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PubMed1990

Nicotinic actions of oxotremorine on murine skeletal muscle. Evidence against muscarinic modulation of acetylcholine release.

The effects of oxotremorine, arecoline and muscarine on neuromuscular transmission of mouse or rat phrenic nerve-diaphragm were investigated. For some studies of endplate potentials (e.p.p.s) the preparation was immobilized by cutting muscle fibers. Oxotremorine (0.3-10 microM) depolarized endplate membranes, reduced miniature e.p.p. amplitudes but increased frequency, induced spontaneous neural discharges and muscle fasciculations, and produced contracture of denervated mouse diaphragm. In mouse and young rat preparations pretreated with Mn2+, Co2+, Ni2+, Cd2+ or low Ca2+ Tyrode to depress evoked acetylcholine release, oxotremorine 0.3-1 microM increased indirect twitches as well as amplitudes and quantal contents of e.p.p.s. These increases were not observed when the synaptic transmission was not depressed, nor in adult rat preparations. The augmentation by oxotremorine of evoked acetylcholine release persisted in preparations pretreated with neostigmine (1 microM) and tetrodotoxin (20 nM), which inhibited acetylcholinesterase and oxotremorine-induced spontaneous neural discharges. These effects of oxotremorine were mimicked by arecoline but not by muscarine and were antagonized by tubocurarine (0.3 microM) but not by atropine (0.1-10 microM). Atropine alone did not affect indirect twitches, synaptic transmission, tetanic responses evoked by direct stimulation of diaphragms, nor the durations of muscle action potential. The direct twitch responses were only slightly increased by oxotremorine at 2-3 microM. Oxotremorine at high concentrations (greater than 2 microM), depressed indirect twitches and e.p.p. amplitude, and accelerated the run-down of trains of e.p.p.s. The IC50 on indirect twitches was reduced by pretreatment with diltiazem or proadifen, which are known to promote receptor desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1987

Pharmacological evidence for the involvement of 1-(2-pyridinyl)-piperazine (1-PmP) in the interaction of buspirone or gepirone with noradrenergic systems.

We investigated in mice the effects of one of the principal metabolites of buspirone and gepirone, 1-(2-pyridinyl)-piperazine (1-PmP), on hypothermia and reduced locomotion induced by clonidine (0.25 and 0.06 mg/kg, respectively), tests related to brain alpha-adrenergic function. Both effects were antagonized dose dependently by 1-PmP (1-16 mg/kg i.p.). Moreover, pretreatment with proadifen (50 mg/kg) prevented the reversal by buspirone and gepirone of clonidine-induced hypothermia. This suggests that 1-PmP could be responsible for some of the apparent noradrenergic effects of buspirone and gepirone.

Animals↗
PubMed1991

Gonadectomy changes the pituitary-adrenocortical response in mice to 5-HT1A receptor agonists.

The effects of 5-HT1A receptor agonists such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), BP-554 and buspirone on the serum corticosterone level were significantly more pronounced in female than in male mice. A similar sex difference was observed for the effect of 8-OH-DPAT on the plasma ACTH level. Pretreatment with proadifen, an inhibitor of microsomal drug-metabolizing enzymes, did not affect the sex difference in the effect of 8-OH-DPAT. The corticosterone response to 8-OH-DPAT in female mice was attenuated by ovariectomy. The effect of 8-OH-DPAT in ovariectomized mice was enhanced by chronic estradiol and the enhancement was blocked by testosterone. In male mice the corticosterone response to 8-OH-DPAT increased at 5 weeks after castration but had not changed at 2 weeks. Chronic estradiol enhanced the corticosterone response to 8-OH-DPAT in castrated mice. There was no difference between sexes in [3H]8-OH-DPAT binding to membranes and in the contents of 5-HT and 5-hydroxyindoleacetic acid in the hypothalamus. Accumulation of 5-hydroxytryptophan after decarboxylase inhibition in the hypothalamus was however greater in female than in male mice.

5-Hydroxytryptophan↗
PubMed1995

Effects of cigarette smoke and disulfiram on tumorigenicity and clastogenicity of ethyl carbamate in mice.

