ABSTRACT Insulin signaling includes generation of low levels of H 2 O 2 ; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H 2 O 2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H 2 O 2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N -acetyl L-cysteine and Trolox, the p47 phox –NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47 phox knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H 2 O 2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca 2+ release by promoting RyR1 S -glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP 3 )-activated Ca 2+ channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca 2+ transfer to the mitochondria. An inhibitor of IP 3 receptors, Xestospongin B, reduced both insulin-dependent IP 3 production and GLUT4myc translocation. We propose that, in addition to the canonical α,β phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca 2+ release and IP 3 -receptor-mediated mitochondrial Ca 2+ uptake, and that these signals jointly stimulate glucose uptake.

ABSTRACT The GTPase Ras is a molecular switch engaged downstream of G-protein-coupled receptors and receptor tyrosine kinases that controls multiple cell-fate-determining signalling pathways. Ras signalling is frequently deregulated in cancer, underlying associated changes in cell phenotype. Although Ca 2+ signalling pathways control some overlapping functions with Ras, and altered Ca 2+ signalling pathways are emerging as important players in oncogenic transformation, how Ca 2+ signalling is remodelled during transformation and whether it has a causal role remains unclear. We have investigated Ca 2+ signalling in two human colorectal cancer cell lines and their isogenic derivatives in which the allele encoding oncogenic K-Ras (G13D) was deleted by homologous recombination. We show that agonist-induced Ca 2+ release from the endoplasmic reticulum (ER) intracellular Ca 2+ stores is enhanced by loss of K-Ras G13D through an increase in the Ca 2+ content of the ER store and a modification of the abundance of inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP3R) subtypes. Consistently, uptake of Ca 2+ into mitochondria and sensitivity to apoptosis was enhanced as a result of K-Ras G13D loss. These results suggest that suppression of Ca 2+ signalling is a common response to naturally occurring levels of K-Ras G13D , and that this contributes to a survival advantage during oncogenic transformation.

In the ciliate Paramecium , a variety of well characterized processes are regulated by Ca 2+ , e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca 2+ signaling, although the molecular identity of the channels responsible for the Ca 2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5) P 3 ) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5) P 3 -binding domain binds [ 3 H]Ins(1,4,5) P 3 , whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5) P 3 -receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca 2+ ( Paramecium cells rapidly adjust their Ca 2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca 2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5) P 3 . Considering the ongoing expulsion of substantial amounts of Ca 2+ by the osmoregulatory system, we propose here that Ins(1,4,5) P 3 receptors serve a new function, i.e. a latent, graded reflux of Ca 2+ to fine-tune [Ca 2+ ] homeostasis.

Although ventricular cardiomyocytes express inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3 ] receptors, it is unclear how these Ca 2+ channels contribute to the effects of Gq-coupled agonists. Endothelin-1 augmented the amplitude of pacing-evoked Ca 2+ signals (positive inotropy), and caused an increasing frequency of spontaneous diastolic Ca 2+ -release transients. Both effects of endothelin-1 were blocked by an antagonist of phospholipase C, suggesting that Ins(1,4,5) P 3 and/or diacylglycerol production was necessary. The endothelin-1-mediated spontaneous Ca 2+ transients were abolished by application of 2-aminoethoxydiphenyl borate (2-APB), an antagonist of Ins(1,4,5) P 3 receptors. Incubation of electrically-paced ventricular myocytes with a membrane-permeant Ins(1,4,5) P 3 ester provoked the occurrence of spontaneous diastolic Ca 2+ transients with the same characteristics and sensitivity to 2-APB as the events stimulated by endothelin-1. In addition to evoking spontaneous Ca 2+ transients, stimulation of ventricular myocytes with the Ins(1,4,5) P 3 ester caused a positive inotropic effect. The effects of endothelin-1 were compared with two other stimuli, isoproterenol and digoxin, which are known to induce inotropy and spontaneous Ca 2+ transients by overloading intracellular Ca 2+ stores. The events evoked by isoproterenol and digoxin were dissimilar from those triggered by endothelin-1 in several ways. We propose that Ins(1,4,5) P 3 receptors support the development of both inotropy and spontaneous pro-arrhythmic Ca 2+ signals in ventricular myocytes stimulated with a Gq-coupled agonist.

Elementary Ca 2+ signals, such as ‘Ca 2+ puffs’, which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca 2+ signalling. We characterized Ca 2+ puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca 2+ puffs in these cell types suggests that they are a generic elementary Ca 2+ signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca 2+ signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca 2+ puffs. The amplitude and duration of Ca 2+ puffs were substantially reduced. In addition, the number of Ca 2+ puff sites active during the onset of a Ca 2+ wave declined. The consequence of the changes in Ca 2+ puff properties was that cells displayed a lower propensity to trigger regenerative Ca 2+ waves. Therefore, Ca 2+ puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.