Mechanisms of intracellular calcium release during hormone and neurotransmitter action investigated with flash photolysis

To understand the complex time course of cytosolic Ca2+ signalling evoked by hormones and neurotransmitters, it is necessary to know the kinetics of steps in the second-messenger cascade, particularly cooperative and inhibitory interactions between components that might give rise to periodic fluctuations. In the case of inositol trisphosphate (InsP3)-evoked Ca2+ release, fast perfusion studies with subcellular fractions or permeabilised cells can be made if sufficient homogeneous tissue is available. Single-cell studies can be made by combining whole-cell patch-clamp techniques and microspectrofluorimetry with flash photolytic release of InsP3 to give quantitative, time-resolved data of Ca2+ release from stores. A technical description is given here of flash photolysis of caged InsP3, and the results of fast perfusion and flash photolytic experiments are reviewed. Studies of kinetics of Ca2+ release have shown that the InsP3 receptor/channel is regulated first by positive and then by negative feedback by free cytosolic Ca2+ concentration, producing a pulse of Ca2+ release having properties that may be important in the spatial propagation of Ca2+ signals within and between cells. The properties of InsP3-evoked Ca2+ release in single cells differ between peripheral tissues, such as the liver, and Purkinje neurones of the cerebellum. Purkinje neurones need 20-50 times higher InsP3 concentrations and release Ca2+ to change the free cytosolic concentration 30 times faster and to higher peak concentrations than in liver. The InsP3 receptors in the two cell types appear to differ in apparent affinity, and the greater Ca2+ efflux from stores in Purkinje cells is probably due to a high receptor density.