Exposure of male Balb/C mice to mainstream cigarette smoke for 4 months, starting 10 or 30 days before the administration of ethyl carbamate (0.3% in drinking water for 3 weeks), resulted in an up to 57.6% (P < 0.05) decrease of lung adenoma multiplicity. However, the number of ethyl carbamate-induced lung tumors was not significantly affected by exposure to cigarette smoke when ethyl carbamate was injected i.p. in single doses of 0.5 or 1.0 g/kg, irrespective of the different treatment schedules used, i.e. (a) 10 days before and 4 days after the ethyl carbamate injection; (b) throughout the experiment starting 10 days before the ethyl carbamate injection, and (c) until the end of the experiment, starting 30 days after the ethyl carbamate injection. Disulfiram (500 mg/kg), given by gavage 24 h and 1 h before the ethyl carbamate injection, decreased by 88.5% (P < 0.001) the multiplicity of lung adenomas but had no effect on tumorigenesis when administered after the carcinogen injection. Proadifen (SKF-525 A, 50 mg/kg) injected i.p. 24 h and 1 h before and 24 h and 48 h after the injection with ethyl carbamate tended to decrease the multiplicity of lung adenomas, but not to a significant extent. Furthermore, disulfiram given 24 h and 1 h before the i.p. administration of ethyl carbamate completely prevented its clastogenicity in mouse bone marrow. On the other hand, cigarette smoke, which was per se a weak clastogen in bone marrow erythroblasts, synergistically potentiated the clastogenic response to ethyl carbamate in a more than additive fashion.

Animals↗
PubMed1992

Differential effects of cytochrome P-450 induction on ligand binding to sigma receptors.

The identity of the sigma receptor as a form of cytochrome P-450 was investigated in rats treated with 3-methylcholanthrene or phenobarbital. The density of [3H]N,N'-di(o-tolyl)guanidine (DTG) binding to sigma 2 receptors in hepatic subcellular fractions increased following both treatments, while [3H](+)-pentazocine binding to sigma 1 receptors was unchanged. Furthermore, proadifen and piperonyl butoxide inhibited [3H](+)-pentazocine and [3H]DTG binding with low potency. The low affinity of cytochrome P-450 inhibitors for sigma receptors, the similar degree of enhancement of [3H]DTG binding by agents with disparate cytochrome P-450 induction profiles and the lack of change in [3H](+)-pentazocine binding are inconsistent with the identity of the sigma receptor as a cytochrome P-450.

Animals↗
PubMed2004

Ca2+ influx is not involved in acute cytotoxicity of arachidonic acid.

Arachidonic acid (AA; 20:4, n-6) has been implicated in cell damage in the brain under ischemia-reperfusion and other pathological conditions. In our experiments, PC12 cells exposed to >10 microM AA died within 1-2 hr, as assessed by the LDH release assay. Since AA is known to induce Ca2+/cation-permeable conductance in the plasma membrane, we investigated whether Ca2+ influx plays a role in this acute cell death. We found that extracellular Ca2+ was not required for the toxic effect of AA. In fact, the removal of extracellular Ca2+ dramatically accelerated its development: the half-time of the toxic effect of 40 microM AA decreased from 70.1 +/- 0.3 min in the presence of 5 mM Ca2+ to 7.4 +/- 0.3 min in the Ca-free solution. The extent of cell killing depended only weakly on AA concentration and ion composition, remaining within the 70-95% range. The AA-induced acute death was not affected by inhibitors of AA metabolism (nordihydroguaiaretic acid, indomethacin, proadifen), whereas some antioxidants tested (deferoxamine and ellagic acid), but not all (melatonin), partially suppressed it. Also, it was not affected by changes in the extracellular ionic strength or mimicked by an acetylenic analog of AA 5,8,11,14-eicosatetraynoic acid. We conclude that lethal injuries sustained by cells during short exposures to AA were caused by the fatty acid itself and were not mediated by the AA-induced influx of Ca2+/cations. Moreover, direct physical effects of AA on the plasma membrane (changes in membrane fluidity or detergent-like action) were also excluded.

Animals↗
PubMed2004

Relaxant effect of oxime derivatives in isolated rat aorta: role of nitric oxide (NO) formation in smooth muscle.

