Fluorescence intensity changes associated with contractile activation in frog muscle stained with Nile Blue A

1. Extrinsic fluorescence intensity changes were studied in frog semitendinosus muscles stained with Nile Blue A in response to electrical stimulation. Muscles were stretched and put into hypertonic solutions to prevent movement. The muscles were illuminated at 90 degrees to their long axis with a narrow beam of light at a central wave-length of 6250 . Fluorescence emission was measured at 90 degrees to the exciting light using a filter which absorbed light of wave-lengths shorter than 6400 . 2. In response to a single stimulus the fluorescence intensity increases briefly. The fluorescence response is propagated at a constant velocity of about 1.5 m/sec. The average ratio of the maximum fluorescence intensity change to the resting fluorescence is 4.5 times 10-3 for supramaximal shocks. The fluorescence intensity change starts early in the falling phase of the action potential. 3. The fluorescence intensity change increases when nitrate replaces chloride and decreases when D2O replaces H2O. The rates of rise and fall of the fluorescence change was unaffected by nitrate replacement of chloride but are slowed where D2O replaces H2O. The rates of rise and fall of the fluorescence change increase with increasing temperature for all solutions used. The peak fluorescence intensity change, however, goes through a maximum at about 17 degrees C for aqueous chloride and nitrate solutions in the range of 10-25 degrees C. With D2O solutions, the peak fluorescence intensity increases monotonically in this range of temperatures. 4. The fluorescence intensity change in response to trains of action potentials are not additive. 5. Depolarization of muscles treated with tetrodotoxin using triangular-shaped fluid electrodes produces an increase in fluorescence at about the same threshold values required to elicit tension in preparations that are not fully stretched. The fluorescence intensity change precedes in time tension development. Near threshold depolarizations, the delay in onset of the fluorescence response can be 80 msec or longer. Byond threshold, delays become shorter and peak responses larger. During maintained depolarization, after the peak response, fluorescence declines to a plateau value. 6. The results suggest that the fluorescence intensity changes are associated with excitation-contraction coupling, possibly with changes in the transmembrane potential of the sarcoplasmic reticulum.