Anion dependence of electrical effects of bicarbonate and sodium on cultured bovine corneal endothelial cells

Abstract

Using intracellular microelectrode technique, the effect of anion substitution on the voltage responses to extracellular bicarbonate and sodium was explored in cultured bovine corneal endothelial cells. The overall amplitude of voltage changes induced by periodic changes of [HCO-3]o (depolarization upon removal of HCO-3 and hyperpolarization upon readdition) was reduced when Cl- was replaced by organic anions (cyclamate, methylsulfate, benzenesulfonate) or by SO4(2-), and to a lesser extent by substitution with Br-. There was a similar effect of anion substitution on the response to changes of [Na+]o. In both cases, in the absence of Cl, the voltage V returned at a slower rate to baseline levels after it had been transiently changed by either an imposed Na- or HCO3- gradient, indicating a slower dissipation of these gradients. The direct response of V to these imposed gradients was affected only to a minor degree. Replacement of Cl- by SO4(2-) or organic anions led to a slow, reversible depolarization of the cell, while substitution with Br- had only a slight effect. The effect of anion substitution on the voltage responses to HCO-3 or Na+ could not be mimicked by a depolarization induced by Ba2+ (1 mM). Furosemide (10(-3) M) led to a slight reduction of the voltage responses to HCO-3, but could not suppress the effect of anion substitution on these reactions. It could neither suppress the depolarization induced by anion substitution and had no effect on steady-state PD. It is suggested, that cultured bovine corneal endothelial cells, in addition to a previously demonstrated electrogenic HCO-3-Na+-cotransport, which is probably not dependent on Cl, possess an electroneutral mechanism for HCO-3 and/or Na+-movement, which depends on Cl. No evidence for a Cl-conductance could be obtained.

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

“…While it is clear from our previous electrophysiological experiments [23,25,26], that bovine corneal endothelial cells express such an electrogenic symport, most of the data shown here (e.g. DIDS-sensitive and HCO3-dependent acidification upon Na § removal, the involvement of a process showing similar sensitivities in the recovery from acid load) may equally well be explained by a Na+-HCO~/CI--H + exchanger (or, equivalently, a NaCO3/CI-exchanger), which is known to be sensitive to DIDS and activated by intracellular acidification [6].…”

“…In previous work, we have shown by intracellular microelectrode measurements [23,25,26] and 22Na+-flux determinations [28], that cultured bovine corneal endothelial cells express an electrogenic sodium-bicarbonate symport which is inhibitable by disulfonic stilbenes (SITS and DIDS) and harmaline [24]. A similar symport had previously been identified in the basolateral membrane of the proximal tubule of the salamander kidney [9], and more recently in mammalian kidney cells of several different species [1,2, 5, 17, t8, 22, 27, 29, 30, 42, 49] and in stomach oxyntic cells [14].…”

“…As suggested by studies of its kinetic properties in BSC-1 cells [30], this symport might actually be a NaCO3-transporter (or a NaCO~--HCO3 symport to account for a 3 : 1 stoichiometry). Also from 22Na+ flux measurements, we postulated earlier the existence of a Na+/H + antiport in the corneal endothelium [28], and electrophysiological data [26] yielded indirect hints at some type of C1-/HCO3 exchange in these cells.…”

Ocular novel drug delivery: impacts of membranes and barriers

“…A solute, based upon molecular properties, is transported across the cell membranes by passive diffusion and/or carrier/receptor-mediated transport [68] . Nearly all ocular tissue displays the Na + /H + exchanger, Na + /HCO 3 -symporter and Cl -/HCO 3 -exchanger that are involved in the regulation of intracellular pH [69,70] . The Na + /H + exchanger is present at the basolateral membranes of both epithelial and endothelial cells, while the Na + /HCO 3 -transporter is predominantly localized at the basolateral side of the corneal endothelium and is faintly expressed in the corneal epithelium.…”

Wavelet analysis of corneal endothelial electrical potential difference reveals cyclic operation of the secretory mechanism

“…The existence of a symport that transports sodium and bicarbonate out of the cell at the apical membrane in this preparation is strongly supported by the electrophysiological evidence of the Wiederholt laboratory [13][14][15][16][17]. Immunocytochemical localization found cotransporters in both the basolateral and apical membranes [18].…”