Chlorpromazine and diltiazem effects in muscle: blockage of acetylcholine-evoked contractures, membrane currents and tracer calcium uptake.

Contractures evoked by 0.1 mmol/l acetylcholine (ACh) in bundles from mouse soleus muscles denervated for 3-7 days were partly inhibited for more than 1 h following 2- to 10-min exposure to 10-100 mumol/l chlorpromazine (CP). The effect was stronger in sucrose than in Na+ solutions. A prolonged ACh-contracture blockage by diltiazem (20 mumol/l) was found only in the sucrose solution. Membrane currents evoked by ACh in the absence of Na+ were blocked by both the drugs, but recovered to more than 60% within 1 min of drug washout. ACh-evoked retention of tracer 45Ca2+ was decreased by more than 90 or 70% when CP or diltiazem, respectively, were applied and washed away 10 min before addition of 45Ca2+ and ACh. The results suggest that the prolonged contracture blockage was not related to the blockage of ionic currents through the ACh-receptor channel but to a drug-induced loss of Ca(2+)-storing capacity.

Comparison of the effects of Ca2+ and those of Mg2+ and La3+ on endplate currents in mouse interosseus muscles.

Acetylcholine-induced currents were measured in dissociated muscle fibres from the mouse interosseus. Fast and slow desensitization could be distinguished, the latter ending in a steady-state current. The time constants of the two phases of current decrease and the steady-state current were decreased when the superfusing solution contained di- or trivalent cations in addition to the 0.2 mM Mg2+ present in the control solution. When Ca2+, Mg2+ or La3+ was added at the time at which the current had settled to a near steady-state, there was a fall of current, the time course of which was much faster than that during desensitization. The amplitude and the time course of current change were nearly the same for Ca2+ and Mg2+, showing that the effect was not specific for any particular cation. No evidence of intracellular action of the cations was found. The [ACh]-current relationship was shifted to a higher [ACh] by raising [Mg2+] or [La3+]. Recovery of the ability to react to ACh was slowed by increasing [La3+], but the initial recovery of fast and slow time constants of desensitization was not much changed. Recovery was impeded by the presence of a low (conditioning) [ACh]; this effect was enhanced by La3+. It was concluded that the effects of polyvalent cations are similar to those of non-specific blockers.

Acetylcholine contractures of skeletal muscles: inhibition by chlorpromazine and diltiazem.

The maximum contractures evoked by 100 microM ACh of mouse soleus muscles denervated for 3-7 days are completely inhibited by a 10 min exposure to 100 microM chlorpromazine (CP). Recovery on washout of CP takes more than 1 hr to complete. ACh evoked contractures are also inhibited by diltiazem (D); washout of D is immediately followed by recovery. Electrically evoked twitches and K evoked contractures are largely unaffected by CP, caffeine evoked contractures are decreased but not abolished. Fast mouse and (non-denervated) frog tonic muscles behave similarly. Depolarization by ACh and ACh-evoked whole cell currents show enhanced desensitization at low, and block at high [CP] and [D]; more than 50% recovery is achieved by less than 1 min washout of CP and D. Currents carried by Na+ and Mg2+ ions behave similarly. It is concluded that activation of ACh evoked contractures may be blocked by CP independent of ionic currents through nicotinic ACh receptors and that it depends on intracellular processes linked to these receptors.

Acetylcholine-evoked currents in juvenile mouse muscles.

Single fibres were obtained from juvenile (8-12 days old) mice by enzymatic dissociation. Whole-cell voltage-clamped soleus and interosseus fibres were jet-superfused by Cl-free solutions. Current responses to ACh (0.5-50 microM) were recorded. At high [Ach], current responses in all fibres showed a transient initial and a prolonged late phase. The reversal potential of the initial phase was about 5 mV and that for the late phase about 18 mV in soleus. No difference between the reversal potentials for the two phases was seen in interosseus. At low [Ach], only the late phase was seen in soleus. Differences in reversal potentials for the two phases were found in adult interosseus fibres denervated for 10-20 days. These differences were smaller, when the pipette was attached to the endplate region, than at the fibre ends. Evidence was presented supporting the view that the diminished difference between the two reversal potentials at the endplate, mentioned above, is due to the non-uniform voltage-clamping of the membrane in the presence of high [ACh].

Acetylcholine-induced currents in denervated mouse soleus muscle: effects of antagonists.

