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1. The sensitivity of cromakalim-activated current (Icrom) to manipulations of extracellular cationic composition was examined in whole-cell voltage clamp recordings from freshly-dispersed, adult guinea-pig ventricular myocytes. In bathing media with different concentrations of K+ (1, 2.5, 5.4 and 12 mM) the Icrom reversal potential (Erev) varied in strict correspondance with the K+ equilibrium potential and inward Icrom amplitude was proportional to the external K+ concentration. 2. Replacement of 12mM K+ with 12mM Rb+ induced a slight positive shift of Erev indicating that PRb+/PK+ = 1.06. K+ replacement with 12mM Cs+ reduced or abolished inward Icrom and produced a negative shift of Erev by at least 50 mV; an upper limit of PCs+/PK+ was fixed at 0.18. 3. Addition of Rb+ (1-30 mM) to 2.5 mM K(+)-containing medium produced a concentration-dependent increase in inward Icrom and positive shift of Erev suggesting that K+ and Rb+ have similar permeabilities and conductivities and do not interfere with each other in the channel. 4. CS+ (0.01-30 mM), added to medium containing 12 mM Rb+, induced a potent, voltage-dependent inhibition of inwardly rectifying current (IK1; IC50 = 0.2-3 mM). Voltage-dependent inhibition of inward Icrom was observed only at considerably higher CS+ concentrations (IC50 = 4-30 mM). Extracellular Rb+ and CS+ did not substantially alter the amplitude of outward Icrom. 5. The results support the contention that the ATP-sensitive K+ channel is the primary target of cromakalim action in ventricular myocytes.

Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na+, Ca2+, and K+ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na+, K+) ATPase in myotonic disorders.

The effects of short term stimulation of beta-adrenergic receptors and elevations in intracellular cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle cells in vitro has been studied using both the 45Ca2+ flux technique and [3H] nitrendipine-binding experiments. Isoproterenol increased the nitrendipine-sensitive 45Ca2+ influx under depolarizing conditions. The effects of isoproterenol were additive to those of depolarization and were antagonized by alprenolol. Half-maximal inhibition of 45Ca2+ influx induced both by depolarization and by isoproterenol occurred at a nitrendipine concentration of 1 nM. Treatments that resulted in an increased level of intracellular cyclic AMP, such as treatment with 1-methyl-3-isobutylxanthine, theophylline, dibutyryl cyclic AMP, or 8-bromocyclic AMP also resulted in an increased rate of 45Ca2+ entry via nitrendipine-sensitive Ca2+ channel. In contrast, long term treatment of myotubes in culture with isoproterenol and other compounds that increased intracellular cyclic AMP led to a large increase in the number of nitrendipine receptors. This increase was accompanied by a 4-10-fold decrease in the affinity of the receptors for nitrendipine. Alprenolol inhibited the long term effects of isoproterenol. In vivo treatment of 7-day-old chicks with reserpine and alprenolol produced a decrease in the number of skeletal muscle nitrendipine receptors. This decrease in receptor number was accompanied by an increase in the affinity of nitrendipine for its receptor by a factor of 4 to 5. These effects on the nitrendipine receptor were prevented by simultaneous injection of isoproterenol. The results are discussed in relation to the role of beta-adrenergic receptors and intracellular cyclic AMP in the regulation of skeletal muscle Ca2+ channels.

The sensitivity of embryonic cardiac cells to tetrodotoxin (TTX) increases with age. At the early embryonic stage, the maximum upstroke velocity is not affected by the presence of TTX. In the course of both in ovo and in vitro development, this velocity reaches an adult-like value of 90-120 V/sec, which is decreased in the presence of TTX to 5-10 V/sec. The differentiation of the Na+ channel has been followed by using three types of specific toxins: (i) TTX or a tritiated derivative of it, (ii) a polypeptide toxin extracted from sea anemone, and (iii) the alkaloidic toxins veratridine and batrachotoxin. Electrophysiological, including voltage-clamp experiments, and biochemical studies have shown (i) that the TTX receptor and the fast Na+ channel machinery exist even when action potentials are insensitive to TTX--the channel is then in a nonfunctional or silent form that is revealed (or chemically activated) by both the alkaloids and the polypeptide toxin--and (ii) that the total number of Na+ channels increases during development by a factor of 4 or 5. In monolayers of cardiac cells insensitive to TTX in which all Na+ channels are in a nonfunctional form, the rate of degradation of the TTX receptor follows first-order kinetics with a half-time of 9 hr. In aggregates fully sensitive to TTX, the number of TTX receptors remains perfectly stable 24 hr after blockade of protein synthesis.

