Effects of calcium antagonists on KCl-evoked calcium uptake by rat cortical synaptosomes

A series of calcium antagonists were used to study their blocking effect on high potassium-induced calcium uptake into rat cortical synaptosomes; these antagonists were classified into five groups: dihydropyridine group (i.e. nifedipine and nitrendipine), benzothiazepine group (i.e. diltiazem), phenylalkylamine group (i.e. verapamil and D600), phenothiazine group (i.e. trifluoperazine) and diphenylpiperazine group (i.e. flunarizine and cinnarizine). Voltage-dependent 45Ca2+-uptake into this fraction was measured after 20 sec KCl-induced depolarization. The ID30 values of the above-mentioned antagonists affecting 45Ca2+-uptake were calculated to be nitrendipine (80 microM), nifedipine (100 microM), verapamil (50 microM), D600 (15 microM), diltiazem (70 microM), trifluoperazine (7 microM), cinnarizine (1.2 microM) and flunarizine (0.7 microM). Our results reveal that in rat brain synaptosomal fractions, calcium influx via the voltage-gated calcium channel appears to be more sensitive to diphenylpiperazine and phenothiazine groups; whereas, phenylalkylamine, benzothiazepine and dihydropyridine groups were relatively insensitive. This contrasts with the well known data obtained from vascular smooth muscle, in which the dihydropyridine group is the most sensitive of all the groups studied. Our results suggest that calcium channels in neuronal tissue are most likely different from those in non-neuronal tissue.     

The effects of strychnine on the regulation of voltage-dependent calcium channels by dihydropyridines in brain and heart.

The effects of strychnine (STR) were investigated on K(+)-stimulated 45Ca2(+)-uptake into mouse brain neurons, the contractile activity of spontaneously beating rat atria and on [3H]nitrendipine and [3H]BAY K 8644 binding to dihydropyridine calcium channel antagonist and agonist binding sites on brain and cardiac membranes. STR (10(-6)-10(-4) M) had no effect on neuronal 45Ca2(+)-uptake. When combined at equimolar concentrations (10(-5) M), STR and nifedipine produced a potent (nM) inhibition (40%) of neuronal 45Ca2(+)-uptake. In the spontaneously beating rat atria, STR produced a dose-dependent (10(-7)-3 x 10(-4) M) decrease in chronotropy but did not affect inotropy. STR (10(-4) M) completely inhibited the positive chronotropic, but did not affect the positive inotropic effects of (-)-S-BAY K 8644. [3H]Nitrendipine and [3H]BAY K 8644 binding to brain and cardiac membranes was enhanced by STR in a concentration-dependent manner (EC50 8 X 10(-6) M). Scatchard analysis revealed that STR increased the affinity (decreased the Kd) of [3H]BAY K 8644 to a greater degree than that of [3H]nitrendipine for dihydropyridine binding sites. STR decreased the Kd of [3H]nitrendipine binding by increasing and decreasing the microassociation and microdissociation constants respectively. STR enhanced [3H]nitrendipine binding to the same extent in the cerebral cortex, striatum, hippocampus, cerebellum, brainstem and spinal cord. The enhancement of [3H]nitrendipine binding in brain was completely inhibited by Ca2+ and partially inhibited by Na+ in a concentration-dependent manner. Glycine (10(-2) M) did not affect the STR enhancement of [3H]nitrendipine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of some cyclohexylaralkylamines related to perhexiline on sodium influx, binding of [3H]batrachotoxinin A 20-alpha-benzoate and [3H]nitrendipine and on guinea pig left atria contractions

