1) Spontaneous muscle contractions taking place under ion-free condition, typically observable in the muscles soaked first in Ca free Ringer and then in sucrose solution, were investigated by assuming that occurrence of the twitching (ST) might be due to a transition of the sarcoplasmic property from linear response type to non-linear one 'in accordance with the change of concentrations of internal free Ca++ as well as sarcoplasmic Ca++. The experimental results obtained were enough to satisfy this assumption. 2) The conditions to produce the spontaneous twitching (ST) were examined when the medium of the muscle was changed from each of various conditioning solutions to isotonic pure sucrose solution. The twitching was discussed with reference to ionic composition of the presoaking solutions. In the muscles showing this peculiar type of contraction (ST), 45Ca efflux or monovalent cation content was determined. 3) Na+ ions more than 37 mM and Ca++ ions less than 1 mM were required as the chemical composition of test solutions using prior to sucrose solution, in order to produce ST. External K+ concentration or membrane polarization was not directly connected to ST which was a particular form of contraction which happened not via usual excitation-contraction coupling process. Among divalent cations used instead of Ca++ ions, Ni++ could effectively prevent ST occurrence after Ca free Ringer, Mg++ and Sr++ were not so effective in preventing the ST, and Ba++, in spite of a divalent cation, showed clearly different effect. 4) Release of organelle-bound calcium by fluoride in the presoaking solution was favorable for ST occurrence. Internal labilization of Ca++, probably from the sarcoplasmic reticulum, was closely related to ST. 5) 45Ca ion release from the muscle was continuously increased in isotonic sucrose solution, but the behaviors of 45Ca ion kinetics across the membrane were without any regards to ST. From these, ST occurrence was independent of membrane phenomena. 6) Presoaking with the solution containing ATP stimulated ST occurrence probably through its chelating activity. Membrane Ca++ portion, removable by ATP, was connected to readily releaseable Ca++ portion within the cell. Considering from the results after K free Ringer, it was reasonable to suppose functional Ca++ compartments. 7) Transient increase in 45Ca efflux by ouabain, and continuous increase in it by iodoacetate or by thyroxine were supposed to connect simultaneously with labilization of stored calcium participating in ST. As far as a specific contraction of ST is concerned, conditions to permit ionization of internal calcium should be considered basing upon the functional Ca++ channel from membrane to cell inside. 8) In general, previous association of Ca++ to the membrane resulted afterward in no ST while dissociation of membrane Ca++, when not transient but continuous in duration, caused ST probably by mobilizing stored calcium. 9) ST was explained by oscillation concept. The oscillation expressing the sarcoplasmic activity to control internal Ca++ was spontaneous repetition of an ability to release Ca++ and to take up it. The repetition was in more detail occurred by transition of the interrelationship between sarcoplasmic effect on ionized Ca++ and sarcoplasmic calcium content from linear response type to non-linear one. The most probable determinative factor for this transition was an amount of constitutional calcium of the sarcoplasmic reticulum.
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