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PubMed1996

Ligand-gated calcium channels inside and out.

Calcium release from intracellular stores occurs through two types of channels associated with intracellular membranes, namely, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. Recently, it has been shown that these channels are regulated by allosteric mechanisms and associated proteins. Release of intracellular calcium induces the opening of calcium-permeable channels on the plasma membrane. Current work has focused on the molecular and functional characterization of these channels which have been identified as store-operated channels or calcium release activated channels.

Calcium↗

PubMed1993

The effect of calcium channel antagonists and BAY K 8644 on calcium fluxes of malignant hyperpyrexia-susceptible muscle.

1. The calcium channel antagonists verapamil (100 microM) and nifedipine (100 microM) inhibited twitch response and KCl induced hypercontractility in malignant hyperpyrexia (MH)-susceptible porcine skeletal muscle. These calcium channel antagonists did not effect hypercontractility induced by 3% halothane or 2 mM caffeine. 2. The calcium channel agonist BAY K 8644 (50 microM) induced contracture in MH-susceptible muscle but did not potentiate contracture response induced by 2 mM caffeine or 3% halothane. BAY K 8644 did not increase the resting tension of control muscle or increase the sensitivity of control muscle to 4 mM caffeine, 3% halothane or 80 mM KCl. 3. The sarcoplasmic reticulum (SR) from MH-susceptible and control porcine skeletal muscle was separated into vesicular fractions enriched in the membrane elements of the terminal cisternae and longitudinal tubules. 4. Verapamil and diltiazem [which has been previously shown to inhibit the hypercontractility of MH-susceptible porcine muscle to caffeine, halothane and KCl (Foster and Denborough, 1989 Br. J. Anaesth. 62, 566-572)] did not effect Ca2+ uptake or Ca(2+)-dependent ATPase activities of SR longitudinal tubule membranes, from MH-susceptible or control muscle. These calcium channel antagonists did not effect Ca2+ release from terminal cisternae preparations. 5. The skeletal muscle relaxant dantrolene inhibited Ca2+ efflux and equilibrium-Ca2+ exchange associated with the terminal cisternae membrane of MH-susceptible and control skeletal muscle. 6. Calcium channel antagonists modify Ca2+ fluxes in MH-susceptible and control muscle by acting at a site distal to the SR. Calcium channel antagonists may inhibit contractile response by modifying events of excitation-contraction coupling associated with the voltage sensor molecule (dihydropyridine-receptor) of the transverse-tubule membrane, whereas dantrolene directly acts on the terminal cisternae membrane to inhibit Ca2+ efflux and equilibrium Ca2+ exchange. Different calcium channel antagonists seem to modify the voltage-sensor mechanism in different ways in MH-susceptible muscle. 7. An abnormality in the coupling mechanism of the voltage sensor-SR calcium release channel may exist in MH-susceptible muscle. This dysfunction may be an adaptation to the elevated levels of myoplasmic Ca2+ and/or the molecular defect described in the Ca2+ release channel of the SR of MH-susceptible porcine muscle. In view of these results it is unlikely that nifedipine or verapamil would be of therapeutic value for the treatment of MH.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1984

Calcium channel 'agonist' BAY K 8644 inhibits calcium antagonist binding to brain and PC12 cell membranes.

BAY K 8644, a drug that elicits calcium-dependent muscle contraction, inhibits binding of the voltage-dependent calcium channel antagonist [3H]nitrendipine to brain and PC12 pheochromocytoma cell membranes. This effect is due to high-affinity (Ki = 4.5 nM) competitive inhibition at the binding site for dihydropyridine calcium antagonists. Allosteric sites that mediate calcium channel blockade by non-dihydropyridine calcium antagonists are not similarly affected. Our findings indicate that BAY K 8644 is active at central, as well as peripheral, calcium channels and are compatible with a multi-state model of the voltage-dependent calcium channel in which antagonist drugs promote a closed state of the channel, while BAY K 8644 promotes an open state.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Characterization of presynaptic calcium channels with omega-conotoxin MVIIC and omega-grammotoxin SIA: role for a resistant calcium channel type in neurosecretion.

