. Superoxide production within the endothelial cell (estimated by ethidium fluorescence) was increased in NTG-treated rabbits and this increased production was normalized by in vivo coadministration of ascorbate with the NTG. It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced hyperpolarization in rabbit AVECs, possibly through chronic actions mediated by superoxide.