Frog Skeletal Muscle Fibers Research Articles

Repriming and activation alter the frequency of stereotyped discrete Ca2+ release events in frog skeletal muscle.

1. Brief localized elevations in myoplasmic [Ca2+] (Ca2+ sparks) in individual sarcomeres of voltage-clamped frog skeletal muscle fibres were examined by laser scanning confocal microscopy. 2. Fibres held at 0 mV were briefly repolarized to -90 mV (repriming pulse) to restore only a small fraction of sarcoplasmic reticulum (SR) calcium release. Subsequent depolarization to 0 mV (test pulse) caused the appearance of small numbers of Ca2+ sparks at different sarcomeres from pulse to pulse. Increasing the repriming time resulted in an increase in the frequency of occurrence of the Ca2+ sparks. 3. The amplitude and spatio-temporal extent of the Ca2+ sparks were independent of the repriming time and test pulse voltage. Ca2+ sparks recorded during small depolarizations of fibres held at -90 mV had a similar amplitude and spatio-temporal extent as those recorded after brief repriming of the same fibre held at 0 mV. 4. We conclude that stereotyped Ca2+ sparks underlie calcium release at all voltages and all extents of repriming. The amplitude of Ca2+ release is thus graded by the frequency but not by the amplitude or spatio-temporal extent of the individual SR Ca2+ release events.

Intracellular pH regulation in detubulated frog skeletal muscle fibers.

Intracellular pH regulation was studied in semitendinosus muscle fibers from frog (Rana pipiens). Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes and membrane potential with conventional microelectrodes. Fibers had their connections between the surface and transverse tubular membrane disrupted (detubulation) with the formamide shock technique. Fibers were approximately 80% detubulated as determined by the decrease in membrane capacitance and the loss of contractile capability. The initial rate of pHi recovery from acidification to approximately 6.8 (no CO2) was dependent on external buffering power, reaching a maximum of approximately 0.6 pH/h at 50 mM HEPES, indicating that the rate of pHi recovery in frog muscle is limited by the diffusion of buffer through an external "unstirred layer". In detubulated fibers, pHi recovery from acidification due to both the amiloride-sensitive Na+/H+ and the 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS)-sensitive (Na+ + HCO3-)/Cl- exchangers was nearly identical to recovery in fully tubulated fibers. This is consistent with these two pH recovery transporters being localized to the surface, and not the transverse tubular, membrane domain in frog skeletal muscle fibers.

Modulation of Ca2+ channels, charge movement and Ca2+ transients by heparin in frog skeletal muscle fibres.

This study is an investigation into the modulatory effects of heparin, a component of the extracellular matrix that binds to dihydropyridine receptors, on contraction and Ca2+ channels in frog skeletal muscle. Using tension and Ca2+ signal measurements in single intact skeletal muscle cells we have found that heparin (100-200 micrograms ml-1) substantially potentiates twitch and tetanic tension (55% and 28%, respectively). In contrast, heparin reduces the amplitude of K+ contractures. Heparin most likely potentiates twitch tension by prolonging action potentials. The ionic basis of this effect was investigated in voltage-clamp experiments. Membrane currents were monitored in voltage-clamped segments of single fibres using the triple Vaseline gap technique. We found that heparin partially blocks delayed rectifier potassium channels. The depressive effects of heparin on K+ contractures prompted us to investigate the effects of heparin on charge movement and Ca2+ currents (ICa) under voltage-clamp. Charge movement was measured using a subtraction procedure that employed a -20 mV control pulse from a holding potential of -100 mV. Heparin depresses the total charge by 25%. We propose that the reduction in the amplitude of potassium contractures is related to a partial blockade of charge movement. Extracellular heparin shifts the ICa-V relation toward more negative voltages and delays the deactivation of tail currents. Double pulse experiments revealed that conditioning depolarizations speed the activation of ICa during test depolarizations. Heparin does not affect this process. The primary action of heparin is to accelerate the activation of ICa during pulses not preceded by conditioning depolarizations. Overall, the kinetic effects of heparin on ICa would increase the Ca2+ influx associated with action potentials. However, mechanical and optical experiments performed in Ca(2+) -free solutions and in the presence of Ca2+ channel blockers revealed that twitch and tetanic potentiation occur even in the absence of Ca(2+) -influx.

