Ca²⁺release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis

Abstract

Intracellular calcium concentration ([Ca(2+)](i)) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP(3)) in the apical region resulted in Ca(2+) release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca(2+) wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca(2+) release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca(2+) failed to evoke a propagating Ca(2+) wave in either cell type. However, the kinetics of the Ca(2+) signal evoked following photolysis of caged-InsP(3) were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP(3) receptor (InsP(3)R) activation. These data suggest that differing expression levels of InsP(3)R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca(2+) signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca(2+)-dependent effectors important for the differing primary physiological role of each gland.

Studying isoform-specific inositol 1,4,5-trisphosphate receptor function and regulation

“…For detailed methods regarding transient and stable expression of constructs in DT40 cells see [29]. In intact cells, Ca 2+ release can be monitored by routine digital imaging of Ca 2+ sensitive fluorescent dyes such as Fluo-4 and Fura-2 [30–32]. In our case, we use a Till Photonics imaging system (Gräfelfing, Germany) consisting of a Polychrome IV monochromator capable of rapid wavelength switching, coupled through a light guide to an Inverted microscope with high NA objective.…”

Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells

“…It is likely that the particular spatial and temporal characteristics of the Ca 2ϩ signal are the key features that lead to the appropriate activation of effectors responsible for secretion (23, 27, 28, 47, 48). In turn, the major step defining the initiation of these processes is the integrated regulation by multiple factors of Ca 2ϩ release through InsP 3 R (10,23,28,47). In this study, we have investigated how cellular levels of adenine nucleotides might contribute to this regulation.…”

“…Single cells or small clusters of parotid acinar cells were isolated from freshly dissected parotid glands from NIH Swiss mice (25 g) by sequential digestion with (single cells) or without (cell clusters) trypsin followed by collagenase as previously described (23,47,48). Following isolation, cells were resuspended in 1% BSA containing BME supplemented with 2 mM glutamine, penicillin/streptomycin and incubated at 37°C and gassed with 5% CO 2-95% O2 until ready for use.…”

“…InsP 3 then acts on inositol 1,4,5 trisphosphate receptors (InsP 3 R) expressed on the endoplasmic reticulum (ER). InsP 3 R are particularly abundant on ER immediately juxtaposed to the apical plasma membrane (28), and rapid Ca 2ϩ release is invariably initiated at the apical Ca 2ϩ release sites followed by the subsequent globalization of the Ca 2ϩ signal (23,28,47). The temporal and spatial information in this signal are ideally suited both for initially activating the apical Cl Ϫ conductance and exocytotic machinery followed by subsequent opening of basolaterally localized K ϩ channels.…”

Bile acids activate ryanodine receptors in pancreatic acinar cells via a direct allosteric mechanism

“…The spatial localization of Ca 2+ release is explained by high inositol trisphosphate receptor (IP 3 R) density in the apical endoplasmic reticulum (ER) (also called Ca 2+ release trigger zone) [8,9]. In contrast, overstimulation of the pancreatic acinar cell by secretagouges and high concentrations of bile acids induce whole-cell, high amplitude, long lasting, (peak-plateau type) Ca 2+ signals.…”