PubMed · 13622756
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T A HARPER. 1959-01-24. A rapid micro-quantitative method for estimating serum calcium.. https://doi.org/10.1038/183232a0
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Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo.
We examined the effects of 4-chloro-m-cresol (4-CmC, a potent and specific activator of ryanodine receptors) on Ca(2+)-release/influx and respiratory burst in freshly isolated human PMN as well as HL60 cells. 4-CmC induces Ca(2+) store-depletion in a dose-dependent manner at concentrations between 400muM and 3mM, however no dose-dependent effect on Ca(2+)-influx was found. 4-CmC depleted Ca(2+) stores that were shared with the GPC agonists such as fMLP and PAF, and therefore 4-CmC presumably depletes Ca(2+) from ER. Since the authentic ligand for RyR is cyclic ADP-ribose (cADPR), we assessed the functional relevance of RyR in PMN by studying the presence and function of membrane-bound ADP-ribosyl cyclase (CD38) in PMN. First, expression of CD38 was confirmed by RT-PCR using cDNA from HL60 cells. Second, PMN from trauma patients showed significantly enhanced CD38 expression than those from healthy volunteers. In addition, although no chemotaxis effect was detected by 4-CmC, it stimulated respiratory burst in PMN in a dose-dependent manner. Our findings suggest that RyRs exist in human PMN and that RyR pathway may play an active role in inflammatory PMN calcium signaling. 8-Br-cADPR and cyclic 3-deaza-ADP did not have inhibitory effects either on 4-CmC-induced Ca(2+) store-depletion or on respiratory burst, on the other hand, PLC inhibitor, U73122, completely attenuated both 4-CmC-induced Ca(2+) store-depletion and respiratory burst. Although it has been used as a specific activator of RyR, 4-CmC has non-specific effects which cause Ca(2+) store-depletion and respiratory burst at least in human PMN.
PMR1, the Ca2+/Mn2+ ATPase of the secretory pathway in Saccharomyces cerevisiae was the first member of the secretory pathway Ca2+ ATPases (SPCA) to be characterized. In the past few years, pmr1Delta yeast have received more attention due to the recognition that the human homologue of this protein, hSPCA1 is defective in chronic benign pemphigus or Hailey-Hailey disease (HHD). Recent publications have described pmr1Delta S. cerevisiae as a useful model organism for studying the molecular pathology of HHD. Some observations indicated that the high Ca2+ sensitive phenotype of PMR1 defective yeast strains may be the most relevant in this respect. Here we show that the total cellular calcium response of a pmr1Delta S. cerevisiae upon extracellular Ca2+ challenge is decreased compared to the wild type strain similarly as observed in keratinocytes. Additionally, the novel magnesium sensitivity of PMR1 defective yeast is revealed, which appears to be a result of competition for uptake between Ca2+ and Mg2+ at the plasma membrane level. Our findings indicate that extracellular Ca2+ and Mg2+ competitively influence the intracellular Ca2+ homeostasis of S. cerevisiae. These observations may further our understanding of HHD.