Modulation by adrenaline of electrophysiological membrane parameters and contractility in intact and internally perfused single muscle fibres of the crayfish.

The effect of catecholamines on basic membrane characteristics (including labeled ionic fluxes) and contractile parameters was followed in current clamp and voltage clamp conditions in intact muscle fibres and internally perfused muscle fibre segments respectively of the crayfish Astacus fluviatilis; i.e. in muscle fibres which spike and activate tension on calcium principle. Both adrenaline and noradrenaline (6.10(-6) mol/l) facilitated twitch tension induced by graded membrane responses or strontium all-or-none spikes. No effect of isoprenaline was observed. Adrenaline (6.10(-6)-6.10(-5) mol/l) produced an inotropic effect, which appeared with a latency of 2 min and reached its maximum in 5 min. The rates of activation and relaxation of contraction were increased, whilst the latency and the threshold depolarization were decreased. The changes persisted (several tens of min) after washout of adrenaline, depending on concentration and duration of adrenaline application. The resting potential and the strontium spike (Ca2+ replaced with Sr2+) were not influenced and the graded responses were facilitated by adrenaline (from 36.4 +/- 1 mV to 40.0 +/- 2 mV; RP = 77.2 +/- 0.5 mV). Extracellular Ca2+ ions are required for the inotropic effect of adrenaline to occur. The decrease of electrical and contractile responses in nominal calcium-free solutions or after a blockade of Ca2+ influx by Ni2+ ions (1 mmol/l) was relieved by adrenaline. The persistence of inotropic effect of adrenaline was absent, when the extracellular concentration of Ca2+ ions, [Ca2+]0 was decreased from 13.5 to 3.4 mmol/l or the Ni2+ ions were added. The influx of 89Sr2+ ions was decreased in the presence of Ni2+ ions from 24.2 +/- 4.7 pmol.cm-2.s-1 to 11.0 +/- 2.8 pmol.cm-2.s-1, but restored to 20.4 +/- 5.8 pmol.cm-2.s-1 in the presence of adrenaline (6 mumol/l). Adrenaline itself decreased the influx of 89Sr2+ ions, and prolonged the time constant of efflux both in resting and stimulated fibres. The effect of adrenaline is dependent on mobilization of Ca2+ ions from the sarcoplasmic reticulum. First, the inotropic effect of adrenaline was absent in the presence of procaine (blockator of the Ca release channel of the SR), in spite of the increase of the active membrane response (all-or-none procaine action potential); second, adrenaline accelerated the uptake of Ca ions by SR as evidenced by shortening of the restitution processes after caffeine contractures by adrenaline. Membrane calcium currents are increased by adrenaline as a rule; mainly at lower depolarizations (-50 to -20 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of Pb2+ ions on calcium currents and contractility in single muscle fibres of the crayfish.

Muscle fibres of crustaceans represent an useful model for studying the mode of action of substances influencing calcium channels as the membrane generates the active responses on a pure calcium principle and the excitation-contraction link is dependent on external calcium. We followed the effect of Pb2+ ions (1-300 mumol/l) on contractile responses in single muscle fibres of the crayfish evoked by massive or intracellular electrical stimulation, by potassium depolarization and caffeine application, as well as on action potential in single intact muscle fibres and on calcium currents in voltage clamp conditions (vaseline gap) in internally perfused muscle fibre segments. All types of contractile responses, single twitches, tetanus, potassium and caffeine contractures were blocked. The strontium action potential was blocked very effectively by Pb2+ ions. The total calcium currents which can be split by means of Hodgkin-Huxley equations into two components (fast and slow respectively) differing in the rate of activation and inactivation were suppressed after addition of Pb(NO3)2 (50-300 mumol/l). The effect of Pb2+ was concentration and time dependent. At lower concentrations (100 mumol/l) the blocking effect was more pronounced on the fast inactivating Ca current component. The Pb2+ ions prolonged the time constant of inactivation tau h of the slow channel, while leaving that of the fast channel intact.

