Effect of cyanide and verapamil on sodium-free contractures in the frog heart

Sodium-free contractures were studied in myocardial strips from R. pipiens with extracellular sodium (Na0+) replaced by choline chloride and extracellular calcium (Ca20+) varied with EGTA buffer. At calculated Ca02+ below 2.8 X 10(-7) mol/l, no contracture occurred in most of the experiments, even in the presence of cyanide. When Ca02+ was above 2.8 X 10(-7) mol/l, relatively short tension transients of up to 80 sec duration could be avoided if the myocardial strip was previously equilibrated for 20 min in a Na+-Ca2+-free solution. Instead, contractures developed slowly within one to several hours. The maximum contracture was dependent on Ca02+ in a dose-response-like pattern. The time-course of contracture development was not affected by verapamil, but KCN significantly increased the rate of resting tension increase. In solutions with normal Na+-Ca2+ content and even in a Na+-Ca2+-free milieu, the cellular ultrastructure was normal. Development of contracture after addition of Ca2+ to the Na+-free solution was combined with ultrastructural damage of the ventricular strip. It is concluded that Na+-free contractures depend on transsarcolemmal net-Ca2+ uptake as a sum of Na-Ca-exchange-dependent Ca2+ uptake and active sequestering of intracellular free calcium Ca2+ mediated by sarcolemmal and probably intracellular Ca2+-ATPases. The negative inotropic effect of the Ca blocker verapamil seems not to be mediated by the Na-Ca exchange.     

Enrichment of fibrillar cytoplasmic actomyosin in protoplasmic strands of Physarum polycephalum for the production of cell-free models

Summary The treatment of isolated protoplasmic strands of Physarum polycephalum with 2.5% ethanol in a physioogical salt solution under isometric conditions induces the formation of a large amount of mostly longitudinally organized actomyosin fibrils in the endoplasmic channel, a region normally free of actomyosin fibrils. The quantity of fibrillogenesis as well as the concomitant force output during the induced contractures are dependent on the Ca^{+ +}- content and the temperature of the test solution. The method was developed to optimize the structure of the plasmodial strands before their subsequent transformation into cell-free models by permeabilization and extraction of the strands.Cryosections of plasmodial strands containing cytoplasmic actomyosin fibrils stained with fluorescently labeled phallotoxins offer a further assay for the study of their contraction physiology under cell-free conditions.     

Tension generation by threads of contractile proteins

Threads of contractile proteins were formed via extrusion and their isometric tensions and isotonic contraction velocities were measured. We obtained reproducible data by using a new and sensitive tensiometer. The force-velocity curves of actomyosin threads were similar to those of muscle, with isometric tensions of the order of 10g/cm2 and maximum contraction velocites of the order of 10(-2) lengths/s. The data could be fitted by Hill's equation. Addition of tropomyosin and troponin to the threads increased isometric tension and maximum contraction velocity. Threads which contained troponin and tropomyosin required Ca++ for contraction and the dependence of their isometric tension on the level of free Ca++ was like that of muscle. The dependence of tension or of contraction velocity upon temperature or upon ionic strength is similar for actomyosin threads and muscle fibers. In contrast, the dependence of most parameters which are characteristic of the actomyosin interaction in solution (or suspension) upon these variables is not similar to the dependence of the muscle fiber parameters. The conclusion we have drawn from these results is that the mechanism of tension generation in the threads is similar to the mechanism that exists in muscle. Because the protein composition of the thread system can be manipulated readily and because the tensions and velocities of the threads can be related directly to the physiological parameters of muscle fibers, the threads provide a powerful method for studying contractile proteins.     

Action of insulin and cell calcium: effect of ionophore A23187.

We have measured the effects of the carboxylic Ca++ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca++ concentration since (a) the contracture was blocked by removing external Ca++ and (b) its size was increased by raising outside Ca++. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca++]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca++ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained.     

