Tension in Isolated Frog Muscle Fibers Induced by Hypertonic Solutions

The effect of hypertonic solutions on the tension of isolated twitch muscle fibers of the frog has been investigated. Increased tonicity up to about 1.7 times normal (1.7 T) caused a very small, graded, maintained tension increase. Above about 1.7 T a large, transient contracture response was superimposed on the small tension. The contracture response was graded with tonicity and reached a maximum at 2.5 T of 108 ± 25 mN·mm² a third of the maximum tetanic tension in isotonic solution. Contracture tension developed with a delay which decreased with increased tonicity. The contracture threshold was lower and the delay shorter in small fibers than in large. Contractures were obtained equally well in depolarized as in polarized fibers. They were completely suppressed by 0.1–0.5 mM tetracaine. The possible mechanism responsible for the tension-inducing effect of hypertonic solutions is discussed in terms of the close similarity between the properties of these contractures and those caused by caffeine, and it is suggested that the effect is due to a release of calcium from internal stores.     

Volume and Twitch Tension Changes in Single Muscle Fibers in Hypertonic Solutions

Single muscle fibers were exposed to solutions made hypertonic (approximately 460 milliosmols/kg water) by addition of either NaCl, glycerol, urea, acetamide, ethylene glycol, or propylene glycol. The changes in either the fiber twitch tension or the volume were measured. In the case of NaCl both fiber volume and twitch tension fall rapidly to 64 and 27% of the respective initial value. These two values were maintained for the duration of the exposure. In the case of the other substances, the fiber volume and twitch tension also decreased but in these cases the effect was transient and the fibers recovered their initial volume and twitch tension. The rate of recovery in the different hypertonic media increased in the order: glycerol < urea < ethylene glycol < propylene glycol < acetamide. In the cases of the last three substances, the initial twitch value was recovered in less than 5 min and even surpassed. However, on returning to normal Ringer the fibers' ability to twitch or to develop potassium contractures was lost. The return of the fibers to normal Ringer after exposure to these hypertonic solutions causes a transient swelling of the fibers. However, when fibers were swelled by exposure to hypotonic media, they did not lose their ability to twitch on return to the normal Ringer.     

Contracture of Slow Striated Muscle during Calcium Deprivation

When deprived of calcium the slow striated muscle fibers of the frog develop reversible contractures in either hypertonic or isotonic solutions. While calcium deprivation continues because of a flowing calcium-free solution the muscles relax slowly and completely. Restoration of calcium during contracture relaxes the muscle promptly to initial tension. When relaxed during calcium lack the return of calcium does not change tension and the muscle stays relaxed. When contractures are induced by solutions containing small amounts of calcium relaxation does not occur or requires several hours. The rate of tension development depends upon the rate at which calcium moves outward since the contractures develop slower in low concentrations of calcium and are absent or greatly slowed in a stagnant calcium-free solution. Withdrawal of calcium prevents the contractile responses to ACh, KCl, or electrical stimulation through the nerve. Muscles return to their original excitability after calcium is restored. Origin of the contractures is unrelated to nerve activity since they are maximal during transmission failure from calcium lack, occur in denervated muscles, and are not blocked by high concentrations of d-tubocurarine, procaine, or atropine. The experiments also indicate that the contractures do not originate from repetitive activity of muscle membranes. The findings are most simply explained by relating the outward movement of calcium as a link for initiating contraction in slow type striated muscle.     

Contractile effects of 4-aminopyridine on isolated frog rectus abdominis muscles

The contractile effects of 4-aminopyridine (4-AP) on isolated frog rectus abdominis muscles were examined, and compared with KCl-induced contractures. 4-AP (1-40 mM) caused slowly developing, concentration-dependent contractures which were not modified by (+)-tubocurarine (2.7-13.3 microM). The contractures were prolonged and very slowly relaxed (greater than 30 min) on washout. KCl-induced contractures developed more rapidly and relaxation was equally rapid, both occurring within 90 s of application and washout, respectively. KCl contractures were slightly but significantly (P less than 0.05) attenuated by (+)-tubocurarine in concentrations that blocked carbachol contractures. In calcium-free Ringer's solution, KCl (10-120 mM) responses were completely abolished, but 4-AP concentration-response curves were shifted to the right three- to four-fold. The results show that 4-AP causes contracture of the frog rectus abdominis. It is suggested that at the lower concentration employed (less than 10 mM), 4-AP increases extracellular calcium entry into the muscle, while larger concentrations produce contractures by a direct intracellular mechanism. 4-AP contractures were independent of postjunctional nicotinic cholinoceptor activation.     

Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

Background

Malignant hyperthermia (MH) is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca²⁺ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca²⁺-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca²⁺ reuptake via the sarcoplasmic Ca²⁺-ATPase (SERCA) enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle.

