Organic Calcium Channel Blockers Research Articles

Pharmacology of L-type Calcium Channels: Novel Drugs for Old Targets?

Inhibition of voltage-gated L-type calcium channels by organic calcium channel blockers is a well-established pharmacodynamic concept for the treatment of hypertension and cardiac ischemia. Since decades these antihypertensives (such as the dihydropyridines amlodipine, felodipine or nifedipine) belong to the most widely prescribed drugs world-wide. Their tolerability is excellent because at therapeutic doses their pharmacological effects in humans are limited to the cardiovascular system. During the last years substantial progress has been made to reveal the physiological role of different L-type calcium channel isoforms in many other tissues, including the brain, endocrine and sensory cells. Moreover, there is accumulating evidence about their involvement in various human diseases, such as Parkinson's disease, neuropsychiatric disorders and hyperaldosteronism. In this review we discuss the pathogenetic role of L-type calcium channels, potential new indications for existing or isoform-selective compounds and strategies to minimize potential side effects.

Cav1.3 L-Type Calcium Channel Dysfunction in Human Disease

Voltage-gated calcium-channels are key molecular regulators of calcium-dependent signaling processes in electrically excitable cells. The family of L-type calcium-channels (LTCCs, Cav1.1-Cav1.4 pore-forming α1-subunits) are sensitive to organic calcium-channel blockers (e.g. dihydropyridines). The in vivo pharmacological effects of these widely used antihypertensives are limited to inhibition of Cav1.2 LTCCs in the cardiovascular system, despite the (often overlapping) expression of both Cav1.2 and Cav1.3 in most excitable tissues. Important insight into the physiological relevance of these channels therefore came from mutant mice and human disease pinpointing their distinct functions not only in the heart but also for hearing, brain and endocrine function. Cav1.2 and Cav1.3 α1 share high structural similarity and are frequently expressed in the same cell but exhibit important functional differences. Their activity is fine-tuned by auto-inhibitory domains within their long C-terminal tails but they use different mechanisms to adjust the extent of auto-inhibition. Moreover, Cav1.3 channels activate at more negative potentials allowing them to serve unique roles for sinoatrial node pacemaking and cochlear inner hair cell neurotransmitter release. Accordingly, a loss-of-function mutation in Cav1.3 α1 (CACNA1D) in humans causes sinoatrial node dysfunction and congenital deafness (SANDD). Human mutations also revealed a novel role for Cav1.3 in adrenal aldosterone-producing adenomas (APA). In APA somatic gain-of-function mutations can drive calcium-dependent aldosterone production causing secondary hypertension. SANDD and APA mutations also provided valuable insight into the structure-function relationship of Cav1.3 α1.Both channels have recently also been implicated as risk genes for psychiatric diseases, including autism spectrum disorders. It will therefore be important to understand how subtle changes in the function of these channels is associated with human disease and how calcium-channel blockers can be further optimized to specifically target them to LTCCs outside the cardiovascular system.(Support: Austrian Science Fund, F44020).

The fourth-generation Calcium channel blocker: cilnidipine.

Several classes of antihypertensive agents have been in clinical use, including diuretics, α-blockers, β-blockers, angiotensin converting enzyme (ACE) inhibitors, angiotensin II type 1 receptor blockers (ARB), and organic calcium channel blockers (CCBs). All these drugs are being currently used in the treatment of Hypertension & various disease conditions of the heart either alone or in combination. Cilnidipine is a new antihypertensive drug distinguished from other L-type Ca(2+) channel blockers or even other antihypertensives, which will be useful for selection of antihypertensive drugs according to the pathophysiological condition of a patient.

Interaction between organic calcium-channel blockers and taurine in vitro and in vivo.

Interaction between organic calcium-channel blockers and taurine in vitro and in vivo.