The antagonist activities of some cyclohexylaralkylamines derived from perhexiline on the fast Na+ channel and slow Ca2+ channel in rat brain and rat heart were examined and compared to the antagonist activities of nifedipine, verapamil, prenylamine and perhexiline. Prenylamine, perhexiline and the cyclohexylaralkylamine derivatives inhibited the [3H]batrachotoxinin A 20-alpha-benzoate binding more than the [3H]nitrendipine binding in rat brain. The nature of the interaction of the cyclohexylaralkylamines with the binding of [3H]batrachotoxinin and [3H]nitrendipine was non-competitive. The synaptosomal 22Na uptake induced by protoveratrine B, a Na+ channel agonist, was also inhibited. Prenylamine, perhexiline and perhexiline derivatives were more potent on the fast Na+ channel than on the Ca2+ channel in contrast to nifedipine and verapamil. The inhibition of Na+ and Ca2+ channels was also shown in guinea pig left atria. Perhexiline, prenylamine and the perhexiline derivatives inhibited the protoveratrine B-induced contraction more than they inhibited that induced by CaCl2, in contrast with nifedipine and verapamil. Our results showed that prenylamine, perhexiline and its related cyclohexylaralkylamines inhibited the fast Na+ channel far more than the slow Ca2+ channel in rat brain, rat heart and guinea pig atria.     

Tissue response selectivity of calcium antagonists is not due to heterogeneity of [3H]-nitrendipine binding sites.

[3H]-nitrendipine binding data and isolated tissue response for five calcium antagonists were evaluated in rabbit myocardium and aorta. The [3H]-nitrendipine binding site was qualitatively identical in myocardium and aorta, as the [3H]-nitrendipine KD, KIS for nicardipine and nifedipine and interactions with verapamil, D600 and diltiazem were not different in aortic and cardiac membranes prepared by similar means. In contrast, the inhibition of the Ca2+-induced contractile response in right ventricular myocardium and aortic ring segments indicated a greater than 10,000 fold selectivity of nicardipine for antagonism of vascular responses. This resulted in a different order of potency for calcium antagonist interaction with the [3H]-nitrendipine binding site in cardiac membranes (nicardipine greater than nifedipine greater than D600 greater than verapamil greater than diltiazem) as compared to antagonism of myocardial tissue response (D600 greater than verapamil greater than or equal to nifedipine greater than nicardipine greater than or equal to diltiazem). In heart the difference between the potency of nicardipine in binding experiments and tissue response approached 4 orders of magnitude. We conclude that tissue response selectivity of calcium antagonists is not explained by heterogeneity of [3H]-nitrendipine binding sites.     

Uptake of calcium antagonistic drugs into muscles as related to their lipid solubilities

Calcium antagonists, e.g. bepridil and verapamil, block the Ca2+-dependent slow action potentials in frog skeletal muscle [L.M. Kerr and N. Sperelakis, J. Pharmac. exp. Ther. 222, 80 (1982)]. To determine whether the calcium antagonistic drugs may enter the fibers and exert an internal action as well, uptake of tritiated bepridil, verapamil, nitredipine, nifedipine, and diltiazem into rat extensor digitorum longus (EDL) muscles was examined. It was found that the uptake values of verapamil, nitrendipine, and bepridil were much higher than those of nifedipine and diltiazem. The order of uptake was: bepridil greater than nitrendipine greater than verapamil much greater than nifedipine greater than diltiazem. The small uptake values of nifedipine and diltiazem may represent primarily binding to the surface membrane. In frog skeletal muscle (sartorius) also, the uptake of bepridil was greater than that of verapamil, and disruption of the T-tubules by the glycerol method did not change them. The same order of drug uptake values was found for monolayer cultures of vascular smooth muscle cells (rat aorta). The order of uptake in isolated sarcoplasmic reticulum (SR) from rat skeletal muscles was: verapamil greater than nitrendipine greater than bepridil greater than nifedipine greater than diltiazem. The lipid solubility values of the calcium antagonists were measured by their partition coefficients in oil/Ringer, octanol/Ringer, and chloroform/Ringer systems. The order of lipid solubility was: bepridil greater than verapamil greater than nitrendipine greater than nifedipine much greater than diltiazem. Thus, the calcium antagonists with the highest lipid solubilities were taken up more by the muscle cells and SR. It is concluded that verapamil, bepridil, and nitrendipine enter and accumulate inside the muscle cells, whereas nifedipine and diltiazem do not permeate readily.     