Acetylcholine-induced currents were measured in partially depolarized mouse soleus muscles, denervated for 3-6 days by using a point voltage clamp. When 0.25 microM d-tubocurarine (d-Tc) was used, the weak currents provoked by 0.1 microM ACh, at a holding potential of -20 mV, were barely affected, while the large currents provoked by 2-5 microM ACh were decreased by more than 50%. By contrast, weak and strong ACh-induced currents were proportionally diminished when, under similar conditions, 20-100 microM ipratropium was used. Currents were proportionally diminished by d-Tc when the holding potential was set at +15 mV, a level corresponding to the reversal potential of the current provoked by small concentrations of ACh. In non-denervated flexor digitorum brevis muscles, d-Tc had the same relative effect at small and at large concentrations of ACh, independent of the holding potential. The reversal potential for the ACh-induced currents was about +14 mV for small concentrations of ACh and decreased to about +3 mV with 4 microM ACh in denervated soleus muscles. It was concluded that denervated soleus muscles, in contrast to the endplate regions of non-denervated mouse muscles, contain a small proportion of highly ACh-sensitive, weakly d-Tc-sensitive, predominantly Na(+)-permeable ACh receptors. These receptors are presumably responsible for the non-fading ACh-induced currents, described before, for the denervated mouse soleus muscle.

Sensitivity of rodent skeletal muscles to dicholines: dependence on innervation and age.

Depolarizations provoked by acetylcholine (ACh) and three dicholines, succinylcholine (SCh), glutarylcholine (GCh) and azelainylcholine (AzCh) were measured in normal and 3-7 days denervated muscles of adult, and in normal muscles of 0-27 days old mice and rats. Soleus and flexor digitorum superficialis muscles were mainly used. To prevent movement during measurement of the membrane potential by microelectrodes, the muscles were bathed in solutions containing 30 mM K+. At the adult endplate region of muscles of the mouse, the -log concentrations required for 7 mV depolarization were 6.5 for AzCh and approximately 5.9 for SCh, GCh and ACh. In extrajunctional areas of denervated muscles the values were 7.1, 6.4, 5.9 and 5.6 for AzCh, ACh, GCh and SCh, respectively. In non-denervated soleus muscles of 1-3 days-old mice, the average difference in sensitivity (estimated as mentioned above) to AzCh and SCh was approximately 2.0 log units, not significantly different from that found in denervated adult muscles. Between days 3 and 24 this difference decreased to about 0.8 at the endplate region but it remained greater in the periphery of soleus muscles. The AzCh-SCh sensitivity differences were smaller in muscles from juvenile rats than in juvenile mice. Sensitivity to ACh and AzCh, but not that to SCh, was by 0.6-0.8 log units greater in the presence than in the absence of anticholinesterases in endplates at any age of the animals. The blocking effectiveness of d-tubocurarine was low in newborn animals (similar to that observed in denervated adult muscles) and it increased with age.

Sensitivity to dicholines of membranes from vertebrate and invertebrate muscles.

1. The depolarizing effectiveness of azelainylcholine (AzCh, a 7-C-chain dicholine) is about 10 times higher than that of succinylcholine (SCh, a 2-C-chain dicholine) in skeletal muscles of chick, frog and fish, and in body muscles of the earthworm. 2. In the chicken anterior latissimus dorsi (ALD) muscle, AzCh is about 100 times more effective than SCh. 3. In contrast to that in mammalian muscles, the AzCh-SCh sensitivity difference is not increased by denervation in frog muscles. 4. d-Tubocurarine is equally effective in the ALD and in other chicken muscles; its effectiveness is not decreased by denervation in frog muscles. 5. Cells containing muscarinic acetylcholine receptors are weakly sensitive to dicholines or not at all.

Permeability to Ca2+ of the acetylcholine receptor channel of denervated mouse muscles in the presence of Na+ and of some other cations.

Calcium uptake caused by exposure to azelainylcholine and the additional membrane slope conductance caused by the same agonist were compared in partially depolarized mouse soleus muscles denervated for 3-5 days. Ca uptake was estimated from the amount of 45Ca retained after a 2 min exposure to the tracer (1 min in the presence of azelainylcholine) and a subsequent 17 min period of tracer washout. The amount taken up in the presence of Na+ was 0.152 m mole/kg fresh muscle. The uptake was by about 60% higher when Na+ was replaced by N-methyl-D-glucamine. For 10 other monovalent cations Ca uptake was less than with Na. Ca uptake was not related to the molecular weight, size or structure of the cation. The slope conductance in the presence of 10 microM azelainylcholine was 4 microseconds and it was 21% of that value when Na+ was replaced by N-methyl-D-glucamine, i.e. conductance was decreased when Ca uptake was increased. This discrepancy points to a major difference in the way cations such as Ca2+ and K+ pass the receptor channel.

