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.     

Basolateral Na(+)-K(+)-2Cl(-) cotransport in cultured and fresh bovine corneal endothelium

PURPOSE: To examine whether Na(+)-K(+)-2Cl(-) cotransport has the potential to contribute to corneal endothelial ion and fluid transport in cultured and fresh bovine corneal endothelial cells. METHODS: Cl- and Na+ sensitive fluorescent dyes were used to measure furosemide-dependent ion fluxes in cultured and fresh endothelial cells. Immunoblot analysis and immunofluorescence were used to determine expression and location of the Na(+)-K(+)-2Cl(-)cotransporter (NKCC1). RESULTS: Application of furosemide (50-100 microM) reduced Cl- and Na+ influx in approximately 50% of trials using cultured cells and only 10% of trials with fresh cells; however, in all cases pretreatment with furosemide slowed Cl- efflux when cells were bathed in Cl(-)-free Ringer's. Double-sided perfusion of cultured cells indicated that furosemide-sensitive Cl- fluxes were located on the basolateral side. Immunoblot analysis revealed 174-kDa bands in both fresh and cultured cells, but the bands were denser in fresh endothelial cells. Immunofluorescence showed distinct lateral membrane staining in addition to significant amounts of perinuclear staining. CONCLUSIONS: The Na(+)-K(+)-2Cl(-) cotransporter is present in both fresh and cultured bovine corneal endothelium, and the expression is apparently higher in the fresh cells. The cotransporter is present on the lateral membrane consistent with a role in loading endothelial cells with Cl-, thereby possibly contributing to a transendothelial Cl- flux. However, in the resting cell, net flux through the transporter is often not apparent.     

Characterization of Cl-/HCO3- exchange in cultured bovine pigmented ciliary epithelium

Many recent data indicate that transport of Cl- across the ciliary epithelium plays an important role in aqueous humor formation. We used 36Cl to investigate the pathways for Cl- transport in confluent monolayers of cultured bovine pigmented ciliary epithelial cells. Cl- uptake mainly occurred via a mechanism with typical characteristics of an anion exchanger, and could be stimulated by an outwardly directed HCO3- gradient. One mM SITS and 1 mM DIDS inhibited Cl- uptake by some 80-90%, the latter with an IC50 of about 20 microM. HCO3- stimulated Cl- uptake could be partly inhibited for furosemide and to a lesser extent by bumetanide, indicating an action of loop-diuretics on the anion exchanger. 36Cl- uptake was cis-inhibited by the halides Cl-, I- and Br-, by NO3-, formate and acetate. Inhibition of Cl- uptake by extracellular HCO3- was less effective in the absence of extracellular Na+, suggesting that not only HCO3- but also NaCO3- binds to the carrier. SO2/4-, cyclamate and gluconate did not significantly reduce Cl- uptake via the anion exchanger. DIDS-senstive Cl- uptake showed saturation kinetics with respect to the Cl- concentration with an apparent Km of 8 mM. Cl- efflux could be stimulated by external Cl- and HCO3- and was inhibited by DIDS. Thus, cultured bovine pigmented ciliary epithelial cells express a Cl-/HCO3- exchanger. A possible role of this carrier system for aqueous humor formation is discussed [corrected].     

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.     

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.     

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 [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 beta-adrenergic receptors in fresh and primary cultured bovine corneal endothelium

Homogenates of fresh bovine corneal endothelium and of cells from primary cultures exhibited high affinity binding of [3H]-dihydroalprenolol, a specific beta-adrenergic antagonist. The binding was rapid and reversible. Specific binding of the radioligand in each preparation was saturable with half-maximal binding occurring at 0.5 nM. Homogenates of fresh tissue consistently showed a higher maximal binding capacity than did those from cell cultures. Both homogenates bound adrenergic agents in a manner consistent with the labelling of beta-adrenergic receptors. The relative affinities of epinephrine and norepinephrine indicated that beta-adrenergic receptors in both preparations are of the beta 2 subtype. The beta-adrenergic agonist, isoproterenol, increased the cAMP content of intact, cultured endothelial cells 27-fold over control. This effect was completely blocked by the beta-adrenergic antagonist, propranolol. Bovine corneal endothelial cells in primary culture contain beta-adrenergic receptors which are essentially identical to those from in vivo sources and appear to be linked functionally to cAMP synthesis in these cells.     

Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium

Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.     

Dependence of cAMP meditated increases in Cl- and HCO(3)- permeability on CFTR in bovine corneal endothelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl(-) and HCO(3)(-) permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl(-) and HCO(3)(-) permeability and steady-state HCO(3)(-) flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by approximately 80% in CFTR siRNA treated cultures. Forskolin (10 microM) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO(3)(-) permeability was increased 2-fold by 10 microM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO(3)(-) permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (DeltapH) at 4h in control cultures was increased approximately 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline DeltapH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO(3)(-) permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO(3)(-) permeability.     

pH homeostasis in the frog retina: the role of Na+:HCO3- co-transport in the retinal pigment epithelium.

