Intracellular pH regulation by a Na+/H+ exchanger in cultured bovine trabecular cells.

Intracellular pH (pHi) of cultured bovine trabecular cells was measured using video-imaging techniques with a pH-sensitive intracellular fluorescent dye, BCECF. In bicarbonate-rich Ringer at pH 7.4, pHi was 7.29 +/- 0.03 (+/- SEM, n = 12 monolayers, 120 cells sampled). Exposure to 20 mM NH4Cl immediately alkalinized pHi: replacement with a Na(+)-rich solution acidified pHi before recovery to resting levels. When NH4Cl was replaced by a low Na+ solution, acidification was sustained but pHi recovery occurred after Na(+)-rich solution. A pHi of 7.11 +/- 0.02 (n = 2 monolayers, 20 cells) occurred in pH 6.8 and pHi was 7.72 +/- 0.03 (n = 2 monolayers, 20 cells) in pH 8.0. Amiloride (1 mM) acidified pHi but DIDS (1 mM) treatment, HCO3(-)-free condition, 1 mM ouabain, 50 mM K+, and 2 mM BaCl2 failed to change pHi. Hydrogen peroxide (1 mM) acidified pHi but no change occurred with 50 microM. Trabecular cells possess an Na+/H+ exchanger similar to that in other cell types.     

Direct stimulation by succinate of Na+:K+ pump in rabbit ciliary epithelium

The effects of succinate on the intracellular potential difference, PDI, were measured in isolated rabbit ciliary processes. Concentration-dependent increases in the hyperpolarization of PDI occurred between 1 and 15 mM succinate in NaCl Ringers. With 5 mM succinate, there was a 6 mV hyperpolarization. Even though the hyperpolarization of PDI was comparable with 10 and 15 mM succinate, it was more sustained at the latter two concentrations. Succinate also elicited comparable hyperpolarizations of PDI in either Cl(-)-free or HCO3(-)-free Ringers. Similarly, following incubation with either 0.1 mM DIDS or 3 mM BaCl2 the effect of succinate on PDI was unchanged. Five mM succinate had no effect if it was added after 5 mM malonate. Malonate (5 mM) rapidly reversed a 5 mM succinate-induced hyperpolarization of PDI which also suggests a metabolically mediated effect on PDI. An isosmotic substitution of Na+ with NMDG Ringers depolarized PDI, whereas PDI depolarized biphasically during exposure to 0.1 mM ouabain. The addition of 5 mM succinate had no effect on either the time course or the magnitude of the depolarization of PDI during blocking of the Na+:K+ pump with either Na(+)-free Ringers or ouabain. Taken together, these results show that succinate selectively stimulates the Na+:K+ pump, but has no effect on any Cl-, HCO3- or a Ba2(+)-sensitive K+ conductance.     

Bicarbonate transport mechanisms in rabbit ciliary body epithelium.

Sections of whole ciliary body dissected from Dutch belted rabbits were incubated with the cell entrappable pH probe BCECEF-AM. This led to a highly specific localization of epifluorescence emission at the exposed, non-pigmented cell layer (npe) of the dual layered epithelium that covers this organ. The BCECF-loaded tissue sections were superfused in a flow-through chamber and the intracellular pH (pHi) of small groups (10-20) of cells was derived from the ratio of the emission intensities derived from excitations at 490 and 440 nm. In CO2/HCO3- Ringer's, npe pHi = 7.09 +/- 0.11. Replacement of CO2/HCO3- by Hepes increased pHi by 0.22 +/- 0.02, indicating alkali secretory activity under the bicarbonate-rich conditions. Replacement of Cl- by gluconate elicited a rapid, 0.6-U increase in pHi. This effect exhibited little dependence on Na+ and was inhibited by 0.5 mM dihydro-4,4'-diisothiocyanatostilbene -2,2'-disulfonate (H2DIDS). These results indicate the presence of an electroneutral Cl-/base exchange activity. Elevation of [K-] (by partial replacement of Na+) also elicited increases in pHi. In Cl(-)-free media pHi reached 7.8-8.0, a condition under which intracellular [HCO3-] is at least twice as high as its extracellular value. This effect did not occur in the absence of Na+. The Na(+)-dependent high [K+]-induced pHi increase was inhibited by H2DIDS. The effects of Ba2+ on pHi, alone and in combination with high [K+], as well as that of full K+ removal, suggested that the link between high [K+] and pHi increase was mainly due to the effect of cell depolarization on an electronegative Na+ dependent HCO3- transporter. Under normal physiological conditions, the two acid/base transport systems are the main determinants of npe pHi.     