Various oxime derivatives were evaluated as nitric oxide (NO) donors in arteries. Relaxation of rat aortic rings was used for bioassay of NO production, and electron paramagnetic resonance spectroscopy for demonstration of NO elevation. In rings with or without endothelium or adventitia, hydroxyguanidine and hydroxyurea were almost inactive, whereas formamidoxime, acetaldoxime, acetone oxime, acetohydroxamic acid and formaldoxime elicited relaxation. Active compounds increased NO levels in endothelium-denuded rings. Formaldoxime was the most potent agent for both relaxation and NO elevation in aortic rings, and it also increased NO in human aortic smooth muscle cells. In endothelium-denuded rings, relaxation was inhibited by a NO scavenger (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) and by inhibitors of soluble guanylyl-cyclase (1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one) or cyclic GMP-dependent protein kinases (Rp-8-bromo cyclic GMP monophosphorothioate). Neither N(omega)-nitro-l-arginine methylester (a NO synthases inhibitor) nor proadifen (a cytochrome P450 inhibitor) decreased the effect of oxime derivatives. However, 7-ethoxyresorufin (7-ER, an inhibitor of P4501A(1) which can also inhibit various NADPH-dependent reductases) abolished the relaxant effect of these compounds, without affecting the one of glyceryl trinitrate (GTN) or 2-(N,N-diethylamino)-diazenolate-2-oxide. 7-ER also abolished formaldoxime-induced NO increase in aortic rings. In rings tolerant to GTN, formaldoxime-induced relaxation and NO elevation were not different from those obtained in control rings. In conclusion, some oxime derivatives release NO by 7-ER-sensitive pathways in aortic smooth muscle, thus eliciting vasorelaxation. Pathways of NO formation are likely distinct from NO synthases and from those responsible for GTN biotransformation. Oxime derivatives could be useful for NO delivery in arteries in which endothelial NO synthase activity is impaired.

Animals↗
PubMed2006

Clozapine bioactivation induces dose-dependent, drug-specific toxicity of human bone marrow stromal cells: a potential in vitro system for the study of agranulocytosis.

Clozapine, an atypical antipsychotic drug effective in treatment of refractory schizophrenia causes potentially life-threatening agranulocytosis. The drug undergoes bioactivation to a toxic, chemically reactive intermediate with capacity to target stromal cells, central components of the bone marrow microenvironment implicated in neutrophil development. To identify possible mechanisms underpinning disruption of stroma as a site of drug bioactivation, toxicity was induced in vitro. Therefore metabolite generation procedures utilizing HOCl or HRP-H(2)O(2) as primary components involved in clozapine metabolism were adapted for stromal culture and coupled with viability determinations. Drug oxidation by HOCl was less toxic to stromal cells than HRP-H(2)O(2) based methods. More specifically, clozapine bioactivation by HRP-H(2)O(2) caused dose-dependent inhibition of stromal viability at therapeutically relevant concentrations. Differences in susceptibility of HAS303 and LP101 cells to the clozapine nitrenium ion were also evident. Stromal cell death was attributed to clozapine in the presence of a complete metabolising system comprising HRP and H(2)O(2). In the absence of a complete metabolising system clozapine was not cytotoxic. For LP101 cells, drug plus HRP (minus H(2)O(2)) also induced toxicity. Importantly, other antipsychotic drugs including risperidone, olanzapine and haloperidol when bioactivated, were not cytotoxic, indicating system specificity for clozapine. Exogenous GSH, N-acetylcysteine, l-ascorbic acid, catalase, and sodium azide afforded protection to cells whereas S-methylGSH, GSSG, ketoprofen and proadifen did not. Thus functional data derived from the in vitro stromal system defined in these studies may enable further investigation of the mechanisms subserving stromal impairment in clozapine-induced agranulocytosis and direct attention to improved methods for its prevention.

Agranulocytosis↗
PubMed1999

K(+) channel blockers and cytochrome P450 inhibitors on acetylcholine-induced, endothelium-dependent relaxation in rabbit mesenteric artery.

Acetylcholine caused an endothelium-dependent relaxation in isolated rabbit mesenteric small artery in the presence of nitro L-arginine and indomethacin. The acetylcholine-induced relaxation was attenuated by high K(+) solution, suggesting that the response is mediated by a membrane potential-sensitive mechanism, presumably an endothelium-derived hyperpolarizing factor. The acetylcholine-induced relaxation was also inhibited with tetraethylammonium, 4-aminopyridine and charybdotoxin, but not with Ba(2+), apamin, iberiotoxin nor glibenclamide. The relaxation was abolished by a combination of apamin and charybdotoxin, but iberiotoxin could not replace charybdotoxin in this combination. The responses to charybdotoxin and 4-aminopyridine were synergistic but neither apamin nor iberiotoxin increased the effect of 4-aminopyridine. Clotrimazole and proadifen inhibited the acetylcholine-induced relaxation, but these drugs also inhibited the cromakalim-induced relaxation, while protoporphyrin IX inhibited the acetylcholine- but not cromakalim-induced relaxation. 17-Octadecynoic acid and 1-aminobenzotriazole did not affect the response to acetylcholine. Four regioisomers of epoxyeicosatrienoic acids did not relax endothelium-denuded artery. A gap junction inhibitor 18alpha-glycyrrhetinic acid attenuated the relaxation to acetylcholine. It is suggested that in rabbit mesenteric artery, the acetylcholine-induced, nitric oxide- and prostacyclin-independent relaxation is mainly mediated by 4-aminopyridine- and charybdotoxin-sensitive K(+) channels and that the relaxation is not mediated through cytochrome P450 enzyme metabolites. The contribution of heterocellular gap junctional communication to the relaxation is discussed.

