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-.     

Intracellular pH regulation in basal corneal epithelial cells measured in corneal explants: characterization of Na/H exchange

Intracellular pH (pHi) was measured in basal corneal epithelial cells from fresh corneal explants using the pH sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The overlying superficial and wing cells were removed by mechanical scraping to expose basal cells attached to their basal lamina. Tissue pieces with attached, dye-loaded basal cells were mounted in a microscope-stage-perfusion chamber which allowed rapid changes of Ringer's bathing solutions while measuring BCECF fluorescence. In NaCl-Ringer's (bicarbonate free). pHo 7.40, resting cell pHi was 7.34 +/- 0.03 (+/- S.E.M., n = 31). Buffering capacity measured by NH4Cl treatment was 31 mM pH at pHi 7.34 and increased with decreasing pHi. Recovery from 20 mM NH4Cl-induced acid loads was dependent on the presence of Na and inhibited by 1 mM amiloride. Adding amiloride to resting cells caused a slow, reversible acidification (0.04 pH units min-1). These results indicate the presence of Na:H exchange, its role in responding to acid loads and in maintaining resting cell pHi. Activation of Na:H by Nao showed simple saturation kinetics, with Km = 44 mM. Net proton efflux via Na:H exchange increased with decreasing pHi and was enhanced by depleting cells of Nai, suggesting roles for both pHi and Nai in control of Na:H activation.     

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)     

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)     

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.     

Comparative studies of furosemide effects on membrane potential and intracellular chloride activity in human and rabbit ciliary epithelium.

Furosemide (1 mM), a potent loop diuretic, caused a 10-mV (n = 14) depolarization of the intracellular potential difference (PDI) of isolated rabbit ciliary epithelium (CE), but produced a 9-mV (n = 5) hyperpolarization of PDI of isolated human CE. In rabbit CE, furosemide consistently depolarized PDI by 13, 7 and 8 mV in HCO3(-)-free Ringer, Na(+)-free Ringer and after BaCl2 treatment, respectively. The depolarization of PDI was reduced to 2 mV (n = 11) in Cl(-)-free conditions. A hyperpolarization of PDI caused by furosemide that was quantitatively similar to that seen in normal Ringer also occurred in human CE during immersion in HCO3(-)-free Ringer, Na(+)-free Ringer and after BaCl2 treatment. There was a small hyperpolarization (3 mV) of PDI in Cl(-)-free conditions. Human or rabbit tissue-cultured nonpigmented ciliary epithelial cells were loaded with the Cl(-)-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) in hypotonic solution (145 mosm) for 4 min at 37 degrees C. Furosemide decreased intracellular Cl- fluorescence activity of both human and rabbit ciliary epithelial cells by 30 +/- 5 (n = 8) and 25 +/- 7% (n = 13), respectively, when the cells were immersed in Cl(-)-rich solution. It is suggested that a furosemide-sensitive Cl- movement exists in both rabbit and human CE, although the mode of Cl- movement to the aqueous across CE may differ between these species.     

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.     

Intracellular pH regulation in fresh and cultured bovine corneal endothelium. I. Na+/H+ exchange in the absence and presence of HCO3-.