Reactivation of a cell-free model from Physarum polycephalum: studies on cryosections indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction

Cell-free models should offer "in situ conditions" to study the physiology of cytoplasmic actomyosin in its natural environment, while, if possible, still associated with its regulatory control proteins and other cytoplasmic components. Detergents and glycerol as the usual media to permeabilize the plasmalemma and to extract a portion of the cytoplasmic components, are accompanied by several disadvantages. We investigated a cell-free model consisting of cryosections of plasmodial strands that were previously enriched with "stress fibrils" and fluorescently labelled with phallotoxins and that contain the non-denatured structures that are to be reactivated in situ. The contraction reaction can be directly observed in the fluorescence microscope. This procedure allows the study of contraction conditions in the natural environment of the fibrils. The aim of these reactivation experiments was to identify the role of calcium ions. According to our results, a reactivation of cryosections is not Ca++ dependent but is partly inhibited at concentrations of 10(-4) to 5 X 10(-2)M Ca++. Complete inhibition occurs at 10 to 20 mM Ca++. Electron microscopical investigations revealed that the fluorescently labelled contracting structures were identical to actomyosin fibrils.     

Early hypoxic contracture of the myocardium in adult and newborn rats]

The effect of 30 min substrate free hypoxia (H) on isometric tension was studied in isolated myocardium (M) of adult (A) and newborn (N) rats. The perfusion with 50% Na+ H solution caused in AM the development of H contracture which was more than 50% higher than control contracture. H perfusion with 0.1 mM Ca2+, 1.0 mM La3+, and 10.0 mM of caffeine provides the discrimination of control and hypoNa+ contractures. It is assumed that early H contracture in AM is a result of inability of Ca-sequestering system to accumulate intracellular Ca2+ and Ca2+ influxing through the sarcolemma. In myocardium of N rats Na-Ca exchange is proposed as a main source of Ca2+ for H contracture development.     

Reactivation of cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength

The effect of calcium ions on the reactivation of cytoplasmic actomyosin contraction in cell-free models of endoplasmic drops from Physarum polycephalum after glycerol extraction at low ionic strength depends on the duration of the extraction procedure: Ca++ prevents contraction in 20-h extracted specimens, whereas after several days of extraction this Ca++-sensitivity is lost. These results indicate an inhibitory effect of Ca++ on cytoplasmic actomyosin contraction.     

Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution

Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.     

Effects of cobalt, magnesium, and cadmium on contraction of rat soleus muscle.

The effects on isometric tension of three divalent ions that block calcium channels, magnesium, cobalt, and cadmium, were tested in small bundles of rat soleus fibers. Cobalt, at a concentration of 2 or 6 mM, reversibly depressed twitch and tetanic tension and the depression was much greater in solutions containing no added calcium ions. Magnesium caused much less depression of tension than cobalt. The depression of tension was not accompanied by membrane depolarization or a reduction in the amplitude of action potentials. A reduction caused by 6 mM cobalt in the amplitude of 40 or 80 mM potassium contractures was not accompanied by a comparable reduction in tension during 200 mM potassium contractures, and could be explained by a shift in the potassium contracture tension-voltage curve to more positive potentials (by +7 mV on average). Similar effects were not seen with 2 or 6 mM magnesium. At a concentration of 20 mM, both cobalt and magnesium depressed twitch and tetanic tension, cobalt having greater effect than magnesium. Both ions shifted the potassium contracture tension-voltage curve to the right by +5 to +10 mV, caused a small depression of maximum tension, and slowed the time course of potassium contractures. Cadmium (3 mM) depressed twitch, tetanic, and potassium contracture tension by more than 6 mM cobalt, but experiments were complicated by the gradual appearance of large contractures that became even larger, and sometimes oscillatory, when the solution containing cadmium was washed out. It was concluded that divalent cations affect both activation and inactivation of tension in a manner that cannot be completely explained by a change in surface charge.     

Relationships between the sarcoplasmic reticulum and sarcolemmal calcium transport revealed by rapidly cooling rabbit ventricular muscle