8,11,14-Eicosatrienoic Acid↗
PubMed1999

Ascorbate-mediated electron transfer in protein thiol oxidation in the endoplasmic reticulum.

Addition of, or gulonolactone oxidase-dependent in situ generation of, ascorbate provoked the oxidation of protein thiols, which was accompanied by ascorbate consumption in liver microsomal vesicles. The maximal rate of protein thiol oxidation was similar upon gulonolactone, ascorbate or dehydroascorbate addition. Cytochrome P450 inhibitors (econazole, proadifen, quercetin) decreased ascorbate consumption and the gulonolactone or ascorbate-stimulated thiol oxidation. The results demonstrate that the ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum, even in gulonolactone oxidase deficient species.

Animals↗
PubMed1999

Role of cytochrome P4501A in biotransformation of the potential anticancer drug oracin.

Metabolic fate of the potential anticancer drug oracin (I), was studied at microsomal level in rat using enzyme induction and inhibition. One of the main metabolites arising during incubation of hepatic microsomal fraction with oracin is 3-hydroxyoracin (III). Cytochromes P450 non-specific inhibitors (carbon monoxide, aminobenzotriazole, 1-benzylimidazole, proadifen hydrochloride, n-octylamine) diminished amount of III. Among several specific inducers of rat cytochromes P450 isoforms used, only 3-methylcholanthrene, inducer of cytochrome P4501A, caused a significant stimulation of 3-hydroxyoracin production. The amount of III was decreased to the level of controls when the microsomes prepared from 3-methylcholanthrene treated rats were incubated with substrate in the presence of specific P4501A inhibitor alpha-naphthoflavone. From the above mentioned results we can assume that metabolite III is formed from oracin by cytochrome P450 belonging to subfamily 1A.

Aniline Hydroxylase↗
PubMed1979

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions.

Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.

Acetylcholine↗
PubMed1991

Structural effects on the binding of amine drugs with the diphenylmethyl functionality to cyclodextrins. I. A microcalorimetric study.

Solution calorimetry has been employed to evaluate the stability constants and enthalpy changes associated with complex formation between alpha-, beta, or gamma-cyclodextrin (CD) and a group of amine compounds having the diphenylmethyl functionality. Data from thermal titrations of the compounds were analyzed using nonlinear least squares. The standard free energy decrease accompanying the formation of inclusion complexes is generally due to a negative standard enthalpy change (delta H degrees). The standard entropy change (delta S degrees) was negative, except in the case of complexes formed with gamma-CD. Of the 13 compounds studied, only 2 formed complexes with 1:2 (compound:beta-CD) stoichiometry, terfenadine . HCl and cinnarizine . 2HCl. All the others formed 1:1 complexes. The structural effect on the stability constants, thermodynamics, and inclusion geometry was explored by relating the calorimetric results to the chemical structures of the guest molecules and the cavity sizes of the CD molecules. The results suggest that one of the phenyl groups of the diphenylmethyl functionality resides in the CD cavity and is in van der Waals contact with the inside wall of the CD cavity. In the case of alpha- and beta-CDs, van der Waals interaction dominates in the stabilization. On the other hand, the interaction between these compounds and gamma-CD is largely entropically driven. Adiphenine . HCl forms a more stable complex with beta-CD than proadifen . HCl, suggesting that hydrogen bonding to the carbonyl oxygen by the hydroxyl group on the rim of the CD ring can influence the strength of the binding interaction.

Calorimetry↗
PubMed1992

Platelet activating factor inhibits Cl and K transport in the medullary thick ascending limb.

