| Abstract: | This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25--30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic Ca2+]free resulting from Ca2+ release from the SR; (c)fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of CA2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of Ca2+]free outside the SR triggers Ca2+ release from the SR. |
