Search PubMed⌕ Search

Biomedical subjects

N A Delamere

Publications and source records attributed to N A Delamere.

At least 19 recordsLinked to original sources
PubMed2001

H+-ATPase-mediated cytoplasmic pH-responses associated with elevation of cytoplasmic calcium in cultured rabbit nonpigmented ciliary epithelium.

Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H+-ATPase activity in cultured rabbit nonpigmented ciliary epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 microm), by exposure to a mixture of epinephrine (1 microm) + acetylcholine (10 microm) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 nm bafilomycin A1, a specific H+-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H+-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H+-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO2. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H+-ATPase-mediated protein export under acid conditions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2001

The inhibitory influence of endothelin on active sodium-potassium transport in porcine lens.

PURPOSE: Endothelin (ET)-1 is known to inhibit active NaK transport by as much as 50% in kidney tubule and other tissues. The presence of low levels of ET-1 in aqueous humor combined with the potential for release of ET-1 from ciliary processes suggests that the lens could be exposed to ET-1 in vivo. In this study, experiments were conducted to examine the influence of ET-1 on active NaK transport in porcine lens. METHODS: The rate of Na,K-adenosine triphosphatase (Na,K-ATPase) dependent potassium transport was determined by measurements of ouabain-sensitive potassium (86Rb) uptake by intact lenses. Lens sodium content was measured by atomic absorption spectrophotometry. Cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. Cytoplasmic calcium concentration in cultured porcine lens epithelium was measured by a fluorescence technique using fura-2. RESULTS: In the presence of ET-1 (0.1 nM or higher concentration), the rate of ouabain-sensitive potassium (86Rb) uptake was diminished. The ET receptor antagonist PD145065 (2 microM) suppressed the inhibitory effect of ET-1 (100 nM) on 86Rb uptake. Sodium content was detectably increased in lenses exposed to ET-1 for 24 hours. Forskolin (1 microM) caused an eightfold increase of cAMP in the lens epithelium, but no increase of cAMP was detected in the epithelium of lenses treated with ET-1. Genistein (150 microM), an inhibitor of tyrosine kinases, abolished the inhibitory effects of ET-1 on lens 86Rb uptake. ET-1 caused an increase of cytoplasmic calcium concentration in cultured porcine lens epithelium. The cytoplasmic calcium response to ET-1 was inhibited by PD145065 and genistein. CONCLUSIONS: The results of the present study suggest that ET-1 causes inhibition of lens active Na-K transport by a mechanism that involves activation of ET receptors. Activation of ET receptors also causes an increase of cytoplasmic calcium concentration in cultured lens epithelial cells. Both responses to ET-1 appear to have a tyrosine kinase step, because they could be prevented by genistein. The physiological purpose of an ET-1-induced reduction in the rate of active Na-K transport by the lens is unknown at this time.

Animals↗
PubMed2000

Studies on H(+)-ATPase in cultured rabbit nonpigmented ciliary epithelium.

Studies were conducted to examine the influence of the H(+)-ATPase inhibitor bafilomycin A(1) on cultured rabbit nonpigmented ciliary epithelial cells (NPE). Cytoplasmic pH and sodium concentrations were measured by digital fluorescence microscopy using BCECF and SBFI respectively. In some experiments, cell sodium content was measured by atomic absorption spectroscopy. Added alone, bafilomycin A(1) (100 nm) failed to change cytoplasmic pH but it caused an increase of cytoplasmic sodium concentration which occurred within 10 min. It is likely that the rise of cytoplasmic sodium concentration was responsible for the stimulation of active sodium-potassium transport which occurred in bafilomycin A(1)-treated cells as judged by a 50% increase of ouabain sensitive potassium ((86)Rb) uptake. In bafilomycin A(1)-treated cells, but not in control cells, dimethylamiloride (DMA) inhibited ouabain-sensitive potassium ((86)Rb) uptake in a dose-dependent manner with an IC(50) of approximately 2 microm. DMA (10 microm) also prevented the increase of cytoplasmic sodium caused by bafilomycin A(1). Added alone, DMA (10 microm) failed to change cytoplasmic sodium content but reduced cytoplasmic pH by approximately 0.4 pH units. In cells that first received 10 microm DMA, the subsequent addition of bafilomycin A(1) (100 nm) caused a further cytoplasmic pH reduction of approximately 0.3 pH units. Taken together, the results suggest H(+)-ATPase might contribute to the regulation of basal cytoplasmic pH in cultured NPE. In the presence of bafilomycin A(1), Na-H exchanger activity appears to be stimulated, so stabilizing cytoplasmic pH but resulting in an increase of cytoplasmic sodium concentration and consequent stimulation of active sodium-potassium transport.