The chloride conductance of intermediate fibres from frog muscles.

Sheets of muscle fibres dissected from surface portions of frog ileofibularis and semitendinosus muscles were soaked in solutions with elevated K and Cl concentrations. The KCl-loaded muscles were then bathed in low [Cl-] solutions, whereby the membrane potential became transiently inside positive. The repolarization of the twitch fibres from the tonus bundle ("intermediate fibres") was faster than that of the fibres adjacent to it ("fast fibres") when the preceding exposure to high KCl was brief (7-15 min), and it was slower than that of the fast fibres when KCl was applied for 4 hours. Measurements of the voltage displacement at constant current and of the current in a point voltage clamp showed that inwardly rectifying K channels were present in the membranes of both types of fibres. The ionic conductance ratio, gK/gCl, was 191/523 in fast fibres and 335/230 in "intermediate" fibres. The different repolarization rates may thus be explained by differences in the chloride conductance of the fast and intermediate fibre membranes. The smaller diameter of the latter fibres may be another factor.

Electrogenic Na-K pump in denervated mouse soleus muscles: prolonged activation by briefly applied acetylcholine.

Thin preparations of mouse soleus muscles denervated for 3-11 days were bathed in Cl-free solutions. The membrane potential (microelectrode technique) was an average of -65.6 mV. On application of 10 microM acetylcholine (ACh) the membrane potential fell to -2 to -8 mV. Following the washout of ACh it returned to values 9-24 mV more negative than before ACh. The membrane potential gradually returned toward the initial level during the ensuing 40-60 min. No hyperpolarization occurred when Na ions were absent during the application and washout of ACh or in the presence of 0.1 mM ouabain. The hyperpolarization was enhanced by replacing the Na ions by Li or Tris ions following an application of ACh in the presence of Na+. The hyperpolarization was suppressed by ouabain irrespective of whether the drug was applied in the presence or absence of Na+. The membrane potential was diminished by reducing [K+] from 4 to 1 mM in the absence of Na+ before ACh, but it was increased by the same procedure by up to 20 mV following the application of ACh. This indicates that the hyperpolarization was not entirely due to a K-depleting action of the Na-K pump at the membrane surface.

Force and membrane potential in acetylcholine and potassium contractures of denervated mouse muscles.

Depolarization and contracture force (P) provoked by acetylcholine (ACh) and by K ions were studied in bundles dissected from mouse soleus muscles that had been denervated for 4-7 days. Cl-free solutions were used. The muscle fibres were depolarized by solutions containing 150 mM K or 10 microM ACh to nearly zero mV resulting in maximum P (Pmax). Threshold P was produced when the membrane was depolarized to more than about -60 mV by both agents. 50% Pmax was produced by [K] causing the membrane to depolarize to -42 mV, whereas a potential more positive than -20 mV was required for 50% Pmax to be produced when ACh was used. The rate of depolarization was always higher for ACh than for K. Pretreatment by 0.05 microM ACh (about threshold for P) did not affect the P-[K] relation appreciably showing that ACh did not "stabilize" the membrane. Nearly equal P was provoked by successive applications of just suprathreshold agent concentrations when the order of application was ACh----K but not with the reverse order. Hypertonicity (by addition of 300 mM sucrose to all solutions) caused PACh to decrease and PK to increase. It was concluded that the ACh receptors are located in the surface membrane of the muscle fibres, not in the T-system membranes.

Manganese ions inhibit acetylcholine receptor synthesis in cultured mouse soleus muscles.

Thin bundles dissected from mouse soleus muscles were kept in culture media containing 10% calf serum. After 3 days of culture at 37 degrees C the muscles were tested for acetylcholine (ACh) sensitivity by measuring the force of the contracture produced by 10 microM ACh. The ACh-receptor density was measured by the [125I]alpha-bungarotoxin method. When the [Ca2+] of the culture medium was 0.02 mM (instead of the normal 2 mM), ACh sensitivity and ACh-receptor density were not different from control. Replacement of 99% Ca2+ by Sr2+ or by Mg2+ was also without effect. The muscles did not survive in solutions containing Ni ions. ACh sensitivity and ACh-receptor density were at levels characteristic of non-cultured muscles when the culture medium contained 0.4-1.8 mM Mn2+. Since the incorporation of ACh receptors into the membrane was not affected by Mn ions these results indicate that Mn ions inhibit the synthesis of ACh receptors.