Effects of cardiac glycosides on excitation-contraction coupling in frog skeletal muscle fibres.

1. The effects of digoxin and ouabain on the calcium release flux from the sarcoplasmic reticulum (SR), isometric tension and intramembrane charge movement were studied in voltage clamped skeletal muscle fibres of the frog. 2. Both cardiac glycosides increased both calcium transients and simultaneously recorded tension at all membrane potentials, showing different effects on the peak and on the steady components of the calcium release flux. These effects were attained at an extracellular digoxin concentration of 5 nM and an estimated intracellular ouabain concentration of 1-2 nM. Digoxin and ouabain thus exerted their effects at the same concentration on calcium release in skeletal muscle as previously observed in isolated cardiac-type ryanodine receptor (RyR) calcium release channels. 3. The peak of SR calcium release increased at all voltages, with the largest potentiation at intermediate membrane potentials. This increase in calcium release flux was attained despite an unchanged SR calcium content. The attenuated release rate therefore reflected an increased number of open RyR channels rather than increased SR loading. 4. These effects could be attributed to an increase in calcium release activation and not a decrease in the rate of inactivation. Rather, the rate of inactivation was enhanced at all voltages as expected from the increased calcium concentration in the triadic junction. 5. In contrast, CMA (17 alpha-acetoxy-6-chloro-4, 6-pregnadiene-3,20-dione; 5 microM), a Na(+)-K(+)-ATPase inhibitor with no positive inotropic effects on the heart, neither influenced SR calcium release nor antagonized the effects of ouabain. 6. Both digoxin and ouabain preserved total intramembrane charge apart from a small negative shift in the mid-point voltage and increase in slope factor. 7. Both digoxin and ouabain induced calcium release from heavy SR vesicles at rates comparable to that induced by ryanodine or caffeine. 8. It is concluded that at least part of the inactivating component of SR calcium release involves distinct RyR calcium release channels that resemble the cardiac RyR isoform in its specific sensitivity to cardiac glycosides.

A gap isolation method to investigate electrical and mechanical properties of fully contracting skeletal muscle fibers

We describe here a single-gap isolation method that allows the simultaneous measurement of electrical activity and tension output from fully contracting segments of frog skeletal muscle fibers. By using single pulses and pulse trains of varying frequency (5-100 Hz), records obtained for both electrical and mechanical fiber response demonstrate that the physiological properties of the fiber segments have been preserved. Action potentials could be recorded free of movement artifacts, even while segments were in fused tetani and developing maximum tensions of more than 600 kN/m2. Single current pulses evoked action potentials that averaged 144 +/- 16 mV (mean +/- SD, n = 8) in amplitude and twitches that averaged 285 +/- 66 kN/m2 and 55 +/- 5 ms (mean +/- SD, n = 20) in magnitude and time to peak, respectively. Trains of action potentials elicited patterns of tension development that exhibited summation, unfused tetani, and fused tetani in a frequency-dependent manner. The AC and DC electrical properties of the single grease gap were modeled with a simple Thévenin equivalent circuit, which satisfactorily predicted the experimental results. Our methodology is easily implemented and potentially applicable to any muscle preparation in which fiber segments with an intact end attached to a piece of tendon can be dissected.

Mechanism of action of K channel openers on skeletal muscle KATP channels. Interactions with nucleotides and protons.

