Calcium channel blockers: effect on morphine-induced hypermotility

Acute morphine treatment has been shown to cause a uniform calcium depletion in various brain regions and to evoke hypermotility in mice. On the other hand, it has been reported previously that calcium channel blockers reduce the behavioral stimulation induced by different methods in mice, and it is known that these drugs increase the morphine analgesia and reduce the abstinence syndrome. The effect of calcium channel blockers, nifedipine and diltiazem, on the morphine- and amphetamine-induced hypermotility were evaluated. Mice activity was measured with photocell motility meters. The results show that neither nifedipine nor diltiazem decrease significantly the motility in control and amphetamine-treated mice; however, when they were administered to morphine-treated mice the hypermotility was significantly reduced. The mechanism responsible for this interference is still unknown.     

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.     

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.     

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     

Calcium channel antagonists: clinical uses--past, present and future

The calcium channel antagonists are a mature group of drugs directed at cardiovascular diseases including hypertension, angina, peripheral vascular disorders and some arrhythmic conditions. Their sites and mechanisms of actions have been well explored over the past two decades and their interactions at the alpha(1) subunit of L-type channels (Ca(V)1.1-1.4) have made them valuable molecular tools for channel classification and localization. With the realization that other members of the voltage-gated calcium channel family exist--Ca(V)2.1-2.3 and Ca(V)3.1-3.3--considerable effort has been directed to drug discovery at these channel types where therapeutic prospects exist for a variety of disorders including pain, epilepsy, affective disorders, neurodegenerative disorders, etc. In contrast to the situation with the L-type channel antagonists success in developing small molecule antagonists of therapeutic utility for these other channel types has thus far been lacking. The reasons for this are explored and potential new directions are indicated including male fertility, bone growth, immune disorders, cancer and schistosomiasis.     

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.     

Role of Ca channel in the renal autoregulatory vascular response analysed by the use of BAY K 8644

Summary The role of Ca channel and extracellular Ca²⁺ on autoregulation of renal blood flow was investigated in the perfused kidney of the anesthetized dog. The perfusion pressure was changed in the range between 60 and 200 mm Hg. Intra-arterial infusion of nifedipine (5 g/min) increased renal blood flow at a perfusion pressure above 100 mm Hg and inhibited autoregulation. Simultaneous infusion of 5 g/min of BAY K 8644 antagonized the effect of nifedipine. Renal blood flow was increased and autoregulatory relationship between flow and perfusion pressure was inhibited by EDTA (30 mg/min) infusion. The inhibitory effect of EDTA on renal autoregulation was counteracted by simultaneous infusion of CaCl₂ at 30 mg/min, but not counteracted by that of BAY K 8644 (5 g/min). BAY K 8644 also could not antagonize the inhibitory effect of a vasodilator, papaverine (5 mg/min) on renal blood flow autoregulation. These results provide the evidence that the renal autoregulation involves the process of Ca²⁺ influx into the vascular smooth muscle cell through the Ca channels. Send offprint requests to N. Ogawa at the above address     

Evidence for verapamil-induced functional inhibition of noradrenergic neurotransmission in vivo

Summary Contractions of the cat nictitating membrane have been used to explore the effects of calcium channel blockers on neurotransmission in vivo, by comparing the effects of verapamil and nifedipine on contractions of nictitating membrane following either electrical stimulation of the superior cervical ganglion or intravenous injection of the -adrenoceptor agonist phenylephrine. Verapamil (0.3, 0.6 and 1.2 mg/kg, iv) produced a dose related and reversible inhibition of stimulation induced contractions but did not affect phenylephrine responses of nictitating membrane. Intravenous nifedipine (10, 20 and 40 g/kg) produced inconsistent effects on both stimulation- and phenylephrine-induced contractions of the nictitating membrane. Thus only verapamil appears to selectively affect noradrenergic neurotransmission in this model, possibly by altering the neurotransmitter release from the terminals innervating the nictitating membrane in the cat. Send offprint requests to S. Gurtu 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     

