dexniguldipine and chlorethylclonidine

Canine atrial cardiomyocytes display a constitutively active, acetylcholine-regulated, time-dependent K+ current (IKH) that contributes to atrial repolarization and atrial tachycardia-induced atrial-fibrillation promotion. Adrenergic stimulation favors atrial arrhythmogenesis but its effects on IKH are poorly understood.. Adrenergic modulation of IKH was studied in isolated canine atrial cardiomyocytes with whole-cell patch-clamping, and action-potential consequences were assessed in multicellular preparations with fine-tipped microelectrodes. Isoproterenol increased IKH in a concentration-dependent manner (maximum 103+/-22% increase), an effect mimicked by forskolin and 8-bromo-cyclic AMP. Isoproterenol effects were prevented by propranolol and the selective beta1-adrenoceptor blocker CGP-20712A, but not the beta2-blocker ICI-118551. Isoproterenol enhancement was prevented by pipette-administered protein kinase A (PKA) inhibitor peptide or by superfusion of H89 (PKA blocker). Phenylephrine decreased IKH in a reversible, concentration-dependent way. This effect was blocked by the alpha-antagonist prazosin and the selective alpha1A-blocker niguldipine, but not the alpha1B-blocker chloroethylclonidine or the alpha1D inhibitor BMY-7378. Phenylephrine effects were prevented by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor bisindolylmaleimide. The PKC-activating phorbol ester PDD (but not its inactive analogue alpha-PDD) mimicked phenylephrine effects. Action potential recordings in the presence and absence of the selective IKH blocker tertiapin indicated a functional role of alpha- and beta-adrenergic actions on IKH. Adrenergic regulation of cholinergic agonist-induced K+ current paralleled that of IKH.. IKH is under dual regulation by the adrenergic system: beta1-adrenergic stimulation enhances IKH via cAMP-dependent PKA pathways, whereas alpha1A-adrenergic stimulation inhibits IKH via PLC-mediated PKC activation. Modulation of constitutive acetylcholine-regulated K+ current is a novel potential mechanism for adrenergic control of atrial repolarization.

Topics: Acetylcholine; Action Potentials; Adrenergic Agents; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Bee Venoms; Clonidine; Cyclic AMP-Dependent Protein Kinases; Dihydropyridines; Dogs; Dose-Response Relationship, Drug; Estrenes; Heart Atria; Imidazoles; Indoles; Isoproterenol; Isoquinolines; Maleimides; Myocytes, Cardiac; Patch-Clamp Techniques; Phenylephrine; Phorbol 12,13-Dibutyrate; Piperazines; Potassium Channels; Prazosin; Propanolamines; Propranolol; Protein Kinase C; Pyrrolidinones; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, beta-1; Sulfonamides; Type C Phospholipases

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.

Topics: Adrenergic alpha-Antagonists; Astrocytoma; Calcium; Cell Membrane; Clonidine; Dihydropyridines; Dioxanes; Dose-Response Relationship, Drug; Humans; Inositol Phosphates; Norepinephrine; Phenylephrine; Piperazines; Prazosin; Receptors, Adrenergic, alpha-1; Thymidine; Tumor Cells, Cultured