Mechanical adaptability of a sponge extracellular matrix: evidence for cellular control of mesohyl stiffness inChondrosia reniformisNardo

The marine sponge Chondrosia reniformis Nardo consists largely of a collagenous tissue, the mesohyl, which confers a cartilaginous consistency on the whole animal. This investigation was prompted by the incidental observation that, despite a paucity of potentially contractile elements in the mesohyl, intact C. reniformis stiffen noticeably when touched. By measuring the deflection under gravity of beam-shaped tissue samples, it was demonstrated that the flexural stiffness of the mesohyl is altered by treatments that influence cellular activities, including [Ca2+] manipulation, inorganic and organic calcium channel-blockers and cell membrane disrupters, and that it is also sensitive to extracts of C. reniformis tissue that have been repeatedly frozen then thawed. Since the membrane disrupters and tissue extracts cause marked stiffening of mesohyl samples, it is hypothesised that cells in the mesohyl store a stiffening factor and that the physiologically controlled release of this factor is responsible for the touch-induced stiffening of intact animals.

Comparison of the effects of nitric oxide donors and calcium channel blockers on the intrinsic myogenic tone of sheep isolated internal anal sphincter

Chronic anal fissure is associated with considerable pain and anal hypertonia. Numerous clinical studies attest to the effectiveness of individual nitro-containing drugs and organic calcium channel blockers in this condition but there are few comparative studies. Isolated segments of sheep internal anal sphincter were prepared for isometric tension recording. The effect of various drugs on myogenic tone was examined in the absence or presence of sodium orthovanadate (SOV), an agent used to mimic anal hypertonia by increasing myogenic tone. All the drugs tested produced concentration-dependent inhibition of myogenic tone, with the maximum effect ranging from 66.4 per cent (verapamil) to 100 per cent (sodium nitroprusside). Sodium nitroprusside and diltiazem were the most potent, followed by glyceryl trinitrate (GTN), nifedipine and verapamil, which had similar potency, and finally nicorandil. The potency of GTN and diltiazem was reduced threefold in the presence of 1 mmol/l SOV. The combined effect of GTN and diltiazem was greater than the effect of either agent alone, even in the presence of 3 mmol/l SOV. Nitro-containing drugs and organic calcium channel blockers are potent inhibitors of anal sphincter myogenic tone that may be used in combination to treat chronic anal fissure.

Reduction of human neocortical and guinea pig CA1-neuron A-type currents by organic calcium channel blockers

In epilepsy models, organic calcium antagonists regularly induce a transient activity increase before suppression of epileptiform discharges. This action was speculated to be mediated by a modulation of potassium currents. Since A-type currents potently regulate neuronal excitability, their modulation by calcium channel blockers was investigated in acutely isolated human neocortical temporal lobe neurons and CA1 neurons of guinea pigs using the whole-cell voltage-clamp technique. In human neurons, 40 μM nifedipine caused an amplitude reduction by 28% at a command potential of −6 mV and produced a biexponential, markedly accelerated current inactivation with time constants of 8.4 ± 1.1 ms ( n = 6) and 62.9 ± 6.4 ms ( n = 5). The time constant under control conditions was 50.1 ± 8.5 ms ( n = 6). Verapamil (40 μM) did not affect the current amplitude, but accelerated the monoexponential current inactivation from 40.2 ± 7.1 ms to 13.3 ± 0.8 ms ( n = 9). Accordingly, verapamil accelerated the inactivation from 42.3 ± 5.9 ms to 15.0 ± 1.3 ms ( n = 11) in guinea pig CA1 neurons, without affecting the current amplitude. In this preparation, it was shown that the two enantiomers of verapamil do not differ in their actions. The results show that the A-type current in human neocortical and in guinea pig hippocampal neurons is reduced by organic calcium channel blockers.

Lowering of the potassium concentration induces epileptiform activity in guinea-pig hippocampal slices

Extra- and intracellular recording techniques were used to study the epileptiform activity generated by guinea-pig hippocampal slices perfused with low potassium containing artificial cerebrospinal fluid. Extracellular field potentials were recorded in CA1 and CA3 regions along with intracellular recordings in CA3 subfield. Reduction of the extracellular potassium concentration [K +] o from 4 to 2 mM caused a transient neuronal hyperpolarisation which was followed by a repolarisation and subsequent depolarisation period. Paroxysmal depolarisation shifts occurred during the transient hyperpolarisation period while epileptic field potentials (EFP) appeared in the late repolarisation or early depolarisation phase. EFP elicited by reduction of [K +] o were neither affected by blockade of N-methyl- d-aspartate (NMDA) glutamate-subreceptor or gamma aminobutyric acid receptor, nor by application of the organic calcium channel blocker nifedipine or the anticonvulsant drugs carbamazepine and valproic acid. Upon application of non-NMDA glutamate-subreceptor blocker the EFP were abolished in all trials, while application of the organic calcium channel blocker verapamil only suppressed the EFP in some cases. The results point to a novel mechanism of epileptogenesis and may provide an in vitro model for the development of new drugs against difficult-to-treat epilepsy.

