Interactions between diltiazem and ethanol: differences from those seen with dihydropyridine calcium channel antagonists

It has previously been shown that dihydropyridine calcium channel antagonists prevent the ethanol withdrawal syndrome and potentiate the acute effects of ethanol and other central depressant drugs. We now report that, in contrast, the benzothiazepine calcium channel antagonist, diltiazem, gave no protection against the behavioural hyperexcitability seen during ethanol withdrawal, when given either acutely, on withdrawal, or chronically, during the ethanol treatment. A significant increase in convulsive behaviour on handling was seen during the withdrawal period when diltiazem was given on cessation of a mild chronic ethanol treatment schedule. Diltiazem decreased the acute general anaesthetic effects of ethanol, and did not appear to potentiate the ataxic action of ethanol. Centrally administered diltiazem did not enhance the hypothermic action of ethanol, but this effect was significantly increased by diltiazem when the calcium channel antagonist was given peripherally. When given alone by the intraperitoneal route, diltiazem decreased spontaneous locomotor activity and lowered body temperature. When the intracerebral route was used for administration of diltiazem, a significantly decrease in body temperature was seen when this compound was given alone, accompanied by a brief hyperexcitability. The interactions between ethanol and diltiazem therefore appear to differ from those seen with other calcium channel antagonists.     

Phencyclidine increases the affinity of dihydropyridine calcium channel antagonist binding in rat brain

Summary Phencyclidine (PCP) significantly reduces the apparent dissociation constant (K _D) of the dihydropyridine (DHP) calcium channel antagonist, [³H]nitrendipine, in synaptosomal membranes of rat and mouse brain without significantly effecting the maximum binding capacity (B _max). At an optimum concentration of PCP (10 M) the apparentK _D of [³H]nitrendipine was reduced from 178±9 pM to 112±9 pM in rat forebrain, a 58% increase in affinity. The structural derivatives of PCP, P-Br-PCP {1-[1-(4-bromophenyl-cyclohexyl)piperidine]}, m-NH₂-PCP {1-[1-(3-anilo)-cyclohexyl]piperidine}, (±)-PCMP [1-(1-phenyl)-cyclohexyl-3-methylpiperidine] also increased the apparent affinity of [³H]nitrendipine in the following order, p-Br-PCP PCMp>PCP>m-NH₂-PCP. Local anesthetics either reduced the apparent affinity of [³H]nitrendipine or had no effect. Kinetic analysis revealed that PCP both increased the microassociation rate constant and decreased the microdissociation rate contant of [³H]nitrendipine. The magnitude of this enhanced binding varied with the brain region studied; the greatest increase in apparent affinity of [³H]nitrendipine was observed in striatum, while no significant increase in affinity was observed in brainstem. In some brain areas, PCP was more effective in reducing theK _D in crude homogenates than in washed tissue. PCP (10 M) did not alter theK _D of [³H]nitrendipine to rat cardiac tissue. Both Ca²⁺ and Mg²⁺ inhibited the effect of PCP, while monovalent ions were ineffective in this regard. These data are consistent with an allosteric modulation of DHP calcium channel antagonist binding sites by PCP and structural derivatives that is not mediated through the brain PCP binding site. This modulation of DHP binding sites may account for some of the psychopharmacologic actions PCP and related compounds in vivo.     

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

Rationale

Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca^2 + channels and their renowned antioxidant properties.

Methods

We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca^2 + channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca^2 + channel-blocking activity and antioxidant properties. The Ca^2 + channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR.

Results

Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca^2 + channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca^2 + channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties.

Conclusions

Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring.     

The behavioral effects of the calcium agonist BAY K 8644 in the mouse: Antagonism by the calcium antagonist nifedipine

Summary Mice injected with the calcium agonist BAY K 8644 (2–4 mg/kg, i. p.) displayed profound behavioral changes including ataxia, decreased motor activity, Straub tail, arched back, limb clonus and tonus, and an increased sensitivity to auditory stimulation. BAY K 8644 significantly impaired rotorod performance in mice with an ED₅₀ of 0.8 mg/kg. The behavioral effects of BAY K 8644 were antagonized by nifedipine, but not by the non-dihydropyridine calcium channel antagonist verapamil or the -adrenoceptor antagonist prazosin. Further, the actions of BAY K 8644 were not mimicked by the -adrenoceptor agonist methoxamine at doses up to 4.5 mg/kg. These observations, coupled with the findings that BAY K 8644 is a potent, competitive inhibitor of [³H]nitrendipine binding to the dihydropyridine binding site in mouse brain (K _i=7.0×10^–9M), suggests that BAY K 8644 may produce its behavioral actions via an interaction with the DHP binding site, which has been linked to the control of calcium flux across membranes in peripheral tissues.     