Effects of ruthenium red on excitation and contraction in muscle fibres with Ca2+ electrogenesis.

The effect of ruthenium red (RR) on the electrical and contractile responses, membrane Ca currents, staining patterns of the external and internal membrane system were tested in intact and mechanically skinned muscle fibres of the crayfish Astacus fluviatilis. The following results were obtained: 1. Depression of the contractile responses following membrane depolarization (twitch, tetanus, potassium contractures). 2. Caffeine contractures were unaffected in intact (100 mumol/l - 1 mmol/l RR) and blocked in skinned fibres (30 mumol/l RR). 3. Mechanical threshold and mechanical latency were increased and/or prolonged. 4. The rate of depolarization of the action potentials (AP) was decreased and decremental spread of AP was recorded. 5. Both fast and slowly inactivating Ca ionic currents were decreased and the time constants of activation (tau(m] and inactivation (tau(h] were prolonged after RR (100 mumol/l) pretreatment. 6. The penetration of RR into the T-system was inversely related to its binding to the sarcolemma. The depression of depolarization-induced contractions was most pronounced in fibres with unstained sarcolemma and stained T-tubules. In intact fibres, neither terminal cisternae nor other elements of SR were stained. On the contrary, all internal membrane structures were stained in skinned fibres. There was a gradient of staining intensity from surface toward the interior.

[Excitation-contraction coupling in isolated muscle fibers with calcium electrogenesis preserved in a culture medium]. / Väzba excitácie a kontrakcie v izolovaných svalových vláknach s kalciovou elektrogenézou prechovávaných v kultivacnom médiu.

The aim of the present work was to check changes in functional characteristics of isolated muscle cells, operating on the calcium electrogenesis principle, while kept in culture media for several days. Skeletal muscle cells of the crayfish Astacus fluviatilis were used to study potassium/caffeine contractures and single/tetanic contractions; simultaneous electrical and mechanical responses were recorded by the microelectrode technique, and kinetics of calcium ionic currents was studied under vaseline-gap voltage clamp. In cultured fibers, active membrane responses and calcium current kinetics remained unchanged, or slightly increased, whereas contractile responses were substantially reduced. A gradual excitation-contraction decoupling was observed. The fiber maintained the ability to respond to direct activation (by caffeine) of the contractile apparatus. Subthreshold caffeine concentrations (0.2-0.5 mmol/l) and adrenaline (6.0(-6), 6.10(-5) mol/l) enhanced the inhibited (due to the culturing) single contractile responses.

Calcium channels in skeletal muscle fibres of the frog.

The vaseline gap voltage clamp technique was used to study slow and fast calcium currents in phasic and tonic muscle fibres of the frogs Rana temporaria (R.t.) and Xenopus laevis (X.l.). At physiological Ca2+ and Na+ concentrations three inward currents could be recorded in R.t. twitch muscle fibres: a fast Ca current, a slow Ca current, and a non-specific (INS) current carried by Na ions. The last current appeared as a sole component at a low threshold membrane potential (MP -55 mV) and was present until the slow Ca current appeared. In tonic R.t. muscle and twitch X. laevis fibres only the fast and the slow Ca currents could be recorded; however the inactivation of the slow Ca current was very prolonged in the presence of Na ions. A modulatory effect of Na ions on Ca2+ currents was observed. In phasic fibres the fast Ca currents inactivated either very slowly or rapidly.

Selectivity of sodium channels in denervated tonic muscle fibre membrane of the frog.