Animals↗
PubMed1999

Stimulation of active sodium-potassium transport by hydrogen peroxide in cultured rabbit nonpigmented ciliary epithelium.

PURPOSE: Studies were conducted to examine the effect of hydrogen peroxide on active sodium-potassium transport in a cell line derived from nonpigmented ciliary epithelium of the rabbit eye. METHODS: Studies were carried out using a rabbit nonpigmented ciliary epithelium cell line. 86Rb uptake by intact cells was measured in the presence or absence of ouabain. The ouabain-sensitive potassium (86Rb) uptake rate was used as an index of the rate of active sodium-potassium transport. Cell sodium content was measured by atomic absorption spectrophotometry. Na,K-ATPase activity was determined by measuring ATP hydrolysis in the presence or absence of ouabain, using membrane material isolated by centrifugation of cell homogenates. RESULTS: Ouabain-sensitive potassium (86Rb) uptake rate measured in cells that had been preincubated with 200microM hydrogen peroxide for either 30 min or 60 min was increased to 196% and 181% of the control uptake rate, respectively. Lesser concentrations of hydrogen peroxide caused lesser degrees of stimulation. 200microM hydrogen peroxide caused an increase of cell sodium content. Such a change of cell sodium content is likely to be responsible, at least in part, for the observed stimulation of active sodium-potassium transport. However, the response may also be partly dependent on activation of a protein kinase since the serine/threonine protein kinase inhibitors staurosporine (1microM) and H-89 (20microM) were both found to prevent the stimulatory effect of 200microM hydrogen peroxide on ouabain-sensitive potassium (86Rb) uptake. Interestingly, neither H-89 nor staurosporine prevented the elevation of sodium content in cells that received 200microM hydrogen peroxide. CONCLUSIONS: Taken together, these findings suggest a low concentration of hydrogen peroxide causes increased sodium entry into the cell and also activates a protein kinase-dependent mechanism for sodium pump stimulation. The protein kinase-dependent mechanism does not appear to be triggered by an increased rate of sodium entry since staurosporine did not prevent the stimulation of ouabain-sensitive potassium (86Rb) uptake elicited by an increase in sodium permeability caused by amphotericin B.

Animals↗
PubMed1999

Genistein inhibits the regulation of active sodium-potassium transport by dopaminergic agonists in nonpigmented ciliary epithelium.

PURPOSE: To determine whether dopamine receptor stimulation regulates Na,K-ATPase-mediated ion transport in cultured nonpigmented ciliary epithelium (NPE). METHODS: Using a rabbit NPE cell line, active Na-K transport activity was determined by measuring ouabain-sensitive potassium (86Rb) uptake in cell monolayers. Western blot analysis of membrane material obtained from cell homogenates was conducted to examine tyrosine phosphorylation of membrane proteins. RESULTS: Ouabain-sensitive potassium (86Rb) uptake was inhibited in the presence of either dopamine or the D1-selective agonist SKF82958. The response was suppressed by SCH23390, a D1 antagonist, but not by sulpiride, a D2-selective antagonist. Quinpirole, a D2-selective agonist, did not cause inhibition of ouabain-sensitive potassium (86Rb) uptake. Cyclic adenosine monophosphate (cAMP) was detectably increased in SKF82958-treated cells, although the concentration of SKF required to elevate cell cAMP was higher than the concentration needed to inhibit ouabain-sensitive potassium (86Rb) uptake. The protein kinase A inhibitor H89 prevented the 86Rb uptake response to SKF82958. Genistein, an inhibitor of tyrosine kinases, also prevented the 86Rb uptake response to SKF82958. Membrane material isolated from cells exposed to SKF82958 showed an increase in the density of several phosphotyrosine bands. These changes in phosphotyrosine immunoblot density were not observed in material isolated from cells that received either genistein or SCH23390 before SKF82958 treatment. CONCLUSIONS: The results of this study suggest D1 agonists cause a reduction of Na,K-ATPase-mediated ion transport by a mechanism that could involve a tyrosine kinase step.