Since the kidney medulla was reported to generate platelet activating factor (PAF), we investigated a possible effect of this agent on the reabsorptive function of in vitro microperfused medullary thick ascending limbs from mouse kidney (mTAL). PAF, 10(-7) M in the bath, significantly decreased the net chloride flux (JCl) from 48.8 +/- 7.1 to 27.4 +/- 5.7 pmol/min. This effect was reversible, blocked by the antagonist BN 50730, and not reproduced by the inactive metabolite lyso-PAF. PAF inhibited the transepithelial potential difference with a threshold at 10(-9) M. In the presence of isoproterenol, the PAF-induced decrease of JCl was not significantly different from that observed in basal conditions; moreover, PAF did not modify the adenylate cyclase activity in isolated mTALs, either in basal condition or under stimulation by isoproterenol. The effect of PAF on JCl was not prevented by mepacrine, NDGA associated with proadifen, or adenosine desaminase. When the apical Na-K-2Cl cotransport was blocked by furosemide or bumetanide, a net K secretion occurred (-1.1 +/- 0.2 pmol/min), which was significantly decreased by PAF (-0.06 +/- 0.3 pmol/min). Moreover, it was verified on isolated mTALs that PAF did not modify the Na,K-ATPase activity. It is concluded that PAF inhibits the reabsorptive function of the mTAL, as indicated by the decrease of Cl reabsorption and K secretion. This effect could not be accounted for by adenosine or arachidonic acid metabolite action, and was not mediated by an inhibition of the adenylate cyclase activity.

Absorption↗
PubMed1997

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells.

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca2+ store depletion and Ca2+ entry was studied further. 2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca2+ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody. 3. Depletion of cellular Ca2+ pools with inositol 1,4,5-trisphosphate (IP3), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca2+ store depletion, chelating of intramicrosomal Ca2+ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca2+ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP3-sensitive stores. 4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca2+ entry pathways, membrane hyperpolarization due to K+ channel activation or tyrosine kinase activity. 5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca2+/Mn2+ entry to Ca2+ store depletion by 43%, while it did not interfere with intracellular Ca2+ release by IP3 or thapsigargin. 6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization. 7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca2+/Mn2+ entry (by 105%) and membrane hyperpolarization (by 40%). 8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium. 9. These data demonstrate that endothelial Cyp450 MO is activated by Ca2+ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells. 10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca2+ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca2+ entry activation and increase the driving force for Ca2+ entry by membrane hyperpolarization in porcine aortic endothelial cells.

8,11,14-Eicosatrienoic Acid↗
PubMed1998

Enhanced acetylcholine induced relaxation in small mesenteric arteries from pregnant rats: an important role for endothelium-derived hyperpolarizing factor (EDHF).

1. Small mesenteric arteries from pregnant rats demonstrated greater sensitivity (pEC50 : P<0.001) and maximum relaxation (P<0.01) to acetylcholine (ACh) than those of control non-pregnant animals. 2. Maximum relaxation, but not sensitivity, to ACh remained greater (P<0.01) in pregnant animals when evaluated in 25 mM KCl, which prevents relaxation dependent upon hyperpolarization. ACh induced relaxation in the presence of 25 mM KCl was completely inhibited in pregnant and non-pregnant groups by N(omega)-nitro L-arginine methyl ester (L-NAME, 100 microM), indomethacin (INDO, 10 microM) and oxadiazole quinoxalin (ODQ, 1 microM), suggesting pregnancy associated enhancement of dilator prostanoid and/or nitric oxide (NO) synthesis. 3. ACh induced relaxation in 5 mM KCI was only partially inhibited by a combination of N(omega)-nitro L-arginine methyl ester (L-NAME, 100 microM), indomethacin (INDO, 10 microM) and oxadiazole quinoxalin (ODQ, 1 microM). The residual relaxation, which was greater in arteries from pregnant rats (maximum relaxation: P<0.01), was prevented by 25 mM KCl, indicating pregnancy associated enhanced synthesis/ reduced degradation of a hyperpolarizing factor. Residual relaxation to ACh in 5 mM KCl was inhibited by the cytochrome P450 inhibitor, proadifen (1 microM) in the pregnant group (P<0.001). 4. Relaxation to spermine NONOate was similar in pregnant and non-pregnant groups and totally inhibited by ODQ (in the presence of L-NAME). 5. This study suggests that, in addition to enhanced endothelium dependent NO/dilator prostanoid synthesis, a hyperpolarizing factor may contribute to the vascular adaptation to pregnancy.

