MECHANISMS OF INTRACELLULAR CALCIUM-RELEASE DURING HORMONE AND NEUROTRANSMITTER ACTION INVESTIGATED WITH FLASH-PHOTOLYSIS

To understand the complex time course of cytosolic Ca2+ signalling evo ked by hormones and neurotransmitters, it is necessary to know the kin etics of steps in the second-messenger cascade, particularly cooperati ve and inhibitory interactions between components that might give rise to periodic fluctuations. In the case of inositol trisphosphate (InsP 3)-evoked Ca2+ release, fast perfusion studies with subcellular fracti ons or permeabilised cells can be made if sufficient homogeneous tissu e is available. Single-cell studies can be made by combining whole-cel l patch-clamp techniques and microspectrofluorimetry with flash photol ytic release of InSP3 to give quantitative, time-resolved data of Ca2 release from stores. A technical description is given here of flash p hotolysis of caged InSP3, and the results of fast perfusion and flash photolytic experiments are reviewed. Studies of kinetics of Ca2+ relea se-have shown that the InSP3 receptor/channel is regulated first by po sitive and then by negative feedback by free cytosolic Ca2+ concentrat ion, producing a pulse of Ca2+ release having properties that may be i mportant in the spatial propagation of Ca2+ signals within and between cells. The properties of InSP3-evoked Ca2+ release in single cells di ffer between peripheral tissues, such as the liver, and Purkinje neuro nes of the cerebellum. Purkinje neurones need 20-50 times higher InSP3 concentrations and release Ca2+ to change the free cytosolic concentr ation 30 times faster and to higher peak concentrations than in liver. The InSP3 receptors in the two cell types appear to differ in apparen t affinity, and the greater Ca2+ efflux from stores in Purkinje cells is probably due to a high receptor density.