Animals↗
PubMed1999

Thrombin inhibits active sodium-potassium transport in porcine lens.

PURPOSE: Although thrombin is best known for its role in blood coagulation, it has been reported to change the activity of ion motive ATPases in some tissues. In the present study, experiments were conducted to determine the influence of thrombin on active sodium-potassium transport in porcine lenses. METHODS: Ouabain-sensitive potassium (86Rb) uptake by intact porcine lenses was used as an index of Na,K-ATPase-mediated active sodium-potassium transport. Na,K-ATPase activity was measured by determining ouabain-sensitive ATP hydrolysis in isolated membrane material. RESULTS: In the presence of thrombin (1 unit/ml) the rate of ouabain-sensitive potassium (86Rb) uptake was reduced by 40% to 60%, but ouabain-insensitive potassium (86Rb) uptake was unchanged. The inhibitory effect of thrombin on ouabain-sensitive potassium (86Rb) uptake was suppressed in the presence of hirudin (an antagonist for thrombin receptors) but persisted in the presence of amphotericin B (a pseudo ionophore that effectively clamps plasma membrane sodium permeability at a high value). Enzyme measurements showed ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was significantly inhibited in membrane material isolated from the capsule-epithelium of lenses, which had been pretreated with thrombin for 30 minutes. However, thrombin failed to exert a direct inhibitory effect on Na,K-ATPase activity when added directly to membrane fragments isolated from the epithelium of control (nonincubated) lenses. Both genistein and herbimycin (tyrosine kinase inhibitors) suppressed the effect of thrombin on the 86Rb uptake response. Results from Western blot studies suggested that tyrosine kinases are activated in the epithelium of lenses exposed to thrombin. CONCLUSIONS: The results suggest the inhibitory effect of thrombin on lens active sodium-potassium transport could involve the activation of a receptor-second-messenger mechanism in intact lens cells. The response appears to involve a tyrosine kinase-mediated step. The functional significance of the thrombin-mediated change of lens active sodium-potassium transport is unclear since appreciable amounts of thrombin may only be presented to the lens during instances of blood-aqueous-barrier breakdown. It is possible that lens receptors are functionally activated by other proteases, possibly cathepsins, which may enter aqueous humor from the ciliary body.

Animals↗
PubMed1998

Cytoplasmic pH responses to carbonic anhydrase inhibitors in cultured rabbit nonpigmented ciliary epithelium.

Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pHi) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pHi was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pHi. We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pHi 0.52 +/- 0.10 pH units. In the presence of DBI, the magnitude of pHi reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 +/- 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by approximately 40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pHi alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pHi changes that might alter the function of other ion transporters and channels in the NPE.

Acetazolamide↗
PubMed1998

Partial inhibition of Na,K-ATPase activity in cultured rabbit non-pigmented ciliary epithelium following an episode of cytoplasmic ATP depletion.

Ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured in digitonin-permeabilized monolayers of cultured cells derived from rabbit non-pigmented ciliary epithelium. Diminished Na,K-ATPase activity was observed in cells that had been pre-treated 10 min with the protein kinase C activator, PDBu, as well as in cells that had been cooled to 4 degrees C for 4 h then rewarmed to 37 degrees C for 30 min (cool-rewarm manoeuvre). In the intact cells, ouabain binding was not decreased either by PDBu treatment or the cool-rewarm manoeuvre. However, both PDBu and the cool-rewarm manoeuvre increased the rate of ouabain-sensitive potassium (86Rb) uptake measured in intact cells. Cell ATP content was diminished in PDBu-treated cells and cells subjected to the cool-rewarm manoeuvre. We suggest that an episode of ATP depletion might initiate a mechanism which causes lasting, partial inhibition of Na,K-ATPase activity. In keeping with this suggestion, diminished Na,K-ATPase activity was observed in cells that had been pre-treated 20 min with the metabolic inhibitors CCCP or rotenone and in cells pre-treated 2.5 h in dextrose-free medium. This study illustrates that Na,K-ATPase activity measured in the permeabilized cell is a complex parameter which is not necessarily a reliable indicator of sodium pump responses in the intact cell.

