ryanodine and taurolithocholic-acid-3-sulfate

Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 μM) or carbachol (1 mM). Ryanodine (100 μM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 μM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 μM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.

Topics: Acinar Cells; Animals; Calcium; Carbachol; Cell Death; Humans; L-Lactate Dehydrogenase; Mice; Pancreas; Protein Isoforms; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Taurolithocholic Acid

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.

Topics: Acinar Cells; Animals; Bile Acids and Salts; Calcium Signaling; Dantrolene; Male; Mice; Pancreatitis; Ryanodine; Ryanodine Receptor Calcium Release Channel; Taurolithocholic Acid

Single rat hepatocytes show repetitive oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated by agonists acting through the phosphoinositide signalling pathway. We have studied the effect of a natural bile acid, taurolithocholate (TLC), and its sulphated form, taurolithocholate 3-sulphate (TLC-S), on [Ca2+]i in single isolated rat hepatocytes. Although these bile acids are believed to act through a common mechanism to permeabilize the intracellular Ca2+ pool, the [Ca2+]i responses induced by the two compounds were different. Whereas TLC induced a sustained elevation of [Ca2+]i, TLC-S evoked repetitive [Ca2+]i oscillations. In addition, we show that ryanodine, which blocks the Ca(2+)-induced Ca2+ release ('CICR') mechanism, blocked TLC-S-induced oscillations in 50% of hepatocytes, but did not affect the TLC-induced rise in [Ca2+]i.

Topics: Adenosine Triphosphate; Animals; Calcium; Chromatography, Thin Layer; Cytosol; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Liver; Male; Rats; Rats, Wistar; Ryanodine; Taurolithocholic Acid