A detailed comparison of intracellular pH (pHi) regulatory mechanisms was made between fresh (FBCE) and cultured (CBCE) bovine corneal endothelium to: (1) identify the ion transport mechanisms that could directly or indirectly affect transendothelial HCO3- transport; and (2) determine if cultured cells could serve as a model for studying transendothelial bicarbonate transport. We used the pH-sensitive fluorescent probe BCECF-AM to measure pHi. FBCE and CBCE readily incorporated the dye and showed pHi calibration curves that were not significantly different with respect to pK (7.39 for FBCE and 7.35 for CBCE). Resting pHi in bicarbonate free Ringer's (pH 7.5) was significantly lower in cultured cells (7.17 +/- .02, n = 50) than in fresh cells (7.30 +/- .02, n = 54). Steady-state pHi was reduced by addition of 0.5 mmol/l amiloride, a Na+/H+ exchange blocker (-.16 pH U for FBCE, -.18 for CBCE) or removal of Na+ (-.47 pH U for FBCE, -.51 for CBCE). Recovery from an (NH4)2SO4-induced acid load was blocked by Na+ removal, and the rate of recovery was inhibited 74% and 79% in the presence of amiloride for FBCE and CBCE, respectively. The dependence of proton efflux on Na+0 showed simple saturating kinetics (apparent Km = 30 and 31 mmol/l for FBCE and CBCE, respectively), consistent with the presence of Na+/H+ exchange in FBCE and CBCE. Na+/H+ exchange activity, as measured by amiloride-sensitive acid recovery, was inversely proportional to pHi. The activity in FBCE was about twice that in CBCE. Furthermore, the zero flux point for Na+/H+ exchange was at least 0.1 pH U higher in FBCE. Changing from bicarbonate-free Ringer's to bicarbonate Ringer's (5% CO2/28 mmol/l HCO3-, pH 7.5) induced a rapid and short acidification followed by an alkalinization .09 and .18 pH U above the starting pHi for FBCE (final pHi 7.37) and CBCE (final pHi 7.33), respectively. This transition was unaffected by amiloride. Similarly, amiloride had no effect on resting pHi in bicarbonate Ringer's for FBCE or CBCE, indicating that Na+/H+ exchange does not contribute to the maintenance of the steady-state resting pHi in bicarbonate Ringer's. Although most of the characteristics of Na+/H+ exchange for FBCE and CBCE were similar, the differences in overall activity and the low levels of activity in resting cells must be considered when using CBCE to model ion coupled fluid transport in BCE.     

Chloride dependent intracellular pH effects of external ATP in cultured human non-pigmented ciliary body epithelium

PURPOSE: To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. RESULTS: In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient. CONCLUSION: We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.     

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.     

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)     

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)     

The electroretinogram, standing potential, and light peak of the perfused cat eye during acid-base changes

DC recordings of light-evoked responses were made in the isolated, arterially perfused cat eye during four acid-base changes designed to alter intracellular pH (pHi) without appreciably altering extracellular pH (pH0). Two acid-base changes were designed to decrease pHi: substitution of high pCO2, high [HCO3-] perfusate for control perfusate and injection of NaHCO3 solution (pH 7.4) into the control perfusate. The initial effects of these two changes were similar: standing potential decreased, the b-wave amplitude decreased, and the c-wave amplitude increased. Subsequent effects, which included rebounds, were complex. The two other acid-base changes were designed to increase pHi: substitution of low pCO2, low [HCO3-] perfusate for the control perfusate and injection of NH4Cl solution into the control perfusate. The initial effects of these two changes were similar; the effects were opposite to those described above for acid-base changes (i) and (ii). The effects of all four acid-base changes were reversible. From these and previously published findings on the effects of pH0, we conclude that during acid-base changes, the initial change in the standing potential varies directly with pHi/pH0, the initial change in b-wave amplitude varies directly with pHi, and the initial change in c-wave amplitude varies inversely with pHi. We also studied the effects of the four acid-base changes on the light peak, a slow voltage response to light generated by the retinal pigment epithelium. Under acid-base changes (i), (ii), and (iii) the light peak was severely depressed. Injection of 2 mM NH4Cl, acid-base change (iv), had little effect on the light peak; however, injection of 5-10 mM NH4Cl did depress the light peak. These results may be interpreted in several ways, for example, the light peak may be sensitive to changes in [HCO-3]0 or to pHi. In any case, we conclude that pH0 is a relatively minor factor influencing the amplitude of the light peak.     

Evidence for a Na(+)-Cl(-)-H(+)-HCO3- exchange system in the mammalian lens.

