Roles Of Purinoceptor Pathways In Activation Of Background Chloride Channels In Nasopharyngeal Carcinoma Cells

【OBJECTIVE】1) To study the roles of the P2Y purinoceptor signal transduction pathway in activation of background chloride channels in nasopharyngeal carcinoma cells(CNE-2Z cells).2) To investigate the roles of background chloride channels in regulation of CNE-2Z cell volume.3) To clarify the involvement of purinoceptor pathways in volume regulation of CNE-2Z cells.【METHODS】1) The whole-cell patch clamp technique was use to record the background chloride current.Current density was cauculated and analyzed.2) The role of the P2Y purinoceptor signal transduction pathway in activation of background chloride channels was investigated using purinergic antagonists and ATPase.3) Cells were perfused with a special perfusion system.The Q500MC image processor and the Scion image analysis software were used to detect the diameter and area of CNE-2Z cells.The standardized cell volume and the volume changes caused by perfusion of isotonic bath solutions with chloride channel blockers or P2Y purinoceptor antagonists and by depletion of extracellular Cl~- were calculated. 【RESULTS】1) A stable background cloride current was recorded under isotonic conditions in CNE-2Z cells.The current density was(5.19±0.83) pA/pF(at +80 mV) and(-5.11±0.64) pA/pF(at -80 mV),without significant differences between outward(at +80 mV) and inward(at -80 mV) currents.2) Extracellular application of reactive blue 2(RB2),a relatively selective P2Y purinoceptor antagonist,depressed the outward and inward background chloride currents of CNE-2Z cells in a concentration dependent manner under isotonic conditions.Isotonic bath solutions with 50,100 and 200μmol/L RB2 inhibited significantly the background chloride currents respectively.The inhibition of the outward current was stronger than that of the inward current (P＜0.01).Washout of RB2 recovered the currents.3) Apyrase(20 units/mL) depressed the background chloride currents under isotonic conditions.The outward and inward background chloride currents were decreased by(72.21±10.51)%(t=3.27,P＜0.05) and(62.35±14.08)%(t=-3.00, P =0.058,n=4) respectively,without significant differences(P＞0.05) between the outward and inward currents.The inhibition of the currents was reversible.4) Extracellular application of the chloride channel blocker 5-nitro-2-3-phenylpropyl-amino benzoic Acid(NPPB,50μmol/L) increased the cell volume by(8.45±1.65)%(t=7.02,P＜0.01,n=8) under isotonic conditions.5) Depletion of the excellular Cl~- decreased the cell volume by(7.66±1.34)%(t=11.99,P＜0.01,n=8) under isotonic conditions.6) Extracellular application of 100μmol/L RB2 increased the volume of the cells by(16.03±1.21)%(t=11.80,P＜0.01,n=10) under isotonic conditions.【CONCLUTION】Nasopharyngeal carcinoma cells express a stable background chloride current under isotonic conditions.The P2Y purinoceptor signal transduction pathway plays an important role in activation of background chloride channels.The background chloride channels are involved in the volume regulation under isotonic conditions in CNE-2Z cells.CNE-2Z cells may release ATP,which activate indirectly the background chloride channels through the P2Y purinoceptor signal transduction pathway and regulate cell volume.