Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca2+ concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca2+ sensitivity in endocrine cells, accurate and fast determination of Ca2+ dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a robust exocytotic activity. Slow increases in the [Ca2+] i revealed exocytotic phases with different Ca2+ sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca2+ sensitivity ‘classes’ of secretory vesicles have been observed: a highly Ca2+ -sensitive, a medium Ca2+ -sensitive and a low Ca2+ - sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca2+ sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca2+ sensor protein., J. Dolenšek, M. Skelin, M. S. Rupnik., and Obsahuje bibliografii a bibliografické odkazy
In vitro produced β-like cells can provide promising cell therapy for curing the epidemic of diabetes. In this context, we aimed to investigate the effects of different concentrations of γ-aminobutyric acid (GABA) on the differentiation of rat pancreatic ductal epithelial-like stem cells (PDESCs) into β-like cells. The PDESC line cells were cultured in the basal media (DMEM/F12 + 10% FBS + 1% penicillinstreptomycin) supplemented with 0 µM, 5 µM, 50 µM, 500 µM, and 5 mM of GABA for 28 days to induce their differentiation. The differentiated cells were detected by cell morphology, dithizone (DTZ) staining, immunofluorescence staining, real-time polymerase chain reaction (qPCR), and glucose-stimulated insulin secretion (GSIS) assay to validate their identity. At the end of 28 days, compared with the control group, enrichment of induced cells was high among the 5 μM, 50 μM, 500 μM, and 5 mM GABA induction groups. The formation of islet-like cell clusters (ICCs) began at 14 days, and the cell clusters showed a growth trend with the culture time. The induced ICCs were positive for DTZ staining, while the control group showed negative results for DTZ staining and the differentiated cells were also positive for β-cell-specific markers (Ins1 and Pdx1). GSIS assay of 50 μM induction group cells at 28 days showed significantly higher levels of C-peptide and insulin secretion than the control, 5 μM, 500 μM, and 5 mM GABA-treated groups (P < 0.01). At the same time, the 50 μM induction group cells also showed significantly higher levels of Ins1, Pdx1 and Nkx6.1 mRNA as compared to the 5 μM, 500 μM and 5 mM GABA groups (P < 0.01). Thus, the addition of GABA to the basal medium effectively induced differentiation of adult rat PDESCs into insulin-secreting β-like cells, and 50 μM was the most effective concentration for the induction.