Portuguese Man-of-war ( Physalia physalis) venom induces calcium influx into cells by permeabilizing plasma membranes

Portuguese Man-of-war ( Physalia physalis) nematocyst venom dose-dependently stimulates calcium ( 45Ca 2+) influx into L-929, GH 4C 1, FRL, and embryonic chick heart cells. Venom-induced calcium influx is not blocked by ouabain, vanadate, nor organic calcium channel blockers, but is blocked by transition metal cations, such as lanthanum and zinc. Venom-induced calcium influx is accompanied in a dose-dependent manner by the release of intracellular lactate dehydrogenase, indicating a loss in plasma membrane integrity and cytolysis. Concentrations of zinc that block 45Ca 2+ influx also block lactate dehydrogenase release. Lanthanum, which also blocks 45Ca 2+ uptake, does not neutralize the cytolytic activity of the venom, but rather inhibits the venom's cytolytic action at the level of the target cell plasma membrane. Our findings indicate that Man-of-war venom causes an influx of calcium into several different cells types, not just those of the cardiovascular system, and this influx likely occurs by permeabilizing the plasma membranes of cells.

Changes of the membrane potential profile induced by verapamil and propranolol

The effects of the organic calcium channel blocker verapamil and the β-receptor blocker propranolol on dipole (φd) and surface (φs) potentials of bilayer lipid membranes were studied. The boundary potentials (φb=φd+φs) of black lipid membranes, monitored by conductance measurements in the presence of nonactin and by capacitive current measurements were compared with φs calculated from the electrophoretic mobility of lipid vesicles. It was shown that the increase of boundary potential, induced by the adsorption of the positively charged propranolol, was caused solely by an increase in surface potential. Although φs also increases due to the adsorption of verapamil, φb diminishes. A sharp decrease of the dipole potential was shown to be responsible for this effect. From Langmuir adsorption isotherm the dissociation constant Kd of verapamil was estimated. The uncharged form of verapamil (Kd=(0.061±0.01) mM at pH 10.5) has a tenfold higher affinity to a neutral bilayer membrane than the positively charged form. The alteration of membrane dipole potential due to verapamil adsorption may have important implications for both membrane translocation and partitioning of small or hydrophobic ions and charged groups of membrane proteins.

Spontaneous sharp waves in human neocortical slices excised from epileptic patients.

Human neocortical temporal lobe tissue resected for treatment of pharmacoresistant epilepsy was investigated. In slices prepared from this tissue, field potentials sometimes superimposed by population spikes were found to appear spontaneously. In individual slices, they were generalized or highly localized to a field of approximately 200 microns in diameter. Synchronous with these potentials, hyperpolarizing and depolarizing postsynaptic potentials were recorded from neurons in the vicinity of the field potential electrode. Hyperpolarizing postsynaptic potentials appeared to be mainly chloride mediated. All potentials, i.e. sharp field potentials as well as postsynaptic potentials, were reversibly suppressed by blockade of the non-NMDA (non-N-methyl-D-aspartate) glutamate-subreceptor and of the GABAA (gamma-aminobutyric acid) receptor, and by application of the organic calcium channel blocker verapamil. By contrast, all potentials remained unaffected by blockade of the NMDA glutamate-subreceptor and the GABAB receptor. The antiepileptic drugs carbamazepine and phenytoin failed to suppress the spontaneous potentials at therapeutic concentrations. Washout of Mg2+ from the superfusate left the spontaneous potentials unchanged or converted them to ictal-type discharges. This epileptiform activity was not suppressed, but augmented by blockade of the GABAA receptor. As a whole, the spontaneously appearing field potentials may be assumed to reflect a state of increased neuronal synchronization.

125I-Labelled mapacalcine: a specific tool for a pharmacological approach to a receptor associated with a new calcium channel on mouse intestinal membranes.