Methods

With informed consent, surplus muscle bundles of 7 MHS (susceptible), 7 MHE (equivocal) and 16 MHN (non-susceptible) classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA) 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM) and halothane (0.11; 0.22; 0.44; 0.66 mM) was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p < 0.05.

Results

Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles.

Conclusion

Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.     

On the Inhibition of Muscle Contraction Caused by Exposure to Hypertonic Solutions

The evidence supporting a site of inhibition of excitation contraction (E-C) coupling near the plasma membrane (the "glycerol effect," the K⁺-potentiating effect) for muscle in hypertonic solution was reinvestigated. It was found, using whole frog sartorii, that there was a rehydration of muscle soaked in glycerol Ringer after 30 min and a large swelling (to 140% after 1 hr soaking) upon return of the muscle to normal Ringer, suggesting that significant amounts of glycerol enter the fibers during this time. While contrary to the original report of the glycerol effect, this finding was consistent with other studies involving the use of single fibers. Also reexamined was the potentiating effect of K⁺ on the hypertonic inhibition of muscle contraction. It was found that muscles exposed to this KCl pretreatment swell so that they are less dehydrated in hypertonic solutions, thus accounting for the observed potentiation. After being treated instead with a K₂-tartrate Ringer solution, muscles did not swell and, as determined with twitch recordings, did not display any potentiation in hypertonic solutions—even though the [K₊] was higher than an osmotically equivalent KCl solution. The evidence was thus consistent with alternative hypotheses in which inhibition of contraction occurs at a later stage in E-C coupling or involves the contractile process itself.     

Inhibition by hypertonic solutions of Ca-dependent electrogenesis in single crab muscle fibers

This study describes the effect of hypertonic solutions on isolated muscle fibers of Callinectes danae. Solutions of twice normal tonicity (2.0 T) inhibit both the normal graded membrane responses and the spikes induced by procaine, tetraethylammonium, or barium. The inhibition is maintained throughout exposure to hypertonic solutions prepared by addition of impermeant solutes such as NaCl, sucrose, or Tris-propionate, but is reversible on their withdrawal. In the presence of permeant solutes such as glycerol or acetamide, the inhibition is transient. In both cases the onset of inhibition of the depolarizing Ca electrogenesis is correlated with shrinkage of the fiber. In the case of permeant solutes, the time course of recovery of the graded responses or the spikes follows the recovery of the fiber volume. Changes in the passive electrical characteristics of the fibers due to hypertonic solutions were unrelated to the blockade of membrane Ca activation. The current-voltage relationship in hypertonic sollution revealed no increase in depolarizing K activation. Inhibition of the graded membrane responses and spikes appears to be associated with depression of Ca conductance. Hypertonic solutions might affect the activation of Ca conductance through reduction of the electric field generated by fixed negative surface charges and/or morphological changes in the T tubules. Membrane depolarization elicited little or no tension in 2.0 T solutions while caffeine contracture (10 mM) with an ampliture of 76% of the maximal contractile ability could still be elicited. This indicates that direct effects of hypertonic solutions on the contractile apparatus were not responsible for loss of tension. The latter is attributed to the inhibition of the transmembrane Ca currents.     

Chemical modification of calcium release from the sarcoplasmic reticulum of mechanically skinned skeletal bullfrog muscle fibers

Several types of reagents that react with amino acid side chains induced repetitive phasic contracture of skinned skeletal muscle from frogs. The presence of 10 mM procaine or 5 mM magnesium in the medium or disruption of the sarcoplasmic reticulum (SR) eliminated this contracture, indicating that the calcium-induced calcium-release mechanism of SR is involved in the contraction. Dithiothreitol inhibited the contracture induced by chloramine T, N-acetylimidazole, or p-chloromercuriphenylsulfonic acid (pCMPS) but not in the case of carbodiimide, phenylglyoxal, trinitrobenzenesulfonic acid, diethylpyrocarbonate (DEP), or N-chlorosuccinimide (NCS). Therefore, modification of groups other than the sulfhydryl ones seems to induce contractures under such conditions. The amplitude of the caffeine-induced contracture decreased after treatment with pCMPS, DEP, or NCS. NCS shifted the pCa-tension curve toward low pCa in the SR-disrupted fibers. This shift would explain the decrease in the caffeine contracture. It is tentatively concluded that pCMPS and DEP release a large amount of calcium from SR.     

