Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+signals consist of both Ca2+release and Ca2+influx. We have investigated the contribution of these processes to membrane-localized Ca2+signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+release in the TIRF zone would be expected to saturate the Ca2+indicator (Fluo-4), these data suggest that Ca2+release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+release, followed by a substantial, maintained plateau phase indicative of Ca2+entry, was observed. The contribution of Ca2+influx and Ca2+release in isolation to these near-plasma membrane Ca2+signals was investigated by using a Ca2+readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+influx was isolated by subsequent Ca2+readmission, the Ca2+signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+signals mediated by Ca2+influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.