Adenosine Triphosphate↗
PubMed1998

The influence of ascorbic acid on active sodium transport in cultured rabbit nonpigmented ciliary epithelium.

PURPOSE: Cultured rabbit nonpigmented ciliary epithelium (NPE) transports ascorbic acid (ASC) inward through a sodium-dependent mechanism. This study was conducted to test whether Na-K transport is activated to export the additional sodium, which enters the cell in cotransport with ASC. METHODS: Studies were conducted using a cell line derived from rabbit NPE. ASC uptake was measured using [14C]ascorbic acid. The ouabain-sensitive potassium (86Rb) uptake rate was measured as an index of active Na-K transport. Cellular sodium was measured by atomic absorption spectrophotometry or SBFI fluorescence. RESULTS: In the presence of 200 microM ASC, ouabain-sensitive potassium (86Rb) uptake rate increased approximately 70%; lesser concentrations of ASC produced lesser increases. Phloridzin (100 microM) inhibited ASC uptake and inhibited the stimulatory effect of external ASC on 86Rb uptake. Dehydroascorbic acid (DHA) did not increase 86Rb uptake. Neither DHA nor ASC altered the Na,K-ATPase activity measured in isolated membrane material. External ASC appeared to stimulate active sodium transport through a mechanism involving an increase of cytoplasmic sodium. In the presence of 200 microM ASC, cellular sodium increased approximately 26%; studies with cells, sodium loaded by nigericin treatment, suggested that this sodium increase could account for the degree of 86Rb uptake stimulation observed in ASC-treated cells. However, the cellular sodium increase could not be explained simply on the basis of sodium entry through the ASC transporter. An additional sodium-entry pathway seemed to be activated in cells that accumulated ASC. Dimethylamiloride (DMA) abolished both the cellular sodium increase and the 86Rb uptake stimulation caused by ASC. DMA did not prevent ASC uptake. CONCLUSIONS: ASC significantly stimulated active Na-K transport in cultured NPE. The mechanism appeared to involve activation of a DMA-sensitive sodium entry pathway, which caused cytoplasmic sodium concentration to increase.

Amiloride↗
PubMed1998

Na,K-ATPase polypeptide upregulation responses in lens epithelium.

PURPOSE: In a previous study, an increase in Na,K-ATPase alpha 2 expression was detected in the epithelium of porcine lenses exposed to amphotericin B, an ionophore that also increases lens sodium and stimulates active sodium transport. The purpose of the present study was to determine whether an increase of Na,K-ATPase alpha 2 synthesis is a response to an episode of rapid Na-K transport or whether the increase in lens sodium alone can initiate the response. METHODS: Western blot analyses were conducted to probe for Na,K-ATPase alpha polypeptides in membrane material isolated from porcine lens epithelium. Ouabain-sensitive adenosine triphosphate hydrolysis was used as an index of Na,K-ATPase activity, and lens ion content was determined by atomic absorption spectrophotometry. 86-Rubidium (86Rb) uptake was measured as an indicator for active potassium transport. RESULTS: 86Rb uptake was markedly diminished in lenses exposed to dihydro-ouabain (DHO), signifying inhibition of active sodium-potassium transport. Consistent with this, the sodium content of DHO-treated lenses increased. By western blot analysis, a marked increase of Na,K-ATPase alpha 2 polypeptide could be detected in the epithelium of DHO-treated lenses. To rule out the possibility that apparent stimulation of Na,K-ATPase alpha 2 synthesis stemmed from binding of DHO to Na,K-ATPase sites, experiments were conducted to confirm an increase of Na,K-ATPase alpha 2 polypeptide in the epithelium of lenses exposed to low-potassium medium to inhibit active sodium-potassium transport. Consistent with the apparent increase of Na,K-ATPase polypeptide, Na,K-ATPase activity was detectably increased in epithelial material isolated from lenses pretreated with DHO or low-potassium medium. CONCLUSIONS: An increase in Na,K-ATPase alpha 2 polypeptide can occur in the epithelium of lenses subjected to an episode of sodium pump inhibition. This suggests the response could be triggered by an increase in cell sodium and does not necessarily require a period of stimulated active sodium-potassium transport.

