An efficient method is required to transfect non-dividing cells with genetically encoded optical probes for molecular imaging.

Genetically encoded fluorescent and bioluminescent reporters are now widely used for imaging and understanding of intracellular signaling in response to extracellular stimuli in real time in single living cells. Primary cultures are a valuable tool, and are often preferred over transformed or immortalized cell lines, since they are biologically more relevant and important in biomedical research and therapeutic development. To incorporate genetically encoded reporters into the primary culture of non-dividing cells, such as mouse or human pancreatic acinar cells, is not an easy task.

Dynamic changes in cytosolic and mitochondrial ATP levels in pancreatic acinar cells.

Background & Aims: Previous studies of pancreatic acinar cells characterized the effects of Ca(2+)-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca(2+), transmembrane potential, and NAD(P)H, but dynamic measurements of the crucial intracellular adenosine triphosphate (ATP) levels have not been reported. Here we characterized the effects of these agents on ATP levels in the cytosol and mitochondria.

Methods: ATP levels were monitored using cytosolic- or mitochondrial-targeted luciferases.

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1-ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP.

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta-structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and a slow calcium leak from the ER followed by formation of STIM1 puncta.

ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile.

Here, we describe novel mechanisms limiting a toxic cytosolic Ca(2+) rise during adenosine 5'-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca(2+) signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate.

Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles.

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK).

Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells.

The intracellular activation of trypsinogen, which is both pH- and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin.

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors.

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+).

Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells.

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca(2+) gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization.

Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration.

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells.

Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating DeltaPsim.

In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin.