Verapamil interaction with the muscarinic receptor: stereoselectivity at two sites.

Verapamil, in addition to blocking calcium channels, exhibits such "non-specific" effects on myocardium as inhibition of sodium and potassium conductances and modifications of muscarinic receptor-ligand interactions. To characterize further the effects of verapamil on the cardiac muscarinic receptor, we examined the abilities of the enantiomers of verapamil to modify the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) to purified canine sarcolemmal vesicles. Membranes were incubated with [3H]QNB and various concentrations of racemic, (+)-, or (-)- verapamil (25 or 37 degrees, pH 7.4), and reactions were terminated by rapid filtration. (-)-Verapamil (Ki of 5.3 +/- 0.2 microM) was twice as potent an inhibitor of equilibrium binding as (+)-verapamil (Ki of 11.4 +/- 0.6 microM), and this effect resulted from the ability of each enantiomer to slow [3H]QNB-receptor association. This degree of stereoselectivity, albeit at nanomolar concentrations, was similar to that observed for each enantiomer to compete for the specific phenylalkylamine site in this preparation. Verapamil also inhibited [3H]QNB-receptor dissociation, but this effect required high concentrations and demonstrated stereoselectivity opposite to that observed for association. These findings support the view that verapamil interacts with two distinct sites, possibly within membrane lipid, each with a different affinity and preference for (+)- and (-)-verapamil, to modify the muscarinic receptor.

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells.

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.

Thyrotropin-releasing hormone causes loss of cellular calcium without calcium uptake by rat pituitary cells in culture. Studies using arsenazo III for direct measurement of calcium.

Thyrotropin-releasing hormone (TRH) may act to stimulate prolactin secretion by increasing the intracellular free Ca2+ concentration. This notion is supported by the finding that TRH acutely enhances 45Ca2+ efflux from pituitary cells which may reflect alterations in Ca2+ influx or efflux, or both. To differentiate among these possibilities, we measured loss and uptake of nonradioactive Ca2+ by GH3 cells, a cloned strain of rat pituitary cells that produce prolactin, during TRH action using the metallochromic indicator arsenazo III. Cells were perfused in medium containing 2.8 microM Ca2+ and nonradioactive Ca2+ was measured in the perfusion effluent. Under these conditions, there was a sustained loss of Ca2+ from the cells for at least 30 min. TRH caused a transient, marked increase in the amount of Ca2+ released into the medium which occurred in parallel with enhancement in 45Ca2+ efflux and stimulation of prolactin secretion. There was no measurable decrease in Ca2+ concentration in the medium at the onset of the TRH effect which would have been consistent with Ca2+ influx into the cells. Furthermore, an identical response to TRH was observed in cells perfused with medium containing 50 microM verapamil, an agent which blocks Ca2+ influx. In static incubations performed in parallel, TRH caused a decrease in total cellular Ca2+ of 23 +/- 5%. These data provide direct evidence that TRH causes loss of Ca2+ from GH3 cells without causing measurable Ca2+ uptake and support the contention that TRH acts by mobilizing Ca2+ from a sequestered cellular pool (or pools).