Animals↗
PubMed1997

Calcium ATPase activity and membrane structure in clear and cataractous human lenses.

PURPOSE: Maintenance of calcium homeostasis is imperative for the clarity of the lens. Ca(2+)-ATPase is essential for the removal of cytosolic calcium, either across the plasma membrane or through intracellular organelles such as the endoplasmic reticulum. In this study, membranes prepared from clear lens epithelium were compared to membranes prepared from cataractous lens epithelium. METHODS: Human lens membranes were prepared by a protocol utilizing homogenization and centrifugation. Ca(2+)-ATPase activity was measured biochemically using Gamma-32P labeled ATP. Lipid order was measured using infrared and Raman spectroscopy. RESULTS: Ca(2+)-ATPase activity was similar in membranes prepared from cataractous lenses that were classified as nuclear subcapsular, nuclear and brunescent cataracts. Ca(2+)-ATPase activity was approximately 50% less in membranes prepared from cataractous lenses in comparison to clear lenses. Because clear lenses from Indian donors was unavailable, clear human lenses were used as a qualitative control for the measurement for Ca(2+)-ATPase activity. Lipid order was measured in lens fibers from cataractous and clear lenses from the United States donors. Lipid order increased from 55% in the hydrocarbon chains from clear lens fibers to 84% in cataractous lens fibers. CONCLUSIONS: These findings support the hypothesis that membranes are deranged in cataractous tissue, which should lead to altered levels of calcium.

Adult↗
PubMed1997

Influence of bafilomycin A1 on pHi responses in cultured rabbit nonpigmented ciliary epithelium.

Aqueous humor secretion is in part linked to HCO3- transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H(+)-ATPase, the present study was conducted to examine whether vacuolar H(+)-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H(+)-ATPase expression as judged by H(+)-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K(+)-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 microM; an inhibitor of vacuolar H(+)-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Cl- medium, HCO3(-)-free medium, or Na(+)-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H(+)-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Indomethacin alters the Na,K-ATPase response to protein kinase C activation in cultured rabbit nonpigmented ciliary epithelium.

PURPOSE: To test whether prostaglandin E2 (PGE2) is generated by cultured nonpigmented ciliary epithelial (NPE) cells treated with the phorbol ester, phorbol dibutyrate (PDBu), an activator of protein kinase C. In addition, the authors tested whether indomethacin, a cyclooxygenase inhibitor, influences the stimulation of active sodium-potassium transport observed in PDBu-treated cells. METHODS: A cell line derived from rabbit NPE was used in this study. PGE2 was measured by an enzyme-linked immunosorbent assay technique. Ouabain-sensitive potassium (86Rb) uptake was measured as an index of active sodium-potassium (Na, K-ATPase-mediated) transport. Ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) also was measured. Cell sodium and potassium content was determined by atomic absorption spectrophotometry. RESULTS: Marked PGE2 generation was observed in PDBu-treated cells. Indomethacin abolished the PGE2 response. Ouabain-sensitive potassium (86Rb) uptake was stimulated approximately 40% in cells exposed to PDBu, but a stimulation of > 100% was observed in cells exposed to PDBu in the presence of indomethacin. Added alone, indomethacin did not alter ouabain-sensitive potassium (86Rb) uptake. Neither nordihydroguaiaretic acid (a lipoxygenase inhibitor) nor ethoxyresorufin (a cytochrome P450 inhibitor) altered the 86Rb uptake response to PDBu. Sodium and cyclic adenosine monophosphate content was unchanged in cells treated with PDBu + indomethacin. CONCLUSIONS: In PDBu-treated cells, there may be generation of cyclooxygenase metabolites of arachidonic acid that inhibit Na, K-ATPase activity, suppressing the stimulatory effect of PDBu on active sodium-potassium transport. Based on the observation that PGE2 can inhibit Na, K-ATPase activity and also inhibit ouabain-sensitive potassium (86Rb) uptake, the authors suggest PGE2 may influence the Na,K-ATPase response to the activation of protein kinase C in NPE cells.

