Abstract
Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K+ and Cl− containing solutions. An outward current detected at 0 mV, corresponded to a K+ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca2+-activated K+ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl− current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4′-isothiocyanostilbene), and its reversal potentials corresponded to the Cl− equilibrium potentials at high and low external Cl− concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K+ and Cl− currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K+ and Cl− conductances in these cells, and that the activation pathway for the K+ conductance apparently involves [Ca2+], while the activation pathway for the Cl− conductance does not involve [Ca2+] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.
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We thank Mr. Louis Stamps for excellent technical support. Thanks also to Dr. Mitsunobu Sato from the Second Department of Oral and Maxillofacial Surgery, Tokushima University, Japan for sending us the HSG-PA cells, and to Dr. Englert from Hoechst company for providing us with NPPB. This work was supported by National Institute of Dental Research grants R01 DE09812 and R03 DE10535.
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Fatherazi, S., Izutsu, K.T., Wellner, R.B. et al. Hypotonically activated chloride current in HSG cells. J. Membarin Biol. 142, 181–193 (1994). https://doi.org/10.1007/BF00234940
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DOI: https://doi.org/10.1007/BF00234940