Under low shear conditions (shear 0.08 dyne/cm2) no increase in ATP release was observed; however, increasing

shear to 0.64 dyne/cm2 caused a rapid relative increase in ATP release in both MLCs and MSCs, and again the magnitude of the peak response was significantly greater in MSCs versus MLCs (P < 0.05, Fig. 5B,C). No difference was noted in lactate dehydrogenase measurements before or after stimulus, for either hypotonic or shear exposure, excluding cell lysis as contributing to measured ATP (data not shown). In other biliary models, ATP release has been linked to exocytosis.18 To determine if exocytosis contributes to ATP release in MLCs and MSCs, studies were performed in the presence or absence of monensin, a carboxylic ionophore known to dissipate the transmembrane pH gradients in Golgi and lysosomal compartments and disrupt vesicular trafficking. In both MLCs and MSCs, monensin significantly inhibited RG-7388 concentration swelling-induced (33% hypotonic exposure) ATP release (Fig. 5D). Thus, both MSCs and MLCs exhibit mechanosensitive ATP release which is dependent on intact vesicular trafficking pathways. Additionally, the magnitude of mechanosensitive ATP release is significantly greater (∼two-fold) in MSCs compared to MLCs. To determine if the difference in ATP release Deforolimus order observed between MSCs and MLCs are the result of generalized

differences in total cellular exocytosis, rates of exocytosis were measured Sodium butyrate in response to mechanical stimuli in both cell types. After equilibration with FM1-43, cells were exposed to hypotonic buffer (33%) which was associated with a rapid increase in fluorescence, reflecting an increase in exocytosis (Fig. 6). In separate studies, exposure to shear (0.64 dyne/cm2) also resulted in an increase in exocytosis (Fig. 6). These findings suggest a functional link between exocytosis and ATP release in both MLCs and MSCs. There was no significant difference noted in the

rate or magnitude of exocytosis between MLCs and MSCs in response to either of these mechanical stimuli. The concentration of extracellular ATP in bile is regulated not only through the rate of ATP release, but also through degradation pathways.23 To determine if differences exist in the kinetics of ATP degradation between MSCs and MLCs, the media bathing confluent cells was loaded with exogenous ATP (10 nM). Changes in bioluminescence were monitored continuously until relative ALU returned to basal levels. As shown in Fig. 7, addition of ATP (10 nM) to MLCs increased relative bioluminescence 2.7-fold. The time course of degradation was described by a single exponential (y = ae−0.038 min, r = 0.99). By comparison, addition of ATP to MSCs increased bioluminescence 2.5-fold with a similar rate of degradation described by a single exponential (y = ae−0034min, r = 0.99). Thus, MLCs and MSCs display functionally similar ATP degradation pathways.