Three major ion transport systems, the nitrendipine-sensitive Ca2+ channels, the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase, have been studied in skeletal muscle from rat and chick after chronic denervation. It is shown that the situation found for the Ca2+ channel differs dramatically from that found for the Na+ channel and the (Na+ + K+)-ATPase and that regulation of the nitrendipine-sensitive Ca2+ channel in denervated muscle also differs widely from that of the tetrodotoxin-sensitive Na+ channel and the ouabain-sensitive (Na+ + K+)-ATPase which show a quite similar evolution.

This paper describes how neurotoxins specific of the fast Na+ channel are used to study its differentiation in embryonic cardiac cells during heart ontogenesis. Structural and functional differentiation of the fast Na+ channel have been followed using both electrophysiological and biochemical techniques.

The two dihydropyridines Bay K8644 and CGP 28392 increase 45Ca2+ influx in cultured rat cardiac cells with half-maximal effects at 2 nM and 30 nM respectively at a membrane potential of -75 mV. This stimulation of Ca2+ uptake is inhibited by nitrendipine, verapamil and bepridil. Ca2+ channel activation produced by Bay K8644 and CGP 28392 has been compared with Ca2+ channel activation produced by depolarization. There is no addition between the effects of drugs activating the Ca2+ channel and the effects of depolarization suggesting that Bay K8644 and CGP 28392 work preferentially on polarized membranes. 45Ca2+ flux experiments yielded results which are in excellent agreement with electrophysiological and contraction data obtained with the same cells in culture. Dose-response curves for the physiological effects of the drugs are observed over the same range of concentrations as their inhibition of [3H]nitrendipine binding to its receptor.

Two distinct and interdependent binding sites for inhibitors of voltage-dependent Ca2+ channels have been identified. They include one site for molecules of the 1,4-dihydropyridine serie such as nitrendipine, nifedipine or PN200-110 and one site for a chemically heterogenous group of compounds comprising verapamil, D600 and desmethoxyverapamil, bepridil and diltiazem. Ca2+ binds to its own coordination site which is distinct from the receptor site for organic Ca2+ channel inhibitors. The molecular size of the native [3H] nitrendipine receptor of transverse tubule membrane, brain and heart, have been determined using the radiation inactivation technique. The [3H] nitrendipine receptor is found to have a Mr of 210,000 +/- 20,000. CHAPS solubilization and purification indicate that the dihydropyridine receptor contains polypeptides of apparent molecular weights of 142,000, 32,000 and 33,000 which copurifie with (+) [3H] PN200-110 binding activity. Two stages in which there is an increased binding of [3H]nitrendipine have been observed during chick myogenesis. The first one occurs during embryonic life and has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors. This increase is accompanied by a decrease in the affinity of nitrendipine for its receptor by a factor of 4 to 10. The second stage of development is partly under innervation control and its expression is modulated by the intracellular cyclic AMP content. The two dihydropyridines Bay K8644 and CGP 28932 work preferentially on polarized membranes. 45Ca2+ flux experiments yielded results which are in good agreement with electrophysiological, contraction and binding data obtained with rat cardiac cells and skeletal muscle cells.

The nitrendipine-sensitive Ca2+ channel of chick skeletal myotubes in culture has been studied using both the 45Ca2+ flux technique and [3H]nitrendipine binding experiments. Ca2+ uptake is insensitive to nitrendipine when chick myotubes in culture are polarized. Whereas depolarization reveals a new component of 45Ca2+ influx which is inhibited by nitrendipine. Half-maximal inhibition occurs at a nitrendipine concentration of 0.7 nM. This value is similar to the dissociation constant Kd = 0.4 nM found in [3H]nitrendipine binding experiments. During myogenesis in vitro the nitrendipine receptor is absent in myoblasts and appears in parallel with the fusion process. Two stages of increased binding have been observed in vivo. The first one, which occurs during embryonic life, has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors. This increase is accompanied by a decrease of affinity of nitrendipine for its receptor by a factor of 4 to 10. Chronic denervation produces a further increase in the number of nitrendipine receptors which reaches a factor of about 2 at 15 days of denervation. Results are discussed in relation to the particular localization of these channels in transverse tubules and with the innervation.

