Molecular cloning, genomic characterization and expression of novel human alpha1A-adrenoceptor isoforms.

We have isolated and characterized from human prostate novel splice variants of the human alpha1A-adrenoceptor, several of which generate truncated products and one isoform, alpha(1A-4), which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the alpha(1A-4) exon is located between those encoding the alpha(1A-1) and alpha(1A-3) variants. CHO-K1 cells stably expressing alpha(1A-4) showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that alpha(1A-4) is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Effects of K+ on the interaction between cardiac glycosides and Na,K-ATPase.

Inhibition of Na,K-ATPase by cardiac glycosides is at least partially antagonized by K+. The kinetics of the antagonism, however, appear complicated because K+ is capable of reducing both association and dissociation rate constants for the glycoside-enzyme interaction. In order to better understand the effect of K+, inhibition of partially purified Na,K-ATPase obtained from rat brain, guinea-pig heart and rat heart by ouabain, digoxin, digoxigenin, dihydrodigoxin and cassaine were compared in the presence of 1, 3 or 10 mM K+. Higher concentrations of K+ caused a parallel shift to the right in the concentration-inhibition curves for these compounds. For ouabain or digoxin, the extent of the shift was minimal with rat brain enzyme, intermediate with guinea-pig heart enzyme and more substantial with rat heart enzyme. For digoxigenin, dihydrodigoxin or cassaine, the extent of the shift was substantial in all enzyme preparations. These results could not be explained from either the affinity of the enzyme for the compound or its lipid solubility alone. The concentrations of these compounds required to cause a 50 percent inhibition of enzyme activity were markedly different with rat brain enzyme, but relatively similar with rat heart enzyme. The effects of K+, which depend on the source of the enzyme and chemical structures of the compounds, have to be considered in studies on comparative effects of various compounds on Na,K-ATPase, [3H]ouabain binding, sodium pumping and the force of myocardial contraction.

Possible mechanisms for inotropic actions of vanadate in isolated guinea pig and rat heart preparations.

The mechanism of inotropic actions of vanadate studied in isolated, electrically stimulated atrial and ventricular muscle preparations of rat or guinea-pig heart. Vanadate produced a negative inotropic effect in guinea-pig left atrial preparations associated with a marked shortening of the action potential plateau. In guinea-pig papillary muscle, or rat atrial or ventricular muscle preparations, vanadate produced a positive inotropic effect, which was not affected by either propranolol, phentolamine or metiamide. The positive inotropic effect was observed when action potential duration was either increased or decreased. Inotropic concentrations of vanadate failed to significantly alter the ouabain-sensitive 86Rb+-uptake, an estimate of sodium pump activity, or tissue concentration of cyclic AMP in electrically stimulated preparations. In partially depolarized rat atrial preparations in which fast sodium channels were inactivated in the presence of a high concentration of K+ (22 mmol/l), vanadate restored electrical activity (calcium-dependent action potentials) and the contraction, similar to isoproterenol. This action of vanadate was abolished by Mn2+, a slow channel inhibitor, but not by tetrodotoxin. The characteristic of vanadate- and isoproterenol-restored preparations, however, were substantially different. Moreover, vanadate failed to restore the contraction or action potential in partially depolarized guinea pig atrial preparations unlike isoproterenol. These results indicate that vanadate may either enhance or inhibit slow channels in cardiac muscle.