Effects of the Ca2(+)-antagonists nifedipine, verapamil, flunarizine and of the calmodulin antagonist trifluoperazine on muscarinic cholinergic receptors in rat cerebral cortex

1. Studies were made of [3H]QNB binding to muscarinic receptors in a membrane fraction from the cerebral cortex of rats treated orally for 13 days with the Ca2(+)-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin antagonist trifluoperazine (3 mg/kg). 2. The [3H]QNB binding capacity (Bmax) was decreased by three Ca2(+)-antagonists: nifedipine, verapamil and flunarizine, the decrease was most pronounced with nifedipine. 3. The decrease in the number of muscarinic receptors after nifedipine and flunarizine was accompanied by an increase in their affinity; verapamil decreased both the number and the affinity of muscarinic receptors. 4. The calmodulin antagonist trifluoperazine changed neither the number nor the affinity of muscarinic receptors. 5. It is suggested that continuous treatment with different Ca2+ or calmodulin antagonists leads to difference in character and degree of alterations in the basic characteristics of muscarinic receptors in rat cerebral cortex.     

Long-term treatment with different calcium- and calmodulin-antagonists induces changes in rat brain alpha-adrenoceptors.

1. The binding characteristics (Bmax and Kd) of the alpha-adrenoceptor radioligand [3H] WB4101 in crude membrane fraction (fraction P2) from cerebral cortex were studied after 13-day oral treatment of male Wistar rats with the Ca(2+)-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin-antagonist trifluoperazine (TFP) (3 mg/kg). 2. A significant reduction of the binding sites (Bmax) for [3H] WB4101 was established after the three Ca(2+)-antagonists as well as after TFP treatment. 3. Different changes in the affinity constant (Kd) of brain adrenoceptors were observed depending on the type of the Ca2+ or CaM-antagonist used: nifedipine did not change the Kd value, verapamil and TFP decreased whereas flunarizine increased the Kd value. 4. Relationships between Ca ions and alpha-adrenoceptor functions are suggested.     

Effects of dihydropyridine derivatives and anticonvulsant drugs on [3H]nitrendipine binding and calcium and sodium fluxes in brain

The binding of [3H]nitrendipine to rat cortical membranes was reduced by phenytoin, phenobarbital, and pentobarbital. The IC50 values were 0.09, 0.40, and 0.76 mM respectively. The drugs reduced the apparent binding affinity of [3H]nitrendipine with little effect on the maximum number of binding sites. The inhibitory effects of the drugs were similar in the absence and presence of calcium (4.5 mM). Neither nimodipine (10(-8) to 10(-5) M) nor nifedipine (10(-8) to 10(-7) M) altered the voltage-dependent uptake of 45Ca2+ by synaptosomes from rat cortex. Phenytoin inhibited 45Ca2+ influx, and this inhibition was not altered by nifedipine. Nimodipine and nifedipine (10(-6) M) produced a small inhibition of the voltage-dependent uptake of 24Na+ by synaptosomes. Ethanol, phenytoin or pentobarbital reduced 24Na+ influx, and this action was not altered by nimodipine. Thus, sedative-anticonvulsant drugs reduced the binding of dihydropyridines to brain membranes, but these interactions did not appear to involve either calcium or sodium channels.     

Calcium antagonism by KB-2796, a new diphenylpiperazine analogue, in dog vascular smooth muscle.

The effects of KB-2796, a new diphenylpiperazine analogue, on [3H]nitrendipine ([3H]NTD) binding, KCl-induced contraction and 45Ca influx has been examined in dog vascular smooth muscle, and compared with those of other diphenylpiperazines. In the binding study, [3H]NTD was found to bind with a high affinity to a single class of sites on aortic membranes (Kd = 0.41 nM and Bmax = 31 fmol (mg protein)-1). KB-2796 inhibited specific [3H]NTD binding in a concentration-dependent manner, with a Ki value of 0.34 microM. The other diphenylpiperazine derivatives such as flunarizine and cinnarizine also inhibited binding in the same manner. Also, in the contraction study, all the diphenylpiperazines antagonized the 50 mM KCl-induced contraction of isolated mesenteric arteries concentration-dependently. The IC50 values of the compounds for KCl-induced contraction correlated strongly with the respective Ki values obtained in the [3H]NTD binding study. In the 45Ca influx study, KB-2796 also effectively inhibited KCl-induced 45Ca influx in mesenteric arteries, with an IC50 value of 0.14 microM. This was close to the IC50 value found in the KCl-induced contraction study. These findings suggest that calcium antagonism by KB-2796 is responsible for its vasorelaxing action in vascular smooth muscle.     

