Comparative effects of bepridil, its quaternary derivative CERM 11888 and verapamil on caffeine-induced contracture in ferret hearts.

1. The effects of bepridil, its quaternary derivative: CERM 11888 (methyl-pyrrolidinium bromide) (10(-7)-10(-5) M), and verapamil (10(-7)-10(-6) M) were compared on caffeine-induced contracture of isolated ventricular trabeculae of the ferret. 2. Bepridil diminished the amplitude of contracture in a concentration-dependent fashion, and this effect was significantly different from that of CERM 11888 which, like verapamil, only reduced the amplitude at the highest concentration used. 3. Bepridil (10(-6) M) significantly shortened the time to peak tension and accelerated the relaxation phase of contracture. This latter effect was different from that of CERM 11888. Verapamil (10(-6) M) also tended to accelerate the relaxation phase. At 10(-5) M these actions of bepridil on the time to peak and relaxation tended to reverse. 4. At all concentrations bepridil and verapamil reduced the rate of repriming of contracture and this effect of bedpridil was significantly different from that of its quaternary derivative which only showed a significant effect at 10(-5) M. 5. These results demonstrate a clear intracellular effect of bepridil in the ferret heart. Verapamil and CERM 11888 had only weak intracellular effects even at high concentrations. 6. Analysis of the results suggests that the main sites of action of bepridil in this model are the sarcoplasmic reticulum and one or two calcium compartments in the sarcolemma.

Hypogravity increases cyclopiazonic acid sensitivity of rat soleus muscle.

The functional capacity of skeletal muscle sarcoplasmic reticulum was explored in slow rat soleus muscle after 21 days of hindlimb suspension. The sarcoplasmic reticulum function was assessed in intact and saponin-skinned fibers by using cyclopiazonic acid, a specific Ca(2+)-adenosinetriphosphatase inhibitor. After hindlimb unweighting, the sensitivity to cyclopiazonic acid of intact and skinned soleus fibers becomes similar to that found in fast-twitch muscles. This change could be related to the expression of fast Ca2(+)-adenosinetriphosphatase-pump protein in unloaded soleus muscles and agrees with a transformation of soleus muscle from slow- to fast-twitch type. These results also indicate that specific pharmacological tools, like cyclopiazonic acid, could be used to analyze subcellular functional changes due to hindlimb unweighting.

Ciliary neurotrophic factor prevents unweighting-induced functional changes in rat soleus muscle.

The purpose of the present work was to see whether changes in rat soleus characteristics due to 3 wk of hindlimb suspension could be modified by ciliary neurotrophic factor (CNTF) treatment. Throughout the tail suspension period, the cytokine was delivered by means of an osmotic pump (flow rate 16 microg. kg(-1). h(-1)) implanted under the hindlimb skin. In contrast to extensor digitorum longus, CNTF treatment was able to reduce unweighting-induced atrophy in the soleus. Twitch and 146 mM potassium (K) tensions, measured in small bundles of unloaded soleus, decreased by 48 and 40%, respectively. Moreover, the time to peak tension and the time constant of relaxation of the twitch were 48 and 54% faster, respectively, in unloaded soleus than in normal muscle. On the contrary, twitch and 146 mM K contracture generated in CNTF-treated unloaded and normal soleus were not different. CNTF receptor-alpha mRNA expression increased in extensor digitorum longus and soleus unloaded nontreated muscles but was similar in CNTF-treated unloaded muscles. The present results demonstrate that exogenously provided CNTF could prevent functional changes occurring in soleus innervated muscle subject to unweighting.

Effects of cyclopiazonic acid on Ca(2+)-activated tension production in skinned skeletal muscle fibres of the ferret.