The aim of this study was to assess the contribution of alpha 1-adrenoceptor subtypes to noradrenaline (NA)-induced inositol phosphate formation in rat striatum. In cross-chopped slices and in the presence of 10 mM LiCl, NA stimulated the accumulation of [3H]inositol phosphates. After 60-min incubation with 100 microM NA, [3H]IP1 was the major product detected (82 +/- 3% of total [3H]inositol phosphates). Best-fit values for the concentration-response curve for NA-induced [3H]IP1 accumulation yielded an EC50 of 9.4 +/- 1.1 microM, maximum effect of 210 +/- 3% of basal, and Hill coefficient (nH) of 1.1 +/- 0.1. Pre-treatment of the slices for 30 min with the alkylating agent chloroethylclonidine (100 microM) failed to decrease significantly the response to 100 microM NA. Inhibition curves for four alpha 1-antagonists, (+)-niguldipine, prazosin, WB-4101 and 5-methylurapidil (5-MU), best-fit to a single-site model with pKi values of 9.4 ((+)-niguldipine), 9.2 (prazosin and WB-4101) and 8.8 (5-MU). The putative alpha 1 D-selective antagonist BMY 7378 reduced the response to NA only partially (30 +/- 3% inhibition at 1 microM: pKi 7.24). NA-induced [3H]IP1 accumulation was significantly reduced (to 20 +/- 9% of controls) by Ca2+ removal and increased as the extracellular Ca2+ concentration was raised from nominally zero (no added Ca2+) to 1 mM Ca2+. NA-induced [3H]IP1 accumulation was reduced by both the non-selective Ca2+ channel blocker Ni2+ (58 +/- 3% inhibition at 2 mM) and nimodipine, an antagonist of L-type voltage-operated Ca2+ channels (77 +/- 4% inhibition at 3 microM). Taken together these results indicate that NA-induced inositol phosphate formation in striatal slices is mediated by activation of alpha 1A-adrenoceptors coupled to Ca2+ entry and Ca2+ activation of phospholipase C.

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Alkylating Agents; Animals; Calcium; Calcium Channel Blockers; Clonidine; Dihydropyridines; Dioxanes; In Vitro Techniques; Inositol Phosphates; Male; Neostriatum; Norepinephrine; Piperazines; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1

Recent evidences from molecular biology, radioreceptor binding and functional studies indicate that the alpha 1-adrenoceptor population is heterogeneous and can at least be divided into two subclasses named alpha 1A and alpha 1B. The present study was designed to obtain, a selective enrichment of rat brain cortical membranes with each subtype of alpha 1-adrenoceptor using alkylating agents. [3H]prazosin binding to rat cortical membranes was saturable and of high affinity (KD = 0.11 +/- 0.02 nM; Bmax = 132.5 +/- 7.2 fmol/mg protein). All ligands competed for specific [3H]prazosin binding in a statistically significant biphasic manner (%Rhigh = 30-40%; %Rlow = 60-70%). These sites meet generally accepted and recently described pharmacologic criteria for their identification as the alpha 1A- and alpha 1B-adrenoceptors. After pretreatment of membranes with benextramine (1 microM) in the presence of clonidine (1 microM), the antagonists, WB4101, (+)-niguldipine and phentolamine, displaced the radioligand with an inhibition curve steeper than in control membranes and with Ki values that agree with those obtained for the low affinity site present in control membranes. On the other hand, after pretreatment with chloroethylclonidine (10 microM) in the presence of WB4101 (1 nM), Hill coefficients for the displacement of the radioligand by WB4101, (+)-niguldipine, and phentolamine, were also increased, but in contrast to the situation described above, the Ki values agree with those obtained for the high affinity site present in control membranes. In conclusion, this method of partial alkylation of receptors could be a valuable tool for separately studying the pharmacological characteristics of the alpha 1-adrenoceptor subtypes in native membranes of cerebral tissue.

Topics: Adrenergic alpha-Antagonists; Alkylating Agents; Alkylation; Animals; Binding Sites; Cerebral Cortex; Clonidine; Cystamine; Dihydropyridines; Dioxanes; Phenoxybenzamine; Phentolamine; Prazosin; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha

The alpha 1-adrenoceptors present in liver membranes from rats, hamsters and mice were characterized using [3H]prazosin. In the liver membranes from the three species a relatively large number of receptors was observed (500-900 fmol/mg of protein) and the affinities for [3H]prazosin were very similar (0.2-0.3 nM). Membrane preincubation with 10 microM chloroethylclonidine markedly decreased [3H]prazosin binding and higher concentrations essentially abolished specific binding of this radioligand. Binding competition experiments indicated the following orders of potency: a) for agonists: oxymetazoline > epinephrine > or = norepinephrine >> methoxamine and b) for antagonists: prazosin > WB 4101 > or = phentolamine = benoxathian > 5-methyl urapidil. The affinity for (+)niguldipine was also low but there was variation between the three species. Total RNA obtained from the liver of these species hybridized with the alpha 1B-adrenergic cDNA probe. The data suggest that these receptors correspond to the alpha 1B subtype.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Binding Sites; Binding, Competitive; Cell Membrane; Clonidine; Cricetinae; Dihydropyridines; Liver; Male; Mesocricetus; Mice; Prazosin; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Species Specificity

