Mechanisms of regulatory volume decrease of human lens epithelial cells associated with hypotonic stimulation

Abstract

Background It is widely appreciated that many animal cells rely on the mechanism of regulatory volume decrease (RVD) after swell under the hypotonic environment,which involved in some processes of cellular physiology.But the RVD of lens epithelial cells(LECs) still is being further researched.Objective Present study was to clarify the possible mechanisms and influencing factors in the RVD of LECs.Methods Human LECs line (HLE B-3)were cultured and passaged in DMEM/F12 containing 10％ fetal bovine serum(FBS),and before volume measurement,cells were stuck to the base of a perfusion chamber,Ringer solution osmolality was decreased from 15％Hypo to 45％ Hypo,and the cells stimulated by 45％ Hypo Ringer solution were used as the control group.Some experiments were performed in the presence of high extracellular K+ concentration,chloride or potassium channel inhibitor,experiments were also carried out in the nominal absence of Ca2+,Cl-or HCO-3 to test the effect of a decrease in intracellular concentration of these ions on the cell volume response.The volume changes of living cells were measured by lag-time microphotograph acquisition and analysis system (IPP6.0).Results Time course of cell volume change after hypotonic shock in HLE B-3 cells was observed.The cell swelling was followed by a gradual volume recovery,indicating the presence of RVD was influenced by the hypotonic stress.Under the stimulation of 45 ％Hypo Ringer solution,the rates of RVD were (59.1 ±7.8)％.RVD was correlated positively to the maximum cell volume (r =0.99,P＜0.05)in S shape,and RVD changes were sensitive to alter maximum cell volume in the range of 115％-135％.RVD reduced to (16.5 ± 1.6) ％,(14.7 ± 2.3) ％,respectively after acted by potassium channel inhibitor,TEA(10 mmol/L)and BaCl2(5 mmol/L)as well as chloride channel inhibitor,NPPB(100 μmol/L)and DIDS(100 μmol/L),with significant differences in comparison with the control group(all P＜0.01).RVD decreased by(5.8±1.6)％ and(2.7±0.8)％ in high concentration of K+ in extracellular fluid and the absence of Cl-(P＜0.01).RVD was significantly inhibited under the absence of Ca2+.When the 45％ Ringer solution was pH6.8,the process of RVD delayed.The rate of RVD in the first ten minutes was (0.86±0.24)％/min,showing a significant decline in comparison with (3.24±0.84) ％ / min of pH 7.4 (P ＜0.05).Conclusions HLE B-3 have RVD ability under the hypotonic stress stimulation.A certain intracellular Ca2+ concentration is the premise of RVD activation,and Cl efflux and K+ efflux are the key mechanism of RVD of HLE B-3.Acidic environment of hypotonic solution delays the occurrence of RVD. Key words: Lens epithelial cells; Regulatory volume decrease; Hypotonic stress stimulation; Cellular ion channel