The effect of cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, was investigated on force generation by the contractile apparatus in Triton-skinned fibres from extensor digitorum longus and soleus. Concentrations of cyclopiazonic acid lower than 10 microM were without effect on Ca(2+)-activated tension in both types of muscles. In contrast, in soleus, cyclopiazonic acid (20, 50, 100 microM) was found to shift reversibly the relation-tension pCa (-log[Ca2+]) towards lower free Ca2+ and to decrease maximal Ca(2+)-activated tension, in a dose-dependent manner. In extensor digitorum longus, the Ca2+ sensitivity was significantly increased only at a high cyclopiazonic acid concentration (100 microM) and for all the concentrations tested between 5 to 100 microM maximal Ca(2+)-activated tension was unchanged. These results suggest that cyclopiazonic acid has a direct effect on contractile proteins, in a dose-dependent manner. Ca2+ sensitivity and Ca(2+)-activated maximal tension of the contractile apparatus were differentially affected in fast- (extensor digitorum longus) and slow-twitch (soleus) fibres from ferret skeletal muscle.

Sarcoplasmic reticulum function in newborn ferret cremaster skeletal muscles.

A study of the properties of the sarcoplasmic reticulum was performed with newborn ferret cremaster muscles at two different development stages: at 8 and 21 days. The effects of extracellular Ca2+, caffeine and cyclopiazonic acid, a specific sarcoplasmic reticulum Ca(2+)-ATPase inhibitor, were examined on intact cremaster skeletal muscles. The uptake and release of Ca2+ were explored on saponin-skinned fibres with or without cyclopiazonic acid and some results obtained were compared with those obtained with adult cremaster muscle. The results have shown that skeletal muscle sarcoplasmic reticulum of newborn animals possesses the ability to accumulate and release Ca2+. Furthermore, application of cyclopiazonic acid modified the twitch, the caffeine responses and decreased the amount of Ca2+ taken up by the sarcoplasmic reticulum in saponin-skinned fibres. In contrast to adult skeletal muscle, in newborn cremaster muscles, the Ca2+ dependence of the twitch suggests that the Ca2+ influx at the sarcolemma level was mainly involved in the activation of the contraction. Furthermore, the results obtained in the presence of cyclopiazonic acid were in favour, as in adult muscle, of a participation of the sarcoplasmic reticulum in the relaxation process.

Dual effects of charged amphiphiles on depolarization-contraction coupling in denervated rat soleus muscle.

Ionic currents and contraction were recorded under voltage clamp conditions in single fibres isolated from rat soleus muscles denervated for more than 20 days. The effects of amphiphiles on depolarization-contraction (d.c.) coupling in Na-free TEA-containing solutions were analyzed. An anionic amphiphile, sodium dodecyl sulfate (1-10 microM), caused a dose-dependent reduction of the contractile response at all amplitudes of depolarization while a cationic amphiphile, dodecyltrimethylamine (1-10 microM), increased the maximum developed tension with a shift in the contractile threshold. A neutral amphiphile, lauryl acetate (20 microM), induced no significant variation. The effects of charged amphiphiles were found to be strongly dependent on the external calcium concentration and on membrane potential. The effect of sodium dodecyl sulfate to decrease tension was reduced or changed to positive inotropy following hyperpolarization of the membrane by, respectively, +10 and +20 mV. In hyperpolarized (+20 mV) cells, dodecyltrimethylamine reduced the amplitude of the contraction. The results demonstrated that changes in Ca-binding properties of surface membrane modified d.c. coupling in denervated slow twitch skeletal muscle.

Which active forms of physostigmine generate contractions in diaphragm muscles of mice?

Physostigmine-induced contractions of isolated small bundles (100 micron diameter) of muscle fibres isolated from the diaphragm of C57 BL mice were studied under various conditions. At different external pH the changes in the maximal amplitude of the contraction were related to the external activity of the permeant neutral form of the drug. However changes in the internal pH showed that the amplitude of the contractile response depended directly on the internal activity of the protonated form of the drug. The amplitude of the contraction was enhanced by caffeine treatment and depended on the external calcium concentrations. In mammalian skeletal muscle, physostigmine appeared to produce contractions due to its anticholinesterase property. The release of calcium seemed to occur from an intracellular store different from that involved in the action of caffeine.

Negative inotropic effect of heparin on tension development in rat skinned skeletal muscle fibres.