The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATP channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyran KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP gamma s, with and without Mg2+, but they had no effects in the absence of internal nucleotides, even after channel activity had significantly declined because of rundown. The effects of KCOs could therefore be attributed solely to a competitive interaction between KCOs and nucleotides, as confirmed by observations that ATP decreased the apparent affinity for KCOs and that, conversely, KCOs decreased ATP or ADP sensitivity. Protons antagonized the action of the non-benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissociation rate. This effect resembled the effect of acidification on benzopyran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325:276-280, 1993), suggesting that, in spite of their structural diversity, KCOs could act through the same binding sites. Detailed analysis of the inhibitory effects of protons on channel activity induced by levcromakalim or SR 47063 revealed that, in the presence of 100 microM ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allosteric model of the KATP channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg(2+)-free nucleotides, and one benzopyran KCO site. The structural implications of this model are discussed.

11-Oxo-tetrodotoxin and a specifically labelled 3H-Tetrodotoxin

Tetrodotoxin was oxidized to a hydrated aldehyde, 11-oxo-tetrodotoxin, which shares the specificity of tetrodotoxin for the Na + channel of the isolated voltage-clamped frog skeletal muscle fiber, but is four to five times more potent. It binds to the solubilized Na + channel of eel electroplax with a similarly higher potency, because of an equilibrium dissociation constant about 0.25, and a dissociation rate constant 2.4 times slower than those for tetrodotoxin. 11-Oxo-tetrodotoxin can be reduced to regenerate a tetrodotoxin, which is chemically and biologically indistinguishable from the original tetrodotoxin. By reducing with tritiated sodium borohydride, a 3H marker can be inserted regiospecifically to yield 11-[ 3H]-tetrodotoxin. Because it has a defined specific activity of > 2.5 Ci/mmole, and a 3H marker which does not exchange with solvent proton, 11-[ 3H]-tetrodotoxin is an ideal tracer for tetrodotoxin. It may enable studies of problems which require higher signals and/or better stability of the marker than those obtainable from currently available tracer Na +-channel ligands.

Reversible depression of action potentials and force production in frog single muscle fibres by calmodulin-inhibitors.

The effects of the calmodulin-inhibitors trifluoperazine, thioridazine and zaldaride maleate on the responses to electrical stimulation in isolated frog skeletal muscle fibres were investigated. All three drugs initially reduced the amplitude of the action potentials but potentiated twitch force. This was followed by a total loss of action potentials and force production. However, the resting membrane potential was not changed. The effects were completely reversible upon removal of the drugs. These results suggest that an intact calmodulin system is required for normal function of the sarcolemmal sodium channels of frog skeletal muscle.

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle.

The effects of the ryanodine receptor (RyR) antagonists ryanodine and daunorubicin on the kinetic and steady-state properties of intramembrane charge were investigated in intact voltage-clamped frog skeletal muscle fibers under conditions that minimized time-dependent ionic currents. A hypothesis that RyR gating is allosterically coupled to configurational changes in dihydropyridine receptors (DHPRs) would predict that such interactions are reciprocal and that RyR modification should influence intramembrane charge. Both agents indeed modified the time course of charging transients at 100-200-microM concentrations. They independently abolished the delayed charging phases shown by q gamma currents, even in fibers held at fully polarized, -90-mV holding potentials; such waveforms are especially prominent in extracellular solutions containing gluconate. Charge movements consistently became exponential decays to stable baselines in the absence of intervening inward or other time-dependent currents. The steady-state charge transfers nevertheless remained equal through the ON and the OFF parts of test voltage steps. The charge-voltage function, Q(VT), shifted by approximately +10 mV, particularly through those test potentials at which delayed q gamma currents normally took place but retained steepness factors (k approximately 8.0 to 10.6 mV) that indicated persistent, steeply voltage-dependent q gamma contributions. Furthermore, both RyR antagonists preserved the total charge, and its variation with holding potential, Qmax (VH), which also retained similarly high voltage sensitivities (k approximately 7.0 to 9.0 mV). RyR antagonists also preserved the separate identities of q gamma and q beta species, whether defined by their steady-state voltage dependence or inactivation or pharmacological properties. Thus, tetracaine (2 mM) reduced the available steady-state charge movement and gave shallow Q(VT) (k approximately 14 to 16 mV) and Qmax (VH) (k approximately 14 to 17 mV) curves characteristic of q beta charge. These features persisted with exposure to test agent. Finally, q gamma charge movements showed steep voltage dependences with both activation (k approximately 4.0 to 6.5 mV) and inactivation characteristics (k approximately 4.3 to 6.6 mV) distinct from those shown by the remaining q beta charge, whether isolated through differential tetracaine sensitivities, or the full approximation of charge-voltage data to the sum of two Boltzmann distributions. RyR modification thus specifically alters q gamma kinetics while preserving the separate identities of steady-state q beta and q gamma charge. These findings permit a mechanism by which transverse tubular voltage provides the primary driving force for configurational changes in DHPRs, which might produce q gamma charge movement. However, they attribute its kinetic complexities to the reciprocal allosteric coupling by which DHPR voltage sensors and RyR-Ca2+ release channels might interact even though these receptors reside in electrically distinct membranes. RyR modification then would still permit tubular voltage change to drive net q gamma charge transfer but would transform its complex waveforms into simple exponential decays.

