Verapamil Derivatives Research Articles

Effects of temperature and allosteric modulators on [3H] nitrendipine binding: methods for detecting potential Ca2+ channel blockers.

The effects of incubation temperature and allosteric modulators were studied on [3H]nitrendipine binding to guinea-pig cardiac membranes. Incubation temperature only slightly affected the ability of nifedipine and verapamil derivatives to inhibit binding. By contrast, the Ca2+ channel blockers d-cis-diltiazem and fostedil (KB-944) stimulated [3H]nitrendipine binding in a temperature-dependent manner (37 degrees greater than 25 degrees greater than 4 degrees C). The stimulatory effect of fostedil could be related to a decrease (2.3-fold at 37 degrees C) in the rate of radioligand binding site dissociation, without significant effects on association kinetics. Both fostedil and d-cis-diltiazem caused a shift to the right of the concentration-inhibition curve of tiapamil, a negative allosteric modulator of [3H]nitrendipine binding. Neither compound affected the ability of nifedipine, a competitive antagonist, to inhibit radioligand binding. This selective effect of fostedil or d-cis-diltiazem may be useful for testing whether potential Ca2+ channel blockers interact in a competitive as opposed to allosteric manner with the dihydropyridine site. Varying the incubation temperature may also be useful in detecting compounds which act as positive allosteric modulators (stimulators) of dihydropyridine binding.

Calcium transport by primary cultured neuronal and glial cells from chick embryo brain.

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca2+ and 5.4 mM-K+. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 mM. The effect of high external potassium was to increase the Vmax value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl2, but 0.5 microM-tetrodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.

Investigations on the structure-activity relationships of verapamil.

An investigation was carried out towards a qualitative and quantitative structure-activity relationship of verapamil based on an analysis of the frequency-dependent negative inotropic action exerted in cat papillary muscles by various groups of verapamil derivatives. (1) Substituents of the benzene ring near the asymmetric carbon atom and the isopropyl group were found to be no essential substituents for the frequency-dependent negative inotropic action of verapamil but to have a strong influence on the potency of the drug. (2) Both the tertiary amino nitrogen and the two benzene rings are essential for the frequency-dependent negative inotropic action of verapamil (3) The molecular importance of the N-methyl group is probably based on steric effects. (4) Investigations of the verapamil derivative H 1 revealed that a quaternization of the drug is followed by a total loss of effectiveness. (5) No significant correlation of the biological activity of verapamil derivatives with the partition coefficient P has been obtained. (6) Hansch analysis with verapamil derivatives of group A (= different substitution of the benzene ring near C) shows that the variance of biological activity can be optimally correlated to a combination of the Hammett constant and the molar volume. Hansch analysis of group B (= exchange of isopropyl group) led to the conclusion that hydrophobic effects are responsible for the influence of the isopropyl substituent.

CALCIUM AND STIMULUS-SECRETION COUPLING IN THE NEUROHYPOPHYSIS

ABSTRACT Groups of 10 hemilobes of rat neurohypophyses were stimulated for 10 min in a medium containing 56 mm K+ or by application of a field current (20 Hz, 2 msec, 40 mA). Both stimulations resulted in a 5 to 8 fold increase in the release of vasopressin which was inhibited (in a dose-related manner) by addition to the medium of various blockers of calcium transport: LaCl3 (4 × 10−4, 4 × 10−3 m/l), prenylamine (2 × 10−5, 1 × 10−4 m/l) and D600, a verapamil derivative (4 × 10−6, 1 × 10−5, 2 × 10−5 m/l). Increasing the Ca2+ concentration in the medium to 14 mm abolished the inhibitory effect of D600. D600 also inhibited vasopressin release caused by introducing Ca2+ into a Ca2+-free medium during continued electrical stimulation. Efflux of 45Ca2+ from neural hemilobes labelled during incubation in a 56 mm K+ containing medium was markedly increased during stimulation with a 56 mm K+ medium or electrical current. Lanthanum (4 × 10−3 m/) and D600 (2 × 10−5 m) inhibited this increased efflux as well as hormone release. When the hemilobes had been pre-labelled in a medium with 4.8 mm K+, stimulation during efflux, by 56 mm K+ or electrical current, caused hormone release comparable to that seen after labelling in 56 mm K+, but with neither type of stimulation was there any significant increase in 45Ca2+ efflux. These findings would appear to be in agreement with a hypothesis that very small amounts of Ca2+ enter the endings of the neurosecretory neurons on stimulation (thereafter possibly releasing intracellular calcium) and that calcium entry is essential in inducing hormone release. The uptake of calcium over the neurosecretory nerve membrane apparently has several of the characteristics shown for calcium entry on stimulation of the squid axon.