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. 1999 Aug 1;341(Pt 3):813–820.

Enhanced purinoceptor-mediated Ca2+ signalling in L-fibroblasts overexpressing type 1 inositol 1,4,5-trisphosphate receptors.

R J Davis 1, J Challiss 1, S R Nahorski 1
PMCID: PMC1220422  PMID: 10417348

Abstract

Mouse L-fibroblast cells stably transfected with either type 1 Ins(1, 4,5)P(3) receptor (InsP(3)R) cDNA (L15) or the vector control (Lvec) have been used to investigate the functional consequences of increased InsP(3)R density on receptor-mediated Ca(2+) signalling. L15 cells express approx. 8-fold higher levels of the type 1 InsP(3)R compared with Lvec cells, which endogenously express essentially only the type 1 InsP(3)R protein. Stimulation of Lvec and L15 cells with UTP or ATP increased cytosolic Ca(2+) concentration to a greater extent in L15 cells at all agonist concentrations. UTP and ATP were equipotent, suggestive of the presence of endogenous cell-surface metabotropic P2Y(2)-purinoceptors. In both cell clones the purinoceptors were coupled via pertussis-toxin-insensitive G-protein(s) to phospholipase C activation, resulting in similar concentration-dependent accumulations of InsP(3). Single-cell microfluorimetry revealed that overexpression of InsP(3)Rs reduced the threshold for purinoceptor-mediated Ca(2+) signalling. L-fibroblasts also exhibited temporally complex sinusoidal cytosolic Ca(2+) oscillations in response to submaximal agonist concentrations, with significant increases in oscillatory frequencies exhibited by cells overexpressing InsP(3)Rs. Sustainable oscillatory responses were dependent on Ca(2+) entry and, at higher agonist concentrations, cytosolic Ca(2+) oscillations were superseded by biphasic peak-and-plateau Ca(2+) responses. Overexpression of InsP(3)Rs in L15 cells resulted in a 4-fold reduction in the threshold for this change in the temporal pattern of Ca(2+) mobilization. These data provide the first direct evidence demonstrating that altering the expression of the type 1 InsP(3)R significantly affects receptor-mediated InsP(3)-induced Ca(2+) mobilization.

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Selected References

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