PubMed · 10736831
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D Pescovitz. 2000. Bioagent chip.. https://doi.org/10.1038/scientificamerican0300-35
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Calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER) takes place through ryanodine receptors (RyRs) and it is often revealed by an increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by caffeine. Using fura-2-loaded cells, we find such an effect in bovine adrenal chromaffin cells, but not in cerebellar granule neurones or in HEK-293 cells. In contrast, a caffeine-induced [Ca(2+)](c) increase was clearly visible with either fluo-3 or cytosolic aequorin. Simultaneous loading with fura-2 prevented the [Ca(2+)](c) increase reported by the other Ca(2+) probes. Caffeine-induced Ca(2+) release was also measured by following changes of [Ca(2+)] inside the ER ([Ca(2+)](ER)) with ER-targeted aequorin in HEK-293 cells. Fura-2 loading did not modify Ca(2+) release from the ER. Thus, fura-2, but not fluo-3, antagonises the generation of the cytosolic Ca(2+) signal induced by activation of RyRs. Cytosolic Ca(2+) buffering and/or acceleration of Ca(2+) diffusion through the cytosol may contribute to these actions. Both effects may interfere with the generation of microdomains of high [Ca(2+)](c) near the ER release channels, which are essential for the propagation of the Ca(2+) wave through the cytosol. In any case, our results caution the use of fura-2 to study CICR.
Mutants of norpA, encoding phospholipase C beta (PLC beta), and itpr, encoding inositol (1,4,5)-trisphosphate receptor (IP(3)R), both attenuate response to diuretic peptides of Drosophila melanogaster renal (Malpighian) tubules. Intact tubules from norpA mutants severely reduced diuresis stimulated by the principal cell- and stellate cell-specific neuropeptides, CAP(2b) and Drosophila leucokinin (Drosokinin), respectively, suggesting a role for PLC beta in both these cell types. Measurement of IP(3) production in wild-type tubules and in Drosokinin-receptor-transfected S2 cells stimulated with CAP(2b) and Drosokinin, respectively, confirmed that both neuropeptides elevate IP(3) levels. In itpr hypomorphs, basal IP(3) levels are lower, although CAP(2b)-stimulated IP(3) levels are not significantly reduced compared with wild type. However, CAP(2b)-stimulated fluid transport is significantly reduced in itpr alleles. Rescue of the itpr(90B.0) allele with wild-type itpr restores CAP(2b)-stimulated fluid transport levels to wild type. Drosokinin-stimulated fluid transport is also reduced in homozygous and heteroallelic itpr mutants. Measurements of cytosolic calcium levels in intact tubules of wild-type and itpr mutants using targeted expression of the calcium reporter, aequorin, show that mutations in itpr attenuated both CAP(2b)- and Drosokinin-stimulated calcium responses. The reductions in calcium signals are associated with corresponding reductions in fluid transport rates. Thus, we describe a role for norpA and itpr in renal epithelia and show that both CAP(2b) and Drosokinin are PLC beta-dependent, IP(3)-mobilising neuropeptides in Drosophila. IP(3)R contributes to the calcium signalling cascades initiated by these peptides in both principal and stellate cells.
The concentration of free calcium within the lumen of the endoplasmic reticulum ([Ca2+]L) fluctuates between 100 and 1000 microM. High [Ca2+]L provides an electro-driving force for Ca2+ release and supports high Ca2+ diffusion rate within the endoplasmic reticulum lumen. Fluctuations in [Ca2+]L also regulate numerous chaperones, responsible for postranslational protein processing. Thus, [Ca2+]L integrates various signalling events and establishes a link between fast signalling, associated with the endoplasmic reticulum Ca2+release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. This paper overviews modern approaches for the direct monitoring of [Ca2+]L which rely on three classes of low-affinity Ca2+ probes: ER-targeted aequorin, synthetic fluorescent Ca2+ dyes and GFP-based ER-targeted Ca2+ probes. These techniques, especially as applied to neurones, may substantially widen our appreciation of the endoplasmic reticulum as a universal signalling organelle.