Differential actions of calcium antagonists on calcium binding to cardiac sarcolemma

The action of four calcium antagonistic drugs, including verapamil, bepridil, nifedipine, and diltiazem, on calcium binding to cardiac sarcolemma from guinea pig was tested. It was found that verapamil (10^?6 to 10^?5 M) inhibited calcium binding to a great extent. Bepridil at the same concentrations was less potent than verapamil in the depression of calcium binding. Nifedipine and diltiazem did not affect sarcolemmal calcium binding. The differential action of the calcium antagonistic drugs was discussed.     

Differential effects of dihydropyridine calcium antagonists on doxorubicin-induced nephrotoxicity in rats

The aim of this study was to compare the roles of dihydropyridine calcium antagonists nifedipine, nitrendipine, amlodipine on doxorubicin (DXR)-induced nephrotoxicity in rats using biochemical, histopathological and immunohistochemical approaches. Male Sprague-Dawley rats were randomly divided into five experimental groups: control; DXR; DXR+nifedipine (15 mg/kg); DXR+nitrendipine (10 mg/kg); DXR+amlodipine (5 mg/kg). Results showed that treatment with DXR alone caused significant changes in the levels of urinary protein, serum creatinine (SCr), and blood urea nitrogen (BUN). Co-administration with amlodipine effectively reversed the effect of DXR on these parameters. In contrast, nifedipine and nitrendipine either had no effect or worsened DXR induced changes in the levels of urinary protein, SCr and BUN. Furthermore, DXR treatment caused significant increases in the levels of malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOS) and significant decreases in the levels of reduced glutathione (GSH), glutathione-S-transferase (GST), and superoxide dismutase (SOD). These effects were significantly reduced by co-administration with amlodipine but not affected by nifedipine and worsened by nitrendipine. In addition to the biochemical changes, histopathological studies showed that DXR caused significant structural damages in the kidneys. Glomerular cell apoptosis, a decrease in Bcl-2 expression and an increase in Bax expression were observed in all rats treated with DXR. Co-administration with amlodipine effectively reversed the effect of DXR while nifedipine and nitrendipine had no effect. In conclusion, this study clearly indicated that amlodipine protected against DXR-induced nephrotoxicity while nifedipine and nitrendipine had no effect.     

Effect of calcium agonists and antagonists on cerebellar granule cells

In cerebellar cultures, comprising predominantly granule neurones, dihydropyridine (DHP) Ca²⁺ agonists were potent stimulators of voltage-sensitive ⁴⁵Ca²⁺ uptake. Their effect was maximal in partially depolarized cells; at 15 mM K_e⁺ half maximal stimulation occurred at about 5 × 10⁻⁸ M BAY K 8644 and 10⁻⁷ M(+)-(S)-202 791. Organic Ca²⁺ antagonists were effective inhibitors of voltage-sensitive calcium entry into granule cells: the order of potency in blocking uptake induced by sub-maximal concentration of K⁺ and BAY K 8644 was nifedipine > (−)-202 791 > D600. BAY K 8644 also stimulated the release of glutamate, the transmitter of the granule cells, from depolarized cells. Granule cells are therefore a class of neurones whose responsiveness to organic Ca²⁺ effectors is similar to that of cardiac and smooth muscle. The discrepant findings on the effect of calcium effectors in various preparations of nervous tissues may thus reflect a differential distribution of voltage-sensitive Ca²⁺ channels in different neuronal cell types.     

