Abstract
A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation.
Highlights
Many extracellular stimuli trigger increases in the cytosolic concentration of Ca2+ ([Ca2+]) that control a wide range of physiological processes, including fertilization, proliferation, development, learning and memory, contraction, and secretion [1,2]
These cellspecific Ca2+ patterns were reproducible upon repetitive histamine additions (Fig. 1B), as observed in different cell types [5,13,14,15], suggesting that individual HeLa cells have characteristic Ca2+ dynamics
The IP3 dynamics were reproducible (Fig. 1B). These findings indicate that the IP3 dynamics are characteristic of individual cells, and that the IP3 dynamics may be involved in the determination of cell-specific Ca2+ responses. [Ca2+] and [IP3] Dynamics Observed in Cells Stimulated with Different Concentrations of Histamine
Summary
Many extracellular stimuli trigger increases in the cytosolic concentration of Ca2+ ([Ca2+]) that control a wide range of physiological processes, including fertilization, proliferation, development, learning and memory, contraction, and secretion [1,2]. The versatility of Ca2+ signals is considered to be dependent on the spatiotemporal patterns of intracellular Ca2+ signals [8]. Extracellular stimuli, such as growth factors, hormones, and neurotransmitters, activate an intracellular effector molecule, phosphoinositide-specific PLC, that catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5trisphosphate (IP3) and diacylglycerol [9]. IP3 acts as a second messenger in many cell types, where its primary effect is to trigger the release of Ca2+ ions from intracellular stores and generate cytosolic Ca2+ signals. The forms of the Ca2+ signals vary from cell to cell, even in clonal populations of genetically identical cells, while individual cells show characteristic and reproducible Ca2+
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