Heparin inhibits inositol trisphosphate receptors, particularly in smooth muscle, but its effect on skeletal muscle is controversial. Our study showed that heparin induced a decrease in the amplitude of 10 mM caffeine-induced contracture in slow and fast saponin-skinned fibres. Moreover, measurements on Triton X-100-skinned fibres in soleus muscle showed that heparin alone decreased maximal Ca2(+)-activated tension and Ca2+ sensitivity of contractile proteins, whereas no significant effect was observed in extensor digitorum longus muscle. However, in the presence of caffeine, heparin decreased maximal Ca2(+)-activated tension in both muscles. It would appear that the heparin-induced decrease in the amplitude of caffeine contracture in rat skeletal muscle was not related to a direct inhibition of Ca2+ release from sarcoplasmic reticulum but to a desensitising effect of heparin and caffeine on myofilaments.

Potentiation of the twitch responses by inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase in frog atrial fibres.

In frog atrial fibres, cyclopiazonic acid as well as thapsigargin, which are inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase, induced a significant increase in the twitch amplitude without detectable changes in its kinetics. The measurements performed on chemically skinned fibres show that cyclopiazonic acid has no effect on the properties of contractile proteins. In the presence of a T-type Ca2+ channel blocker or L-type Ca2+ channel blocker, cyclopiazonic acid still induced a potentiation of the twitch while no effect was found in the presence of a Na(+)-Ca2+ exchange blocker. The effect of cyclopiazonic acid was not related to any modification in myofibrillar Ca2+ sensitivity or in Ca2+ influx through Ca2+ channels. It is proposed that the inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase resulted in a potentiation of the effect of the Ca2+ influx and that the major role of the sarcoplasmic reticulum was to limit the intracellular Ca2+ concentration.

Inhibition of caffeine-induced Ca2+ release by adenosine in mammalian skinned slow- and fast-twitch fibres.

The present study performed on chemically skinned skeletal fibres was designed to compare the effects of adenosine on the Ca2+ sensitivity of contractile proteins and on caffeine-induced Ca2+ release in rat slow- (soleus) and fast-twitch (edl) muscles. The tension-pCa relationships were obtained by exposing triton X-100 (1% v/v) skinned fibres sequentially to solutions of decreasing pCa in the presence or in absence of adenosine. Then, changes in caffeine contracture due to adenosine were recorded on saponin (50 microg/ml) skinned fibres. The results show that the sensitivity to Ca2+ of contractile proteins in the presence of different concentrations of caffeine was not significantly modified by adenosine. However, it was proposed that adenosine (0.1-2 mM) reduced the Ca2+ released by caffeine (0.1-10 mM) from the sarcoplasmic reticulum in slow- and fast-twitch fibres and that the soleus was more sensitive to adenosine than edl muscle. The effects of specific A2a and A1 agonists and antagonists were also tested on caffeine contractures. It was found that the A1 antagonist reduced adenosine effect on caffeine response. Then it is proposed that adenosine modulates the sarcoplasmic reticulum Ca2+ release by a direct effect on the RyR1 receptors and/or by an indirect effect mediated by A1 receptors located at the sarcoplasmic level.

Differential effects of nandrolone decanoate in fast and slow rat skeletal muscles.

PURPOSE: We studied the effects of high doses of an anabolic-androgenic steroid, exercise training, and a combination of steroid and training on mammalian fast- and slow-twitch skeletal muscles at the cellular level. METHODS: Thirty-two male rats were divided into sedentary and treadmill-trained groups (increased speed and time: 18 m.min-1, 0.5 h.d-1, 5 d.wk-1). Eight animals of each group were treated with nandrolone decanoate (ND) (15 mg.kg-1.wk-1), and others received the same doses of solvent. The animals were killed after 5 wk, and the contractile parameters for isolated small bundles of soleus and extensor digitorum longus (edl) fibers were estimated. RESULTS: Muscle mass, twitches, and K+ contractures were increased in soleus and edl muscles after the drug treatment and after the exercise training. Caffeine contractures were increased only after the exercise training. The combination of exercise with ND treatment produced greater effects, particularly a significant increase in sensitivity to caffeine and the amplitude of K+ contractures as well as a shortening of the time required to restore contracture. These modifications were more marked in slow than fast muscle. CONCLUSION: These results show that 5 wk of exercise training produced changes in the contractile responses developed by isolated skeletal muscle cells. The combination of exercise training with ND treatment potentiated these effects, suggesting that there was some modification in the excitation-contraction coupling mechanism. ND treatment also produced a more potent effect in soleus than edl sedentary muscle.