Differential effects of L-type calcium channel blockers and stimulants on naloxone-precipitated withdrawal in mice acutely dependent on morphine

The effects of L-type calcium channel blockers and stimulants on naloxone-precipitated withdrawal in miceacutely dependent on morphine were evaluated. Verapamil (10–80 mg/kg), diltiazem (20–120 mg/kg) and nicardipine (20–160 mg/kg), when administered subcutaneously, produced a dose-dependent reduction in forepaw tremor and weight loss during the abstinence reaction; jumping was also reduced by all three drugs, although the effect was not statistically significant in the case of nicardipine. By contrast, the calcium agonist Bay K 8644 (0.5–2 mg/kg, SC) increased forepaw tremor and weight loss, although this latter effect did not reach statistical significance. The effects of the calcium channel active drugs on the rotarod test were also explored, no correlation appearing with the results observed in abstinence (except for the jumping response), which suggests that the withdrawal results are not influenced by motor incoordination or unspecific CNS depression. These findings suggest that L-type calcium channels probably play an important role in withdrawal after acute morphine dependence. Taken together with other observations in chronic models, these results show that calcium channels are similarly involved in morphine abstinence after acute and chronic dependence, in contrast to the differences in the content and uptake of neuronal calcium induced by morphine under both conditions.     

Synthesis and calcium channel antagonist activities of 3-nitrooxyalkyl, 5-alkyl 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-3, 5-pyridinedicarboxylates

A group of racemic 3-[(2-nitrooxyethyl), (3-nitrooxypropyl), (4-nitrooxybutyl) or (1,3-dinitrooxy-2-propyl)], 5-methyl (ethyl or propyl) 1,4-dihydro-2,6-dimethyl-4-(1-methyl-5-nitro-2-imidazolyl)-3,5-pyridinedicarboxylates (18-29) were synthesized using modified Hantzsch reaction that involved the condensation of 2-nitrooxyethyl (8), 3-nitrooxypropyl (9), 4-nitrooxybutyl (10) or 1,3-dinitrooxy-2-propyl (13) acetoacetate with methyl (14), ethyl (15) or isopropyl (16) 3-aminocrotonate and 1-methyl-5-nitroimidazole-2-carboxaldehyde (17). In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. Compounds 18-29 exhibited superior, or equipotent, calcium antagonist activity (IC50= 10(11) - 10(-13) M range) relative to the reference drug nifedipine (IC50 = 1.07 +/- 0.12 x 10(-11) M), which could serve as potential probes to investigate the in vivo release of nitric oxide which induces vascular muscle relaxation.     

l-2-Chloropropionic acid-induced cerebellar granule cell necrosis is potentiated by L-type calcium channel antagonists

We have used the model of l-2-chloropropionic acid (L-CPA)-induced selective cerebellar granule necrosis to study excitatory amino acid-induced necrotic cell death in vivo produced by the activation of N-methyl-d-aspartate (NMDA) receptors. However, the mechanism for the NMDA receptor activation and the biochemical events which dictate the anatomical selectivity for the L-CPA-induced lesion are as yet unknown. We examined whether blockade of sodium and calcium channels may reduce the neurotoxicity through a reduction of glutamate release from granule cells. None of the sodium channel antagonists examined, i.e. phenytoin, lamotrigine or rilazole nor the mixed sodium/calcium channel blocker, lifarazine, altered the L-CPA neurotoxicity. However, L-type calcium channel blockers, verapamil and nifedipine enhanced the L-CPA-induced granule cell necrosis, assessed by measuring the degree of L-CPA-induced reductions in cerebellar aspartate concentration, increases in cerebellar glycine concentrations and the development of cerebellar oedema. In addition, the locomotor activity of rats receiving both L-CPA and either verapamil or nifedipine was significantly lower than when rats received L-CPA alone, suggesting an enhancement of the neurotoxicity of L-CPA by L-type calcium channel blockade. The data suggest that L-CPA may interfere with non-L-type calcium channels located on granule cell bodies and nerve terminals leading to reduction of the calcium entry into the cells. We suggest that a combination of L-type channel blockade and non-L-type channels which are sensitive to L-CPA produces reductions in intracellular calcium concentrations below that required for neuronal survival. Received: 28 April 1997 / Accepted: 1 July 1997     

Attenuation of the ischaemia-induced fall of electrical ventricular fibrillation threshold by a calcium antagonist,diltiazem