Rabbit right ventricular papillary muscles were cooled from 30 to approximately 1 degree C immediately after discontinuing electrical stimulation (0.5 Hz). This produced a contracture that was 30-50% of the preceding twitch magnitude and required 20-30 s to develop. The contractures were identical in cooling solutions with normal (144 mM) or low (2.0 mM) Na. They were therefore not Na-withdrawal contractures. Contracture activation was considerably slower than muscle cooling (approximately 2.5 s to cool below 2 degrees C). Cooling contractures were suppressed by caffeine treatment (10.0 mM). Rapid cooling did not cause sufficient membrane depolarization (16.5 +/- 1.2 mV after 30 s of cooling) to produce either a voltage-dependent activation of contracture or a gated entry of Ca from the extracellular space. Contractures induced by treating resting muscles with 5 X 10(-5) M strophanthidin at 30 degrees C exhibited pronounced tension noise. The Fourier spectrum of this noise revealed a periodic component (2-3 Hz) that disappeared when the muscle was cooled. Cooling contractures decayed with rest (t1/2 = 71.0 +/- 9.3 s). This decay accelerated in the presence of 10.0 mM caffeine and was prevented and to some extent reversed when extracellular Na was reduced to 2.0 mM. 20 min of rest resulted in a net decline in intracellular Ca content of 1.29 +/- 0.38 mmol/kg dry wt. I infer that cooling contractures are principally activated by Ca from the sarcoplasmic reticulum (SR). The properties of these contractures suggest that they may provide a convenient relative index of the availability of SR Ca for contraction. The rest decay of cooling contractures (and hence the decay in the availability of activating Ca) is consistent with the measured loss in analytic Ca during rest. The results suggest that contraction in heart muscle can be regulated by an interaction between sarcolemmal and SR Ca transport.     

Slow sodium-free contractures in the frog heart mediated by the sodium-calcium exchange

1. Sodium-free contractures were studied in myocardial strips from R. pipiens when extracellular sodium (Na+o) was replaced by choline chloride and extracellular free calcium (Ca2+o) was defined with EGTA-buffer. 2. Resting membrane potentials (RMP) were normal in sodium-free solutions with Ca2+o calculated below 1.0 x 10(-9) mol/l. 3. When Ca2+o was subsequently increased from zero to 1.0 x 10(-3) mol/l Na+-free contractures developed slowly with unchanged RMP even at maximum contracture, at which the intracellular ultrastructure is grossly altered. 4. The contractures developed significantly faster in the presence of 3 x 10(-6) mol/l ouabain. 5. In sodium-free solutions La3+ did not influence Ca2+-dependent contractures, apart from causing an increase in time to maximum contracture. 6. It is concluded that sarcolemmal integrity is maintained in frog myocardium treated initially with Na+/Ca2+-free solutions and then with Na+-free medium containing 1 mmol/l Ca2+. 7. Our experiments indicate that sodium-free, Ca2+o-dependent contractures are mediated by the Na+/Ca2+-exchange, operation at higher rates when Na+i is increased. La3+ (1 mmol/l) probably does not compete with Ca2+ at extracellular binding sites of the exchanger. 8. The Na+/Ca2+-exchange may under certain experimental conditions be able to increase Ca2+i to cytotoxic concentrations.     

Rate-limiting steps in the tension development of freeze-glycerinated vascular smooth muscle

A method for "skinning" arterial smooth muscle is presented which yields isometric tension development typically 60-80% of maximum physiological tension in the presence of micromolar Ca++ and millimolar Mg-ATP, while retaining essentially the native protein content. Using the methods of "CA jump," the time-course of Ca++-activated tension development in the skinned artery can be made identical to, but not faster than, the rate of tension development in the intact artery. In the skinned artery, activating free [Ca++] does not substantially alter the rate at which tension development approaches the final steady tension attained at that free [Ca++] (less than 25% decline in speed for a 10-fold decrease in [Ca++]). These observations are taken to mean that the rate-limiting step in isometric tension development in arterial smooth muscle does not depend directly on Ca++.     

The effect of diazepam on potassium contractures, contraction threshold, and resting tension in rat skeletal muscles

The effects of diazepam on potassium contractures, contraction threshold, and resting tension have been examined in rat soleus muscle fibres. Two actions of the drug were defined that could not be attributed to changes in the resting membrane potential or depolarization in high potassium solutions. The major effect was an increase in the amplitude of submaximal tension during either twitches or potassium contractures and an increase in resting tension. At 400 microM diazepam, there was (a) a fourfold increase in 40 mM potassium contracture tension, (b) a negative shift of 8 mV in the membrane potential for half maximum tension estimated from the best fit of a Boltzmann-type equation to average potassium contracture data, (c) a negative shift of 8 mV in the threshold for contraction measured under voltage clamp conditions, and (d) a contracture of variable amplitude to a level that was occasionally equivalent to maximum tetanic tension. These potentiating actions of diazepam depended on drug concentration within the range of 100-800 microM. In contrast, the second effect of diazepam, depression of maximum tension by 10-15%, was independent of drug concentration between 100 and 400 microM. The results support the idea that diazepam produces an increase in resting myoplasmic calcium concentrations.     