Differential effects of succinylcholine and acetylcholine on endplate and extrajunctional membranes of normal and denervated mouse skeletal muscle fibres.

Depolarization provoked by 10(-7)-10(-4) succinylcholine (SCh) and acetylcholine (ACh) was measured in thin bundles dissected from normal and 1-15 days denervated mouse muscles using the microelectrode technique. The muscles were superfused by Cl- -free solutions containing 30 mM K+ to prevent contracture responses. In endplate areas of normal muscles, sensitivity to SCh was roughly 10 times higher than the sensitivity to ACh. By contrast, the extrajunctional membrane of chronically denervated muscles was less sensitive to SCh than to ACh by about the same factor. The extrajunctional membrane of non-denervated soleus muscles was also more sensitive to ACh than to SCh but, unlike the response of the denervated membrane, the depolarization was transient.

Mammalian skeletal muscle: long-lasting contractures and potentiated tetani produced by conditioning with weak acid anions.

Reversible contractures can be induced in slow mammalian muscles by manipulations that probably generate a long-lasting alkalinization of the muscle cell interior. Such contractures reach about 1/4 of the tetanic force, P0, and last 10 times longer than potassium contractures. While in contracture, the muscle fibers have high resting potentials so that they can be electrically stimulated. Tetanic force is then increased and added to that of the contracture so that total force may reach 2 P0. This level of potentiation has not been reached by any previously-known method.

Potassium contractures in mouse limb muscles.

The force of contractures produced by 14-400 mM-K+ (as methanesulphonate) was measured in whole mouse soleus (sol.), extensor digitorum longus (e.d.l.), and in bundles from these muscles. Frog semitendinosus muscles were used for comparison. Whole mouse muscles displayed biphasic contracture responses lasting more than 5 min when provoked by 150m M-K+. Contractures of bundles dissected from these muscles were monophasic and had a short duration. The time required for the muscle bundles to contract and relax to 1/2 maximum force (T) was an inverse function of [K+]. T was increased by lowering [K+] from 400 to 50 mM by a factor of 8.3 and 7.0 in proximal and distal portions of sol. and by a factor of 4.2 and 2.8 in proximal and distal portions of e.d.l., respectively. The force-[K+] relation was steeper for sol. than for e.d.l., and the proximal portions were more sensitive to K+ than the distal portions, particularly in e.d.l. The capability of the muscles to produce force in response to 400 mM-K+ after a 10 min exposure to 30 or 50 mM-K+ was high in sol., somewhat lower in proximal parts of e.d.l. and in frog semitendinosus, and lowest in distal parts of e.d.l. It was concluded that K contractures of mouse muscles are basically monophasic, and that biphasic contractures of whole muscles arise because of K+-diffusion delays, slow responses to intermediate [K+], and differences in responsiveness of the fibres contained in a particular muscle.

Regulation of acetylcholine receptor density in membranes of denervated mouse muscles.

The ACh sensitivity of denervated muscles of rats stimulated in situ or in culture for several days decreases to low levels characteristic of normal muscles. Possible causes are the electrical activity, depolarization per se, contraction and intracellular [Ca2+] changes. To test the last three hypotheses, isolated bundles of denervated mouse soleus muscles were placed in narrow chambers (internal diameter 2 mm) which were periodically perfused with standard mixtures of Minimal Essential Medium (MEM) and calf serum. At 10-15-min intervals the chambers were filled for 15-43 s with a Ringer solution containing 55 microM ACh or 8-10 mM caffeine or with a solution containing 200 mM KCl. After 3-7 days the muscles were tested for ACh sensitivity by comparing the force developed in response to 110 microM ACh and that to 400 mM K methanesulphonate. In addition, the ACh receptor density was measured with 125I alpha-bungarotoxin. The results showed no difference in ACh sensitivity or receptor density between treated and untreated muscles. Small differences produced by caffeine were probably caused by muscle damage. Ca influx, but not efflux, was strongly elevated during exposure to ACh. It was concluded that neither depolarization per se nor contractile activity or the associated calcium movements have any effect on ACh receptors.

Influence of divalent cations on potassium contracture duration in frog muscle fibres.

