Chloride current activated by hypotonicity in cultured human astrocytoma cells

Abstract

Cultured astrocytoma cells were voltage clamped with pipettes where [Ca2+] in the pipette was buffered to 10(-7) M with 10 mM 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and membrane currents recorded. In isotonic solution predominantly K+ currents were evoked by depolarizing or hyperpolarizing commands and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 1 mM) had little effect on inward and outward currents evoked by voltage steps to negative and positive potentials. If the osmolarity of the bathing solution was reduced from 280 to 200 mosmol l-1, a large current developed, which rectified outwardly and reversed close to the equilibrium potential for chloride ions, ECl. Upon stepping to potentials positive to about +30 mV in hypotonic solution, this outward current inactivated over 2-3 s; this decline was greater with N-methyl-D-glucamine chloride (NMDG-Cl) in the pipette than when KCl or sodium glutamate was present. Holding at various positive potentials inactivated the current, with half-inactivation occurring around +50 mV. The current reactivated over 2-3 s at potentials negative to about +30 mV. The current evoked by hypotonic solution was inhibited by various putative chloride channel blockers with the order of potency DIDS > SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) approximately NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) > niflumic acid > 9-AC (anthracene 9-carboxylic acid). The currents inhibited by these compounds reversed close to the calculated ECl. It is concluded that hypotonic solution evokes a large anionic current in these cultured astrocytoma cells largely carried by chloride ions. This current is absent in isotonic solution, when currents are carried mainly by potassium ions. It is likely that the current elicited by hypotonic solution is part of the mechanisms regulating astrocyte volume in vivo.