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.

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 mu-mol/l - 1 mmol/l RR) and blocked in skinned fibres (30 mu-mol/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 mu-mol/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.

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.

The excitation-contraction (E-C) coupling in crayfish muscle fibres can be dissociated at low temperatures (5CC—3°C) if Sr ions are substituted for Ca ions. The decoupling becomes irreversible if the fibre was exposed to low temperatures and strontium salines for more than 45 min. The dissociation depends on the Ca2+/Sr2+ ratio in the extracellular medium. The potentiating effect of Ca2+ on E-C coupling dominates over the inhibitory action of Sr 2+ at Ca/Sr around 0.3. The dissociation of the strontium action potential from the contraction is also weakened by increasing the intracellular Ca2+ concentration, (Ca) with subthreshold con­ centrations of caffeine (0.2 mmol/1). Procaine action potentials initiated by low concentrations of procaine (2 mmol/1) in the presence of Ca 2+ are not dissociated. The dissociation appearing at higher procaine concentrations may be explained by the action of procaine on the sarcoplasmic reticulum and may be abolished by subthreshold caffeine doses, i.e. by increasing (Ca)*. Action potentials induced by the blockade of K channels with TEA+ (tetraethylammonium) in Ca 2+ solutions cannot be dissociated from contraction by cooling. The results show that in this kind of dissociation a decisive role is played by strontium ions which enter the cell through the calcium channel. Sr2+ are supposed to be less effective in activating contraction as compared with Ca2+; this difference becomes accentuated at low temperatures.