The appearance of the voltage-dependent Na+ channel during the fetal and post-natal development of rat brain, cerebellum and skeletal muscle has been followed using a highly radiolabelled derivative of tetrodotoxin. The number of Na+ channels is low at the fetal stage and increases drastically during post-natal development. The time-course of this increase is different in brain, cerebellum and skeletal muscle. Changes in affinity of the Na+ channel for tetrodotoxin occur during brain and cerebellum development. The results are discussed in relation with the maturation of the three types of excitable tissues.

[3H]Desmethoxyverapamil, (S/-)[3H]bepridil, and d-cis-[3H]diltiazem bind to skeletal muscle transverse-tubule (T-tubule) membranes with affinity constants in the range of 2-50 nM and with Bmax values of the order of 50-80 pmol/mg of protein. These drugs are mutually competitive and modulate allosterically the 1,4-dihydropyridine binding. The molecular size of the native [3H]nitrendipine receptor of T-tubule membranes has been determined using the radiation inactivation technique. The [3H]nitrendipine receptor was found to have a Mr of 210,000 +/- 20,000. Photoaffinity labelling using (+)[3H]PN 200-110 indicates the presence in the dihydropyridine receptor of a polypeptide (or polypeptides) of Mr 170,000 +/- 5,000. The 3-[(3-cholamidopropyl)-dimethylammonio]-1 propane sulfonate (CHAPS) solubilization of the T-tubule membranes preserved the binding properties of the different drugs to the calcium channel. The dihydropyridine-sensitive calcium channel has been substantially purified by a combination of ion exchange chromatography, lectin affinity chromatography, and gel filtration. The purified material contained polypeptides of apparent molecular weights of 142,000, 32,000, and 33,000, which copurified with (+)[3H]PN 200-110 binding activity. Two stages in which there is an increased binding of [3H]nitrendipine have been observed during chick myogenesis in ovo. The first one occurs during embryonic life and has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The appearance of specific receptors for the Ca2+ channel antagonist nitrendipine has been followed during the fetal and post-natal development of rat brain without cerebellum, cerebellum, skeletal muscle and cardiac muscle. The number of nitrendipine receptors is low at the fetal stage and increases drastically during post-natal development of brain, cerebellum, skeletal muscle and cardiac muscle. The time course of this increase is different for each type of tissue studied. No significant change in receptor ligand dissociation constant (Kd) can be detected over the development period studied. The results are discussed in relation with the known properties of the differentiation process in the four types of excitable tissues studied.

This paper describes the interaction of several polypeptide neurotoxins isolated from sea anemone toxins and scorpion venom with the tetrodotoxin-resistant Na+ channel of rat cardiac cells. The 22Na+ flux and tension development were measured to examine in parallel the cardiotonic and cardiotoxic effects of these polypeptides. Inotropic effects and arrhythmias were seen in the concentration range in which an action of the toxins on the Na+ channel was observed. The maximal inotropic effect was systematically observed at toxin concentrations below the concentration value observed for half-maximal stimulation of 22Na+ flux through the Na+ channel. Arrhythmias began at concentrations near the value for half-maximal stimulation of 22Na+ flux by the toxins. Toxins extracted from the sea anemones Anemonia sulcata and Anthopleura xanthogrammica were more active than scorpion toxins and sea anemone Radianthus paumotensis toxins. The most interesting among all the toxins tested for potential use in cardiotherapy was toxin II from Anthopleura xanthogrammica.

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.

The long-lasting after-hyperpolarization(s) (AHP) that follows the action potential in rat myotubes differentiated in culture is due to Ca2+-activated K+ channels. These channels have the property to be specifically blocked by the bee venom toxin apamin at low concentrations. Apamin has been used in this work to analyze, by electrophysiological and biochemical techniques, the role of innervation in expression of these important channels. The main results are as follows: (i) Long-lasting AHP that follows the action potential in rat myotubes in culture disappears when myotubes are cocultured with nerve cells from the spinal cord under the conditions of in vitro innervation. (ii) Extensor digitorum longus muscles from adult rats have action potentials that are not followed by AHP but AHP are systematically recorded after muscle denervation and they are blocked by apamin. (iii) Specific 125I-labeled apamin binding is undetectable in innervated muscle fibers but it becomes detectable 2-4 days after muscle denervation to be maximal 10 days after denervation. (iv) Apamin receptors detected with 125I-labeled apamin are present at fetal stages with biochemical characteristics identical to those found in myotubes in culture. The receptor number decreases as maturation proceeds and 125I-labeled apamin receptors completely disappear after the first week of postnatal life, in parallel with the disappearance of multi-innervation. All these results taken together strongly suggest an all-or-none effect of innervation on the expression of apamin-sensitive Ca2+-activated K+ channels.

