Bulk ATP release was studied from confluent cells using the luciferin-luciferase (L-L) assay as previously described.13, 19, 20 Cell swelling was induced by adding water to dilute media 33% and defined shear stress was applied to confluent cells in a parallel plate chamber. All luminescence values are reported as relative change from basal luminescence per total protein level in the sample (measured in micrograms per milliliter) to control for any potential differences in luciferase activity or confluency between samples, respectively. Detailed protocols for measurements of ATP release, ATP degradation, protein levels, and lactate dehydrogenase

are described in Supporting Information Methods. MLCs and MSCs were grown on collagen-coated polycarbonate filters with a pore size of 0.4 μm (Costar, Cambridge, MA) and the transmembrane resistance was measured daily (Evohm voltmeter; World Precision

Selleckchem Tanespimycin Instruments, Lorlatinib price Sarasota, FL).21 Filters were mounted in an Ussing chamber, filled with standard buffer solution, and transepithelial short-circuit current response (Isc) was measured under 0 mV voltage-clamp conditions through agar bridges connected to Ag-AgCl electrodes using an epithelial voltage clamp amplifier (model EC-825; Warner Instruments, MRA International, Naples, FL). The Isc represents the net sum of the transepithelial fluxes of anion and cation and the level of ion secretion.11 Studies included paired, same-day monolayers to minimize any potential effects of day-to-day variability. Detailed

descriptions of the reagents, buffer solutions, experimental protocols, and statistical 上海皓元 analysis are provided in Supporting Information Materials. In both MLCs and MSCs, complementary DNAs were probed with oligonucleotide primers specific to the seven P2X subtypes and seven P2Y subtypes in mouse (shown in Supporting Information Table 1) and amplified using RT-PCR. Representative studies are shown in MLCs and MSCs (Fig. 1), and in primary isolated cholangiocytes (Supporting Information Fig. 1). In both MLCs and MSCs, clear bands corresponding to P2X4 and all seven P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13) are present. These results are consistent with previous studies of human and rat biliary cells where a predominance of P2X4 and multiple P2Y receptors were observed.11, 14, 15 To establish the functional significance of mouse cholangiocyte P2 receptor expression, MSCs and MLCs were grown to confluence (Fig. 2) and changes in Ca2+ fluorescence measured in response to P2Y and P2X agonists. Exposure to ATP, UTP, a P2Y-preferring agonist, or Bz-ATP, a P2X-preferring agonist, all resulted in significant increases in [Ca2+]i in both MLCs and MSCs (Fig. 3). The ATP-stimulated increase in [Ca2+]i was abolished by the P2Y receptor blocker, suramin (Fig. 3D).