Effect of hypotonic media on the membrane voltage of cultured bovine corneal endothelial cells

The effects of hypotonicity on cultured bovine corneal endothelial cells were investigated using standard microelectrode and superfusion techniques. Confluent monolayers of cells were superfused with an isotonic (305 +/- 5 mosm/kg) control solution until a stable membrane voltage (V) was obtained, then with a hypotonic (240 +/- 5 mosm/kg) solution. Under control conditions, V was - 51.4 +/- 0.8 mV (means +/- SEM, n = 154). Decreasing solution osmolality resulted in an immediate depolarization: mean maximal delta V = 18.7 +/- 0.9 mV at 2.6 +/- 0.2 minutes with a gradual recovery to a new but still depolarized steady-state V (delta v = 11.1 +/- 0.9 mV at 8.2 +/- 0.3 minutes, n = 25). The depolarizing response to hypotonicity persisted in the presence of amiloride (10(-3)M), DIDS (10(-3)M), bumetanide (10(-4)M) or ouabain (10(-4)M) as well as in the absence of extracellular Cl-, Na+, HCO3- or Ca2+. Relative K+ conductance was estimated by the effect on V of increased extracellular [K+] - this was significantly reduced at 5, 10 and 20 mM K+ under hypotonic conditions. The depolarization induced by 1mM Ba2+ was also reduced from 19.6 +/- 0.5 mV (n = 8) under isotonic conditions to 15.4 +/- 0.4 mV (n = 6) under hypotonic conditions (p less than 0.001). The conductive HCO3- pathway - as judged by the hyperpolarization of V induced by increasing extracellular [HCO3-] from 28 to 60 mM, was also reduced under hypotonic conditions (delta V = 17.2 +/- 0.8 mV, n = 13 (isotonic) compared to delta V = 9.5 +/- 0.3 mV, n = 15 (hypotonic].(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular pH regulation in fresh and cultured bovine corneal endothelium. II. Na+:HCO3- cotransport and Cl-/HCO3- exchange.