Selective antagonism of calcium channel activators by fluspirilene.

1. Fluspirilene has been claimed to bind to a high affinity site in the calcium channel in skeletal muscle. We have investigated its calcium-antagonistic effects in smooth muscle and affinity for the channel in radioligand binding assays. 2. Fluspirilene was weakly active as an antagonist of Ca2(+)-induced contractions in K(+)-depolarized taenia preparations from the guinea-pig caecum, with threshold antagonism starting from concentrations of 30 nM. Nitrendipine, nicardipine and nimodipine were very potent antagonists in this model (threshold antagonism, greater than 1 nM). 3. In contrast, fluspirilene (10-1000 nM) was a potent non-competitive antagonist of the effects of Bay K 8644 (1-3000 nM) on Ca2(+)-induced contractions and, at 10 nM, selectively antagonised the effects of Bay K 8644, abolished the Ca2(+)-channel activator effects of CGP 28392, without changing the calcium antagonist effects of nitrendipine, or modifying the sensitivity of the tissues to Ca2+. In contrast, the dihydropyridines were more effective as antagonists of Ca2+ than of Bay K 8644. Fluspirilene therefore selectively antagonised the effects of dihydropyridine Ca2+ channel activators without affecting the antagonist potency. 4. In radioligand binding experiments, fluspirilene was a potent displacer of [3H]-PN-200-110 binding to rat cerebral cortical membranes (EC50 30 nM), albeit with a low Hill slope (0.66), and was more potent than other lipophilic diphenylalkylamines such as flunarizine and lidoflazine. Fluspirilene interacted non-competitively with [3H]-PN-200-110 and increased dissociation of the radioligand.     

Effects of anipamil on myocardial sarcolemmal and mitochondrial calcium transport, comparison with verapamil and nifedipine

The calcium antagonists anipamil, verapamil and nifedipine inhibited, dose dependently, passive and ATP-driven 45Ca2(+)-uptake in purified rabbit ventricular sarcolemmal vesicles exposed to a wide range of free calcium concentration (from 0 to 200 microM). The IC50 values for passive binding were virtually identical for all calcium antagonists and the inhibition was relatively independent of the amount of free calcium employed. On the contrary, the order of potency for inhibition of the ATP-driven calcium uptake was: anipamil greater than verapamil greater than nifedipine. The inhibition of nifedipine, at free calcium concentrations lower than 80 microM, was preceded by a slight stimulation. The inhibitory effects of anipamil and verapamil, but not those of nifedipine, on the ATP-driven calcium uptake were more evident with increasing external calcium concentration. Verapamil and nifedipine failed to modify the initial rate of mitochondrial calcium transport either in the presence or in the absence of ADP; on the contrary, anipamil induced a dose-dependent inhibition of mitochondrial calcium transport. The inhibition occurred over the whole range of calcium concentrations tested, independent of the presence of ADP. The effects of anipamil, but not those of verapamil and nifedipine, on sarcolemmal and mitochondrial calcium transport were long lasting and survived membrane isolation.     

Comparison of negative inotropic potency, reversibility, and effects on calcium influx of six calcium channel antagonists in cultured myocardial cells.