Mevinolin (MK803), a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) (Ki, 30 X 10(-9) M), depressed de novo synthesis of cholesterol in 11-day chicken embryonic cardiac cells cultured in lipoprotein-deficient serum (LPDS). Cardiac cells exposed to different concentrations of mevinolin for 1-3 days presented different electrophysiological and mechanical properties: The resting membrane potential, the rate of increase, and the shape of the action potential and contractile properties were changed at concentrations as low as 0.1 microM mevinolin. At a concentration of 1 microM mevinolin, the cardiac cells became quiescent and electrical stimulation induced action potentials of short duration without contraction. Isoproterenol and Bay K8644 were unable to restore excitability and contraction. Although the number of receptors for the tritiated Ca2+ channel blocker nitrendipine was the same in control and in mevinolin-treated cells, voltage-clamp data on isolated cardiac cells and 45Ca2+ flux experiments on monolayers showed that most of the slow Ca2+ channel activity was lost in mevinolin-treated cells. These results suggest that the disappearance of Ca2+ channel activity is most probably at the origin of the loss of cardiac contractility.

The properties of interaction of the Ca2+ channel antagonist [3H]nitrendipine have been investigated in chick hearts at various stages of in ovo and post-natal development and in cultured cells. The dissociation constant of the [3H]nitrendipine-receptor complex is between 0.4 nM and 0.5 nM for intact ventricle and cultured cells. [3H]Nitrendipine binding is antagonized by nitrendipine analogs. The order of efficacy of the different dihydropyridine molecules is nitrendipine greater than nimodipine greater than nifedipine greater than nisoldipine with Kd values ranging from 0.5 to 4 nM. Inhibition of [3H]nitrendipine binding by other antiarrhythmic molecules like amiodarone, F13004 and bepridil was observed. Half-maximum inhibitions (K0.5) were found for verapamil and D600 at concentrations between 0.23 and 0.26 microM. The potency of organic Ca2+ blockers to depress by 50% the maximum amplitude of spontaneous beating of heart cells is closely related to K0.5 values obtained from [3H]nitrendipine binding experiments. Electrophysiological results indicate that the slow channel is insensitive to nitrendipine at the younger stage of development (3-day-old) whereas, in adult like cells, nitrendipine (50 nM) abolished both slow action potential due to the slow Ca2+ channel and contraction. The maximum binding capacity for [3H]nitrendipine is found to increase during development of the embryonic heart from 40 fmol/mg protein at day 3 to 100 fmol/mg protein at day 14, to stay relatively stable until day 18. Then the number of sites increases rapidly to reach a second plateau at 210 fmol/mg protein on day 4 after hatching. Treatment with 6-hydroxydopamine results in 35% increase in [3H]nitrendipine binding, whereas reserpine treatment is without effect. Developmental properties of nitrendipine-sensitive Ca2+ channels have been compared with those of tetrodotoxin-sensitive Na+ channels and muscarinic receptors. These results indicate that nitrendipine receptors exist at the early stage of development (3-day-old-hearts) but that they do not correspond to functional slow Ca2+ channels, that in ovo development corresponds both to an increase of the number of [3H]nitrendipine receptors and to the transformation of silent Ca2+ channels into functional Ca2+ channels, and that there is a regulation of the level nitrendipine-sensitive Ca2+ channels by innervation.

Ca2+ current and tension have been simultaneously recorded from single twitch fibres of the semi-tendinosus of Rana esculenta in a medium containing a physiological Ca2+ concentration (1.8 mM). Under appropriate conditions it can be shown that tension develops in two phases. The first is rapid and reaches its maximum before activation of the inward Ca2+ current. The second phase is slower and with a time course which appears to be correlated with that of the inward current. Nifedipine, a specific Ca2+ channel inhibitor greatly reduced ICa2+ and the slower component of tension. Bay K8644 a Ca2+ channel activator, which has receptors on T-tubule, increased ICa2+ and the slow component of tension. These results indicate that a slow component of skeletal muscle contraction is related to the inward Ca2+ current flowing through dihydropyridine sensitive voltage-dependent Ca2+ channels.

