Differential effects of Na-K-ATPase pump inhibition, chemical anoxia, and glycolytic blockade on membrane potential of rat optic nerve.

Na(+)-K(+)-ATPase pump failure during either anoxia or ouabain perfusion induces rapid axonal depolarization by dissipating ionic gradients. In this study, we examined the interplay between cation and anion transporting pathways mediating axonal depolarization during anoxia or selective Na(+)-K(+)-ATPase inhibition. Compound resting membrane (V(m)) potential of rat optic nerve was measured in a grease gap at 37 degrees C. Chemical anoxia (2 mM NaCN or NaN(3)) or ouabain (1 mM) caused a loss of resting potential to 42 +/- 11% and 47 +/- 2% of control after 30 min, respectively. Voltage-gated Na(+)-channel blockade was partially effective in abolishing this depolarization. TTX (1 microM) reduced depolarization to 73 +/- 10% (chemical anoxia) and 68 +/- 4% (ouabain) of control. Quaternary amine Na(+) channel blockers QX-314 (1 mM) or prajmaline (100 microM) produced similar results. Residual ionic rundown largely representing co-efflux of K(+) and Cl(-) during chemical anoxia in the presence of Na(+)-channel blockade was further spared with DIDS (500 microM), a broad-spectrum anion transport inhibitor (95 +/- 8% of control after 30 min in anoxia + TTX vs. 73 +/- 10% in TTX alone). Addition of DIDS was slightly more effective than TTX alone in ouabain (74 +/- 5% DIDS + TTX vs. 68 +/- 4% in TTX alone, P < 0.05). Additional Na(+)-entry pathways such as the Na-K-Cl cotransporter were examined using bumetanide, which produced a modest albeit significant sparing of V(m) during ouabain-induced depolarization. Although cation-transporting pathways play the more important role in mediating pathological depolarization of central axons, anion-coupled transporters also contribute to a significant, albeit more minor, degree.

Mechanisms of regulatory volume decrease in nonpigmented human ciliary epithelial cells.

To study the net solute and water efflux pathways of the ciliary epithelium we employed a cultured human NPE cell line. Because of the possible relationship between transepithelial ion and water flux and cell volume regulation, the ion efflux pathways mediating regulatory volume decrease (RVD) were investigated. Osmotic swelling of NPE cells was followed by a volume recovery. Volume recovery was K+ dependent and inhibited by K+ channel blockers such as quinine (1 mM). After osmotic swelling, a Cl(-)-dependent membrane depolarization occurred that was inhibited by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) or Ca2+ chelators such as ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2.0 mM). Cell swelling was also accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) of approximately 200 nM. The swelling-induced rise in [Ca2+]i and RVD were diminished in the presence of 10 microM La3+, 50 nM 12-O-tetradecanoylphorbol 13-acetate, and nominally Ca(2+)-free medium. Near total blockage of RVD occurred after pretreatment of NPE cells with Ca(2+)-free EGTA-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester-containing solutions. The inhibition of RVD by EGTA-BAPTA treatment was overcome by increasing K+ conductance with gramicidin. The above findings indicate that RVD in NPE cells is mediated by separate K+ and Cl- conductances (channels). These data also show that swelling-induced increases in [Ca2+]i help modulate net ion efflux during regulation.

Regulatory volume decrease in frog retinal pigment epithelium.

To measure changes in cell water during cell volume regulation, retinal pigment epithelial cells were loaded with tetramethylammonium (TMA). Regulatory volume decrease (RVD) in TMA-loaded retinal pigment epithelial (RPE) cells was measured using double-barreled K(+)-specific microelectrodes. Hyposmotic removal of 12.5 mM NaCl from the apical bath caused bullfrog RPE cells to rapidly swell by approximately 10% and to recover to control level within 10-15 min. Hyposmotic RVD was inhibited by 5 mM basal but not apical BaCl2. Raising K+ in the basal bath from 2 to 12 mM also inhibited RVD. Hyposmotic swelling was accompanied by an increase in the ratio of apical to basolateral membrane resistance (Ra/Rb). The swelling-induced increase in Ra/Rb was inhibited by 5 mM BaCl2. Together, the above findings suggest that hyposmotic swelling enhances basolateral K+ conductance such that K+ and presumably anion efflux mediate net solute and water loss during RVD. RPE cells can also regulate their volume when swollen in isosmotic Ringer solution under certain conditions. When urea or apical HCO3- was used to induce cell swelling, RPE cells underwent an RVD. In contrast, isosmotic elevation of apical K+ from 2 to 5 mM resulted in an increase in RPE cell volume with no subsequent RVD. Thus the method used to swell RPE cells is an important determinant of RVD. Because changes in RPE cell volume in vivo may alter the volume and composition of the extracellular (subretinal) space surrounding the photoreceptors, isosmotic volume regulation may play an important physiological role in maintaining the integrity and health of the neural retina under normal and pathophysiological conditions.

Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells.