Effects of Cannabinoids on Tension Induced by Acetylcholine and Choline in Slow Skeletal Muscle Fibers of the Frog

We investigated the effects of cannabinoids on acetylcholine (ACh) or choline contractures in slow skeletal muscle fibers from Rana pipiens. Bundles of cruralis muscle fibers were incubated with the cannabinoid receptor 1 (CB₁) agonist, arachidonylcyclopropylamide (ACPA), which diminished the maximum isometric tension by 10 % and the total tension by 5 % of the ACh contracture, and 40 and 22 % of the choline contracture, respectively. Preincubation with the CB₁ antagonist, AM281, or with pertussis toxin (PTX) completely blocked the effect of ACPA on the ACh contracture. On the other hand, the decrease in choline contracture by ACPA was only partially blocked by AM281 (~16 % decrease), PTX (20 %), or by dantrolene (~46 %). Our results show that ACPA modulates ACh and choline contractures, and suggest that this effect involves the participation of CB₁, the ACh receptor, and ?RyR in ACh contractures. For choline contractures, ACPA may also be acting through cannabinoid receptor-independent mechanisms.     

L-glutamate and potassium-induced contractures in denervated cockroach muscles

1. The effects of denervation on the mechanical responses to various concentrations of L-glutamate in retractor unguis muscle of cockroach (Perilpaneta americana) was examined, comparing them with contractures induced by high potassium saline. 2. The dose-response curve was shifted to the lower concentrations of L-glutamate after 9 days of denervation. 3. Following a transient increase in the maximum contracture tension, it decreased with days after denervation and reached a constant level by several days. However, from 16 to 20 days after denervation, the tension ratios of the maximum glutamate to potassium contractures were significantly higher than that of the innervated muscles. 4. A sustained contracture was observed on and after treatment of L-glutamate in the denervated muscle. Pretreatment of the muscle by concanavalin A facilitated to induce L-glutamate contracture. 5. It was suggested that the sensitivity of the muscle membrane to L-glutamate was increased in the denervated muscle.     

Cytoplasmic actomyosin fibrils in tissue culture cells

Summary A special cell line derived from a rat mammary adenocarcinoma (RMCD cells) displays a distinct pattern of actomyosin fibrils (AM fibrils) visible with phase contrast, Nomarski interference and polarized light optics. It was shown that the cytoplasmic AM fibrils are arranged as bundles of highly parallel F-actin filaments. The chemical nature of the filaments was identified by incubation with heavy meromyosin from rabbit skeletal muscle.These cytoplasmic actomyosin fibrils actively contract under isotonic conditions. This was shown by contraction experiments under polarized light optics, by cinematographic analysis and by direct proof of the contractility of AM fibrils isolated by laser micro-dissection. Thus, cytoplasmic AM fibrils can be assumed to represent structures essential for motive force generation in contraction processes in non-muscle cells.We thank Dr. W. Meier-Ruge and Mr. W. Bielser (Basic Medical Research Departments, Sandoz AG, Basle, Switzerland) for making the laser equipment available to us and for their kind cooperation during this investigation. Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Fragmin induces tension reduction of actomyosin threads in the presence of micromolar levels of Ca2+

Fragmin was able to reduce the isometric tension of Physarum actomyosin threads to 15-30% of the control tension at the Ca2+ concentrations greater than 10(-6) M. However, fragmin had no effect on the tension of threads when the Ca2+ concentration was lowered below 10(-7) M. The tension once reduced by fragmin could not be recovered by the removal of Ca2+. The remaining tension was shown to be still active from the experiment with quick release or stretch of the thread. This tension reduction is parallel to the decrease in viscosity of F-actin solution by fragmin. Electron microscopy showed that F-actin filaments became shorter in the thread after the tension was reduced by fragmin. Therefore, the severing of F-actin by fragmin in micromolar concentration of calcium resulted in the relaxation of tension by actomyosin threads.     