Extracellular action potentials of skeletal muscle fibre affected by 4-aminopyridine: a model study.

A new specially designed analytical function approximating the intracellular action potentials (ICAPs) for calculation of the extracellular potentials (ECAPs) at various radial and axial distances from the active fibre is proposed. 4-Aminopyridine (4-AP) was used to obtain ICAPs with a prolonged repolarization phase in order to investigate the influence of changes in ICAP shape on the ECAPs. From the experimentally recorded ICAPs before and after treatment of frog skeletal muscle fibres with 4-AP, approximated by the new function, the ECAPs were calculated applying the line-source model in a finite fibre. Using this function allowed calculation of the ECAPs at distances not accessible for the experimental recordings. The total ionic current (Ii) during the action potential was calculated using the cable equation. Our results showed that the ratio of the first positive to the negative phases of the ECAPs of treated fibres increased at large radial distances (3000 microns and more) and the terminal positive phase was asymmetric with an abrupt initial deflection followed by a slow inverse deflection. The calculated ECAPs at various axial distances from the fibre end (cylindrical and conical part) and at radial distances from the fibre membrane ranging from 0 to 5000 microns, corresponded in shape to the experimentally recorded potentials of untreated and 4-AP-treated muscle fibres.

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.

Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited.

Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ signal. In the presence of TBQ the rate of relaxation and the rate of fall of Ca2+ became progressively slower in a series of twitches until relaxation failed. Relaxation rate decreased with a time course (approximately 2 s-1) similar to the Mg2+ off rate from purified parvalbumin (PA; 3.6 s-1). TBQ slowed the rate of fall of Ca2+ (5-fold) and force (8-fold) in a 0.3-s tetanus so that the rate of fall of Ca2+ (approximately 2.5 s-1) was similar to the Mg2+ off rate from PA. TBQ caused a near total failure of both Ca2+ sequestration and relaxation in a 1.1-s tetanus, during which PA would be saturated with Ca2+ and could not contribute to relaxation. Thus, when the SR Ca(2+)-ATPase is inhibited, Mg(2+)-PA can sequester Ca2+ and produce relaxation at a rate that is defined by the Mg2+ off rate from PA.

Charge movement in overstretched amphibian skeletal muscle cut fibers.

Membrane current experiments were done in the absence of any permeable ion (except calcium) and the presence of TTX (tetrodotoxin) and TEA+ (tetraethylammonium) in the external solution and caesium in the internal solution at two different sarcomere lengths (3.8 and 4.56 microns) in skeletal muscle fibers of frog (pipiens). Single cut fibers were voltage-clamped in a double Vaseline gap apparatus, and intramembrane charge movement and slow calcium currents were measured. In order to obtain the charge movement, a conventional control was used (the response to a negative pulse) obtained in normally polarized fiber. A descriptive analysis of the results shows us that at 3.8 microns the fibers respond with slow calcium current and charge movement with the characteristics of charge 1 (Q1), but at a sarcomere length of 4.56 microns, just a slow calcium current is obtained. The absence of intramembrane charge movement in overstretched fibers could suggest a modification in the transverse tubular system geometry obscuring the charge movement phenomenon (the linear membrane capacity in stretched fibers was 13.10 micro F/cm2 as average for 5 fibers and in overstretched ones was 13.61 micro F/cm2 as average for 11 fibers). The presence of slow calcium current in this condition (overstretched fiber) means an electrical connection of the tubule with superficial membrane and suggests that the charge movement does not correspond to gating of the "L" type calcium channel.