Measurement of sarcomere length during fast contraction of muscle fibers by digital image analysis.

A low-cost, high-resolution (spatial and temporal) image analysis system was developed to measure sarcomere length (Sl) during fast twitch of isolated striated muscle fibers at different temperatures. Fiber images were examined during twitch with an imaging rate of 220 Hz. To increase temporal resolution beyond 220 Hz, consecutive temporally shifted image sequences (N sequences) were acquired. Individual or average Sl was directly measured from a horizontal profile without spatial-frequency assessment. Measurement precision (E) was determined and expressed as: E(%) = 100xPs/(IsxSl), where Ps is the pixel size and Is the involved sarcomere number. At 18 degrees C during isometric twitch, Sls were measured with 220 Hz temporal and 0.2% spatial resolutions. Sl shortened in the central region (0.21+/-0.12 microm) as tension developed, reaching a maximal shortening of 8.09 + 2.05% (at rest, Sl = 2.59+/-0.05 microm, n = 4) in 32.5+/-1.96 ms. At 30 degrees C, Sl variations were examined with 880 Hz temporal resolution, in which case maximal S1 shortening was reached in 15.74+/-1.99 ms, and then decreased to 5.19+/-1.97% (at rest, S1 = 2.6+/-0.06 microm). The twitch tension developed by the whole fiber was recorded and compared with sarcomere length behavior. Sarcomere length variations in the central region were representative of overall developed tensions at 18 and 30 degrees C.

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle.

Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twitch muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased 45Ca-binding. These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.

Sarcoplasmic reticulum Ca(2+) content affects 4-CmC and caffeine contractures of rat skinned skeletal muscle fibers.

This study investigated whether the sarcoplasmic reticulum Ca(2+) content of rat skeletal muscle fibers affected contractile responses obtained by an application of 4-chloro-m-cresol (4-CmC) and caffeine. Contractures were elicited on saponin-skinned fibers under different Ca(2+) loading conditions. The amplitude of 4-CmC and caffeine contractures of fast-twitch muscle fibers (edl, extensor digitorum longus) differed between the different loading conditions, and this is associated with a greater change in sensitivity to 4-CmC. When the sarcoplasmic reticulum was loaded with a low Ca(2+) concentration for a short period, the 4-CmC concentration providing half-maximal response was tenfold higher than with a larger sarcoplasmic reticulum Ca(2+) loading for a longer period, whereas for caffeine this concentration was only twofold higher in the same conditions. These findings indicate that 4-CmC contractile responses of edl muscle fibers are more dependent on luminal Ca(2+) activity than those of caffeine are. Thus 4-CmC would appear to be of greater interest than caffeine for the study of muscle contractile responses where variations in intracellular Ca(2+) activity exist.

Effects of cyclopiazonic acid, an inhibitor of the Ca++ ATPase of sarcoplasmic reticulum, on Ca++ transport, contraction and relaxation in cardiac muscle.

Cyclopiazonic acid is a potent inhibitor of cardiac sarcoplasmic reticulum Ca++ ATPase. It scarely affects inotropism but significantly impairs lusitropism suggesting a greater role for cardiac sarcoplasmic reticulum in the control of cardiac relaxation than in the control of cardiac contraction.

Sodium withdrawal contractures in rat slow twitch skeletal muscle.