(+)-Niguldipine inhibited specific 125I-BE 2254 binding more potently in membrane preparations from rat tissues enriched in the alpha 1A subtype (hippocampus and vas deferens) than those with the alpha 1B subtype (liver and spleen). Inhibition curves for (+)-niguldipine were better fit by a two-site model in most tissues, although Kl values for each site varied markedly between tissues. The potency of this lipophilic drug was highly dependent on tissue concentration, probably accounting for most of this variability. Pretreatment of membranes with chloroethylclonidine (CEC) to inactivate the alpha 1B subtype did not completely eliminate the low affinity sites for (+)-niguldipine, particularly in heart. Saturation analysis showed that (+)-niguldipine competitively inhibited both alpha 1A and alpha 1B subtypes. However, substantial non-competitive inhibition was also observed in several tissues. Analysis of inhibition curves for 5-methylurapidil gave similar proportions of alpha 1A and alpha 1B receptor sites as were calculated for (+)-niguldipine in various tissues. Although (+)-niguldipine and 5-methylurapidil revealed variable proportions of low affinity sites in CEC-pretreated hippocampus and heart, this was not observed with inhibition curves for WB 4101 and phentolamine. These results are generally consistent with the previously defined alpha 1A and alpha 1B subtypes. 5-Methylurapidil currently appears to be the best antagonist for discriminating these subtypes; (+)-niguldipine shows similar selectivity but is complicated by a high lipophilicity. However, the persistence of low affinity sites for 5-methylurapidil and (+)-niguldipine after CEC pretreatment and the noncompetitive effects of (+)-niguldipine in some tissues raise the possibility of an additional subtype(s) of alpha 1-adrenergic receptors in rat tissues.

Topics: Adrenergic alpha-Antagonists; Animals; Binding Sites; Binding, Competitive; Calcium Channel Blockers; Clonidine; Dihydropyridines; Dioxanes; Kinetics; Male; Membranes; Phentolamine; Piperazines; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Time Factors

In rat ventricular myocytes, the effects of alpha adrenoceptor stimulation on outward currents were studied by means of the whole cell voltage-clamp technique. Phenylephrine (30 microM) in the presence of propranolol (1 microM) to block beta adrenoceptors reduced voltage-activated transient outward current. Both components of transient outward current were affected, i.e., peak current (Ipeak) was reduced by 25.3 +/- 1.8%, the outward current at the end of a clamp step (Ilate) was reduced by 39.1 +/- 3.5% (n = 5; holding potential -40 mV, clamp step to +20 mV). In order to describe the alpha-1 adrenoceptor subtypes involved in this action, the effect of phenylephrine was also investigated after pretreatment of the cells with various antagonists. Pretreatment with prazosin (0.3 microM) abolished completely the phenylephrine effect. The alpha-1A adrenoceptor subtype-selective antagonists 5-methylurapidil and (+)-niguldipine (0.1 microM each) and the irreversible alpha-1B adrenoceptor subtype antagonist chloroethyl-clonidine (100 microM) blocked the phenylephrine effect on Ipeak, but merely attenuated the effect on Ilate, whereas pretreatment with a combination of chloroethylclonidine and (+)-niguldipine suppressed the phenylephrine-induced effect on both outward current components just like prazosin did. In conclusion, stimulation of both adrenoceptor subtypes is required for reduction of Ipeak, but stimulation of either alpha-1A or alpha-1B subtype is sufficient for reduction of Ilate. Therefore, stimulation of both alpha-1 adrenoceptor subtypes contributes to the phenylephrine-induced reduction in transient outward currents of isolated rat myocytes.

Topics: Adrenergic alpha-Antagonists; Animals; Clonidine; Dihydropyridines; Electrophysiology; Heart; Membrane Potentials; Myocardium; Phenylephrine; Piperazines; Propranolol; Rats; Receptors, Adrenergic, alpha; Stimulation, Chemical