Simple freezing apparatus for resolving rapid metabolic events associated with smooth muscle activation

A method is described for freezing thin strips of smooth muscle by replacing physiological saline in the muscle chamber with cold organic solvent in <100 ms. Calculations suggest that, with a perfectly stirred boundary at the tissue surface, freezing could occur within approximately 15 ms at the center of a 200-microm-thick piece of tissue by use of acetone coolant at -78.5 degrees C and in approximately half the time with either isopentane at its freezing point (-160 degrees C) or aluminum chilled with liquid nitrogen. Myosin light chain phosphorylation in muscles frozen with cold acetone began to rise approximately 200 ms earlier than force and increased at a much more rapid rate. The difference in onsets of the two processes reflects the delay in arresting phosphorylation plus two lags associated with force generation, attachment of phosphorylated bridges followed by force generating movements of the attached bridges. The much more rapid rise of phosphorylation, once it began, suggests that most of this delay is due to physiological lags and not to slow arrest of metabolism.