Abstract
The activation of intracellular calcium release and calcium entry across the plasmalemma in response to intracellular application of inositol 2,4,5-trisphosphate and adenophostin A, two metabolically stable agonists for inositol 1,4,5-trisphosphate receptors, was investigated using Xenopus laevis oocytes and confocal imaging. Intracellular injection of inositol 2,4,5-trisphosphate induced a rapidly spreading calcium signal associated with regenerative calcium waves; the calcium signal filled the peripheral regions of the cell in 1-5 min. Injection of high concentrations of adenophostin A (250 nM) similarly induced rapidly spreading calcium signals. Injection of low concentrations of adenophostin A resulted in calcium signals that spread slowly (>1 h). With extremely low concentrations of adenophostin A (approximately 10 pM), stable regions of Ca2+ release were observed that did not expand to peripheral regions. When the adenophostin A-induced calcium signal was restricted to central regions, compartmentalized calcium oscillations were sometimes observed. Restoration of extracellular calcium caused a rise in cytoplasmic calcium restricted to the region of adenophostin A-induced calcium mobilization. The limited diffusion of adenophostin A provides an opportunity to examine calcium signaling processes under spatially restricted conditions and provides insights into mechanisms of intracellular calcium oscillations and capacitative calcium entry.
Highlights
A metabolite of Penicillium brevicompactum, adenophostin A, has been isolated and demonstrated to be an agonist for the (1,4,5)IP3 receptor and to have a potency ϳ100fold greater than that of (1,4,5)IP3 (7)
Much is known about the role of (1,4,5)IP3 in mobilizing intracellular Ca2ϩ, and a working model for the Ca2ϩ entry process is described by the capacitative model (5, 6) according to which the depletion of intracellular (1,4,5)IP3-sensitive Ca2ϩ stores signals the activation of plasma membrane calcium channels
We examined the activation of Ca2ϩ entry in oocytes injected with low concentrations of adenophostin A
Summary
A metabolite of Penicillium brevicompactum, adenophostin A, has been isolated and demonstrated to be an agonist for the (1,4,5)IP3 receptor and to have a potency ϳ100fold greater than that of (1,4,5)IP3 (7). Our results confirm that diffusion of adenophostin A throughout the oocytes is slower than that of IP3 and indicate that low concentrations of adenophostin A cause a confined mobilization of intracellular Ca2ϩ that is capable of supporting spatially restricted Ca2ϩ oscillations and spatially restricted Ca2ϩ entry. This unique action of adenophostin A provides new insights into the role of IP3 receptor binding in [Ca2ϩ]i oscillations and into the spatial relationships between intracellular Ca2ϩ release and activation of capacitative calcium entry. Much is known about the role of (1,4,5)IP3 in mobilizing intracellular Ca2ϩ, and a working model for the Ca2ϩ entry process is described by the capacitative model (5, 6) according to which the depletion of intracellular (1,4,5)IP3-sensitive Ca2ϩ stores signals the activation of plasma membrane calcium channels
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