Transport of acid/base equivalents across the isolated frog retinal pigment epithelium (RPE) was studied by means of pH selective microelectrodes. Abrupt changes in retinal extracellular pH, from 7.40 to values between 6.66 and 7.66, were found to cause rapid changes in both intracellular pH and choroidal extracellular pH. The initial rates of these changes were reduced by more than 60%, when the cellular Na+:HCO3- co-transport system was inhibited by Na+ removal, or by administration of 1 mM SITS. It is concluded that RPE transcellular HCO3- transport changed in response to changes in retinal extracellular pH. If present in vivo these changes in RPE HCO3- transport would tend to stabilize retinal extracellular pH. It is suggested that cellular Na+:HCO3- cotransport plays a significant role for the retinal pH homeostasis.     

Sodium and potassium saturation kinetics of Na+K+-ATPase in plasma membranes from corneal endothelium: fresh tissue vs. tissue culture

The maximal velocity (Vmax) and the apparent dissociation constant (K0.5) of Na+K+-ATPase have each been estimated with respect to sodium and potassium ion activation. These estimations were made from the enzymatic activity of plasma membrane preparations derived from bovine corneal endothelial cells. The determinations were made on cells obtained from fresh tissue and from secondary tissue cultures. Two methods were used to obtain the estimates: the first used a combination of Eadie-Hofstee and Hill plots; the second used Eisenthal-Cornish-Bowden plots. The Vmax for sodium was 5.58-5.60 mumol Pi/mg protein/30 min for fresh tissue vs. 2.00-1.80 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 62-57 mM (fresh tissue) vs. 7.9-6.7 mM (tissue culture). Vmax for potassium was 4.28-4.00 mumoles Pi/mg protein/30 min for fresh tissue vs. 1.37-1.34 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 3.3-3.1 mM (fresh tissue) and 1.7-1.7 mM (tissue culture). The results indicate a lowered activity and change in affinity of the enzyme for the two ions in tissue cultures compared to fresh tissues. The detergent Lubrol W-X increased activity in both tissue sources. Sonication had no significant effect on the activity. Variations in pH (7-9) indicated that the highest activity was obtained at pH 7.8 for the enzyme in tissue culture while activity was highest at pH 8.0 for the enzyme in fresh tissues. These differences in kinetic activity suggest a response to changes in the ion requirements of these cells due to their environment, developmental stage or some other parameter.     

Pharmacological regulation of morphology and mitosis in cultured rabbit corneal endothelium

Cultured rabbit corneal endothelial cells elongate when grown in the presence of epidermal growth factor (EGF) and indomethacin (INDO); whereas maintenance of the differentiated polygonal cell shape is apparently dependent upon endogenous synthesis of prostaglandin E2 (PGE2). In the current study, the authors demonstrate morphological changes in phenotypically altered cells and identify two intracellular pathways which interdependently regulate endothelial cells. Morphometric and mitotic analyses of cultures treated with a variety of pharmacological agents indicate that both protein kinases A- and C-dependent pathways regulate cell shape and cell division in corneal endothelial cells. Marked intracellular reorganization is associated with the morphological changes in the endothelial cells. When stained with rhodamine conjugated phallicidin, polygonal endothelial cells have circumferential bands of f-actin at their borders. EGF and/or INDO induce elongation and redistribution of f-actin into a diffuse cytoplasmic reticulum. Transmission electron microscopy demonstrates loss of several characteristic morphological markers for endothelial cells in response to pharmacologically induced elongation. The elongated cells lose intracellular junctions, apical/basal polarity and rough endoplasmic reticulum. These ultrastructural markers and circumferential f-actin bands are restored in cultures supplemented with exogenous PGE2. Modulation of these pathways in vivo may regulate cellular migration and mitosis during wound closure, stress, trauma and with age.     

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.     