Temperature dependence of mammalian muscle contractions and ATPase activities

Isolated rat and mouse extensor digitorum longus (EDL) and soleus muscles were studied under isometric and isotonic conditions at temperatures from approximately 8 degrees -38 degrees C. The rate constant for the exponential rise of tension during an isometric tetanus had a Q10 of approximately 2.5 for all muscles (corresponding to an enthalpy of activation, delta H = 66 kJ/mol, if the rate was determined by a single chemical reaction). The half-contraction time, contraction time, and maximum rate of rise for tension in an isometric twitch and the maximum shortening velocity in an isotonic contraction all had a similar temperature dependence (i.e., delta H approximately 66 kJ/mol). The Mg++ ATPase rates of myofibrils prepared from rat EDL and soleus muscles had a steeper temperature dependence (delta H = 130 kJ/mol), but absolute rates at 20 degrees C were lower than the rate of rise of tension. This suggests that the Mg++ ATPase cycle rate is not limiting for force generation. A substantial fraction of cross-bridges may exist in a resting state that converts to the force-producing state at a rate faster than required to complete the cycle and repopulate the resting state. The temperature dependence for the rate constant of the exponential decay of tension during an isometric twitch or short tetanus (and the half-fall time of a twitch) had a break point at approximately 20 degrees C, with apparent enthalpy values of delta H = 117 kJ/mol below 20 degrees C and delta H = 70 kJ/mol above 20 degrees C. The break point and the values of delta H at high and low temperatures agree closely with published values for the delta H of the sarcoplasmic reticulum (SR) Ca++ ATPase. Thus, the temperature dependence for the relaxation rate of a twitch or a short tetanus is consistent with that for the reabsorption rate of Ca++ into the SR.     

Influence of homologous n-alcanoic acids on the function properties of isolated skeletal muscles. III. Contractures by fatty acids and relations to the effects of caffeine]

The influences of octanoic, decanoic, and hexadencanoic acid were tested on the contracture capability of isolated skeletal muscle of frogs and rats. 1. 100 mM octanoic or 10mM decanoic acid induce contractures in skeletal mucles after 20-30 min of exposure. 2. The time of exposure necessary for induction of contractures is shortened by an increase of bath temperature, electrical stimulation or KCl-depolarization of muscles. 3. Simultaneous addition of fatty acid and caffeine (10 mM) effects a depression and a delay of the caffeine contracture. The contractures evoked by 5 mM caffeine are inhibited by lower concentrations of fatty acids (1 mM octaonoic acid, 0,1 mM hexadecanoic acid). 4. After the complete development of a caffeine (or fatty acid) contracture the muscle is not able to develop an identical contracture by a second application of the same drug, even after intermediate treatment during one or two hours in Ringer solution. If the contracture is interrupted one minute after the caffeine application by changing the solution, the tension returns quickly to the resting level. A subsequent addition of caffeine (10 mM) after about 10 minutes effects an identical contracture. Thus the effect of fatty acids on caffeine contracture may be studied on the same muscle which served as its own control. 5. As mechanisms involved in the development of fatty acid contractures and in the inhibition of caffeine contractures, interactions of free fatty acids and lipids of biological membranes are disucssed. Especially, there may be changes of the calcium affinity of cellular membranes.     

Enhanced permeability to sugar associated with muscle contraction. Studies of the role of Ca++

When contractures were induced in isolated frog sartorius muscles with 4 mM caffeine, there was an increase in permeability of the muscle cells to 3-methylglucose. This observation suggests that the changes in permeability to sugar that are known to occur in electrically stimulated muscles may not be intimately related to the depolarization phase of the tissue response. Contractures that were elicited by exposing the muscles to a high concentration of K+ were also associated with an increased permeability to sugar. As the concentration of 45Ca in the medium was raised, more 45Ca entered the muscles during potassium contractures, and the contractures lasted longer, in agreement with the observations of other investigators. There was also a greater change in permeability to sugar when potassium contractures were elicited in the presence of higher concentrations of Ca++. The possibility that the enhanced permeability to sugar may be related to changes in the intracellular concentration of Ca++ is discussed.