Mapacalcine is a small protein (Mr=19041) composed of two homologous chains purified from the marine sponge Cliona vastifica. Recently, we demonstrated that it was able to specifically block a Ca2+ channel which could not be related to already described channels on mouse intestinal myocytes. This Ca2+ current was insensitive to the known peptidic and organic calcium channel blockers. Mapacalcine was ineffective on T-type and L-type Ca2+ currents present on rat portal vein myocytes [Morel, Drobecq, Sautière, Tartar, Mironneau, Qar, Lavie, and Hugues (1997) Mol. Pharmacol. 51, 1042-1052]. We report here the preparation and purification of a monoiodo-derivative of mapa-calcine which retains its biological properties. Binding parameters of mapacalcine to its receptors have been characterized on mouse intestinal membranes. It binds to its receptors with a Kd=0. 8 nM, and a maximal binding capacity of 171 fmol/mg of protein on membrane preparations. Our data show that we have prepared a tool that is usable for pharmacological studies of a receptor associated with a new type of calcium channel for which no ligand was available until now.

A calcium antagonistic effect of the new antiepileptic drug lamotrigine

The new antiepileptic drug lamotrigine (LTG; 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine) has been shown to be effective in the treatment of focal epilepsies with or without secondary generalization. Furthermore, some case reports indicate an efficacy in the treatment of bipolar affective disorders. It has been suggested that the main mechanism of action of LTG is the inhibition of glutamate release through blockade of voltage sensitive sodium channels and stabilisation of the neuronal membrane. Since some antidepressant drugs and the antiepileptic substance carbamazepine have calcium antagonistic properties, which may be of significance in the pathophysiology of epilepsies and affective disorders, the interaction of lamotrigine with carbamazepine and the organic calcium channel blocker verapamil was analyzed in the low Mg 2+-induced model epilepsy which has been shown to be suppressed specifically by organic calcium antagonists. Lamotrigine reduced the frequency of occurrence of low-magnesium induced field potentials in CA1 and CA3 areas of the hippocampus slice preparation (guinea pigs) in a dose-dependent manner. The subthreshold concentrations which yielded no effect were 1 μmol/l for lamotrigine, 10 μmol/l for carbamazepine and 2 μmol/l for verapamil. Combinations of these subthreshold concentrations elicited a reduction in the repetition rate of field potentials. The results indicate that lamotrigine behaves additive with verapamil and carbamazepine what can be due to a common action on the same subtype of calcium channels. It can be assumed that lamotrigine may have besides its action on high-frequency sodium dependent action potentials also effects on calcium channels.

Alteration of loosely bound calcium in the guinea pig organ of Corti after treatment with diltiazem as calcium channel blocker.

After oral administration of the organic calcium channel blocker diltiazem to guinea pigs for 7 days, calcium ions were precipitated with potassium antimonate in the cochleae. The spatial distribution of the precipitates was studied by energy-filtering transmission electron microscopy and the amount of the ultrastructural reaction products formed was determined semiquantitatively by an image processing system. Compared with untreated control ears, the number of the formed precipitates was reduced drastically in the inner hair cells after diltiazem treatment. In addition, electron microscopic analysis revealed that the number of calcium precipitates attached at the basolateral membrane of the outer hair cells was clearly reduced when compared with untreated control specimens. A large number of histochemical reaction products could be identified in the basilar membrane and were also observed in the untreated control specimens. The spatial distribution of the calcium precipitates in the lamina reticularis was not affected by diltiazem treatment and calcium precipitates could be identified within different cell membranes. The techniques used was considered to be helpful for identifying calcium channels ultrastructurally in intact undissected tissues and to support light microscopic analyses and patch-clamp electrophysiological measurements.

Myoblast fusion requires cytosolic calcium elevation but not activation of voltage-dependent calcium channels