The comparison of intracellular pH (pHi) regulation between fresh (FBCE) and cultured (CBCE) bovine corneal endothelium was extended to HCO3- transport mechanisms. Upon introduction of CO2/HCO3- Ringer's solution, there was a small, sharp acidification followed by an alkalinization .09 and .18 pH U above the HCO3- free resting pHi for FBCE and CBCE, respectively. This increase in pHi totally depended upon the presence of Na+, independent of Cl- and blocked by the anion transport inhibitor, H2DIDS (0.5 mmol/l). Recovery from an (NH4)2SO4-induced acid load also was blocked by Na+ removal and inhibited 62% and 84% by 0.5 mmol/l H2DIDS for FBCE and CBCE, respectively. These results indicate the presence of a Na+ dependent HCO3- transporter. Additions of 22 mmol/l K+ or 5 mmol/l Ba2+ led to substantial H2DIDS-inhibitable base influx in HCO3- Ringer's, which was significantly reduced in the absence of HCO3- for FBCE and CBCE, consistent with the presence of electrogenic Na+:nHCO3- cotransport. Using the Na+ sensitive intracellular dye, SBFI, we confirmed that the addition of HCO3- resulted in a H2DIDS-sensitive Na+ influx. The mean steady-state [Na+i] = 14 +/- 3 mmol/l in bicarbonate-free Ringer's and 31.5 +/- 2 mmol/l in CO2/HCO3-. HCO3- induced Na+ influx was reduced 74% by 0.5 mmol/l H2DIDS. Removal of Cl- from bicarbonate Ringer's alkalinized FBCE and CBCE by .12 +/- .01 and .21 +/- .03, respectively. The increased pHi was completely blocked by 0.5 mmol/l H2DIDS. Similar alkalinizations were seen when Cl- was removed from air equilibrated bicarbonate-free Ringer's. However, because of the lowered buffering capacity in the absence of HCO3-, the flux was reduced by 80%. Cl- free alkalinizations were eliminated by equilibrating the bicarbonate-free Ringer's with 100% nitrogen gas, indicating that residual CO2 can act as a substrate. Bicarbonate efflux on readdition of Cl- was Na+ independent and pHi sensitive (inactivated by low pHi), and showed simple saturating kinetics with respect to bath Cl, K1/2 = 22 and 16 mmol/l for FBCE and CBCE, respectively. These data are consistent with the presence of Cl-/HCO3- exchange in FBCE and CBCE. Application of 0.5 mmol/l H2DIDS to resting cells in HCO3- Ringer's significantly reduced pHi by .23 +/- .03 and .18 +/- .02 in FBCE and CBCE, respectively, indicating net HCO3- influx in resting cells and suggesting that the stoichiometry of the Na+:nHCO3- cotransporter favors Na+ and HCO3- uptake, ie, n < or = 2.     

Intracellular pH of tissue-cultured bovine corneal endothelial cells.

Intracellular pH (pHi) of bovine tissue-cultured corneal endothelial cells has been measured under several experimental conditions. Determinations were made on individual cells using video-imaging techniques that allowed assessment of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence at 440 and 490 nm. Each experiment had a calibration performed on a cell monolayer: this was performed using a high K(+)-nigericin solution. Resting pHi was 7.25 +/- 0.03 (n = 18) in bicarbonate solution at pH 7.4. Amiloride (1 mM) caused an acidification of approximately 0.2 U within 2 min: replacement with normal Ringer allowed a return to normal pHi after an alkali overshoot. Exposure to 20 mM NH4Cl caused alkalinization that became acidic upon washout of NH4Cl. In Na(+)-rich solution pHi returned to normal after acidification but pHi remained low in Na(+)-free solution until substituted by Na(+)-rich solution. Removal of HCO3- from the bathing solution caused a nonsignificant acidification of pHi by 0.1 U at 2 and 4 min, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1 mM) acidified pHi by 0.14 U at 2 min and 0.24 U at 4 min. Addition of DIDS (1 mM) in a HCO3(-)-free solution had no effect on pHi. Hydrogen peroxide acidified pHi by 0.3 U at 50 microM and 1 mM. These results indicate that a Na+:H+ antiport exists that regulates pHi even at normal ambient pH in the presence of bicarbonate: this process becomes highly activated after an acid load. There is a DIDS-sensitive HCO3- movement that is probably coupled to Na+ or Cl-.     

