An antibody to dihydropyridine calcium entry blockers. A comparison with the calcium channel receptor in skeletal muscle.

Antibodies that recognize dihydropyridine (DHP) calcium entry blockers were elicited from rabbits. A sensitive and specific radioimmunoassay for dihydropyridines was developed and its specificity compared to the DHP binding site in skeletal muscle membranes. The antibody bound [3H]nitrendipine with a higher affinity (KD = 0.155 nM) than did the DHP receptor of skeletal muscle (KD = 1-3 mM); however, in contrast to the DHP receptor, the antibody recognized only those DHP drugs with meta-nitrophenyl substituents at the 4-position on the DHP ring. Both the antibody and receptor exhibited stereospecificity, with each site recognizing the (+)-isomer of nicardipine as the more potent. This antibody should prove useful in our studies of some potentially irreversible DHP molecules.

Effects of dihydropyridine calcium channel blocking drugs on rat brain muscarinic and alpha-adrenergic receptors.

The dihydropyridine (DHP) Ca2+ channel blocking drugs nicardipine, nitrendipine, nimodipine, felodipine, nifedipine and nisoldipine were examined for activity in inhibiting specific (-)-[3H] QNB and [3H]WB4101 binding to the muscarinic and alpha-adrenergic receptors, respectively, of rat brain. Muscarinic receptor binding was affected most by nicardipine, with felodipine having less activity; the other DHP drugs were essentially inactive at 3 X 10(-5) M. The (+)-stereoisomer nicardipine (KI = 4.07 X 10(-7) M) was 27 times more potent than the (-)-isomer in inhibiting [3H]QNB binding, and this inhibition was found to be competitive. This inhibitory effect of nicardipine was not mediated via interaction with the high-affinity DHP binding site assumed to be associated with a Ca2+ channel. (+)-Nicardipine inhibited the binding of [3H]nitrendipine to this DHP binding site of brain, with a K1 of 9.01 X 10(-11) M, and was 10 times more potent than the (-)-isomer. Thus, the muscarinic receptor was 4200 times less sensitive to (+)-nicardipine than was this DHP binding site. Nicardipine was also the most potent DHP drug inhibiting [3H]WB4104 binding to the alpha-adrenergic receptor, although the other drugs were also somewhat active, in the rank order sequence listed above. This effect of nicardipine on the adrenergic receptor was also stereoselective, with (+)-nicardipine (KI = 3.46 X 10(-7) M) being about 3 times more potent than the (-)-isomer, in producing competitive inhibition of radioligand binding. These data suggest that the effects on brain receptors occur as a result of direct, stereospecific effects of DHP drugs on these receptors and are not due to Ca2+ channel blocking activity of these drugs.

Calmodulin inhibition of brain membrane phosphorylation.

Calmodulin has been found to inhibit the phosphorylation of rat brain membrane proteins of molecular weight 14,900-18,900 in a dose-dependent manner. This phenomenon was seen under conditions in which calmodulin simultaneously produced a stimulatory effect on the phosphorylation of proteins of molecular weight 51,000 and above. This inhibition required calcium, but was not sensitive to cyclic AMP or increasing ATP concentration and was not due to activation of a phosphatase. These results suggest either that calmodulin induces its inhibitory effects on phosphorylation by an indirect mechanism via a presently unknown pathway, or that in addition to the kinase stimulated by calmodulin, there exists another distinct kinase which is inhibited by calmodulin.

The interaction of dihydropyridine calcium channel blockers with calmodulin and calmodulin inhibitors.

The calmodulin inhibitors R24571 and trifluoperazine were found to inhibit competitively the binding of [3H]nitrendipine to a 48,000 X g particulate fraction of rat brain with IC50 values of 1.0 and 18.8 microM, respectively. Equilibrium dialysis was used to test the ability of the dihydropyridines nitrendipine, felodipine, and nicardipine to inhibit the binding of [3H]chlorpromazine, [14C]pimozide, and 45Ca2+ to calmodulin. At dihydropyridine concentrations near the limit of solubility (10 microM), the only significant effect in these three binding experiments was a 26% inhibition of [14C]pimozide binding to calmodulin by nicardipine, indicating that the dihydropyridines do not bind to the same site on calmodulin as chlorpromazine, pimozide, or calcium. Equilibrium dialysis was also used to determine the ability of the dihydropyridines to interact directly with calmodulin. [3H] Nitrendipine bound to calmodulin in a calcium-dependent manner; however, this binding was of a low-affinity, unsaturable nature. These results suggest that the dihydropyridine drugs do not interact with calmodulin at concentrations that are pharmacologically significant.

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: evidence for a calcium channel.

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [3H]-nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (KD) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited KD values of 0.2-0.3nM. The concentration of binding sites per mg. protein (Bmax) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [3H]-nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5-trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 X 10(-5)M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 X 10(-5)M nitrendipine was seen on net 45Ca uptake by HSR. These results suggest that [3H]-nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [3H]-nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [3H]-nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.

Clofibrate, calcium and cardiac muscle.

The anti-hyperlipidemic drug clofibrate produces negative inotropic effects and arrythmias in isolated perfused rabbit heart Langendorff preparations. In electrically stimulated rat left atria, clofibrate produces negative inotropic effects, the speed of onset and extent of which are decreased by raising the Ca concentration of the bathing medium. Sensitivity of isolated rat atria to clofibrate is not increased when the tissues are stimulated under slow Ca channel conditions, in which the tissues are activated by either isoproterenol or dibutyryl cyclic AMP, although sensitivity to clofibrate is decreased when atria are exposed to increasing concentrations of norepinephrine. Increasing the stimulation frequency of isolated guinea-pig atria to produce a positive treppe also decreases the inhibitory effect of clofibrate, while in rat atria the typical negative treppe is altered towards a positive treppe in presence of clofibrate. The effects of paired electrical stimulation are not diminished by the drug, suggesting that Ca release from the sarcoplasmic reticulum is not affected by clofibrate, although the drug inhibits the rate of Ca uptake by isolated cardiac sarcoplasmic reticulum and mitochondria. These results suggest that clofibrate has multiple effects on Ca functions in cardiac muscle.

