PHOTOLYSIS OF CAGED COMPOUNDS CHARACTERIZED BY RATIOMETRIC CONFOCAL MICROSCOPY - A NEW APPROACH TO HOMOGENEOUSLY CONTROL AND MEASURE THE CALCIUM-CONCENTRATION IN CARDIAC MYOCYTES

Here we describe the subcellularly uniform control of the intracellula r Ca2+ concentration ([Ca2+](i)) by flash photolysis of caged Ca2+ or a caged Ca2+ buffer. A mixture of the two Ca2+ indicators Fluo-3 and F ura-red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells wit h the indicator mixture together with either DM-nitrophen or diazo-2 a nd to measure changes in the membrane current. An in vivo calibration was performed to convert the Fluo-3/Fura-red fluorescence ratios to [C a2+] values. The resulting calibration curve suggested an apparent K-D of 1.6 mu M, R(max) of 2.15, R(min) of 0.08 and a Hill-coefficient of 0.75 for the indicator mixture. Controlled rupture of the cell membra ne revealed a large fraction of immobile intracellular Fura-red fluore scence that may account for the reduced in vivo R(max) value when comp ared to the in vitro value of 3.1. In cardiac myocytes, flash photolyt ic release of Ca2+ from DM-nitrophen generated inwardly directed Na+/C a2+ exchange currents and Ca2+ signals that were graded with the disch arged flash-energy. Rapid line-scans revealed subcellularly homogeneou s [Ca2+] jumps regardless of the discharged flash energy. Ca2+ signals evoked by L-type Ca2+ currents (I-Ca) could be terminated rapidly in a spatially homogeneous manner by UV flash photolysis of diazo-2. No s ide-effects of the photolytic products of DM-nitrophen or diazo-2 with the mixture of Fluo-3/Fura-red were detectable in our experiments. Th e combination of UV flash photolysis and laser scanning confocal micro scopy enabled us to control [Ca2+], homogeneously on the subcellular l evel. This approach may improve our understanding of the subcellular p roperties of cardiac Ca2+ signalling. The technique can also be applie d in other cell types and with other signalling systems for which cage d compounds are available.