Stimulation of toad lens epithelial Na+/H+ exchange activity by hypertonicity

Incubation of toad lenses with the acetoxymethyl ester of 2',7'-biscarboxyethyl-5(6)-carboxy-fluorescein led to a highly selective accumulation of the de-esterified, pH-sensitive form of the dye in the epithelial cells, enabling the continuous fluorometric monitoring of epithelial intracellular pH (pHi) in intact lenses. The effects of changes in extralenticular [Na+] and of amiloride-addition indicated that the epithelium contains an amiloride-sensitive Na+/H+ antiport. Exposure of lenses to hypertonic conditions (by the addition of sucrose to the medium) resulted in a biphasic change in pHi; a rapid initial, 'spike-like' decrease was immediately followed by a persistent reversal that raised pHi in CO2/HCO3- -rich and -free media by 0.13 and 0.18 units, respectively. Under CO2/HCO3- -free conditions, the hypertonic exposure raised pHi to a value near the calculated equilibrium position for a lens Na+/H+ exchanger. At this point, monensin addition did not affect pHi, suggesting that the tonicity shift had induced a rapid endogenous Na+/H+ exchange activity. In contrast, in the presence of 1 mM amiloride or in the absence of extralenticular Na+, sucrose addition induced only a persistent pHi decrease, which could be reversed (in the 'amiloride' case) by monensin addition. These results demonstrate that the hypertonic exposure induced an epithelial cell acidification as well as a stimulation of the Na+/H+ exchange activity which reverted the acidification. The hypertonic exposure also elicited pHi increases in lenses that had been preacidified by the 'NH4+ loading' or 'pCO2 raise' methods, indicating that the onset of the stimulation could not be attributed to a pHi decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells

Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.     

Evidence for parallel Na(+)-H+ and Na(+)-dependent Cl(-)-HCO3- exchangers in cultured bovine lens cells.

BCECF, a cell-entrapable dye with a pH-sensitive fluorescence spectrum, was used to identify transport mechanisms contributing to pH homeostasis of cultured bovine lens epithelial cells. Cells from a spontaneously established lineage were grown on glass coverslips that fit diagonally in a standard curvette and intracellular pH (pHi) was measured. Under perfusion with a CO2-HCO3(-)-free medium (pH 7.45), pHi was 7.19 +/- 0.21 (mean +/- S.D., n = 94 cell preparations). Cell acidifications (pHi to 6.65, n = 8) induced by the 'NH(4+)-loading' method were rapidly followed by a Na(+)-dependent, amiloride-inhibitable pHi recovery. Introduction of a CO2-HCO3(-)-rich medium (pH 7.45) resulted in a small acidification (0.18 +/- 0.04 U, n = 16; P < 0.002) due to rapid CO2 entry and an ensuing slow alkalinization to a pHi near the control CO2-HCO3(-)-free value. Subsequent removal of Cl- resulted in a further alkalinization of 0.18 +/- 0.02 U (n = 13; P < 0.001). This Cl- effect was completely inhibited by the absence of Na+, but was insensitive to amiloride, suggesting the presence of a Na(+)-dependent Cl(-)-HCO3- exchanger. Consistent with this posit, the reintroduction of Na+ to cells perfused in the absence of the cation with a HCO3(-)-containing, amiloride-complemented solution resulted in a gradual recovery from the acidic pHi induced by the baseline conditions (n = 6). The amiloride-insensitive, Na(+)- and HCO3(-)-dependent recovery was completely inhibited in cells pre-incubated with DIDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3- permeability of bovine corneal endothelium

Corneal endothelium secretes HCO(3)(-) from basolateral (stroma) to apical (anterior chamber) compartments. Apical HCO(3)(-) permeability can be enhanced by increasing [Ca(2+)](i). We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca(2+) activated apical HCO(3)(-) permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417-430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical HCO(3)(-) permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [Ca(i)(2+)] by application of ATPgammaS or cyclopiazonic acid (CPA) increased apical HCO(3)(-) permeability in siControl transfected CBCEC, while having no effect on apical HCO(3)(-) permeability in bCLCA1 specific siRNA transfected cells. Baseline HCO(3)(-) permeability, however, was not different between controls and siRNA treated cells. We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and can contribute to Ca(2+) dependent apical HCO(3)(-) permeability, but not resting permeability, across the corneal endothelium.     

Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body

Electronic cell sizing has been used to measure the volume of cells suspended in isosmotic and in hyposmotic solutions of identical ionic composition. Without inhibitors, the cells displayed a regulatory volume decrease (RVD) following anisosmotic cell swelling with a time constant (tau) of 6.3 +/- 0.9 min (mean +/- S.E.). The RVD was markedly impaired by substituting gluconate for external Cl-, and tau was prolonged by: (i) reducing the chemical gradient favoring K+ loss (by elevating the external [K+] and blocking the Na. K-exchange pump), (ii) blocking the K+ channels with Ba2+, (iii) blocking Cl- channels and Cl-/HCO3 = exchange with DIDS, and (iv) removing external HCO3-. Withdrawing HCO3- may have altered the RVD either directly by inhibiting a Cl-/HCO3- antiport, or indirectly by affecting intracellular pH. The regulatory volume response of ODM Cl-2/SV40 cells is in several respects qualitatively similar to that of non-pigmented epithelial cells of the intact ciliary body. These common characteristics suggest that the cultured cells can serve as a useful model for studying solute and fluid transport across the human ciliary epithelium. The basis for the RVD is likely to be activation of separate K+ and Cl- channels, with or without the parallel operation of coupled K+/H+ and Cl-/HCO3- antiports.     

Effect of bicarbonate on intracellular potential of rabbit ciliary epithelium.

Extracellular HCO3- hyperpolarizes the intracellular potential and makes the aqueous medium negative with respect to the stromal surface of the rabbit ciliary epithelial syncytium. The bases for these observations have been unclear. We have been studying the bicarbonate-induced hyperpolarization (BIH) with sustained intracellular recordings for periods as long as 1-2 hrs. The BIH was observed [6.0 +/- 0.4 mV (mean +/- SE, N = 22)] even when the external pH was clamped constant by appropriately changing the CO2 tension. External HCO3- was required since aeration with CO2 at low external pH did not replicate the BIH. DIDS [4,4'-diisothiocyano-2,2'-disulfonic acid] did not abolish the effect. The hyperpolarization is unlikely to reflect the pH dependence of K+ channels alone, since the effect was not reduced by either 2 mM Ba2+ alone or 2 mM Ba2+ together with 50-100 microM quinidine. The BIH depends directly or indirectly on external Na+, since the sign of the polarization response was reversed either by replacing Na+ with N-methyl-D-glucamine or by blocking the Na+,K(+)-exchange pump with 50-100 microM ouabain. Replacement of external Cl- with NO3- or application of the Cl(-)channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate] depolarized the membrane and reversed the sign of the BIH. The response of the ciliary epithelium to HCO3- is complex and may arise from several mechanisms. We suggest that one important element is an anion channel whose conductance is reduced by bicarbonate and whose reversal potential is indirectly dependent on the operations of the Na+,K(+)-pump and a Cl(-)-linked symport.     

Evidence for Na+/H+ exchange and pH sensitive membrane voltage in cultured bovine corneal epithelial cells

Two methods were used to investigate cellular ion transport processes in confluent monolayers of cultured bovine corneal epithelial cells: measurements of membrane voltage (V) using conventional microelectrodes, and intracellular pH (pHi) measurements using the pH sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-4', 5'dimethyl-fluorescein. (1) V averaged -39.2 +/- 0.9 mV (mean +/- SEM, n = 71) with a range of -30 to -59 mV. Increasing extracellular potassium depolarized the cell membrane with a K+-slope of 43.3 mV/decade [K+] (for [K+] between 20 and 80 mM). Intracellular as well as extracellular acidification reversibly depolarized the cell membrane. Depolarization induced by 40 mM K+-pulses was smaller at extracellular pH (pHo) of 6.9 as compared to pHo = 7.9. These findings are compatible with a pH-sensitive K+ conductance. (2) During steady state pHi was 6.96 +/- 0.05 (mean +/- SEM, n = 7). After intracellular acidification, induced by NH4Cl-prepulse technique, pHi was regulated back towards normal steady state pHi. Application of 1 mM amiloride reversibly inhibited pHi recovery. Furthermore, pHi backregulation was inhibited by removing sodium from the extracellular solution. The effect was reversible after readdition of sodium. These findings suggest that a Na+/H+ exchange is present in corneal epithelial cells and participates in pHi backregulation after an intracellular acid load.     