36Cl- efflux was studied in the isolated rat lens under two conditions that are known to decrease internal pH. The first follows exposure to a pulse of ammonium chloride (50 mM) and the second accompanies exposure to an acidified propionate (20 mM) solution. Under acidifying conditions, a stimulation in 36Cl- efflux was observed, that was abolished on removing external Na+ and also on removing external Cl- and HCO3-. In the absence of external Cl-, the presence of HCO3- (16 mM) resulted in an increase in 36Cl- efflux during internal acidification. In the absence of internal acidification, the addition of 0.1 mM dibutyrylcAMP or 0.5 mM IBMX to the external medium produced a rapid increase in 36Cl- efflux. This stimulation was reduced by 0.2 mM SITS. Neither cAMP or IBMX had any significant effect on the electrical resistance of the lens membranes. It is suggested that a coupled SITS-sensitive, Na(+)-Cl(-)-H(+)-HCO3- exchange mechanism is activated when the lens internal pH falls and further that cAMP may play a role in regulating this mechanism.     

Metabolic acidosis-induced retinopathy in the neonatal rat

PURPOSE: Carbon dioxide (CO2)-induced retinopathy (CDIR) in the neonatal rat, analogous to human retinopathy of prematurity (ROP), was previously described by our group. In this model, it is possible that CO2-associated acidosis provides a biochemical mechanism for CDIR. Therefore, the effect of pure metabolic acidosis on the developing retinal vasculature of the neonatal rat was investigated. METHODS: A preliminary study of arterial blood pH was performed to confirm acidosis in our model. In neonatal rats with preplaced left carotid artery catheters, acute blood gas samples were taken 1 to 24 hours after gavage with either NH4Cl 1 millimole/100 g body weight or saline. In the subsequent formal retinopathy study, 150 newborn Sprague-Dawley rats were raised in litters of 25 and randomly assigned to be gavaged twice daily with either NH4Cl 1 millimole/100 g body weight (n = 75) or saline (n = 75) from day 2 to day 7. After 5 days of recovery, rats were killed, and retinal vasculature was assessed using fluorescein perfusion and ADPase staining techniques. RESULTS: In the preliminary pH study, the minimum pH after NH4Cl gavage was 7.10+/-0.10 at 3 hours (versus 7.37+/-0.03 in controls, mean +/- SD, P < 0.01). In the formal retinopathy study, preretinal neovascularization occurred in 36% of acidotic rats versus 5% of controls (P < 0.001). Acidotic rats showed growth retardation (final weight 16.5+/-3.0 g versus 20.2+/-2.6 g, P < 0.001). The ratio of vascularized to total retinal area was smaller in acidotic rats (94%+/-4% versus 96%+/-2%, P < 0.001). CONCLUSIONS: Metabolic acidosis alone induces neovascularization similar to ROP in the neonatal rat. This suggests a possible biochemical mechanism by which high levels of CO2 induce neovascularization and supports the suggestion that acidosis may be an independent risk factor for ROP.     

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.     

Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells

The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.     

Characterization of active ion transport across primary rabbit corneal epithelial cell layers (RCrECL) cultured at an air-interface

