AM-LOADING OF FLUORESCENT CA2-MUSCLE( INDICATORS INTO INTACT SINGLE FIBERS OF FROG)

The AM loading of a number of different fluorescent Ca2+ indicators wa s compared in intact single fibers of frog muscle. Among the 13 indica tors studied, loading rates (the average increase in the fiber concent ration of indicator per first 60 min of loading) varied similar to 100 -fold, from similar to 3 mu M/h to >300 mu M/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compo unds, with the rate increasing steeply as molecular weight decreased b elow similar to 850. Properties of Delta F/F (the Ca2+-related fluores cence signal observed with fiber stimulation) were also measured in AM -loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of Delta F/F was very similar with AM-loading and microinjection; however, the amp litude of Delta F/F was usually smaller with AM-loading, There was a s trong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of Delta F/F in AM-load ed versus microinjected fibers). For indicators with small loading rat es (<10 mu M/h, N = 5), f values were generally small (less than or eq ual to 0.4, N = 4); whereas with large loading rates (>100 mu M/h, N = 4), f values were large (less than or equal to 0.8, N = 4). This sugg ests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely d e-esterified or, if completely de-esterified, not located in the myopl asmic compartment). If the loaded concentration is small, the nonspeci fic indicator will present a significant source of error in the estima tion of [Ca2+](i).