Intracellular voltage recordings in bovine non-pigmented ciliary epithelial cells in primary culture

Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na+ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3 +/- 0.5 mV (n = 36). Relative K+ conductance was increased by extracellular alkalinization. Removing extracellular K+ led to a depolarization and readdition of K+ to K+ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na+/K+ ATPase is inhibited by K+ replacement, and that there is overshoot-activation of the pump when K+ is readded. Extracellular Cl- replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stilbene-sensitive Cl(-)-conductance. Removing HCO3- led to a Na+ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na+ was not influenced by DIDS or the extracellular HCO3(-)-concentration.     

Coupling of 22Na and 36Cl uptake in cultured pigmented ciliary epithelial cells: a proposed role for the isoenzymes of carbonic anhydrase

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.     

Na-dependent pHi regulatory mechanisms in native human retinal pigment epithelium.

This study provides the first information about pHi regulatory mechanisms in human retinal pigment epithelium (RPE). The experiments were carried out on fresh explant tissues from adult donor and fetal eyes, and pHi was measured using fluorescence microscopy techniques and the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In adult donor RPE, the resting pHi is 7.30 +/- 0.14 (mean +/- standard deviation; n = 6) in HCO3 Ringer's solution. In HCO3 Ringer's solution, apical Na removal caused rapid cell acidification with an initial rate of 0.40 +/- 0.10 pH U/min (n = 4). This Na-dependent acidification was partially inhibited by apical amiloride (n = 1) and DIDS (n = 1). In HCO3 Ringer's solution, pHi recovery from an acid load (NH4 prepulse) also was blocked by apical Na removal. In nominally HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells. These results suggest that the apical membrane of adult human RPE contains an Na/H exchanger and possibly a Na-dependent, DIDS-inhibitable pH regulatory mechanism, perhaps a NaHCO3 cotransporter. For the fetal RPE, the resting pHi was 7.16 +/- 0.10 (n = 9) and 7.19 +/- 0.10 (n = 20) in HCO3 and HCO3-free Ringer's solution, respectively. In HCO3 and HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells and the removal of apical Na caused cell acidification with an initial rate of 0.30 +/- 0.08 (n = 32) and 0.58 +/- 0.29 (n = 6) pH U/min, respectively. The pHi recovery from an acid load also was blocked by apical amiloride and apical Na removal. These results suggest that the apical membrane Na/H exchanger is the dominant acid extrusion mechanism in human fetal RPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the Na, K-ATPase pump in cultured bovine corneal endothelial cells

Bovine corneal endothelial cells in culture possess Na, K-ATPase pump sites, as measured by [3H]ouabain binding, and demonstrated an active ouabain-sensitive 86Rb+ uptake. The binding of [3H]ouabain, a specific inhibitor of Na, K-ATPase, was used to quantitative the density of Na, K-ATPase pump sites in bovine corneal endothelial cell cultures. [3H]ouabain binding was time-dependent and reached saturation after 1-2 hr. The specific binding represented more than 99% of the total cell-associated [3H]ouabain, and about 85% of this binding was abolished in the presence of K+ ions. The binding was concentration-dependent and saturated at a ouabain concentration of 2 X 10(-8)M with a dissociation constant (Kd) of 1.0 X 10(-8)M. The number of [3H]ouabain binding sites was maximal in sparse, activity growing cultures and decreased accompanying the development of a confluent monolayer. A pump density of 2.2 X 10(6) pump sites cell-1 was estimated for sparse cultures, declining to 0.8 X 10(6) pump site cell-1 at confluence. The activity of the Na, K-ATPase pump in bovine corneal endothelial cell cultures was evaluated by measuring 86Rb+ influx. Sparse and confluent cultures demonstrated 86Rb+ ouabain-sensitive uptake at a rate of 4.2 nmol (10(6) cells)-1 min-1 and 1.5 nmol (10(6) cells)-1 min-1, respectively. The ouabain-sensitive 86Rb+ uptake was linear for at least 30 min, while the ouabain-insensitive 86Rb+ uptake was slower and declined during the 30-min time period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical membrane properties of a cell clone derived from human nonpigmented ciliary epithelium

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.     

