(full paper is archived in the Miller Library)
Title: The dynamics of Ca2+ release at IP3 receptors studied with flash photolysis
Student Author(s): Kim, Michael S.
Faculty Advisor(s): Thompson, Stuart
Location: Final Papers Biology 175H
Date: June 1997
Abstract: Stimulation of N1E-115 mouse neuroblastoma cells with the neurotransmitter bradykinin (Bk) leads to inositol 1,4,5-trisphosphate (IP3) production and calcium release from IP3-regulated Ca2+ pools located in the endoplasmic reticulum. This involves a specific G-protein second messenger cascade. The release of intracellular Ca2+ is preceded by a latent period of up to 30 seconds. This study investigated the contribution of IP3 diffusion and the dynamics of IP3 receptor activation to the latency between surface receptor binding and Ca2+ release. Intracellular injection of caged IP3 plus the fluorescent calcium indicator fluo-3 was used investigate the basis of the observed latency. Following injection, cells were exposed to a brief (170ms) flash of light (230 joules, 360nm wavelength) to photolyse the caged compound. Fluorescence video imaging was used to follow the change in intracellular [Ca2+]. The study showed that [Ca2+]i begins to rise immediately (within 200ms) following uncaging of IP3 by flash photolysis, and reaches maximum levels within 1.5 seconds. The result indicates that neither IP3 diffusion to its receptors nor delay between IP3R activation and Ca2+ release contribute significantly to overall latency. Analysis of Ca2+ wave kinetics shows that in contrast to cells stimulated with neurotransmitter, injected cells do not exhibit propagation of Ca2+ waves following photolysis of caged IP3. This observation suggests that the IP3 receptor is distributed throughout the cytoplasm of the soma and major neurites. Ca2+ waves apparently travel through this IP3 receptor rich medium. The constant rate in which Ca2+ waves travel shows that propagation is an active process rather than a diffusional process. The mechanism of propagation could involve either IP3 production or Ca2+-induced Ca2+ release at the wave front.