The peptide Ca2+ channel antagonists omega-conotoxin (omega-CTX) MVIIC and omega-grammotoxin (omega-GTX) SIA were studied by measuring their effects on the release of [3H]glutamate from rat brain synaptosomes. The pseudo-first-order association constant for omega-CTX MVIIC (1.1 x 10(4) M-1 sec-1) was small, relative to that for omega-GTX SIA (3.6 x 10(5) M-1 sec-1). Equilibrium experiments showed that omega-CTX MVIIC blocked approximately 70% of Ca(2+)-dependent glutamate release evoked by 30 mM KCl (IC50 approximately 200 nM), whereas omega-GTX SIA virtually eliminated release, with lower potency (IC50 approximately 700 nM). At stronger depolarizations (60 mM KCl), neither toxin (at 1 microM) showed significant block of release, but when these or other Ca2+ channel antagonists (omega-CTX GVIA or omega-agatoxin IVA) were used in combination a substantial fraction of release was blocked. [3H]Glutamate release that was resistant to omega-CTX MVIIC was characterized with respect to its sensitivity to block by omega-GTX SIA and the inorganic blocker Ni2+. Both omega-GTX SIA and Ni2+ were relatively weak blockers of the resistant release. These results suggest that a previously uncharacterized Ca2+ channel exists in nerve terminals and can be distinguished on the basis of its resistance to omega-CTX MVIIC and its weak sensitivity to omega-GTX SIA and Ni2+. Thus, at least three channel types (P, N, and a "resistant" type) contribute to excitation-secretion coupling in nerve terminals.

Animals↗

PubMed1987

Calcium channels, the sodium-calcium exchange and intracellular sodium in the predisposition of the heart to the calcium paradox.

A wide variety of cardiac tissues load with Na+ on exposure to Ca-free fluid. This loading is due to a sustained influx of Na+ through the calcium channels and predisposes the tissues to the changes known as the Calcium Paradox on reperfusion with calcium containing fluid. The cells then load with calcium, most probably via activation of the Na/Ca exchange. This calcium loading may be responsible for the subsequent changes in intracellular pH and cellular metabolism.

Action Potentials↗

PubMed1987

New perspectives in cardiovascular medicine: the calcium channel blocking drugs.

Calcium channel blocking drugs reduce the influx of calcium (Ca++) through cell membrane passageways in excitable tissues. This unique pharmacodynamic action represents an important new addition to cardiovascular therapeutics. Clinically available members of this diverse group of compounds are verapamil, nifedipine, and diltiazem. Beneficial responses to these drugs can be explained by vasodilatation and resulting hemodynamic improvement in tissue perfusion-oxygen demand relationships, by suppression of Ca++-dependent arrhythmogenic mechanisms, by direct reduction of pathologic Ca++ overload in ischemic injured cells, or by a combination of these effects. Calcium channel blockade already has become a therapeutic mainstay in human medicine for management of ischemic heart disease and some forms of cardiac dysrhythmias. Relative to veterinary medicine, Ca++ channel blocking drugs may provide a clinical option for controlling supraventricular tachyarrhythmias. Other indications might include obstructive cardiomyopathies, shock-trauma, and congestive heart failure. Importantly, however, essential issues about adverse cardiovascular side effects of Ca++ channel blocking drugs remain unresolved and controversial. Recent studies especially have raised questions about the tendency for Ca++ channel blockade to exacerbate preexisting or occult myocardial contractile failure. Such pharmacodynamic complexities should be assessed judiciously as the Ca++ channel blocking drugs are appraised for entry into veterinary internal medicine.

Animals↗

PubMed1990

Inotropic and calcium kinetic effects of calcium channel agonist and antagonist in isolated cardiac myocytes from cardiomyopathic hamsters.