This paper shows the presence, in rat myometrial smooth muscles, of low affinity binding sites for tetrodotoxin with a K0.5 value of 2 microM. Electrophysiological experiments using both intact strips and single isolated myometrial cells in culture have shown that veratridine and sea anemone toxins reveal functional Na+ channels. The activity of these channels was blocked by tetrodotoxin (10 microM) or by removal of Na+ ions. Results presented here are the first direct demonstration of the existence in rat myometrium of Na+ channels of the tetrodotoxin-resistant type.

This paper shows the interaction of the cardiotonic agent 4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy]-1H-indole- 2-carbonitrile (DPI 201-106) and its optic enantiomers R-DPI (205-429) and S-DPI (205-430) with the Na+ channel of a variety of excitable cells. Voltage-clamp experiments show that DPI 201-106 acts on neuroblastoma cells and rat cardiac cells. S-DPI (205-430) increases the peak Na+ current, slows down the kinetics of Na+ channel inactivation, and is cardiotonic on heart cells. Conversely, R-DPI (205-429) reduces the peak Na+ current and blocks Na+ channel activity and cardiac contractions. Binding experiments using radioactively labeled toxins indicate that DPI 201-106 and its enantiomers do not interact with sites already identified for tetrodotoxin or sea anemone and scorpion toxins. DPI 201-106 and its enantiomers inhibit binding of a 3H-labeled batrachotoxin derivative, [3H]batrachotoxinin A 20-alpha-benzoate, to brain membranes. The dissociation constant of the complex formed between the Na+ channel and both R-DPI and S-DPI is Kd congruent to 100 nM. 22Na+ uptake experiments using different cell types have shown that R and S enantiomers of DPI 201-106 are active on the different Na+ channel subtypes with similar IC50 values. These results are discussed in relation with the cardiotonic properties of DPI 201-106 that are not accompanied by cardiotoxic effects.

Properties of nitrendipine receptors have been analyzed in skeletal muscle from normal young boys and boys with Duchenne muscular dystrophy (DMD). The dissociation constant (Kd) of the complex formed by nitrendipine with its specific receptors was 0.5 +/- 0.1 nM in dystrophic muscle and 0.4 +/- 0.1 nM in normal muscle. Maximum binding capacities Bmax were 403 +/- 80 and 460 +/- 60 fmol/mg protein in DMD and normal muscle, respectively. These results suggest that nitrendipine binding sites on nitrendipine-sensitive Ca2+ channel binding sites are not altered in Duchenne muscular dystrophy.

The properties of interaction of tetrodotoxin with its receptor site on the voltage-sensitive Na+ channel were analysed in two ways: (a) by titrating Na+ channels with a tetrodotoxin derivative, [3H]ethylenediamine-tetrodotoxin; (b) by studying the physiological properties of interaction of the toxin with its receptor from 22Na flux measurements. Using a variety of cell types in culture, three different kinds of situations were observed. 1. Cells like N1E 115 neuroblastoma, CCl 39 fibroblasts, embryonic chick cardiomyocytes and chick skeletal myotubes only have one family of Na+ channels with high-affinity binding sites (in the nanomolar range) for tetrodotoxin. These Na+ channels are the same ones as those that are activated by the alkaloid and polypeptide toxins that accelerate 22Na+ influx. 2. C9 cells have Na+ channels with low-affinity binding sites for tetrodotoxin. These Na+ channels are also the ones that are activated by alkaloid and polypeptide toxins (the median inhibitory concentration for tetrodotoxin inhibition of 22Na+ influx through these Na+ channels in 300 nM). 3. Rat myotubes that have differentiated in culture in the absence of neuronal influence have both high-affinity binding sites (in the nanomolar range) detected with the tritiated tetrodotoxin derivative and low-affinity binding sites (on the micromolar range) detected by 22Na+ flux experiments. Only low-affinity binding sites correspond to Na+ channels that can be activated with alkaloid and polypeptide toxins.

Action potentials of myotubes in culture prepared from 18-19 day -old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarization (ahp) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action potentials which were never followed by a.h.p.'s. Voltage-clamp experiments have shown that Na+ channel activity was identical in mutant and control muscles and that the activity of fast and slow Ca2+ channels was nearly absent in the mutant.