Ionic selectivity of sodium channels was examined under voltage clamp conditions in normal and denervated twitch fibres and denervated tonic fibres isolated from m. ileofibularis of the frog (R. temporaria). Membrane currents were recorded by means of the Hille-Campbell vaseline-gap voltage clamp method from muscle fibre segments exposed to a potassium-free artificial internal solution. Permeability ratio (PS/PNa) were determined from changes in the reversal potential after replacing all Na ions in the solution bathing the voltage clamped external membrane area with sodium substituting ions (S). The permeability sequence was: Na+ greater than Li+ greater than NH4+ greater than K+. No inward currents were observed for Ca2+. The permeability ratios were as follows. Denervated tonic fibres: 1:0.88:0.23:0.012; control twitch fibres: 1:0.94:0.22:0.076; denervated twitch fibres: 1:0.91:0.14:0.082. The permeability to Li+ ions deviates from independence to a greater extent in tonic than in phasic fibres. Our results are consistent with the Hille model of sodium channel selectivity, and they support the hypothesis that sodium channels formed in denervated tonic muscle fibres of the frog are of the same genetic origin as Na channels expressed under physiological conditions.

Operational amplifier with adjustable frequency response.

The authors describe an operational amplifier with an adjustable frequency response and its use in membrane physiology, using the voltage clamp and current clamp method. The amplifier eliminates feedback poles causing oscillation. It consists of a follower with a high input resistance in the form of a tube and of an actual amplifier with an adjustable frequency response allowing the abolition of clicks by one pole and of oscillation by two poles in the 500 Hz divided by infinity range. Further properties of the amplifier: a long-term voltage drift of 1 mv, a temperature voltage drift of 0.5 mv/degrees K, input resistance greater than 1 GOhm, amplification greater than 80 dB, output +/- 12 v, 25 ma, noise, measured from the width of the oscilloscope track in the presence of a ray of normal brightness, not exceeding 50 muv in the 0-250 kHz band, f1 = 1 MHz. A short report on the amplifier was published a few years ago (Gulísek and Hencek 1973).

Membrane currents in a calcium type muscle membrane under voltage clamp.

Four ionic current components were identified in the total membrane current recorded under voltage clamp conditions from the muscle membrane of the crayfish (Astacus fluviatilis). The early inward current component is dependent on the presence of Ca2+ ions, disappears in Ca2+ free solutions and is insensitive to variaton of external Na+ ions and to tetrodotoxin. The outward current consists of at least three components, an early, a late and a slow outward current. The outward currents are sensitive to TEA and their reversal potentials differ. The early potassium current may be separated in a proportion of fibres by a hump from the later potassium current. An insufficient space clamp as a cause of the hump was excluded by comparing the size of the clamped membrane area with the distribution of large membrane clefts in the fibre. The early outward current is critically dependent on the presence of Ca2+ ions and is relatively more sensitive to TEA ions and to conditioning depolarisation than the late outward current.

Calcium currents and conductances in the msucle membrane of the crayfish.

1. Membrane currents in calcium type muscle membrane of the cray-fish Astacus fluviatilis were analysed by a method in which a membrane microarea was isolated by circulating sucrose rings contacting the fibre perpendicular to the fibre surface.2. The early calcium inward currents were separated from the total membrane currents by subtraction of the early and delayed potassium currents from the total membrane current.3. The isolated calcium currents show a time course characteristic for a transient change of calcium conductance. The presence of inactivation was further checked by the time course of the tail currents at the end of voltage clamp pulses of variable duration.4. The reversal potential of the early calcium currents determined from the current-voltage relations was +85 +/- 4.2 mV. The calcium potentials were used to express the calcium currents in the form of chord conductances.5. Calcium conductances (g(Ca)) as functions of time and voltage were found to be described quantitatively on the assumption that g(Ca) is determined by two variables (m and h), according to the equation g(Ca) = m(6)hg(Ca), where g(Ca) is a constant and m and h obey first order differential equations of the Hodgkin-Huxley type.6. The activation parameters of the g(Ca) were determined by fitting the solutions of the above equations to the experimental values of the g(Ca). This method was also used to check the inactivation parameters.7. The inactivation parameters of the g(Ca) were obtained from the inactivation curves, which were determined for several membrane potentials by variation of the duration of the conditioning step.8. The average calcium conductance constants were tabulated and compared with sodium conductance constants in excitable membranes.