Enhancement of HCO(3)(-) permeability across the apical membrane of bovine corneal endothelium by multiple signaling pathways

PURPOSE: In this study, the involvement of signaling pathways in the regulation of HCO(3)(-) permeability across the apical membrane of the corneal endothelium was examined. METHODS: Cultured bovine corneal endothelial cells (CBCECs) were grown to confluence on permeable membranes. Apical and basolateral sides were perfused with a HCO(3)(-)-rich Cl(-)-free Ringer's solution (28.5 mM; pH 7.5). Relative changes in apical HCO(3)(-) permeability were assayed by pulsing the apical perfusion bath with a low-HCO(3)(-) Cl(-)-free Ringer's solution (2.85 mM; pH 6.5), in the presence or absence of agonists or inhibitors, and comparing the rates of change in intracellular pH (pH(i)), as measured with a pH-sensitive dye. Ca(2+)-activated signaling was measured with the Ca(2+)-sensitive dye Fura-2. Qualitative changes in membrane potential (E(m)) were measured with a voltage-sensitive dye. RT-PCR using calcium-activated chloride channel (CLCA)-specific primers was used to examine the expression of CLCA in the corneal endothelium. RESULTS: The adenoceptor agonist adenosine (20 M) enhanced HCO(3)(-) permeability by a factor of 2. Forskolin (40 microM) exerted a 6.3-fold increase of HCO(3)(-) permeability, which was inhibited by the Cl(-) channel blockers, glibenclamide (50 microM) and niflumic acid (100 microM). Adenosine triphosphate (ATP) and ATPgammaS, P(2) receptor agonists that increased intracellular Ca(2+) in corneal endothelium, enhanced HCO(3)(-) permeability by 87% and 79%, respectively. ATPgammaS induced depolarization of the E(m), consistent with anion channel activation, rather than activation of Ca(2+)-dependent K(+) channels, which could secondarily increase extrusion of anions by E(m) hyperpolarization. Cyclopiazonic acid (CPA), an endoplasmic reticulum (ER) Ca(2+)-pump inhibitor that increased [Ca(2+)](i), also enhanced HCO(3)(-) permeability by 95%. Both the calmodulin kinase II (CaMKII) inhibitor KN-62 and the PKC inhibitor bisindolylmaleimide I (BIMI), decreased HCO(3)(-) permeability induced by ATPgammaS. The PKC activator PMA also increased HCO(3)(-) permeability by a factor of 1.8. RT-PCR using CLCA-specific primers showed the expression of CLCA1 in both fresh and cultured BCECs. CONCLUSIONS: Activation of adenoceptors and purinoceptors enhances HCO(3)(-) permeability across the apical membrane of the cultured corneal endothelium. Multiple signaling pathways (PKA, PKC, and Ca(2+)/CaMKII) contribute to the HCO(3)(-) transport in cultured corneal endothelium. Both cAMP and Ca(2+)-activated Cl(-) channels (possibly CLCA) may be involved in HCO(3)(-) transport.     

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)     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的FasL表达

目的：明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义,材料和方法：采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养,抗FasL染色呈阳性;IPE细胞及对照组均为阴性。结论：在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而PE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非FasL－FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的

目的明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义。材料和方法采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养。抗FasL抗体、S-P试剂盒（DAB显色）对传一代IPE细胞和角膜内皮细胞进行免疫组化染色。结果角膜内皮细胞FasL染色呈阳性;IPE细胞及对照组均为阴性。结论在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而IPE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非Fas-FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

Histamine H1 receptor-mediated Ca2+ signaling in cultured bovine corneal endothelial cells.

The corneal endothelium pumps ions and water from the stroma to the aqueous humor, maintaining corneal transparency. This report investigates the possibility that cultured corneal endothelial cells express neurohormonal Ca2+ signaling pathways employed by other epithelia to regulate transport or other cellular functions. Agonist-stimulated changes in intracellular calcium ([Ca2+]i) in single bovine corneal endothelial cells (BCEC) derived from confluent cultures were measured by microspectrofluorimetry using the Ca(2+)-sensitive probe, fura 2. Mean resting [Ca2+]i in BCEC was 46 +/- 2 nM (n = 124). The muscarinic cholinergic agonist, carbachol, did not mobilize Ca2+, whereas histamine induced a rapid increase in [Ca2+]i to initial peak levels of 549 +/- 22 nM (n = 46) at maximally stimulating doses. The initial rise in [Ca2+]i in response to histamine was dose dependent, with a minimum effective dose of 50 nM, EC50 = 0.84 mumol/l, and a maximum effective dose of 10 mumol/l. [Ca2+]i decreased from the initial peak, but then stabilized to form an agonist-dependent sustained elevation or abruptly fell back to baseline to begin oscillatory fluctuations. The initial peak was insensitive to removal of extracellular calcium (Ca2+o), whereas subsequent elevations in [Ca2+]i or sustained [Ca2+]i oscillations required Ca2+o. The amplitude of the oscillations in [Ca2+]i increased with an increase in [histamine]. However, frequency was independent of [histamine] (mean = 0.62 spikes min-1 +/- 0.06, n = 33). Histamine-induced Ca2+ mobilization was inhibited by the H1 receptor antagonist triprolidine, but was unaffected by ranitidine (H2 antagonist) or thioperamide (H3 antagonist).(ABSTRACT TRUNCATED AT 250 WORDS)