Picrotoxin potentiates contraction while inhibiting Ca current but increasing birefringence signal in frog skeletal muscle fibers.

In single frog skeletal muscle fibers, picrotoxin (5 mM) potentiated the voltage-dependent component of contractions in response to 2-s depolarizing pulses while greatly inhibiting the simultaneously recorded tubular Ca current in a normal-Ca, Na- and CI-deficient solution, provided the contractions were generated at long time intervals (2 min). In normal Ringer's solution, picrotoxin reversibly increased the amplitude of the early large birefringence signal and the amplitude and duration of the simultaneously recorded twitch tension, suggesting that the drug may increase, directly or indirectly, the release of Ca from the SR.

Resting cytoplasmic free Ca2+ concentration in frog skeletal muscle measured with fura-2 conjugated to high molecular weight dextran.

Intact frog skeletal muscle fibers were injected with the Ca2+ indicator fura-2 conjugated to high molecular weight dextran (fura dextran, MW approximately 10,000; dissociation constant for Ca2+, 0.52 microM), and the fluorescence was measured from cytoplasm (17 degrees C). The fluorescence excitation spectrum of fura dextran measured in resting fibers was slightly red-shifted compared with the spectrum of the Ca(2+)-free indicator in buffer solutions. A simple comparison of the spectra in the cytoplasm and the in vitro solutions indicates an apparently "negative" cytoplasmic [Ca2+], which probably reflects an alteration of the indicator properties in the cytoplasm. To calibrate the indicator's fluorescence signal in terms of cytoplasmic [Ca2+], we applied beta-escin to permeabilize the cell membrane of the fibers injected with fura dextran. After treatment with 5 microM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca2+, ATP), whereas the 10-kD fura dextran only slowly leaked out of the fiber. It was thus possible to estimate calibration parameters in the indicator fluorescence in the fibers by changing the bathing solution [Ca2+] to various levels; the average values for the fraction of Ca(2+)-bound indicator in the resting fibers and the dissociation constant for Ca2+ (KD) were, respectively, 0.052 and 1.0 microM. For the comparison, the KD value was also estimated by a kinetic analysis of the indicator fluorescence change after an action potential stimulation in intact muscle fibers, and the average value was 2.5 microM. From these values estimated in the fibers, resting cytoplasmic [Ca2+] in frog skeletal muscle fibers was calculated to be 0.06-0.14 microM. The range lies between the high estimates from other tetracarboxylate indicators (0.1-0.3 microM; Kurebayashi, N., A. B. Harkins, and S. M. Baylor. 1993. Biophysical Journal. 64:1934-1960; Harkins, A. B., N. Kurebayashi, and S. M. Baylor. 1993. Biophysical Journal. 65:865-881) and the low estimate from the simultaneous use of aequorin and Ca(2+)-sensitive microelectrodes (< 0.04-0.06 microM; Blatter, L. A., and J. R. Blinks. 1991. Journal of General Physiology. 98:1141-1160) recently reported for resting cytoplasmic [Ca2+] in frog muscle fibers.

Stereoselective effects of mexiletine enantiomers on sodium currents and excitability characteristics of adult skeletal muscle fibers.

