| Objective:Extracellular acidification plays a promoting role in the development of acute pancreatitis.However,whether extracellular acidification aggravates acute pancreatitis by activating Cl~-channels remains unclear.Here,this study aims to investigate the effect of extracellular acidification on the Cl~-channels in AR42J cells.Methods:In this study,we routine culture the rat pancreatic acinar AR42J cells as the research object.Here,we investigated the effects of extracellular acidification(pH 7.1,6.8,6.2,5.6)on acid-sensitive Cl~-channels in AR42J cells using the whole-cell patch-clamp recording.BCECF-AM fluorescent probe was used to measure the intracellular pH.RNA analysis by quantitative real-time PCR was used to detect Cl~-channel m RNA expression in AR42J cells.Immunofluorescence and Western blot were used to observe the distribution of the cellular chloride channel protein ClC-3.DCFH-DA fluorescent probe were used to detect the level of reactive oxygen species.The expression of ClC-3 was silenced by sh RNA treatment.Statistical analysis used the SPSS 13.0software.Results:We recorded a small and stable background current in control bath solution with the normal pH value(pH 7.4)by using whole-cell patch clamp on AR42J cells.When the rat pancreatic acinar AR42J cells were exposed to four bath solutions,currents were induced.The pH 6.8 activated chlorine current was the largest,with the current density of58.05±6.62 p A/p F at+80 m V and-42.84±4.71 p A/p F at-80 m V(P<0.01).The acid-sensitive currents reversed at the potential approximating to the Cl~-equilibrium potential(-0.9 m V).We investigated extracellular acidification induced a moderately outward-rectified chlorine current,with a selectivity sequence of I~->Br~-≥Cl~->gluconate~-.The acid-activated currents were suppressed significantly when encountered with DIDS.Both outward and inward currents were inhibited,with the inhibition rate of76.99±2.08%at+80 m V and 54.91±6.60%at-80 m V(P<0.01),showing a stronger inhibitory effect on the outward currents(P<0.01).The acid-activated currents were suppressed significantly when encountered with NPPB,with the inhibition rate of 109.19±2.59%at+80 m V and 106.70±5.7%at-80 m V(P>0.05).Compared with the inhibitory effect of DIDS,NPPB showed no obvious difference between the inhibitions of the outward and inward currents.However,intracellular acidification did not induce currents.ClC1～7 m RNA expression of ClC chloride channel family can be detected in the rat pancreatic acinar AR42J cells and the expression of ClC-3 m RNA was the highest.The ClC-3 protein was found on the cell membrane,cell cytoplasm and cell nucleus using immunofluorescence.When the expression of the ClC-3 among ClC chloride channel family in AR42J cells was silenced by ClC-3 sh RNA(P<0.01),the acid-activated chlorine currents were attenuated significantly(P<0.01),indicating that ClC-3 was an important component of the acid-activated Cl~-channel.The ROS production in AR42J cells was increased notably(76.11±0.75%)when treated with the extracellular acid(pH 6.8)for 30 min(P<0.01).When ROS production was cleared,the acid-activated chlorine current was eliminated 95.49±2.49%(P<0.01),indicating the important role of ROS in the induction of acid-activated chlorine currents.On the other hand,the level of acid-induced ROS was not affected by ClC-3 expression silence,indicating that ROS plays a role as the upstream of ClC-3.Conclusions:(1)Extracellular acidification activated chloride current.(2)The ClC-3 chloride channel protein is the key molecule in the acid-induced chloride current.(3)Extracellular acidification leaded to an increase of intracellular ROS. |