The negative inotropic effects of calcium channel antagonists on the myocardium were used as a standard for the definition and determination of potency of this group of drugs. The effects of six calcium channel antagonists (verapamil, methoxyverapamil (D600), nifedipine, lidoflazine, perhexiline and diltiazem) were compared on cultured chick embryo ventricular cells. Drug concentrations producing 50% inhibition of contractile amplitude, derived from linearized concentration-response curves, varied from 2.8 X 10(-8)M for nifedipine to 8.3 X 10(-7)M for perhexiline. Equipotent negative inotropic concentrations of verapamil, D600, perhexiline, diltiazem and lidoflazine produced a similar inhibitory effect on 45Ca uptake into cultured cells. Nifedipine produced no significant inhibition of 45Ca uptake. The time required for recovery of contractility after cessation of drug superfusion varied in the order lidoflazine greater than perhexiline greater than D600 greater than verapamil greater than nifedipine greater than diltiazem. This relative order accords closely with the reported in vivo half-lives of these drugs. It is concluded that while some inhibition of 45Ca2+ uptake into cardiac cells can be demonstrated with five of the six calcium channel blockers studied, the relationship between the degree of inhibition of calcium influx and negative inotropic effects may not be uniform for all calcium channel antagonists.     

Effect of an homologous series of aliphatic alcohols on neuronal and smooth muscle voltage-dependent Ca2+ channels.

The acute inhibitory actions of alcohol on K(+)-stimulated 45Ca2+ uptake into synaptosomes shows regional variation in sensitivity throughout the brain, suggesting the possibility of a selective action on a specific Ca2+ channel subtype. This was examined by comparing the effects of a homologous series of aliphatic alcohols on synaptosomal Ca2+ channels with their actions on K(+)-stimulated Ca2+ channels in guinea-pig intestinal longitudinal muscle, which have been demonstrated to be of the L-type. K(+)-stimulated contraction of and [3H]nitrendipine binding to smooth muscle were both inhibited by the alcohols at similar concentrations, with the potency increasing with chain length. In synaptosomes, however, K(+)-stimulated 45Ca2+ uptake was 5-30 times more sensitive to the inhibitory actions of alcohol than were [3H]nitrendipine and [125I]omega-conotoxin binding. These observations suggest that K(+)-stimulated 45Ca2+ uptake is mediated by a non-L non-N type channel which is more sensitive to the acute effects of alcohols. This is supported by the observation that K(+)-stimulated 45Ca2+ uptake which is insensitive to L- and N-channel antagonists was inhibited by funnel web spider venom.     

Evidence for the presence of regional differences in the calcium antagonist receptors in lower urinary tract smooth muscle

Summary (+)-[³HPN 200-110 (a dihydropyridine calcium channel antagonist) was utilized to characterize calcium channel binding sites in rabbit bladder dome, bladder base, and urethra. Specific binding of (+)-[³H]PN 200-110 to membrane particulates was saturable, reversible, linear to protein concentration, and of high affinity. The density of (+)-[³H]PN 200-110 binding sites (B_max values in fmol/mg of protein) and the affinity constants for (+)-[³H]PN 200-110 (K_D value in pM) in urethra, bladder dome and bladder base were 64.1 ± 7.8 and 179±31; 21.9±3.0 and 213±36; and 18.8±4.2 and 140±28, respectively. Agonists and antagonists inhibited (+)-[³H]PN 200-110 binding with Ki values in the following rank order: nitrendipine < nifedipine < niguldipine Bay K 8644 verapamil. Although carbachol-induced contractile responses were 20–30 times smaller in muscle strips from urethra than from bladder base or bladder dome, KCl-induced contractions were only 3–4 times smaller in urethra than in bladder tissues. Nifedipine inhibited carbachol-induced contractions in urethra, bladder dome, and bladder base by 76%, 64%, and 60%, respectively, and completely inhibited KCl-induced contractions in all three tissues. IC₅₀ values for nifedipine inhibition of both carbachol- and KCl-induced contractions were significantly smaller in urethra than in bladder base or bladder dome. Nitrendipine, niguldipine and verapamil inhibited urethral contractions induced by carbachol and KCl to the same degree as did nifedipine. The IC₅₀ values, obtaines from functional studies, for calcium channel antagonists were in good agreement with Ki values obtained from binding studies. BAY K 8644, a calcium channel agonist, increased both KCl- and carbachol-induced contractions and potentiated CaCl₂-induced contractions in K+-depolarizing media in urethra but not in bladder dome or bladder base. Our data indicate that the density of (+)-[³H]PN 200-110 binding sites is higher in the urethra than in the bladder dome or bladder base and that the urethra is functionally more sensitive to dihydropyridine agonists and antagonists than is either the bladder dome or bladder base.Send offprint requests to R.M. Weiss at the above address     