Contractile responses due to alterations in [Na+]o have been investigated in fast (iliacus) and slow (soleus) twitch muscles of the rat. On exposure to a Na-free solution, the soleus in contrast to the iliacus cells, generated contractile responses without depolarizing the surface membrane. Following glycerol treatment, the twitch and a part of the Na-withdrawal contracture were abolished. The amplitude of the remaining contracture was between 5 and 50% of the original response and the time to peak was 0.4 to 2 times longer. In intact and detubulated preparations, the amplitude of the zero-Na contracture was modified by changes in [Ca2+]o and a linear relationship was found if the reciprocal of tension was plotted against 1/square root[Ca2+]o. In intact and detubulated fibres, a steep dependence of the Na-withdrawal contracture on [Na+]o was found and [Na+]o which induced the half maximal response at each [Ca2+]o was the same, the responses were inhibited by Mg-ions in a competitive way. It is assumed that the activator Ca is triggered at the tubular and sarcolemmal membrane level by lowering [Na+]o and that a calcium-induced calcium release mechanism at the S.R. level may also be involved.

Changes of contractile responses due to simulated weightlessness in rat soleus muscle.

Some contractile and electrophysiological properties of muscle fibers isolated from the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of rats were compared with those measured in SOL muscles from suspended rats. In suspended SOL (21 days of tail-suspension) membrane potential (Em), intracellular sodium activity (aiNa) and the slope of the relationship between Em and log [K]o were typical of fast-twitch muscles. The relation between the maximal amplitude of K-contractures vs Em was steeper for control SOL than for EDL and suspended SOL muscles. After suspension, in SOL muscles the contractile threshold and the inactivation curves for K-contractures were shifted to more positive Em. Repriming of K-contractures was unaffected by suspension. The exposure of isolated fibers to perchlorate (ClO4-)-containing (6-40 mM) solutions resulted in a similar concentration-dependent shift to more negative Em of activation curves for EDL and suspended SOL muscles. On exposure to a Na-free TEA solution, SOL from control and suspended rats, in contrast to EDL muscles, generated slow contractile responses. Suspended SOL showed a reduced sensitivity to the contracture-producing effect of caffeine compared to control muscles. These results suggested that the modifications observed due to suspension could be encounted by changes in the characteristics of muscle fibers from slow to fast-twitch type.

Membrane currents and activation of contraction in rat ventricular fibres.

1. A double sucrose gap technique has been combined with an optical method of measuring the movement of the preparation and applied to rat ventricular trabeculae.2. Experiments performed in normal McEwen and in depleted sodium solutions with and without tetrodotoxin, manganese or predepolarization suggest that the fast inward current is not as important as the slow inward current in determining the contractile strength. Furthermore, there is a marked similarity between potential dependence of the slow inward current and the contraction, particularly in sodium-depleted fluids.3. The appearance of a staircase response associated with a series of depolarizations, in normal McEwen, may mean that there is an intervention of a further, possibly intracellular, stage between inward current and contraction, as suggested by earlier experiments on frog as well as on mammalian heart.

Calcium sensitivity of skinned ferret EDL, soleus, and cremaster fibers.

The properties of the contractile system at different times of the year in the ferret extensor digitorum longus (EDL), soleus and cremaster muscles were examined by using chemically skinned (Triton X-100) preparations. The results show clear differences in calcium sensitivity between these skeletal muscles. The apparent calcium threshold for activation was lower in soleus than in EDL, while calcium concentrations ([Ca2+]) required to obtain the half-maximal tension, expressed as pCa50 (-log[Ca2+]), was lower in EDL than in soleus muscle. In fact, pCa50 obtained in fast and slow fibers by fitting the experimental data points by a modified Hill equation was 5.92 +/- 0.02 (n = 9) and 6.09 +/- 0.03 (n = 11) respectively. So EDL appears to be a typical fast-twitch muscle and soleus a typical slow-twitch muscle. Adult ferret cremaster muscle was composed of two types of fibers during the quiescent period similar to EDL and soleus, and only one type that was intermediate between EDL and soleus in the breeding period, as assessed by pCa50 values. These annual modifications in calcium activation of adult ferret cremaster muscle could be related to changes in the function of these muscles and may be correlated with seasonal variations of sexual activity.