The effects of the enantiomers of mexiletine were tested on sodium currents of frog skeletal muscle fibers recorded by means of the three vaseline gap voltage clamp method and compared with the effects produced by tocainide enantiomers. The R-(-) mexiletine produced a tonic block of the sodium current, elicited by single depolarizing test pulses from the holding potential of -100 mV to -20 mV, with an IC50 of 43.9 +/- 1 microM, whereas the corresponding S-(+) enantiomer produced the same effects at about twofold higher concentrations. A similar steroselectivity was observed with tocainide enantiomers, but at about 5 fold higher concentrations. Both the R-(-) and S-(+) enantiomers of mexiletine and tocainide produced a further use-dependent block of sodium currents when the test pulse was applied repetitively at a frequency of 2 Hz. The use dependent behavior led to a significant lowering of the IC50 values with respect to the tonic block but the eudismic ratios ([IC50S-(+)]/[IC50R(-)]) and the relative potency between mexiletine and tocainide were maintained. All the tested compounds produced a left shift of the steady state inactivation curves (h infinity), suggesting a high-affinity interaction with the inactivated sodium channels. Again a stronger potency of R-(-) vs. S-(+) enantiomers and of mexiletine vs. tocainide was observed. The excitability characteristics recorded from the semitendinosus muscle by the two microelectrode technique were modified by the tested drugs in agreement with their ability to block sodium current. Thus a concentration-related increase in the threshold current required to elicit an action potential as observed along with a decrease in the amplitude and a shortening of the latency of action potential and a decrease in the firing capability of the membrane. Again the R-(-) isomers were more potent than the S-(+) ones and mexiletine was more effective than tocainide. These data corroborate the presence of a stereospecific site for these drugs on adult skeletal muscle sodium channels. The constant eudismic ratios between the enantiomers during both tonic and use-dependent block suggest that the increase in the apparent affinity of the receptor during state-dependent conformational changes of the channel does not enhance its stereospecificity. The decrease in effective concentration upon high frequency stimulation supports the potential usefulness of low doses of R-(-) mexiletine in the treatment of the abnormal hyperexcitability of the myotonic muscles, with a likely reduction of unwanted side effects.

Effects of piperazine derivatives on the activity of frog skeletal muscle fibers

1. 1. This study was undertaken to characterize the effects of some piperazine derivatives on excitable cell membranes. Three original Bulgarian compounds with favorable effects on cardiovascular and nervous system—piperazine derivatives with code names P-II ( N 1-[3-oxo-3-phenyl-2-methyl-propyl]- N 4-[trans-3-hydroxy-1,2,3,4-tetrahydro-2-naphthyl]-piperazine dihydrochloride), AS 2 ( N 1-benzhydryl- N 4-allyl piperazine dihydrochloride) and 35-M (Schiff's base of N 1-benzhydryl- N 4-aminopiperazine with triacetonamine, dioxalate salt) were tested in experiments with conventional microelectrode technique on isolated frog muscle fibers. 2. 2. After 30-min treatment with tested drugs at concentrations of 10–100 μM the recorded intra-(ICAP) and extracellular action potentials (ECAPs) showed an amplitude decrease and duration increase. The total ionic current ( I i ) decreased as the outward phase was almost abolished by P-11. The propagation velocity (PV) of excitation and the twitch amplitude also decreased. These changes were agent- and concentration-dependent. 3. 3. The effect potency of the agents diminished in the following order: P-11 > AS 2 > 35-M. 4. 4. Concentrations higher than 100 μM for all agents completely, but reversibly, inhibited membrane excitability. 5. 5. The results demonstrate compound- and concentration-induced modulation of Ca 2+ current with blockade of Ca 2+-dependent K + and Cl − membrane channels of muscle fiber treated with the compound tested.

Effects of methylene blue and ascorbate on transmembrane potential in frog skeletal muscle

1. l. The effects of methylene blue (MB, 10 −4 M) and ascorbate (ASC, 10 −4 M) on the resting membrane potential of frog skeletal muscle fibers were studied in Cl −-free medium at various external K + concentrations. 2. 2. Muscle fibers formed two distinct populations according to their resting potentials (hyperpolarized to −104 mV or depolarized to −31 mV) after a 90 min period of K + withdrawal. ASC increased the relative contribution of hyperpolarized fibers, while in the presence of MB, all fibers depolarized during the 90 min of K + withdrawal. 3. 3. In the presence of 2.5 mM K +, ASC had no significant effect on the resting potential, while MB moved half of the fibers into the depolarized pool. Elevation of [K +] 0 to 10 mM caused repolarization of all previously depolarized fibers (to −63 mV) in spite of the continuous presence of MB. 4. 4. Ionic currents were measured during a 60 mV depolarization step using the double sucrose-gap technique. MB significantly increased the peak inward current, while ASC had no effect. The steady-state outward current was not affected by MB or ASC. 5. 5. The results suggest that ionic conductances may be under redox control in frog skeletal muscle.