Clofibrate and vascular smooth muscle: actions on rabbit aorta preparations.

Clofibrate (Atromid-S) has been found to inhibit the contractility of isolated rabbit aortic muscle rings produced by exposure to either norepinephrine, histamine or angiotensin. The inhibitory effect of clofibrate is seen optimally in Krebs medium containing low, 5 X 10-5 M, calcium but is not found in medium containing 2.5 X 10-3 M calcium. In contrast, clofibrate does not inhibit contractions triggered by depolarizing concentrations of K+, in either low or normal Ca medium. Since only receptor-mediated contractions are inhibited, it is suggested that the actions of clofibrate in aorta influence an event common to receptor-linked contractions, possibly by interfering with the utilization of membrane-bound calcium.

Modification of ryanodine toxicity by dantrolene and halothane in a model of malignant hyperthermia.

Ryanodine toxicity in animals has been suggested to constitute a model of malignant hyperthermia. Dantrolene is known to block the development of malignant hyperthermia triggered by halothane in susceptible swine. The authors studied the influences of dantrolene and halothane on the effects of ryanodine in vitro in isolated rat diaphragm muscle segments, and in vivo in mice, to explore the validity of this model. In the diaphragm experiments, dantrolene was found to block or delay the development of contractures produced by ryanodine and to delay the potentiation of ryanodine-induced contractures caused by halothane. In mice, ryanodine at various dosages was injected and animals surviving after one hour were examined. Such survivors appeared grossly to be normal, and may constitute a model for the malignant hyperthermia patient. They were found to be susceptible to halothane and to succinylcholine, being killed by treatment with these two agents at dosages that were not lethal to control mice. Pretreatment of mice for 48 hours with orally administered dantrolene, followed by injection of ryanodine and then halothane anesthesia, decreased the lethality of ryanodine but did not reduce the number of deaths caused by the subsequent exposure to halothane. That the effects of ryanodine in vitro and in vivo are diminished and potentiated by dantrolene and halothane, respectively, would suggest that the ryanodine toxicity model of malignant hyperthermia may have validity and is worthy of further study. A prediction from this model is that the terminal cisternae of skeletal muscle sarcoplasmic reticulum may be altered in MH.

Effects of alkanols and halothane on rat brain muscarinic and alpha-adrenergic receptors.

Effects of the primary alcohols ethanol, butanol, pentanol and of halothane were measured on the binding functions of muscarinic and alpha-adrenergic receptor preparations in rat brain homogenates, with the use of the antagonists 3H-quinuclidinyl benzilate and 3H-WB-4101. IC50 concentrations of the alkanols for the muscarinic and alpha-receptors respectively were: ethanol, 2.0 M and 1.4 M; butanol, 0.24 M and 0.16 M; heptanol, 3.7 X 10(-3) M and 2.6 X 10(-3) M. The plot of IC50 values versus number of carbon atoms in the alkanol was linear and of the same slope as the plot of membrane fluidity changes, thus indicating the importance of the membrane/water partition coefficient of the alkanol. Halothane at clinical concentrations had no effect on the receptors, although significant inhibition of radioligand binding was produced by 2.5 mM halothane, and inhibition was complete in presence of 17.5 mM anesthetic. From the correlation of receptor binding inhibitions with membrane fluidity changes reported by other workers, it is suggested that the activity of membrane receptors may be modulated by the fluidity of their membranes.

Effect of carbamazepine on the in vitro uptake and release of norepinephrine in adrenergic nerves of rabbit aorta and in whole brain synaptosomes.

The effects of carbamazepine, in vitro, on adrenergic neuronal and whole brain synaptosomal uptake and release of tritiated norepinephrine (3H-NE) were assessed. At 10(-4) M, carbamazepine inhibited 3H-NE uptake by 22% in rabbit thoracic aorta and in brain synaptosomes. At the same concentration, carbamazepine inhibited stimulation-induced release of 3H-NE by 42.6% and inhibited isometric contraction in rabbit ear artery helical strips by 31.6%. At 10(-5) M, carbamazepine exhibited a 17.6% inhibition of 3H-NE uptake in brain synaptosomes in the absence of effects on transmitter release. Cocaine, 10(-4) M, and imipramine, 10(-4) M, inhibited uptake by 88% and 85%, respectively, in aorta, and cocaine, 10(-4) M, inhibited synaptosomal uptake by 67.7%. Since antiepileptic blood levels of carbamazepine range between 1.3 and 3.0 X 10(-5) M, it was concluded that the observed effects of carbamazepine are insufficient to account for the anticonvulsant action of the drug. However, the blockade of 3H-NE uptake by brain synaptosomes at 10(-5) M serves to explain the recently described analeptic activity of this agent.

Calcium dependence of rat brain tryptophan hydroxylase.

The effects of various concentrations of ionized Ca were examined on the activity of rat brain tryptophan hydroxylase previously treated with EGTA. Within the range of ionized Ca-concentrations though to be physiologically important (10(-8) to 10(-5) M), no significant activation of the enzyme occurred, although activation was observed at higher concentrations of the metal.