The mechanism by which heart cells of cardiomyopathic (CM) hamsters become calcium overloaded is not known. We examined the number of slow calcium channels, calcium uptake via slow calcium channels, calcium pool sizes, and the contractile response to Bay K 8644, verapamil, and nifedipine using isolated cardiac myocytes from 8-9-month-old CM hamsters (BIO 14.6) and age-matched normal controls. The number of dihydropyridine binding sites as assessed by specific binding of [3H]PN200-110 was similar in the two groups (control hearts: Bmax = 333 +/- 89 [mean +/- SD] fmol/mg; CM hearts: Bmax = 357 +/- 75 fmol/mg; n = 5 experiments, p = 0.6). Current density through L-type calcium channels was determined using the whole-cell clamp technique (at -50 mV holding potential and -10 mV test potential) and was the same in CM myocytes (17.8 +/- 1.5 [mean +/- SD] pA/pF) and control myocytes (18.6 +/- 2.1 pA/pF) (n = 5 experiments, p = 0.5). The current-voltage relation (test potentials varied from -40 to +50 mV) was also the same in CM and control cells, as was apparent threshold, peak current, and reversal potential. However, the initial rate of 45Ca influx as well as the size of the rapidly exchangeable calcium pool was significantly greater in myocytes obtained from CM than from normal hamsters. In both myocyte preparations, Bay K 8644 increased the rate of 45Ca uptake by 25% at 60 seconds; verapamil decreased 45Ca uptake at 60 seconds by 16% and 17% in normal and CM hamsters, respectively. A similar inhibitory effect was observed with nifedipine. The amplitude of cell motion in cells driven at 1.5 Hz as assessed by an optical-video system increased progressively with increasing concentrations of extracellular calcium or Bay K 8644 in cardiac myocytes from normal or CM hamsters. However, the concentration-effect curves for the two effectors were shifted to the left in CM cells compared with cells from normal hamsters. Both preparations demonstrated similar contractile responses to verapamil and nifedipine. These findings demonstrate that single enzymatically dissociated cardiac myocytes from CM hamsters have impaired contractile properties analogous to those seen in the intact heart and thus provide a useful experimental system in which to study underlying cellular mechanisms operative in this model of heart failure. Our results further indicate that calcium overload in CM hamster cardiac myocytes may not be due to increased calcium influx via dihydropyridine-sensitive calcium channels, as suggested previously, but rather to abnormalities of intracellular calcium homeostasis.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2001

hKv4.3 channel characterization and regulation by calcium channel antagonists.

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.

Cadmium Chloride↗

PubMedDate not supplied

Dietary sodium intake: influence on calcium channels and urinary calcium excretion in spontaneously hypertensive rats.

Binding of the 1,4-dihydropyridine [3H]PN200 110 was employed as an index of cardiac Ca2+ channels in normotensive (WKY) and spontaneously hypertensive (SHR) rats during 4 weeks of normal (0.73% NaCl) and high (8% NaCl) sodium diets when the rats were between 20 and 24 weeks of age. Binding site density was not different at the beginning of the study but was increased significantly (P less than 0.01) after 1 week in the SHR on a high sodium diet; this difference was not apparent at 2, 3 or 4 weeks of the diet. During this same period, the urinary Ca2+ excretion in SHR was enhanced significantly (P less than 0.01) and the urinary calcium/sodium ratio was elevated during the high sodium intake period.

Animals↗

PubMed2006

Prolonged high-pressure treatments in mammalian skeletal muscle result in loss of functional sodium channels and altered calcium channel kinetics.