Acetylcholine↗
PubMed1999

Involvement of CYP3A-derived arachidonic acid metabolite(s) in responses to endothelium-derived K+ channel opening substance in monkey lingual artery.

1. In monkey lingual artery strips partially contracted with prostaglandin F2alpha, acetylcholine-induced, concentration-related relaxations were abolished by removal of the endothelium. The response was not significantly influenced by indomethacin but attenuated by NG-nitro-L-arginine (L-NOARG); the effect of the nitric oxide (NO) synthase inhibitor was reversed by L-arginine. 2. The response to acetylcholine resistant to L-NOARG was suppressed in the strips exposed to high K+ media. Charybdotoxin partially inhibited the relaxation, and the remaining relaxation was abolished by additional treatment with apamin, whereas glibenclamide, iberiotoxin or apamin alone was without effect. Relaxations induced by sodium nitroprusside were not influenced by charybdotoxin. 3. The L-NOARG-resistant acetylcholine-induced relaxation was inhibited by metyrapone, proadifen and 17-octadecynoic acid, non-selective cytochrome P450 mono-oxygenase (CYP) inhibitors, and progesterone and ketoconazole, inhibitors selective to CYP3A. The inhibitors did not affect the nitroprusside-induced relaxation. Selective inhibitors of other CYP isoforms, such as debrisoquine and lauric acid, did not reduce the response to acetylcholine. 4. Reaction mixture containing human liver microsome rich in CYPs, arachidonic acid and NADPH incubated at 37 degrees C and filtrated relaxed endothelium-denuded monkey lingual artery strips, used as bioassay tissues. This response was abolished in the strips exposed to high K+ media. The response was also suppressed by combined treatment of the assay tissue with charybdotoxin plus apamin, but was not affected by treatment with iberiotoxin. The reaction mixture co-incubated with ketoconazole failed to relax the strips. 5. It is concluded that the monkey lingual arterial relaxation dependent on the endothelium is mediated by NO and also by a charybdotoxin plus apamin-sensitive but iberiotoxin-insensitive Ca2+-activated K+ channel opening substance(s) that may be a CYP3A-derived arachidonic acid metabolite(s).

Animals↗
PubMed2001

Bradykinin attenuates the [Ca(2+)](i) response to angiotensin II of renal juxtamedullary efferent arterioles via an EDHF.

1. Bradykinin (BK) effect on the [Ca(2+)](i) response to 1 nM angiotensin II was examined in muscular juxtamedullary efferent arterioles (EA) of rat kidney. 2. BK (10 nM) applied during the angiotensin II-stimulated [Ca(2+)](i) increase, induced a [Ca(2+)](i) drop (73+/-2%). This drop was prevented by de-endothelialization and suppressed by HOE 140, a B2 receptor antagonist. It was neither affected by L-NAME or indomethacin, nor mimicked by sodium nitroprusside, 8-bromo-cyclic GMP or PGI(2). The BK effect did not occur when the [Ca(2+)](i) increase was caused by 100 mM KCl-induced membrane depolarization and was abolished by 0.1 microM charybdotoxin, a K(+) channel blocker. 3. Although proadifen prevented the BK-caused [Ca(2+)](i) fall, more selective cytochrome P450 inhibitors, 17-octadecynoic acid (50 microM) and 7-ethoxyresorufin (10 microM) were without effect. 4. Increasing extracellular potassium from 5 to 15 mM during angiotensin II stimulation caused a [Ca(2+)](i) decrease (26+/-4%) smaller than BK which was charybdotoxin-insensitive. Inhibition of inward rectifying K(+) channels by 30 microM BaCl(2) and/or of Na(+)/K(+) ATPase by 1 mM ouabain abolished the [Ca(2+)](i) decrease elicited by potassium but not by BK. 5. A voltage-operated calcium channel blocker, nifedipine (1 microM) did not prevent the BK effect but reduced the [Ca(2+)](i) drop. 6. These results indicate that the BK-induced [Ca(2+)](i) decrease in angiotensin II-stimulated muscular EA is mediated by an EDHF which activates charybdotoxin-sensitive K(+) channels. In these vessels, EDHF seems to be neither a cytochrome P450-derived arachidonic acid metabolite nor K(+) itself. The closure of voltage-operated calcium channels is not the only cellular mechanism involved in this EDHF-mediated [Ca(2+)](i) decrease.

Angiotensin II↗