Abstract
Oscillations in intracellular Ca2+ concentration ([Ca2+]i) are thought to play an important role in phosphoinositide-linked Ca2+ signaling events. We demonstrate corresponding inositol 1,4, 5-trisphosphate (IP3)-dependent oscillations of Ca2+ concentration within the lumen of the IP3-sensitive stores ([Ca2+]L) of saponin-permeabilized HSY cells by monitoring [Ca2+]L with the fluorescent Ca2+ indicator Mag-fura-2. The associated openings and closings of the IP3-sensitive Ca2+ release channel were detected via quenching of Mag-fura-2 fluorescence due to the entry of Mn2+, a Ca2+ surrogate, into the stores. Evidence for complimentary Ca2+ oscillations at the external surface of the stores was provided by the membrane-bound Ca2+ probe Calcium Green C18. The permeabilization of saponin-treated HSY cells to macromolecules was confirmed by demonstrating that permeabilized cells readily took up and lost (t1/2 approximately 46 s) a fluorescently tagged 70-kDa dextran. Our results impose a number of constraints on possible mechanisms for [Ca2+]i oscillations. In addition, they support recent proposals that [Ca2+]i oscillations arise directly from the (biphasic) effects of Ca2+ itself on IP3-sensitive Ca2+ channel activity.
Highlights
Oscillations in intracellular Ca2؉ concentration ([Ca2؉]i) are thought to play an important role in phosphoinositide-linked Ca2؉ signaling events
Stimulation of receptors linked to phosphoinositide signaling pathways results in oscillations of intracellular calcium concentration ([Ca2ϩ]i) due to the periodic release and reuptake of Ca2ϩ by intracellular stores [1,2,3,4]
We have previously shown [15] that the luminal calcium concentration ([Ca2ϩ]L) within the IP3-sensitive intracellular Ca2ϩ stores of saponin-permeabilized HSY cells can be monitored with the fluorescent Ca2ϩ indicator Mag-fura-2
Summary
Oscillations in intracellular Ca2؉ concentration ([Ca2؉]i) are thought to play an important role in phosphoinositide-linked Ca2؉ signaling events. We demonstrate corresponding inositol 1,4,5-trisphosphate (IP3)dependent oscillations of Ca2؉ concentration within the lumen of the IP3-sensitive stores ([Ca2؉]L) of saponinpermeabilized HSY cells by monitoring [Ca2؉]L with the fluorescent Ca2؉ indicator Mag-fura-2.
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