Previously, we reported the development of a primary culture model of tight rabbit corneal epithelial cell layers (RCrECL) characterizing bioelectric parameters, morphology, cytokeratin, and passive permeability. In the present study, we specifically evaluated the active ion transport processes of RCrECL cultured from either pigmented or albino rabbits. Primary cultured RCrECL were grown at an air-interface on Clear-Snapwells precoated with collagen/fibronectin/laminin and mounted in a modified Ussing-type chamber for the evaluation of their active ion transport processes under short-circuited conditions. Contribution of active Na(+) and Cl(-) transport to overall short-circuit current (I(sc)) was evaluated by removing Na(+) and Cl(-), respectively, from bathing fluids of RCrECL and measurements of net fluxes of Na(+) and Cl(-) using (22)Na and (36)Cl, respectively. Amiloride and benzamil were used to determine the role of apical Na(+)-channel activities to net Na(+) fluxes. N-phenylanthranilic acid (NPAA), ouabain, BaCl(2) and bumetanide were used to determine the role of basolateral Na,K-ATPase, apical Cl(-)-channel, and basolateral K(+)-channel and Na(+)(K(+))2Cl(-)-cotransporter activities, respectively, in active ion transport across RCrECL. I(sc) of RCrECL derived from pigmented rabbits was comprised of 64+/-2% and 44+/-5% for active Na(+) and Cl(-) transport, respectively, consistent with net Na(+) absorption and Cl(-) secretion of 0.062+/-0.006 and 0.046+/-0.008 muEq/cm(2)/hr estimated from radionuclide fluxes. Apical amiloride and benzamil inhibited I(sc) by up to approximately 50% with an IC(50) of 1 and 0.1 microm, respectively, consistent with participation of apical epithelial Na(+)-channels to net Na(+) absorption across RCrECL cultured from pigmented rabbits. Addition of ouabain to the basolateral, NPAA to the apical, BaCl(2) to the basolateral and bumetanide to basolateral fluid decreased I(sc) by 86+/-1.5%, 53+/-3%, 18+/-1.8% and 13+/-1.9% in RCrECL cultured from pigmented rabbits, while 85+/-0.7%, 36+/-1.6%, 38+/-1.8% and 15+/-3.5% decreases are observed for RCrECL from albino rabbits, respectively. Air-interface cultured RCrECL from either pigmented or albino rabbits exhibited active ion transport properties similar to those present in excised tissues. This primary culture system may be a reliable in-vitro model for mechanistic characterization of corneal epithelial function and regulation of transport properties.     

A Na+/H+ exchanger and its relation to oxidative effects in plasma membrane vesicles from lens fibers.

The existence of a Na+/H+ exchange mechanism and its role in the regulation of lens fiber cell intracellular pH, as well as the effect of oxidants and antioxidants, have been examined in vesicular preparations from spiny dogfish and bovine eyes. The fluorescent probes, SBFI (sodium-binding benzofuran isophthalate) for Na+ and SNARF-1 (seminaphthorhodafluor-1) for H+, were used to determine fluorescent ratios in a dual-wavelength spectrofluorimeter. The results show that: (1) the plasma membrane vesicles can be purified from lens fiber (52.5% of the vesicles were in the right-side out orientation for the fish eyes and 56.3% for the bovine eyes, show enrichment for the membrane marker activities and have an average size of 0.2-0.5 microns); (2) the influx of Na+ was dependent on an outwardly directed pH gradient with the uptake of Na+ sensitive to 500 microM amiloride but not affected by 50 microM valinomycin and 50 mM K gluconate; (3) when the pHo (extravesicular pH) of the vesicles was set at 8.1 and pHi (intravesicular pH) to 6.1, an inwardly directed Na+ gradient caused an increase in intravesicular pH by about 0.3 pH unit, and in bovine lens fiber vesicles the Na+ influx is highly dependent on the intravesicular pH (or on the pHo/pHi gradient); (4) 50 microM H2O2 increases the Na+/H+ exchange rate by 174% in the vesicles derived from fish lens fibers. Similarly, 100 microM H2O2 stimulates the Na+/H+ exchange rate by 194% in bovine eye fibers. This activation is prevented by preincubation with GSH (reduced glutathione) but not with GSSG (oxidizing glutathione). We conclude that a Na+/H+ exchange mechanism is present in the lens fiber membranes. This exchange possibly regulates intracellular pH and controls the intracellular Na+ concentration. The sensitivity of the Na+/H+ exchanger to the oxidant, hydrogen peroxide (H2O2) and the protection by the antioxidant GSH suggests a possible role for the Na+/H+ exchange mechanism in the formation of cataracts.