Ion transport mechanisms in cultured bovine corneal endothelial cells

Intracellular potential measurements of confluent monolayers of cultured bovine corneal endothelial cells were used to define passive ion transport processes in these cells. Previous studies (Jentsch et al., J. Membr. Biol. 78:103 (1984); Jentsch et al., J. Membr. Biol. 81:189 (1984] have provided the experimental basis for a cellular model, in which bicarbonate entry across the basolateral membrane is indirectly driven by a Na+/H+-exchanger, which is inhibitable by amiloride (1mM). Bicarbonate and sodium should leave the cell via an electrogenic bicarbonate sodium cotransport, which is inhibitable by the disulfonic stilbene derivates SITS or DIDS. This model is also consistent with results from transendothelial studies. In this paper, we briefly review the evidence we have obtained for this model and demonstrate, that the electrical response to sodium (depolarization upon Na+-removal) is neither due to an inhibition of Na+/K+-ATPase nor explainable in terms of changes in K+-conductance. This is concluded from the observation of these responses in the presence of ouabain (10(-4)M) or barium (1mM).     

Role of anions in nitric oxide-induced short-circuit current increase in isolated porcine ciliary processes

PURPOSE: To investigate how nitric oxide (NO) modulates short-circuit current (Isc) in isolated porcine ciliary processes. METHODS: Isc changes (Ussing-type chamber) induced either by the NO donors SNP or SIN-1, or by the cGMP analogue 8-pCPT-cGMP were assessed. The effect of inhibitors of guanylate cyclase (10 microM ODQ, 100 microM LY83583), protein kinase G (30 microM Rp-8-pCPT-cGMP, 3 microM KT 5823), protein kinase A (1 microM KT 5720), or protein kinase C (1 microM Go6983) on SNP- or 8-pCPT-cGMP-induced Isc changes were investigated. The effect of inhibitors of anion channel (100 microM niflumic acid, 1 mM DIDS, and 1 mM 9-AC), K+-channel (10 mM TEA, 10 mM BaCl2), Na+-channel blockers (1 mM amiloride), Na+-K+-2Cl- cotransporter inhibitor (0.5 mM bumetanide), or carbonic anhydrase inhibitor (1 mM acetazolamide) was studied. In Cl(-)- or HCO3(-)-free Krebs-Ringer solution, the effect of SNP- or 8-pCPT-cGMP-induced Isc changes was accessed. RESULTS: SNP, SIN-1, or 8-pCPT-cGMP increased Isc with a change in the potential difference that became more negative toward the nonpigmented epithelium (aqueous) side. The Isc increase induced by SNP or SIN-1, but not by 8-pCPT-cGMP, was prevented by ODQ and LY83583. SNP- and 8-pCPT-cGMP-induced Isc increases were prevented by Rp-8-pCPT-cGMP or KT5823 (but not by KT5720 or Go6983), or by niflumic acid, DIDS, 9-AC, or acetazolamide (but not by TEA, BaCl2, amiloride, or bumetanide). The effect of SNP and 8-pCPT-cGMP was abolished in Cl(-)- and reduced in HCO3(-)-free solutions. CONCLUSIONS: NO activates a guanylate cyclase-cGMP-protein kinase G pathway that appears to stimulate stroma-to-aqueous anionic transport, possibly Cl-, in porcine ciliary epithelium.