The solute and water transport properties of the bovine ciliary epithelium were studied using isolated pigmented (PE) and nonpigmented (NPE) cells. It was shown that these cells were functionally coupled by demonstrating dye diffusion between paired PE and NPE cells after microinjection of lucifer yellow. Electronic cell sizing was used to measure cell volume changes of isolated PE and NPE cells in suspension after anisosmotic perturbations and after transport inhibition under isosmotic conditions. The PE cells showed the presence of a regulatory volume increase when subjected to osmotic shrinkage with NaCl, whereas the NPE cells did not demonstrate a regulatory volume increase under these conditions. In contrast, the NPE cells exhibited a regulatory volume decrease when subjected to osmotic swelling, whereas the PE cells did not recover from swelling. The regulatory volume decrease in NPE cells was inhibited by increased bath K or pretreatment with quinine (1 mM). The presence of a bumetanide-sensitive mechanism capable of moving measurable amounts of solute and water, probably Na-K-2Cl cotransport, was demonstrated in the PE cells but absent in the NPE cells. Bumetanide produced a dose-dependent shrinkage of PE cells at concentrations as low as 1 microM. Isosmotically reducing bath Cl, Na, or K concentration caused a rapid shrinkage of PE cells that was bumetanide inhibitable. The asymmetry of transport properties in PE and NPE cells supports a functional syncytium model of aqueous humor formation (39) across the two layers of the ciliary epithelium wherein ion uptake from the blood is carried out by the PE cells and ion extrusion by the NPE cells. Gap-junction coupling between the cells allows the ions taken up by the PE cells to move into the NPE cells. Extrusion of Na by the Na-K pump across the aqueous facing (basolateral) membranes of the NPE cells, most likely accompanied by Cl, determines the formation of the aqueous humor.

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport.

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.

Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium.

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).

Dynamic NMR measurement of volume regulatory changes in Amphiuma RBC Na+ content.

23Na nuclear magnetic resonance (NMR) and conventional chemical methods were employed to measure Na+ fluxes in Amphiuma red blood cells (RBC) during volume regulation. Paramagnetic shift reagents [dysprosium triethylenetetraminehexaacetic acid (DyTTHA) and dysprosium tripolyphosphate (Dy(TPP)2)] were used to alter extracellular Na+ magnetic resonance. Data are presented describing 23Na resonance dependence on shift reagent, sodium and calcium concentration. We confirmed that the shift reagents neither enter the cells nor alter intracellular Na+, K+, and Cl-concentrations under control conditions when extracellular calcium was maintained greater than 0.5 mM. We also confirmed that the shift reagent complexes chelate calcium [Dy(TPP)2 much more so than DyTTHA] and that their toxic effects could be alleviated by adjusting calcium in the cell's suspension medium to control levels. In parallel experiments, where volume-activated Na+ fluxes ranged from 0.3 to 3 mmol Na+/kg dry cell solid (DCS) x minute in cells containing from 30 to 150 mmol Na+/kg DCS, changes in intracellular sodium measured by 32Na NMR were within 4% of those measured by conventional destructive methods. Finally, we present data that are consistent with the interpretation that 6 mmol Na+/kg DCS plus 16% of intracellular Na+ is NMR invisible.

Activation of electroneutral K flux in Amphiuma red blood cells by N-ethylmaleimide. Distinction between K/H exchange and KCl cotransport.

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.

Calcium-induced transient potassium efflux in human red blood cells.

Human red blood cells pretreated with low-ionic-strength solutions and resuspended in saline respond biphasically to extracellular Ca. At first, addition of Ca causes a large transient K efflux of as much as 600 mM . liter cell H2O-1 . h-1; this is followed by a decrease in K flux below control levels. The first phase (phase I) resembles the Gardos effect in several respects. It is inhibited by oligomycin, by external K, and by increased exposure time to Ca. Further, the K permeability of phase I is similar to that of the Gardos effect (5 X 10(-8)-9 X 10(-8) cm/s), and the cells hyperpolarize in a low-K medium when Ca2+ is added. However, phase I is not identical to the Gardos phenomenon. For example, La, which prevents the Gardos response, is ineffective on phase I. Moreover, external Ba prevents the development of phase I but not the Gardos response, whereas internal Ba prevents the Gardos response. Attempts to demonstrate a Ca leak or pump failure during phase I have failed; passive Ca movements of both treated and normal cells are similar. The results suggest that low-ionic-strength solution exposes Ca-sensitive sites to the external medium; these sites are maintained when the cells are returned to saline.

Erythrocyte membrane potentials determined by hydrogen ion distribution.

If the extracellular fluid is left unbuffered, dynamic membrane potential changes in the red blood cell may be determined from external pH readings. For some types of experiments it is necessary to accelerate H+ equilibration by adding minute amounts of hydrogen carriers. The method is independent of hematocrit over a wide range of membrane potential changes. Membrane potential jumps produced by permeability changes or by changes in ionic composition may be measured. The method provides a convenient means of measuring parameters of both the conductive and non-conductive anion pathways in the red cell.