Reversible vacuolation of the transverse tubules of frog skeletal muscle: a confocal fluorescence microscopy study.

A confocal microscope was used to investigate the reversible vacuolation of frog skeletal muscle fibres produced by the efflux and entry of glycerol (80-100 mM). The formation, development and disappearance of vacuoles was observed in the fibres by staining simultaneously with two fluorescent membrane probes, RH414 and DiOC6(3). The styryl dye, RH414, stains only the plasmalemma and the membranes of the transverse tubules. In normal and glycerol-loaded fibres, RH414 revealed regular, narrow dotted bands located at the position of the Z-lines. Glycerol removal produced, within 2-10 min, many empty round vacuoles (0.4-1.5 microns in diameter) that were apparently anchored to the stained bands. Later on, individual vacuoles tended to enlarge and align into longitudinal chains of vacuoles. Neighbouring vacuoles that contacted each other fused to form large vacuoles up to several sarcomeres in length. Neither the T-tubules, nor the vacuoles, were stained by DiOC6(3). However, glycerol efflux was also accompanied by a redistribution of sarcoplasmic reticulum membranes and by changes in mitochondria that were revealed on staining the same fibres with the carbocyanine dye, DiOC6(3). The alterations in staining patterns revealed by RH414 and DiOC6(3) were completely reversible. Within 5-10 min after a second application of glycerol, the pattern of staining returned to normal with the exception of very bright, spots stained with RH414, which appeared in place of many but not all of the vacuoles, and probably correspond to the irregular nets of T-tubules observed under the electron microscope in such fibres. They are considered to be defects in regeneration of the T-system after vacuolation. The vacuolation/devacuolation cycle could be repeated several times following glycerol efflux and entry. The development and disappearance of vacuoles then mainly involved conversion of bright spots to large vacuoles and vice versa. Some possible mechanisms of vacuole formation and disappearance are discussed, and it is suggested that vacuolation of the T-system may be important in relation to regulating the volume of skeletal muscle cells.

Nystatin-, mycoheptin- and levorin-induced conductance in the membrane of frog skeletal muscle fibres.

The effects of the polyene antibiotics nystatin (2 x 10(-5)-10(-4) mol/l), mycoheptin (1.3 x 10(-6)-10(-5) mol/l) and levorin (10(-8)-5 x 10(-5) mol/l) on isolated frog skeletal muscle fibres and whole sartorius muscles of the frog have been investigated. Cation conductance was measured under current clamp conditions using a double sucrose-gap technique. Cation effluxes were studied by means of flame emission photometry. All three antibiotics increased the cation conductance and efflux rates; however, differences between the polyenes were found in the steady state values of induced cation transport at a given concentration. The values of both induced conductance gA infinity and efflux rate constants KA formed the following sequence: levorin > mycoheptin > nystatin, demonstrating a correlation with the order of antifungal activities. The dose-response curves of lg polyene-induced cation transport against lg of antibiotic concentration in our experiments had slope values which were much lower than those in bilayers: 1.7 and 1.3 for nystatin and mycoheptin, respectively, whereas the aromatic heptaene levorin had an even smaller concentration dependence. The decline in the equilibrium conductance caused by nystatin- and mycoheptin removal was very fast (during the first minute tau = 0.74 and 2.39 min, respectively). In contrast, levorin-induced conductance was irreversible. It is proposed that the processes which limit the rate of channel formation are different in biological and model membranes.