Competitive inhibition of [3H]spiperone binding to D-2 dopamine receptors in striatal homogenates by organic calcium channel antagonists and polyvalent cations

The specific binding of [3H]spiperone (30 pM) to D-2 dopamine receptors in homogenates of the rat corpus striatum, as defined by the D-2 antagonist haloperidol (100 nM), was displaced by organic calcium channel antagonists and by polyvalent cations. Both classes of agents were able to totally displace [3H]spiperone binding by an apparently competitive mechanism in that the dissociation constant was increased while the density of binding sites was unchanged. The rank order of inhibition potency for the cations was Zn2+ greater than Cd2+ greater than La3+ greater than Cu2+ greater than Ni2+ greater than Co2+ greater than Mn2+ greater than Mg2+, Ca2+ greater than Ba2+. Of the organic calcium channel antagonists, D600 and verapamil were the most potent displacers of [3H]spiperone binding (IC50 values both 2.0 microM), while diltiazem possessed an IC50 of 33 microM. Nicardipine (IC50 6.0 microM) was the only 1,4-dihydropyridine to inhibit [3H]spiperone binding. The results suggest that sites labelled by [3H]spiperone also bind organic calcium channel antagonists and polyvalent cations.     

SR33557, an indolizinsulfone blocker of Ca2+ channels: identification of receptor sites and analysis of its mode of action

SR33557 belongs to a new class of molecules (indolizinsulfones) that act on the same receptor complex that has been characterized for other classical calcium channel effectors. The main binding properties of SR33557 to rabbit skeletal muscle are as follows. (i) Unlabeled SR33557 completely inhibits the specific binding of all classes of calcium channel antagonists such as dihydropyridines [(+)-[3H]PN200-110], phenylalkylamines ([3H] verapamil), benzothiazepines (d-(cis)-[3H]diltiazem), and diphenybutylpiperidines ([3H]fluspirilene). In all these cases inhibition of binding is of a noncompetitive nature. (ii) [3H]SR33557 binds with high affinity to T tubule membranes (KD = 0.08 nM) and the maximum binding capacity (Bmax = 78 pmol/mg of protein) is the same as that found for other classes of Ca2+ channel antagonists. Photoaffinity labeling confirms that [3H]SR33557 associates with the same protein of Mr 165,000 that binds the classical calcium channel inhibitors. 45Ca2+ uptake experiments performed with the rat aortic cell line A7r5, the insulin-secreting cell line RINm5F, and the pheochromocytoma cell line PC12 demonstrate that SR33557 fully inhibits the 1,4-dihydropyridine-sensitive 45Ca2+ uptake elicited by depolarization. A very good correlation was found between inhibition of 45Ca2+ uptake and of [3H]dopamine release in PC12 cells and between inhibition of 45Ca2+ uptake and of L-type Ca2+ current in A7r5 cells under whole-cell patch-clamp conditions.     

Organic and inorganic calcium antagonists reduce vasoconstriction in vivo mediated by postsynaptic α 2-Adrenoceptors

The influence of various calcium antagonists and divalent metal cations on the pressor responses induced by the selective alpha 1-adrenoceptor agonist methoxamine and the selective alpha 2-adrenoceptor stimulating agent B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]-azepine) was studied in pithed rats. 1. The calcium antagonists verapamil, D 600 and nifedipine, when given intraarterially (i.a.) in doses up to 1 mg/kg did not influence the pressor effects of methoxamine. Only higher amounts of these calcium antagonistic drugs (1 - 3 mg/kg i.a.) somewhat reduced this pressor response. 2. The vasoconstriction due to B-HT 920, as reflected by the increase in diastolic pressure, was markedly inhibited by verapamil, D 600 and nifedipine in a dose-dependent manner. In low doses a parallel displacement to the right was observed, whereas in higher amounts the shift was non-parallel. 3. The divalent cations MN2+, Ni2+ and Co2+ (0.05 - 0.15 mmol/kg i.a.) hardly affected the pressor effect of methoxamine, whereas B-HT 920-induced vasoconstriction was highly sensitive to these metal ions. La3+ and Mg2+ were ineffective. 4. The calcium antagonists verapamil, D 600 and nifedipine displayed only minor affinities for [3H]prazosin (alpha 1) as well as [3H]clonidine (alpha 2) binding sites of rat brain membranes. 5. It is concluded that an influx of extracellular Ca2+ is necessary for the vasoconstriction in vivo initiated by stimulation of vascular postsynaptic alpha 2-adrenoceptors. On the other hand, vasopressor responses to alpha 1-adrenoceptor stimulation are not directly dependent on a transmembrane influx of calcium ions.     