Activation and inactivation of ion channels involve volume changes from conformational rearrangements of channel proteins. These volume changes are highly susceptible to changes in ambient pressure. Depending on the pressure level, channel function may be irreversibly altered by pressure. The corresponding structural changes persist through the post-decompression phase. High-pressure applications are a useful tool to evaluate the pressure dependence as well as pressure limits for reversibility of such alterations. Mammalian cells are only able to tolerate much lower pressures than microorganisms. Although some limits for pressure tolerance in mammalian cells have been evaluated, the mechanisms of pressure-induced alteration of membrane physiology, in particular of channel function, are unknown. To address this question, we recorded fast inward sodium (I(Na)) and slowly activating L-type calcium (I(Ca)) currents in single mammalian muscle fibers in the post-decompression phase after a prolonged 3-h, high-pressure treatment of up to 20 MPa. I(Na) and I(Ca) peak amplitudes were markedly reduced after pressure treatment at 20 MPa. This was not from a general breakdown of membrane integrity as judged from in situ high-pressure fluorescence microscopy. Membrane integrity was preserved even for pressures as high as 35 MPa at least for pressure applications of shorter durations. Therefore, the underlying mechanisms for the observed amplitude reductions have to be determined from the activation (time-to-peak [TTP]) and inactivation (tau(dec)) kinetics of I(Na) and I(Ca). No major changes in I(Na) kinetics, but marked increases, both in TTP and tau(dec) for I(Ca), were detected after 20 MPa. The apparent molecular volume changes (activation volumes) deltaV(double dagger) for the pressure-dependent irreversible alteration of channel gating approached zero for Na+ channels. For Ca2+ channels, deltaV(double dagger) was very large, with approx 2.5-fold greater values for channel activation than inactivation (approx 210 A3). We conclude, that in skeletal muscle, high pressure differentially and irreversibly affects the gating properties and the density of functional Na+ and Ca2+ channels. Based on these results, a model of high pressure-induced alterations to the channel conformation is proposed.

Animals↗

PubMed1987

Calcium channel blockers: correlation among receptor binding, calcium uptake and contractility in vitro.

Ten known calcium channel blockers were studied for inhibition of K+-induced 45Ca++ uptake into rabbit aortic smooth muscle cells in culture, and for displacement of [3H]nitrendipine [2,6-dimethyl-3-carbomethoxy-5-carbomethoxy-4-(3-nitro)phenyl-1, 4-dihydroxypyridine] binding to rat ventricular membrane preparations, in order to relate their effects on receptor binding with their inhibitory activities on 45Ca++ uptake and on contractile responses of vascular smooth muscle. Steady-state 45Ca++ uptake increased with K+ concentration in a dose-dependent manner. With 25 to 50 mM K+, Ca++ uptake was 0.6 nmol of Ca++ per one million cells. All calcium channel blockers inhibited K+-induced 45Ca++ uptake and [3H]nitrendipine binding in a dose-dependent fashion. The enatiomeric dihydropyridines 202-791 [isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-nitro-3-pyridinecarboxylate] exhibited marked stereoselectivity in both studies, the agonist (+)-202-791 significantly enhancing 45Ca++ uptake at 15 to 50 mM K+. The similarity between the order of potency in inhibiting 45Ca++ uptake and displacing [3H]nitrendipine resulted in a highly significant linear (1:1) correlation. An equally significant correlation was also established for the 10 blockers between their inhibitory potencies on 45Ca++ uptake and the contractile response of rabbit aortic strips as cited in the literature. These findings support the hypothesis that calcium channel blockers block contraction of vascular muscle by inhibiting cellular calcium uptake through voltage-dependent calcium channels as a result of binding to receptors associated with these channels. The aortic cells possess channels that are functionally similar to those found in intact vascular tissue.

Animals↗

PubMed1997

Myocardial action potential prolongation by calcium channel activation under calcium free-EGTA conditions in guinea-pigs: developmental and regional variations.

Prolongation of action potentials upon the addition of various cAMP increasing agents under Ca2+ free-EGTA condition, which was previously suggested to be produced by persistent Na+ influx through Ca2+ channel due to lack of Ca(2+)-mediated inactivation, was examined in isolated myocardial preparations from neonatal and adult guinea-pigs. Application of isoproterenol under Ca2+ free-EGTA condition produced prolongation of the action potential duration in both neonatal and adult preparations from various regions of the heart. Both in the neonate and adult, the prolongation in the right ventricular free wall and atrial preparations was smaller than that in preparations from other parts of the ventricle. In all regions of the heart except the right ventricle, the prolongation by isoproterenol was greater in the adult than in the neonate, no developmental difference was observed in the right free wall. In right ventricular free-wall preparations, the prolongation produced by forskolin and isobutyl-methylxanthine in the adult was larger than in neonatal ones. Our present results suggest regional and developmental difference in contribution of Ca(2+)-mediated inactivation of Ca2+ channels to the repolarization of guinea-pig myocardium. The developmental changes were similar to but much smaller than, those observed in the rat myocardium.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions.

