Using cardiomyocytes isolated from mice expressing troponin T mutants (TnT-I79N, TnT-F110I and TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca2+ binding. The underlying cause was an increase in the cytosolic Ca2+ binding affinity, whereas maximal Ca2+ binding capacity was unchanged. The effect was sufficiently large to alter Ca2+ handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca2+] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca2+ content and release after pauses. Basing on the observation that early stage embryonic stem cells-derived cardiomyocytes continuously contracted in high extracellular potassium solution, we provide experimental evidence that the spontaneous activity of these cells is not generated by transmembrane ion currents, but by intracellular [Ca2+]i oscillations. In cardiac muscle, Ca2+ release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca2+ release refractoriness. Gene-targeted ablation of Casq2 (Casq2 KO) abolished SR Ca2+ release refractoriness in isolated mouse ventricular myocytes. Surprisingly, impaired Ca2+-dependent inactivation of L-type Ca2+ current (ICa), which is responsible for triggering SR Ca2+ release, significantly contributed to the loss of Ca2+ release refractoriness in Casq2 KO myocytes. Recovery from Ca2+-dependent inactivation of ICa was significantly accelerated in Casq2 KO compared to wild type (WT) myocytes.
Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca2+ concentration inside pancreatic acinar cells ([Ca2+]i), due to excessive release of Ca2+ stored inside the cells followed by Ca2+ entry from the interstitial fluid. Electrophysiological data show that in the pancreatic acinar cells this store-operated inward current is relatively insensitive to removing external Na+, but sensitive to changes in the external Ca2+ concentration. It is therefore not a transient receptor potential (TRP) type nonselective cation current, but it is a Ca2+-selective CRAC-type current consistent with the very marked current inhibition evoked by GSK-7975A. There is currently no specific treatment of pancreatitis, but the received data show that pharmacological CRAC blockade is highly effective against toxic [Ca2+]i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis. Asparaginase, an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, evoked intracellular Ca2+ release followed by Ca2+ entry and also substantially reduced Ca2+ extrusion because of decreased intracellular ATP levels. In isolated mouse pancreatic acinar cells or cell clusters, removal of extracellular glucose had little effect on this ATP loss, suggesting that glucose metabolism was severely inhibited under these conditions. Surprisingly, we show that replacing glucose with galactose prevented or markedly reduced the loss of ATP and any subsequent necrosis.
Ca2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared. Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. We have compared Ca2+ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca2+ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto-digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca2+ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca2+ signals in PSCs. The initial Ca2+ rise in PSCs was due to inositol trisphosphate receptor-mediated release from internal stores, whereas the sustained phase depended on external Ca2+ entry through Ca2+ release-activated Ca2+ channels. We have, for the first time, recorded depolarization-evoked Ca2+ signals in pancreatic nerves and shown that, although acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells.