Effect of calcium channel antagonists on calcium uptake and release by isolated rat cardiac mitochondria

The effects of calcium channel antagonists on Ca2+ uptake and Na+-induced Ca2+ release were studied in isolated rat cardiac mitochondria. Diltiazem, nitrendipine and nimodipine were more effective inhibitors of Na+-induced Ca2+ release (IC50 = 19-100 microM) than of Ca2+ uptake (IC50 = 0.2-1 mM). Nitrendipine and nimodipine had virtually identical IC50 values for inhibiting Ca2+ uptake, but nitrendipine was 3-4 times more potent than nimodipine at inhibiting Na+-induced Ca2+ release. If these calcium channel antagonists achieve intracellular concentrations in the range of 10(-5)-10(-4) M, our results suggest that calcium channel antagonists would preferentially inhibit mitochondrial calcium release more than mitochondrial calcium uptake.     

Mode of action and comparative efficacy of pharmacological agents that inhibit calcium-dependent dehydration of sickle cells.

1. Selected Ca-channel antagonists were tested at 20 microM as inhibitors of Ca(2+)-uptake in human sickle red cells. Nitrendipine, fendiline, and bepridil (and its stereoisomers), were found to be as effective as methoxyverapamil (D-600) in inhibiting a fraction (25%) of Ca(2+)-uptake. In contrast cetiedil and Org 30701 were ineffective. 2. The drugs were subsequently tested as inhibitors of Ca(2+)-induced K+ efflux (Gardos) from sickle cells. They all showed inhibitory activity, with the order of efficacy nitrendipine greater than fendiline greater than bepridil greater than cetiedil greater than Org 30701. 3. With a 15 h programme of deoxygenation/reoxygenation cycles in a gas exchanger, it was shown that the inhibitors protected against cellular dehydration and loss of filterability in the order nitrendipine greater than fendiline greater than bepridil greater than cetiedil greater than Org 30701. However, significant stomatocytosis occurred at high concentrations of cetiedil, and bepridil (including its stereoisomers and analogues) impairing cell deformability. 4. It is concluded that Ca-antagonists may partially block both Ca(2+)-uptake and Ca(2+)-induced K+ efflux. The latter pathway is significant in contributing to sickle cell dehydration and nitrendipine is the most effective inhibitor of this route.     

Effect of Ca2+ antagonists on high-K+ evoked increase in [Ca2+]i in rat cerebral synaptosomes and hippocampal neurons.

By fura-2 fluorometry, we investigated the direct effects of Ca2+ antagonists including a new benzothiazepine, clentiazem, on the high-K(+)-evoked increase in the concentration of cytosolic free Ca2+ ([Ca2+]i) in rat cerebral synaptosomes and cultured hippocampal neurons. In both preparations, metal ions inhibited the high-K(+)-induced increase in [Ca2+]i, in the following order: La3+ greater than Cd2+ much greater than Ni2+. Although flunarizine and nicardipine inhibited the K(+)-induced increase in [Ca2+]i in synaptosomes, other Ca2+ antagonists, including clentiazem and nitrendipine, had little effect at 10 microM. In hippocampal neurons, clentiazem inhibited the K(+)-induced increase in [Ca2+]i at 10 microM, as did flunarizine and nicardipine. However, nifedipine and nitrendipine had little effect in either cultured neurons or in synaptosomes.