Many studies of in vitro skeletal myogenesis have demonstrated that fusion of myoblasts into multi-nucleated myotubes is regulated by calcium-dependent processes. Calcium ions appear to be necessary at the outer face of the membrane, and an additional internal calcium increase seems required to promote fusion of aligned myoblasts. It has been proposed that a calcium influx could take place prior to fusion and that this may be mediated by voltage-dependent calcium channels. Previously, we showed that two types of voltage-dependent calcium currents were expressed in multinucleated myotubes but not in rat myoblasts growing in primary culture before the withdrawal of the growth medium. We also showed that the previous formation of multinucleated synticia was not a prerequisite of developmental appearance of calcium currents, suggesting that the two events were time-correlated but not sequentially dependent. These features led us to investigate changes in internal calcium activity and the possible appearance of voltage-dependent calcium influx pathways just after the promotion of fusion by the change of culture medium. The results confirm that a rise in cytosolic calcium activity occurs slightly before fusion in confluent myoblasts and remained in newly formed myotubes. Reducing this elevation by internal calcium buffering lowered myoblast fusion and, reciprocally, blocking cell fusion prevented calcium increase. Treatment with the organic calcium channel blockers nifedipine (5 μM) and PN 200-110 (1 μM) did not alter cytosolic calcium changes nor cell fusion, and voltage-dependent calcium currents were never observed by the perforated patch-clamp technique in aligned fusion-competent myoblasts. Other voltage-operated mechanisms of calcium rise were not detected since depolarization with hyper-potassium solutions failed to elicit increases in intracellular calcium. On the contrary, acetylcholine was able to promote extracellular calcium-dependent calcium transients. Our results confirm the requirement of an increase in resting calcium during fusion, but do not support the hypothesis of an influx through voltage-dependent channels or other voltage-operated pathways. The elevation of internal calcium activity may result from other mechanisms, such as a cholinergic action for example.

Modulation of the activity of midbrain central gray substance neurons by calcium channel agonists and antagonists in vitro.

Changes in the background impulse activity of midbrain central gray substance neurons have been studied on slice preparations from the rat midbrain upon application of calcium-free solution, an activator of calcium channels, BAY-K 8644 (10 nM), organic (verapamil, 40 microM; D600, 10 microM; nifedipine, 1-10 microM; amiloride, 1 microM) and inorganic (Co2+, 1.5 mM) calcium channel blockers. Besides BAY-K 8644, all the substances inhibited most of the neurons studied. Verapamil, BAY-K 8644 and Co2+ also revealed facilitatory effects. Facilitatory action of BAY-K was most effective in silent neurons and in those previously inhibited by amiloride. Latent period values of inhibition in calcium-free solution and upon application of organic and inorganic blockers have the following sequence: D600 > amiloride > verapamil > Co2+ > nifedipine > calcium-free solution. Maximum rise time had the following order: amiloride > D600 > nifedipine > verapamil > Co2+ > calcium-free solution. Complete suppression of the neuronal activity induced by amiloride lasted twice as long as that induced by calcium-free solution, Co2+ and nifedipine, and six times as long as verapamil-induced suppression. Preliminary application of calcium channel blockers reduced facilitatory and increased inhibitory effects of serotonin and substance P. Data obtained are discussed with the supposition in mind that inhibition of the function of calcium channels in central gray substance neurons could be one of the mechanisms underlying the analgesic effect of a series of neurotropic agents after their introduction into this structure.

The block of the expressed L-type calcium channel is modulated by the β3 subunit

The α 1C subunit of the L-type calcium channel was stable, expressed alone or in combination with the β 3 subunit in Chinese hamster ovary cells. The β 3 subunit enhanced significantly the inactivation of barium currents indicating that both subunits interacted with each other. The β 3 subunit decreased signinicantly the half-maximal inhibitory concentration of the calcium channel blockers (−)-gallopamil and verapamil, but did not affect significantly the block caused by isradipine and mibefradil at the holding potentials of −80 mV and −40 mV. These results suggest that the β 3 subunit affects distinctly the interaction of the expressed α 1c subunit with different classes of organic calcium channel blockers.

Effects of the serotonin-1A agonists buspirone and ipsapirone on field potentials in the hippocampus slice: comparison with carbamazepine and verapamil

The serotonin-1A agonists buspirone (BU) and ipsapirone (IPSA) have been demonstrated to exert antidepressant and anxiolytic effects. Since some antidepressant drugs and the antiepileptic substance carbamazepine have calcium antagonistic properties, the interaction of BU and IPSA with carbamazepine and the organic calcium channel blocker verapamil was analyzed in the low Mg 2+ induced model epilepsy which has been shown to be suppressed specifically by organic calcium antagonists. BU and IPSA reduced the frequency of occurrence of low magnesium induced field potentials in CA1 and CA3 areas of the hippocampus slice preparation (guinea pigs) in a dose dependent manner. The subthreshold concentrations which yielded no effect were 5 μmol/1 for BU and IPSA, 10 μmol/1 for carbamazepine and 2 μmol/1 for verapamil. Combinations of these subthreshold concentrations elicited a reduction in the repetition rate of field potentials. The results indicate that BU and IPSA behave additively with verapamil and carbamazepine, which may be due to a common action on the same subtype of calcium channels. It may be assumed that besides their action on 5-HT 1A receptors BU and IPSA may also have calcium antagonistic properties.

