Pitfalls in β-cell ion imaging with fluorescent indicators and their use for real-time detection of somatostatin secretion

Abstract: Fluorescent ion indicators have become indispensable tools in cell physiology. Dye indicators can easily be loaded into most types of cells, while genetically encoded indicators are advantageous in allowing specific cellular or subcellular targeting. Comparing responses of dye and protein-based indicators may provide useful insights into indicator properties and cellular processes. Here, a few genetically encoded Ca2+ indicators and fluorescent Ca2+ dyes were compared in insulin-secreting β-cells. Recordings of depolarization-triggered changes of the cytoplasmic Ca2+ concentration ([Ca2+]i) beneath the plasma membrane with total internal reflection fluorescence microscopy demonstrated distinct [Ca2+]i spikes with protein-based indicators, while the dyes mainly reported stable [Ca2+]i elevations. The spikes reflected Ca2+ release from the endoplasmic reticulum, triggered by autocrine purinergic receptor activation from exocytotic release of ATP. The indicator-dependent differences were unrelated to Ca2+ binding affinity and buffering and probably reflected slower Ca2+ dissociation kinetics of the protein indicators. In glucose-stimulated mouse islets, the dye Fura-2 reported the characteristic [Ca2+]i response with an initial lowing followed by rapid increase, which was abolished by hyperpolarization with the K+-channel opener diazoxide. The simultaneously present genetically encoded indicator R-GECO1 failed to detect the lowering and reported a spurious [Ca2+]i elevation also in the presence of diazoxide, responses that could be ascribed to pH sensitivity of the indicator. Recordings with fluorescent H+ indicators demonstrated that glucose increases cytoplasmic pH in β-cells. Elevations of [Ca2+]i counteracted the alkalinization and [Ca2+]i oscillations in glucose-stimulated islets were associated with anti-phasic oscillations of [Ca2+]i and pH. A [Ca2+]i imaging-based reporter cell assay for real-time detection of the islet hormone somatostatin was generated by transfecting HeLa cells with somatostatin receptor 2, the G-protein Gα15 and R-GECO1. The reporter cells detected somatostatin secretion from islets imaged in the same view-field as dose-dependent [Ca2+]i elevations. Mouse and human islets released somatostatin in response to high K+, glucose, and the hormones GLP-1 and ghrelin. In glucose-stimulated mouse islets, bursts of somatostatin release were synchronized with islet [Ca2+]i oscillations. Analyses of islets from human donors indicated that type 2 diabetes is associated with hypersecretion of somatostatin. In conclusion, this thesis highlights potential pitfalls with fluorescent ion indicators in β-cell signalling studies and provides new insights into β-cell regulation of [Ca2+]i and pH. Moreover, it introduces an assay for real-time detection of somatostatin secretion from islets that holds promise for studies of the role of this hormone under normal conditions and in diabetes.