Cell volume regulatory mechanisms are usually disclosed by exposure of cell to anisotonic media. If a cell is suddenly exposed to hypotonic media, it swells initially like an osmometer but within minutes regains its original cell volume. This behavior has been labelled as regulatory cell volume decrease (RVD). RVD is believed to result from the loss of permeable ions through the membrane. In this study, we examined hypotonically induced changes in the membrance currents involved in RVD by using whole cell voltage clamp technique in the unfertilized hamster egg.
At -40㎷ of the holding potential, the stationary current was maintained in the hamster egg exposed to isotonic solution composed of, mainly, 115mM NaCl and 40mM mannitol. Hypotonic solution was prepared by removing mannitol. Therefore, the concentrations of Na^+ and CI^- in this hypotonic media were the same as those in the isotonic solution. Following 30 to 60 sec after applying the hypotonic media to the egg, the inward current was evoked. This inward current was eliminated by 100μM 4-acetamido-4´-isothiocyanostil-bene-2,2´-disulfonic acid(SITS), an anion channel blocker, leaving the small outward current component. Further addition of 2mM Ba^(2+), a broad K^+ channel blocker, completely abolished the small outward current left even in the presence of SITS during hypotonic stress. These results suggest that K^+ and Cl move out of cells, resulting in RVD.
To test the involvement of Na^+ in RVD, 20mM Na-isethionate was substituted for mannitol in isotonic media(135mM Na^+) and Na-isethionate (20mM) was freed the hypotonic solution. Only Cl^- concentration in both isotonic and hypotonic media was kept constant at 115mM, whereas concentration of Na^+ was lowered in hypotonic solution to 115mM from 135mM in isotonic solution. Hypotonic medium induced the outward current in the egg equilibrated isotonically. This current was reduced by 100μM SITS but was augmented by 2 mM Ba^(2+). In terms of RVD, these results imply that Cl^- efflux is coupled with K^+, maybe for electroneutrality during hypotonic stress and/or with Na^+ via unknown transport mechanism(s).
From the overall results, the hypotonic stress facilitates the movement of Cl^- and K^+ out of the hamster egg to regain cellular volume with electroneutrality. If there exist a difference in ［Na^+］_0 between isotonic and hypotonic solution, another transport mechanism concerned with Na^+ may, at least partly, participate in regulatory volume decrease.
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