PHOTOCHEMICAL REGULATION OF CALCIUM IN CELL PHYSIOLOGY

$273,302R01FY2000GMNIH

Mcp Hahnemann University, Philadelphia PA

Investigators

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Abstract

DESCRIPTION: (Applicant's Abstract) Changes in intracellular Ca2+ concentration control a myriad of physiological processes including muscle contraction, secretion, mitosis, channel gating, chemotaxis and stomatal pore closure. During the past 15 years two techniques have revolutionised the study of cell physiology: measurement of Ca2+ concentrations using ratiometric, Ca2+-specific fluorescent indicators such as fura-2; and the rapid increase in concentration of cellular substrates such as ATP, cGMP, etc. from a biologically inert or "caged" form by flash-photolysis techniques. The objective of this proposal is use rapid photochemical control of divalent cation levels to characterise the mechanism and regulation of cellular physiological processes. Ca2+ -specific photolabile chelators will be developed and used to manipulate intracellular Ca2+ concentrations independently of other effectors, such as Mg2+, ATP, GTP, etc. so that the regulatory roles that these species have on Ca2+-dependent cell physiology can be defined. Additionally, a new, ultra-fast caged IP3 will be synthesised. This key second messenger is implicated in the control of Ca2+ concentration in many cell types. The basis of this proposal is a new Ca2+-specific photolabile chelator called DMNPE-4, which has been recently synthesized. The new probes will be used to study the kinetics and regulation of secretory events in neuroendocrine cells; of contraction in cardiac muscle and the kinetics of Ca2+ release in the cerrebellum because Ca2+ is the key intracellular second messenger in these systems. Many of these processes are disturbed in pathological states. Before an adequate description of these disease states can be given, a more complete understanding of non-disease states should be accomplished. The proposed studies will contribute to a greater understanding of the normal functioning of these processes.

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