Summary Calcium antagonists have been reported to decrease the incidence of sudden death in postinfarction management and vulnerability to fibrillation secondary to experimental coronary occlusion. In order to confirm such beneficial results regarding ischaemic ventricular fibrillation, the threshold intensity for fibrillation electrically induced with impulses of 100 ms and 180 beats · min^–1 was measured during the course of ischaemias obtained by total occlusion of the left anterior descending coronary artery near its origin in open-chest pigs. The variations of electrical fibrillation threshold with ischaemia duration (30, 60, 120, 180, 240, 360 s) were compared under control conditions and after i.v. diltiazem (0.50 mg · kg^–1 plus 0.02 mg · kg^–1 · min^–1 over 25 min). Electrical fibrillation threshold was not influenced by diltiazem before, but raised during ischaemia, particularly from the 60th s (1.7 to 4.0 mA), with delay in the triggering of fibrillation which occurs when the fibrillation threshold falls down to the pacing threshold (0.2 to 0.3 mA). In 6 pigs out of 8, fibrillation was even avoided in the longest of the ischaemic periods considered (360 s), for fibrillation threshold ceased falling before reaching the critical level. These experimental results obtained with diltiazem are consistent with the clinical effectiveness of calcium antagonists recently observed in the prevention of postinfarction sudden death, provided that myocardial contractility is not too much adversely affected. But, left ventricular dP/dt_max was not reduced by more than 6.8% in the present experiments. Correspondence to: Q. Timour at the above address     

静脉注射钙拮抗剂对大鼠胰岛B细胞功能影响的实验观察

目的观察不同钙拮抗剂对Sprague Dawley（SD）大鼠胰岛B细胞功能的作用。方法将正常成年SD大鼠随机分成4组（每组8只）：对照组、维拉帕米组、地尔硫卓组及尼卡地平组。利用静脉糖耐量实验技术（intravenous glucose tolerance test,IVGTT）观察静脉注射不同钙拮抗剂对大鼠胰岛B细胞分泌功能的影响。结果与对照组相比,钙拮抗剂维拉帕米、地尔硫卓和尼卡地平组的＋血糖升高,早期时相的胰岛素分泌下降。结论静脉给予钙拮抗剂降压可能影响胰岛B细胞的分泌功能。     

Potentiation of cardiodepressive action among calcium antagonists from different classes: evidence for a mechanism at the single calcium channel level

Summary The ability of calcium antagonists and antiarrhythmic agents to potentiate the negative inotropic effects of calcium antagonists was investigated in guinea-pig left atria. The poteny of nitrendipine was enhanced by several amphiphilic agents by one order of magnitude or more (by pretreatment with quinidine or bepridil). The effect of preincubation with bepridil was investigated for a larger number of dihydropyridines. Only some of them were potentiated like nitrendipine. There was no potentiation between any two members of the same chemical group, i.e. between two dihydropyridines or two catamphiphilic calcium antagonists.The interaction between bepridil and nitrendipine was studied in more detail. In atria, its extent was influenced by several conditions, such as the stimulus frequency, the incubation temperature, or the extracellular K⁺ concentration. In measurements of whole-cell calcium currents in guinea-pig myocytes, the interaction was found to take place in a quantitatively similar manner. At the single channel level, an enhancement of the effects could also be demonstrated. It appears here that both drugs interact by binding to the same channel molecule.We conclude that the interaction may be due to 1.: an amphiphilic drug (like bepridil) binding to the channel very transiently and thus briefly favouring the inactivated channel state, which means that 2.: the other drug (like nitrendipine) has a higher chance to be bound because of its high affinity towards inactivated channels. Alternative explanations are also discussed. Send offprint requests to S. Herzig at the above address     

Decrease in calbindin content significantly alters LTP but not NMDA receptor and calcium channel properties

The contribution of the cytosolic calcium binding protein calbindin D(28K) (CaBP) to the synaptic plasticity was investigated in hippocampal CA1 area of wild-type and antisense transgenic CaBP-deficient mice. We showed that long-term potentiation (LTP) induced by tetanic stimulation in CaBP-deficient mice was impaired. The fundamental biophysical properties of NMDA receptors and their number were not modified in CaBP-deficient mice. We also demonstrated that the physiological properties of calcium channels were identical between genotypes. An insufficient Ca(2+) entry through NMDA receptors or calcium channels, or a decrease in NMDA receptor density are unlikely to explain this impairment of LTP. Interestingly, we showed that the loss of LTP was not prevented by glycine but was restored in the presence of a low concentration of the NMDA receptor antagonist D-APV (5 microM) and of the calcium chelator BAPTA-AM (5 microM). Moreover, we observed a loss of LTP in the wild-type mice when the postsynaptic tetanic-induced [Ca(2+)](i) rise is excessively increased. Conversely, a weaker tetanus stimulation allowed LTP induction and maintenance in CaBP-deficient mice. These results suggest that a higher cytosol [Ca(2+)](i), due to the decrease of CaBP expression may impair LTP induction and maintenance mechanisms without affecting the mechanisms of calcium entry. Thus, CaBP plays a critical role in long term synaptic plasticity by limiting the elevation of calcium rise in the cytosol to some appropriate spatio-temporal pattern.     