Synthesis and docking studies of new 1,4-dihydropyridines containing 4-(5)-Chloro-2-ethyl-5-(4)-imidazolyl substituent as novel calcium channel agonist

1,4-Dihydropyridines have been recognized as calcium channel agonist. Three new analogues of Bay K8644 in which the ortho trifluromethyl phenyl group at position 4 is replaced by the 4-(5)-Chloro-2-ethyl-5-(4)-imidazolyl substituent, were designed and synthesized as calcium channel agonist. For this propose, the structures of designed compounds were drawn by HYPERCHEM program. Conformations of the compounds were optimized through semi-empirical method followed by PM3 calculation. Then the crystalin stucture of L-type calcium channel was obtained from the Protein Data Bank (PDB) server. Docking calculations were carried out using Auto-Dock.4 program. The good interaction of our 1,4-DHP derivatives showed that they can be as possible calcium channel agonist agents. Finally compounds were synthesized according to a modified Hantzsch condensation procedure.     

Differential effects of opiate agonists-antagonists on morphine-induced hyperexcitability and analgesia in mice

The effects of two opiate agonists-antagonists, butorphanol (4.0 and 8.0 mg/kg) and buprenorphine (0.1 and 1.0 mg/kg), were assessed on locomotor activity and analgesia in DBA/2 and C57BL/6 mice. Diferrent behavioral effects were evident in these strains, which might becharacterized by different reactions to the effects of opiates and by differences in endorphin distributions and opiate receptor populations. In particular, buprenorphine acted as an agonist-antagonist to morphine in both strains while a dissociation of buturphanol effects was evident, depending on the strain considered. The clinical implications of these findings are discussed.     

Differential involvement of voltage-dependent calcium channels in apomorphine-induced hypermotility and stereotypy

The involvement of the voltage-dependent calcium channel in behavioral effects of apomorphine was tested in naive rats and in animals which were morphine-abstinent or were subjected to chronic electroconvulsive treatment (ECS). In naive rats a calcium channel blocker, nifedipine, which by itself does not affect locomotor activity, inhibited the locomotor stimulation induced by apomorphine, while it facilitated stereotyped behavior. Morphine-abstinent and ECS-treated rats displayed elevated responsiveness to apomorphine, reflected by hypermotility and stereotyped behavior after a dose of 1 mg/kg IP that does not produce overt behavioral effects in naive animals. Nifedipine, 5 mg/kg IP, significantly reduced hypermotility produced by apomorphine in morphine abstinent or ECS-treated rats. The calcium channel blocker did not, however, antagonize enhanced stereotyped behavior. The results indicate that apomorphine hypermotility is controlled by dihydropyridine calcium channels and that enhancement of calcium channel density produced by morphine abstinence and by chronic ECS potentiates the hypermotility response. Calcium channels seem to be differently involved in control of apomorphine-induced hypermotility and stereotypy.     

Stress-induced opioid analgesia and activity in mice: Inhibitory influences of exposure to magnetic fields

An exposure for 30 min to a 0.5 Hz rotating magnetic field (1.5–90 G) significantly reduced immobilization stress-induced, opioid analgesia and hyperactivity in CF-1 and C-57 BL strains of mice, respectively. The magnetic exposure also eliminated the day-night rhythm in stress-induced analgesia, with maximum inhibitory effects occurring in the dark period. Pre-treatment with naloxone (1.0 mg/kg) had comparable inhibitory effects on immobilization-induced analgesia and activity. These results suggest that exposure to magnetic stimuli can significantly influence stress-induced activation of endogenous opioid systems and their behavioral and physiological consequences.     