Studies of the halothane-cooling contractures of skeletal muscle

The characteristics of transient contractures elicited by rapid cooling of frog or mouse muscles perfused in vitro with solutions equilibrated with 0.5-2.0% halothane are reviewed. The data indicate that these halothane-cooling contractures are dose dependent and reproducible, and their amplitude is larger in muscles containing predominantly slow-twitch type fibers, such as the mouse soleus, than in muscles in which fast-twitch fibers predominate, such as the mouse extensor digitorum longus. The halothane-cooling contractures are potentiated in muscles exposed to succinylcholine. The effects of Ca2+-free solutions, of the local anesthetics procaine, procainamide, and lidocaine, and of the muscle relaxant dantrolene on the halothane-cooling contractures are consistent with the proposal that the halothane-cooling contractures result from synergistic effects of halothane and low temperature on Ca sequestration by the sarcoplasmic reticulum. Preliminary results from skinned rabbit muscle fibers support this proposal. The halothane concentrations required for the halothane-cooling contractures of isolated frog or mouse muscles are comparable with those observed in serum of patients during general anesthesia. Accordingly, fascicles dissected from muscle biopsies of patients under halothane anesthesia for programmed surgery develop large contractures when rapidly cooled. The amplitude of these halothane-cooling contractures declined with the time of perfusion of the muscle fascicles in vitro with halothane-free physiological solutions. It is suggested that the halothane-cooling contractures could be used as a simple experimental model for the investigation of the effects of halothane on Ca homeostasis and contractility in skeletal muscle and for study of drugs of potential use in the management of the contractures associated with the halothane-induced malignant hyperthermia syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of calcium in excitation-contraction coupling of lobster muscle

Potassium contractures were induced in lobster muscle bundles under conditions which produced varying KCl fluxes into the fibers. The presence or absence of chloride fluxes during depolarization by high concentrations of potassium, had no effect on the tensions developed. The curve relating tension to the membrane potential had a typical sigmoid shape with an apparent "threshold" for tension at -60 mv. Soaking the muscles in low (0.1 mM) calcium salines for 30 min completely eliminated the potassium contractures but the caffeine contractures were only slightly reduced under these conditions. The potassium contracture could be completely restored in less than 2 min by return of the calcium ions to the saline. Evidence is presented for independent, superficial, and deep calcium sites; the superficial sites appear to be involved in the coupling mechanisms associated with potassium contractures. These sites are highly selective for Ca⁺⁺, and attempts to substitute either Cd⁺⁺, Co⁺⁺, Mg⁺⁺, Ba⁺⁺, or Sr⁺⁺ for Ca⁺⁺ were unsuccessful. However, K⁺ appeared to compete with Ca⁺⁺ for these sites, and the evoked tension could be reduced by prestimulation of the muscle fibers with high K⁺ salines. The results of studies on the influx of ⁴⁵Ca during potassium contractures were compatible with the view of muscle activation by the entry of extracellular calcium.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Do independent processes control the activation and inactivation of potassium contracture tension in rat skeletal muscle?

Potassium (K⁺) contracture tension, measured in small bundles of rat soleus muscle fibers during maintained depolarization, increases to a peak value and then decays either to the baseline or to a pedestal level. We have tested the hypothesis that the rise and fall of tension are determined by independent activation and inactivation processes. If the “Independence” hypothesis is correct, tension during the decay of K⁺ contractures should equal tension predicted from the product of the activation and inactivation parameters determined from the same K⁺ contractures. Both the measured and predicted tensions decayed to a pedestal level that was increased in amplitude in the presence of perchlorate ions. However, the measured tensions in normal solutions and in the presence of perchlorate were three to five times smaller than the predicted tensions. This result indicates that the activation and inactivation of processes controlling the rise and decay of K⁺ contracture tension are not independent.     

Contracture Coupling of Slow Striated Muscle in Non-Ionic Solutions and Replacement of Calcium, Sodium, and Potassium

The development of contracture related to changes of ionic environment (ionic contracture coupling) has been studied in the slowly responding fibers of frog skeletal muscle. When deprived of external ions for 30 minutes by use of solutions of sucrose, mannitol, or glucose, the slow skeletal muscle fibers, but not the fast, develop pronounced and easily reversible contractures. Partial replacement of the non-ionic substance with calcium or sodium reduces the development of the contractures but replacement by potassium does not. The concentration of calcium necessary to prevent contracture induced by a non-ionic solution is greater than that needed to maintain relaxation in ionic solutions. To suppress the non-ionic-induced contractures to the same extent as does calcium requires several fold higher concentrations of sodium. Two types of ionic contracture coupling occur in slow type striated muscle fibers: (a) a calcium deprivation type which develops maximally at full physiological concentration of external sodium, shows a flow rate dependency for the calcium-depriving fluid, and is lessened when the sodium concentration is decreased by replacement with sucrose; (b) a sodium deprivation type which occurs maximally without external sodium, is lessened by increasing the sodium concentration, and has no flow rate dependency for ion deprivation. Both types of contracture are largely prevented by the presence of sufficient calcium. There thus seem to be calcium- and sodium-linked processes at work in the ionic contracture coupling of slow striated muscle.     