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.

Animals↗

PubMed1998

Distinct calcium channel isoforms mediate parathyroid hormone and chlorothiazide-stimulated calcium entry in transporting epithelial cells.

Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.

Animals↗

PubMed1984

Regulation of cardiac calcium channel current and contractile activity by the dihydropyridine Bay K 8644 is voltage-dependent.

Pharmacological studies have provided considerable information about the molecular structure of ion channels in the membranes of excitable cells. Two classes of drugs, the local anesthetics and the biotoxins, have been used to study sodium channels in nerve, skeletal muscle, and cardiac cells, and tetraethylammonium ion and many of its derivatives have provided structural information about potassium channels in nerve. More recently, organic compounds that block calcium channels (calcium channel antagonists) have begun to be used to probe calcium channels in cardiac and smooth muscle cells (see). One group of calcium channel blockers, the dihydropyridines, has provided considerable information on the structure and function of these channels on the basis of electrophysiological and binding studies. Slight modification of the structure of one of these dihydropyridines, nifedipine, has led to the discovery of a group of compounds that are presumed to act by increasing the influx of Ca2+ into cardiac and smooth muscle cells. One of these novel compounds, Bay K 8644 (methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5 carboxylate) has been reported to act at the same receptor site as the calcium channel antagonist nifedipine, but enhances contractile activity of the perfused heart and aortic strips. Because activation of contraction in cardiac muscle is closely linked to calcium entry via voltage-dependent calcium channels (see for review) these results suggested that Bay K 8644 might act on these channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Chick cerebellar Purkinje cells express omega-conotoxin GVIA-sensitive rather than funnel-web spider toxin-sensitive calcium channels.

Voltage-gated calcium channels form a complex family of distinct molecular entities which participate in multiple neuronal functions. In cerebellar Purkinje cells these channels contribute to the characteristic electrophysiological pattern of complex spikes, first described in birds and later in mammals. A specific calcium channel, the P-type channel, has been shown to mediate the majority of the voltage-gated calcium flux in mammalian Purkinje cells. P-type channels play an essential role in synaptic transmission of mammalian cerebellum. It is unclear whether the P-type calcium channel is present in birds. Studies in chick synaptosomal preparations show that the pharmacological profile of calcium channels is complex and suggest a minimal expression of the P-type channel in avian central nervous system. In the present work, we studied voltage-gated calcium channels in dissociated chick cerebellar Purkinje cells to examine the presence of different calcium channel types. Purkinje cells were used because, in mammals, they express predominantly P-type channels and because the morphology of these cells is thought to be phylogenetically conserved. We found that omega-conotoxin GVIA (omega-CgTx GVIA), a specific antagonist of N-type calcium channel, rather than the synthetic funnel-web spider toxin (sFTX), a P-type channel antagonist, blocks the majority of the barium current flowing through calcium channels in chick Purkinje neurons.

Animals↗

PubMed1998

Genetic analysis of voltage-dependent calcium channels.

Molecular cloning of calcium channel subunit genes has identified an unexpectedly large number of genes and splicing variants, and a central problem of calcium channel biology is to now understand the functional significance of this genetic complexity. While electrophyisological, pharmacological, and molecular cloning techniques are providing one level of understanding, a complete understanding will require many additional kinds of studies, including genetic studies done in intact animals. In this regard, an intriguing variety of episodic diseases have recently been identified that result from defects in calcium channel genes. A study of these diseases illustrates the kind of insights into calcium channel function that can be expected from this method of inquiry.

Animals↗

PubMed1998

Contraction of human hepatic stellate cells activated in culture: a role for voltage-operated calcium channels.

BACKGROUND/AIMS: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca2+]i) and cell contractility. METHODS: Binding studies using [3H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca2+]i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. RESULTS: Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1+/-1.4x10(4) sites/cell with a Kd of 2.1+/-0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca2+]i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca2+]i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. CONCLUSION: Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca2+]i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.

Calcium Channel Blockers↗