The high-affinity immunoglobulin E receptor (FcepsilonRI) regulates mitochondrial calcium uptake and a dihydropyridine receptor-mediated calcium influx in mast cells: Role of the FcepsilonRIbeta chain immunoreceptor tyrosine-based activation motif

A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcepsilonRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcepsilonRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03-3 ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcepsilonRIbeta immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcepsilonRIbeta ITAM is essential for a sustained [Ca2+]m uptake. The FcepsilonRIbeta ITAM was essential for overall calcium response upon weak FcepsilonRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcepsilonRIbeta regulates [Ca2+]m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel.     

Synthesis, QSAR and calcium channel antagonist activity of new 1,4-dihydropyridine derivatives containing 1-methyl-4,5-dichloroimidazolyl substituents

A group of dialkyl and diarylester analogues of nifedipine, in which the ortho-nitrophenyl group at position 4 was replaced by a 1-methyl-4,5-dichloroimidazolyl substituent, were synthesized and evaluated as calcium-channel antagonists using the high K(+)concentration of guinea-pig ileum longitudinal smooth muscle. The structure of all compounds was confirmed by IR,(1)H-NMR, and mass spectra. The calcium-channel antagonist activity of compounds 10a-f demonstrated that compound 10b was the most active and 10f the least active one. With unsymmetrical diesters 12a-k, the most active compound was the ethyl, phenethyl derivative. Structural parameters on the calcium-channel antagonist activity were evaluated by QSAR analysis and a linear correlation was found between the -log IC(50) values of these compounds and their constitutional and topological properties.     

Comparison of electrophysiological effects of calcium channel blockers on cardiac repolarization

Dihydropyridine (DHP) calcium channel blockers (CCBs) have been widely used to treat of several cardiovascular diseases. An excessive shortening of action potential duration (APD) due to the reduction of Ca²⁺ channel current (I_Ca) might increase the risk of arrhythmia. In this study we investigated the electrophysiological effects of nicardipine (NIC), isradipine (ISR), and amlodipine (AML) on the cardiac APD in rabbit Purkinje fibers, voltage-gated K⁺ channel currents (I_Kr, I_Ks) and voltage-gated Na⁺ channel current (I_Na). The concentration-dependent inhibition of Ca²⁺ channel currents (I_Ca) was examined in rat cardiomyocytes; these CCBs have similar potency on I_Ca channel blocking with IC₅₀ (the half-maximum inhibiting concentration) values of 0.142, 0.229, and 0.227 nM on NIC, ISR, and AML, respectively. However, ISR shortened both APD₅₀ and APD₉₀ already at 1 µM whereas NIC and AML shortened APD₅₀ but not APD₉₀ up to 30 µM. According to ion channel studies, NIC and AML concentration-dependently inhibited I_Kr and I_Ks while ISR had only partial inhibitory effects (<50% at 30 µM). Inhibition of I_Na was similarly observed in the three CCBs. Since the I_Kr and I_Ks mainly contribute to cardiac repolarization, their inhibition by NIC and AML could compensate for the AP shortening effects due to the block of I_Ca.     

Reversal of acute theophylline toxicity by calcium channel blockers in dogs and rats

Theophylline, widely used in the treatment of pulmonary diseases, has a narrow therapeutic index; the recommended plasma levels being 10–20 μg/ml in humans. The misuse or abuse of theophylline can cause life-threatening central nervous system and cardiovascular effects. Increased intracellular Ca²⁺ levels are thought to play an important role in theophylline toxicity and death. The objective of this study was to determine whether Ca²⁺ channel blockers, e.g. verapamil, nifedipine, or diltiazem, prevent sudden death caused by theophylline treatment in rats and dogs. Groups of Sprague-Dawley rats were treated with theophylline alone (150 mg/kg i.p.) or with theophylline pretreatment followed by administration of verapamil (0.25 to 0.5 mg/kg i.p.), nifedipine (0.25 to 1.0 mg/kg i.p.), or diltiazem (0.5 to 1.0 mg/kg i.p.), 2.5 to 15 min later. The rats were observed for toxic signs and survival over a period of 15 days. All three calcium channel blockers significantly reduced the theophylline-induced sudden death in rats. In a separate study, neither verapamil (0.5 mg/kg i.p.) nor nifedipine (1.0 mg/kg i.p.) prevented the theophylline-induced myocardial necrosis in the rat. In beagle dogs, verapamil (0.5 mg/kg i.v.) prevented theophylline (15 mg/kg/min i.v. for 10 min)-induced hypotension, arrhythmias, and sudden death. Our results support previously reported findings that calcium plays a major role in theophylline-induced toxicity and death.     