Solute-dependent activation of cell motility in strongly hypertonic solutions in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>, human melanoma HTB-140 cells and walker 256 carcinosarcoma cells

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na⁺ or K⁺, sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na⁺ ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.     

Chloride dependence of the K+-stimulated release of taurine from synaptosomes

Exposure of a crude synaptosomal fraction to K⁺ concentrations ranging from 25 to 100 mM evokes the release of [³H]taurine and [³H]GABA. These high concentrations of K⁺ induce, besides depolarization, a marked synaptosomal swelling, which is prevented by replacing chloride in the solutions with the largely impermeant anion gluconate. The depolarizing effect of K⁺ is unaffected by omission of chloride. The K⁺-evoked release of taurine seems related to K⁺-induced changes in synaptosomal volume rather than to a depolarizing effect, since it is totally calcium-independent but is abolished by reducing chloride and by making solutions hypertonic with mannitol. The release of [³H]GABA, in contrast is unaffected in chloride-free or hypertonic solutions.     

The effect of hypertonic solution on the wet weight and contractions of rat uterus and vas deferens

The role of propagated activity in the responses to agonist drugs was studied for the rat uterus and vas deferens. Hypertonic solutions were used to inhibit propagation of activity by shrinking cells. Tissue weight was used to indicate cell volume. Hypertonic solutions after 10 min caused weight loss and reduced the size of contractions in response to submaximal doses of drugs, to KCl, and to external electrical stimulation. Contractions in response to KCl and drugs were diminished to a similar degree in the vas deferens, but in the uterus, drug contractions were depressed much more. Prolonged action of hypertonic solution also differed for the two tissues. In the uterus, weight changes correlated with changes in size of the drug-induced contractions. Uterine contractions reduced in hypertonic solution could be increased by using supramaximal doses of drug. When stimulation was applied to one end of the uterus in a three compartment bath, propagation of spontaneous drug- and KCl-induced contraction occurred, but it was prevented by placing hypertonic solution in the center compartment. An increase of the KCl to 44 mM in the hypertonic solution restored propagation. These experiments yielded no evidence of propagated responses in the rat vas deferens. It was concluded that propagated activity plays a role in drug-induced contractions in the rat uterus but not in the rat vas deferens. Hyperpolarization of shrunken cells might be involved in inhibition of propagation by hypertonic solutions.     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

Loss of the plateau of the cardiac action potential in hypertonic solutions

The effect of hypertonicity on the electrical properties of vertebrate myocardial cells was studied in ventricular muscle fibers of guinea pig, cat, frog, and chicken. The latter two species do not have a T-tubule system, whereas the former two do. In hypertonic solutions (2 x isotonic) produced by addition of sucrose or excess of NaCl, cell diameter decreased and there was a slight hyperpolarization and decrease in action potential overshoot. In guinea pig and cat, the hypertonic solution caused a decrease in input resistance and the plateau of the action potential to disappear in some of the cells; contractions of the entire ventricle also became depressed. These effects were reversed by returning the muscle fibers to isotonic solution. Addition of 5 mM SrCl₂ to the hypertonic solution also caused the plateau component and contraction to reappear. In frog and chick cells, loss of the plateau component and contraction never occurred in hypertonic solution, and input resistance increased. Urea and glycerol hyperosmolarity (2 x) caused no loss of the plateau component or contraction. If the frog and chicken ventricular, and guinea pig atrial myocardial cells (all of which lack T tubules) were to serve as an adequate control for possible effects of hypertonicity on the surface membrane and on contractile proteins, then the results suggest that swelling of the T tubules of mammalian myocardial cells leads to loss of the plateau component.     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

The Effect of Bathing Solution Tonicity on Resting Tension in Frog Muscle Fibers

Resting tension and short-range elastic properties of isolated twitch muscle fibers of the frog have been studied while bathed by solutions of different tonicities. Resting tension in isotonic solution at 2.3-µm sarcomere spacing averaged 0.46 mN·mm^-2 and was proportional to the fiber cross-section area. Hypertonic solutions, containing 0.1–0.5 mM tetracaine to block contracture tension, caused a small sustained tension increase, which was proportional to the fiber cross-section area and which reached 0.9 mN·mm^-2 at two times normal tonicity (2T). Further increases in tonicity caused little increase in tension. Hypotonic solutions decreased tension. Thus, tension at 2.3 µm is a continuous, direct function of tonicity. The dependence of tension on tonicity lessened at greater sarcomere lengths. At 3.2 µm either a very small rise or, in some fibers, a fall in tension resulted from an increase in tonicity. Hypertonic solutions also decreased the tension of extended sarcolemma preparations. In constant-speed stretch experiments the elastic modulus, calculated from the initial part of the stretch response, rose steeply with tonicity over the whole range investigated (1–2.5T). The results show that tension and stiffness of the short-range elastic component do not increase in parallel in hypertonic solutions.