A pharmacological analysis of calcium channels involved in phasic and tonic responses of the guinea-pig ureter to high potassium.

1. Previous studies have established a marked difference in sensitivity to organic calcium channel blockers of the phasic compared with tonic component of the contraction to potassium chloride (KCl) in the guinea-pig ureter. The mechanisms responsible for this difference have remained unsettled. In particular, the possible involvement of non-L-type calcium channels in contractility of the ureter has not been determined. In this study we have re-addressed this problem and, to eliminate any possible contribution of sensory neuropeptides released by KCl from peripheral endings of afferent nerves, all experiments were performed in ureters pre-exposed to the sensory neurone blocking agent, capsaicin (10 microM for 15 min). 2. Increasing concentrations of KCl (10-160 mM) produced phasic and tonic contractions of the guinea-pig isolated ureter: the L-type calcium channel agonist, Bay K 8644 (1 microM), enhanced both components of the contraction to KCl. 3. Nifedipine (1 microM) abolished all responses to increasing concentrations of KCl after 60 min contact time; after a shorter incubation period (15 min), the phasic contractions to low KCl concentrations were still observed, while the tonic responses were abolished. 4. The effects of nifedipine (0.1 nM-1 microM) on the phasic and tonic components of the response to 80 nM KCl were assessed after 15-120 min contact time. Nifedipine was equipotent in inhibiting the tonic response at all times tested, while a marked time-dependency of inhibition toward phasic responses was observed. After 15 min contact time, nifedipine was 181 times more potent in inhibiting tonic than phasic response to KCl, while after 120 min contact time the difference between EC50 values was only 5.4 times. 5. Cadmium chloride (3-30 microM) was equi-effective in inhibiting the phasic and tonic responses to KCl while nickel chloride was ineffective at 10-fold higher concentrations. omega-Conotoxin (0.1 microM) and tetrodotoxin (0.3 microM) were ineffective. 6. The present findings indicate that L-type voltage-dependent calcium channels mediate both phasic and tonic components of the response of the guinea-pig ureter to KCl while neither T-type nor N-type voltage-dependent calcium channels are involved. The marked time-dependency of inhibitory action of nifedipine suggests that L-type voltage-dependent calcium channels which are responsible for the generation of phasic contraction of the ureter are in a low affinity state for interaction with nifedipine.

Picrotoxin-induced epileptic activity in hippocampal and neocortical slices (guinea pig): suppression by organic calcium channel blockers

Epileptic activity induced by the GABAA receptor antagonist bicuculline is known to be blocked by organic calcium antagonists. To further analyse the mechanism underlying convulsant activity induced by substances reducing GABA-mediated synaptic transmission, the effect of organic calcium channel blockers on epileptic activity induced by the GABAA channel blocker Picrotoxin in hippocampal and neocortical slices of guinea pigs were investigated. Verapamil and flunarizine suppressed paroxysmal depolarization shifts (PDS) of single neurons and accompanying epileptic field potentials (EFP). As a measure of drug action the repetition rate of epileptic events was used. The depression down to 10% the initial value (90% depression) is indicated. In the hippocampus verapamil suppressed PDS/EFP within 70 ± 16 min (40 μmol/l) and within 39 ± 5 min (60 μmol/1). This suppression was reversible with washout of verapamil. Flunarizine irreversibly blocked EFP/PDS within 108 ± 14 min (18 μmol/l). In the neocortex verapamil reversibly suppressed EFP within 146 ± 6 min (40 μmol/1) and 127 ± 26 min (60 μmol/l). Flunarizine irreversibly blocked EFP within 181 ± 30 min (3 μmol/1) and 109 ± 13 min (18 μmol/l). The results suggest that voltage dependent calcium channels are essentially involved in picrotoxin-induced epileptic activity.