Rate of L-type calcium channels on yohimbine-precipitated clonidine withdrawal in vivo and in vitro

Summary This study was designed to elucidate the possible participation of L-type calcium channels in the expression of clonidine-withdrawal precipitated by yohimbine in clonidine-dependent animals. Mice implanted for 5 days with osmotic minipumps containing the ₂-adrenoceptor agonist clonidine showed symptoms of a withdrawal syndrome (jerks, headshakes, defecations and weight loss) when yohimbine, an ₂-adrenoceptor antagonist, was injected. Similarly, isolated rat ilea incubated with clonidine in vitro showed a withdrawal contracture when yohimbine was added to the organ bath. The effects of L-type calcium channel blockers (verapamil and diltiazem) and the stimulant Bay K 8644 on these two different types of withdrawal responses were evaluated. A dose-dependent decrease in yohimbine-precipitated clonidine withdrawal in vivo was observed when verapamil (10–40 mg/kg, s.c. and 120 g/mouse, i.cv.) or diltiazem (5–20 mg/kg, s.c. and 160 g/mouse, i.c.v.) were administered to mice dependent on clonidine. No effect was found after Bay K 8644 (0.5–5 mg/kg, s.c. and 1–5 g/mouse) was injected under these conditions. In vitro, both verapamil (0.1–5 M) and d-cis-diltiazem (1–50 M) concentration-dependently reduced the height of the yohimbine-precipitated withdrawal contracture in rat ileum incubated with clonidine. Furthermore, the effect of diltiazem was stereospecific, as d-cis-diltiazem 10 M markedly inhibited clonidine withdrawal, whereas the same concentration of l-cis-diltiazem had no effect. In contrast, the calcium channel stimulant Bay K 8644 (0.1–1 M) increased the height of the ileum withdrawal contrature. These results confirm that yohimbine-precipitated clonidine withdrawal can be obtained both in vivo and in vitro, and suggest that the expression of these abstinence responses involves activation of L- type calcium channels. The present results, together with those of previous studies of the effects of calcium channel-acting drugs on ethanol-, opiate- and benzodiazepine-withdrawal, suggest that L-type calcium channels play an important role in the expression of the withdrawal responses to CNS depressant drugs.Correspondence to: J. M. Baeyens at the above address     

Effects of calcium channel blockers on the development of early rat postimplantation embryos in culture

Rat embryos (9.5-day-old) were cultured for 48 h in the presence of nifedipine (NIF), nimodipine (NIM), nitrendipine (NIT), gallopamil HCl (GAL), verapamil HCl (VER) and diltiazem HCl (DIL). The effects on growth and morphogenetic differentiation in vitro were monitored. Dose-response relationships were evaluated, including an assessment of the no-observed-effect-level (NOEL) or the lowest-observed-effect-level (LOEL), and the lowest concentration tested inducing abnormalities in 100% of the embryos (100% EL). The morphological alterations observed at the highest concentrations were very similar for all six drugs. The abnormalities concerned yolk sac circulation and morphology, as well as heartbeat, the morphology of the heart, head, neural tube, or fore-limbs, and the shape of the embryo. The abnormal embryos were also growth retarded (decrease in protein content and crown-rump length). Interference with calcium channel functions seems to represent an interesting model for studying a special kind of abnormal prenatal development, especially the differentiation of certain mesenchymal structures. The concentration ranges between NOELs and 100% ELs were found to be: NIM=0.1–1 g/ml; NIT and VER=1–10 g/ml; DIL=1–30 g/ml, and LOELs-100%ELs were: GAL=1–10 g/ml; NIF=10–30 g/ml.On leave of absence from: Industrial Toxicology Research Centre, Lucknow, India