Single fibres or small fibre bundles were dissected from twitch muscles of frogs and washed in low Cl-solutions. Contractures were provoked by 122.5 mM K+. At room temperature (17-20 degrees C) the contracture duration was about 1.5 s in the absence of divalent cations and about 3 s in the presence of 2 mM Ca2+. Contractures were prolonged when Ca2+ was replaced by Ni2+ showing that inward Ca current was not the factor responsible for the contracture prolongation. K contracture duration was prolonged in the cold (3 degrees C) by a factor of about 4 in the presence of non-permeating divalent cations (Ni, Co), when 0.1 mM La3+ was applied together with 2 mM Ca2+, and in the virtual absence of divalent cations. The contractures were prolonged in the cold by a factor of 6 or more in the presence of permeant divalent cations (Ca, Sr, Ba, Mg, and Mn at 8 mM). Diffusion of divalent cations in the transverse tubules of the muscle fibres was shown to have a Q10 similar to that in free solution. It was concluded that inward current of divalent cations may shorten contracture duration by causing ionic depletion of the transverse tubules.

Two cases of adynamia episodica hereditaria: in vitro investigation of muscle cell membrane and contraction parameters.

Membrane potentials, current-voltage relationships, and contractile parameters were studied in intact muscle cell bundles obtained from two patients with adynamia episodica hereditaria. In a normal extracellular medium, the cell membranes had resting potentials of about -80 mV and their current-voltage relationships were not significantly different from control curves. In contrast to normal muscles the afflicted cells were paralyzed in a medium having 6-10 mmol/liter potassium. The mechanisms of paralysis in the two specimens were different from each other. Many fibers from one patient were spontaneously active even in normal solution. In high potassium solution spontaneous activity was increased and the cells gradually depolarized to values at which excitatory sodium current is normally inactivated. This depolarization was connected with an increased sodium conductance and was reversed by the application of tetrodotoxin (TTX). The fibers from the other patient were not spontaneously active. In high potassium solution they were paralyzed at membrane potential values at which normal fibers would still contract. The reason for this paralysis was a reduced excitability.

Desensitization in denervated mouse muscles.

In denervated mouse soleus (DSOL) muscle preparations washed in Na methanesulfonate solutions containing 30 mmol X 1(-1) K+, bath-applied ACh (55 mumol X 1(-1) ) caused the resting potential to decrease from about -36 to -3.2 mV within 2-4 s; the potential remained stable or became slightly more negative when ACh was applied for 1- 2 min. Two components of the membrane current change (ACh current) were found in DSOL when using a point voltage clamp, an initial current component declining to 1/2 the initial amplitude in 11-15 s (desensitization) and a steady late current comprising 16-47% of the maximum ACh current. Membrane conductance (in microseconds X cm-2) was 0.35 in the absence of ACh, 20.3 at the peak of the initial current, and 1.57 during late current. The late current saturated at 0.55 to 5.5 mumol X 1(-1) ACh, whereas the initial current required 55 or more mumol X 1(-1) ACh to saturate. The null (reversal) potential was 6-13 mV more positive for the late current than for the initial current. The late current was masked when Na+ was replaced by Tris+, sucrose, or K+. An initial and a late current could also be distinguished in non-denervated endplates. The late current was more sensitive to ACh than the initial current, but the null potential was more negative than that for the initial current in endplates. In denervated membrane, the half time of desensitization was increased when Mg2+ was replaced by Ca2+ but the changes were on the average less than 15% the control values. It was concluded that desensitizing and non-desensitizing receptors may exist in extrajunctional membranes of denervated muscles and in endplates, the two being attached to different ionic channels.

Membrane defects in paramyotonia congenita with and without myotonia in a warm environment.

Three patients with paramyotonia congenita and 3 control persons were biopsied for an in vitro investigation of the sarcolemmal membrane parameters and of the contractile properties of paramyotonic muscle. At 37 degrees C, paramyotonic muscle fibers had normal resting potentials, but on cooling to 27 degrees C they depolarized. Depolarization to -60 mV caused spontaneous activity, and further depolarization to -40 mV caused inexcitability. Depolarization could be prevented by the application of tetrodotoxin, a finding suggesting a defect in the Na channels. Analysis of the membrane current densities using voltage clamps with 3 microelectrodes revealed that in paramyotonic patients at 37 degrees C all component conductances were normal, except for a decreased Cl conductance in the patient who had myotonia in a warm environment. At 27 degrees C, the Na and Cl conductances were abnormally high. The K conductance was always normal. The results explain the clinical symptoms of weakness and paralysis. Potassium- and caffeine-contracture experiments gave normal results. The clinical symptom of paramyotonic stiffness, therefore, has not been explained by these studies.