Inhibition of morphine-induced analgesia and locomotor activity in strains of mice: a comparison of long-acting opiate antagonists

The long-acting opiate antagonistic potency of naloxazone (NXZ), beta-chlornaltrexamine (beta-CNA) and beta-funaltrexamine (beta-FNA) was compared using three inbred strains of mice, in which morphine induces either analgesia (DBA/2), locomotion (C57BL/6), or both responses (C3H/He). The antagonists were applied SC 24-120 hr before morphine (10 or 20 mg/kg, IP), followed by the tests after 30 min. The minimal dose which completely antagonized morphine-induced analgesia in DBA and locomotion in C57 mice during 24 hr were: for NXZ 50 and 100 mg/kg, for beta-CNA 0.8 and 6.2 mg/kg, for beta-FNA 1.6 and 12.5 mg/kg, respectively. beta-FNA and beta-CNA more potently blocked morphine-induced analgesia in DBA mice than the activity response in the C57 strain. In contrast, beta-FNA prevented morphine-induced locomotion at a lower dose (6.2 mg/kg) than analgesia (greater than 50 mg/kg) in C3H mice, while beta-CNA was equipotent (1.6 mg/kg). In general, beta-CNA turned out to be the most reactive compound, antagonizing morphine effects in low doses up to 120 hr. beta-FNA selectively antagonized either morphine-induced analgesia or locomotion, depending on the strain used. This suggests that a given morphine response might be caused by a genetically determined multiplicity of opiate receptor types and their mutual interactions.     

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.     

Influence of depolarization on vasorelaxant potency and efficacy of Ca2+ entry blockers, K+ channel openers, nitrate derivatives, salbutamol and papaverine in rat aortic rings

We examined the effects of various KCl concentrations on the actions of some vasodilators belonging to different pharmacological classes in rat aortic rings. In some experiments, tissues were precontracted with noradrenaline after blocking voltage-dependent channels to assess the effects of depolarisation unaccompanied by the entry of extracellular Ca²⁺ into the cytosol. Concentration/response curves for the vasorelaxant effect of calcium entry blockers (e.g. diltiazem), K⁺ channel openers (e.g. aprikalim), nitrate derivatives (e.g. nitroglycerin), a β₂-adrenergic agonist (salbutamol) and papaverine were obtained by using endothelium-denuded rat aortic rings precontracted with KCl (20–60 mM) to determine the potencies and efficacies of the drugs. The efficacies and potencies of calcium entry inhibitors were virtually independent of the [KCl]. A reduction in the potency (up to 18-fold) of papaverine occurred without changes in efficacy when the [KCl] was raised from 20 to 60 mM. The decline in potency was even greater for nitrate-like compounds. The potency of K⁺ channel openers in aortic rings precontracted with 30 mM KCl decreased by three- to sixfold compared with those precontracted with 20 mM KCl. With the exception of pinacidil, the efficacy of these agents already started to decline in preparations precontracted with 25 mM KCl and was virtually zero in preparations precontracted with 60 mM KCl. In contrast to other K⁺ channel openers, the vasorelaxant action of pinacidil was relatively resistant to glibenclamide. Salbutamol produced only a slight relaxation even in preparations precontracted with 20 mM KCl. In nitrendipine-pretreated, noradrenaline-precontracted aortic rings, the vasorelaxant effects of aprikalim, but not those of linsidomine or papaverine, declined when the [KCl] of the bathing medium was increased. In conclusion, the vasorelaxant potency and efficacy of calcium entry blockers is independent of the [KCl] used to precontract rat aortic rings, and thus, of the degree of membrane depolarisation. In contrast, increasing the [KCl] strongly reduces the potency and the efficacy of K⁺ channel openers not only in this preparation but also in noradrenaline-precontracted rings in which the entry of extracellular Ca²⁺ was prevented with nitrendipine. This indicates that, with the exception of pinacidil, the vasorelaxant activity of K⁺ channel openers depends on the degree of membrane depolarisation. Finally, the vasorelaxant potency and efficacy of nitrate-like compounds and papaverine are independent of depolarisation per se but they are markedly affected by the influx of Ca²⁺ accompanying elevated [KCl]. Thus, the degree of vessel depolarisation should be taken into consideration when attempting to compare potencies and efficacies among vasorelaxant agents. Received: 4 May 1998 / Accepted: 6 July 1998