Animals↗
PubMed1997

Membrane-associated carbonic anhydrase in cultured rabbit nonpigmented ciliary epithelium.

PURPOSE: To measure the activity of membrane-associated carbonic anhydrase (CA) in cultured rabbit nonpigmented epithelial (NPE) cells, determine its identity and its sensitivity to extracellular trypsin, and compare the ability of acetazolamide and a cell-impermeant dextran-bound CA inhibitor to change cytoplasmic pH. METHODS: Studies were conducted using a cell line derived from rabbit NPE. The cells were lysed and separated into soluble and insoluble fractions by differential centrifugation. CA activity in these fractions was determined using a CO2 hydration assay. In studies with intact cells, a membrane-impermeable high-molecular-weight dextran-bound inhibitor (DBI) was synthesized and used to selectively bind and inhibit the extracellular-facing membrane-bound CA. Measurements of CA activity in intact red blood cells were conducted to confirm DBI remains extracellular. Acetazolamide, a membrane-permeable CA inhibitor, was used to inhibit total CA activity. Intracellular pH was determined using the pH-dependent absorbance of the fluorescent dye BCECF-AM. RESULTS: A low-speed pellet enriched with plasma membrane material accounted for 22.3 +/- 6.1% (n = 18) of the total CA activity in the cultured NPE. When intact cells were exposed to trypsin-EDTA, a 28% reduction of membrane-associated CA activity was observed; DBI inhibited this CA activity loss. Cytosolic CA activity was inhibited by 0.2% sodium dodecyl sulfate (SDS). In contrast, membrane-associated CA was SDS resistant, a characteristic of the CA-IV isozyme. By Western blot, CA-IV immunoreactive polypeptide was detected in the cultured cells and also in native rabbit and porcine ciliary epithelium. Inhibition of total CA activity with acetazolamide and inhibition of extracellular-facing membrane-associated CA with DBI caused an identical intracellular pH decrease in intact NPE cells. CONCLUSIONS: Expression of the CA-IV isozyme could account for the significant fraction of CA activity in the cultured NPE, which is membrane associated and SDS resistant. Sensitivity to tryptic hydrolysis suggests the membrane-associated CA partially faces extracellularly. As judged by responses to an extracellular CA inhibitor, the membrane-associated CA has a functional role in maintaining cytoplasmic pH.

Acetazolamide↗
PubMed1996

Studies on lipids and the activity of Na,K-ATPase in lens fibre cells.

Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase alpha1 and beta1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the NA,K-ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase alpha1 and beta1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase alpha1 beta1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase alpha1 beta1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K-ATPase alpha1 and beta1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes.

Animals↗
PubMed1996

Influence of amphotericin B on the sodium pump of porcine lens epithelium.

Active transport by Na(+)-K(+)-ATPase in the monolayer of lens epithelium is vital for the regulation of sodium and potassium levels within the mass of fiber cells that make up the bulk of the lens. In this study, experiments were conducted using porcine lenses to test whether Na(+)-K(+)-ATPase activity in the epithelium is altered when the permeability of lens cell plasma membranes is increased by the ionophore amphotericin B. After 24 h, sodium was significantly (P < 0.01) elevated in lenses exposed to 5 or 10 microM amphotericin B. Amphotericin B stimulated 86Rb uptake, probably through an increase of cytoplasmic sodium concentration due to increased inward sodium leak; the rate of ouabain-sensitive potassium (86Rb) uptake by intact lenses was significantly increased by amphotericin B at 5 microM (P < 0.05) and 10 microM (P < 0.01). After 24 h, the epithelium from lenses exposed to amphotericin B had an Na(+)-K(+)-ATPase activity that was more than twofold higher (P < 0.01) than the Na(+)-K(+)-ATPase activity in control lenses. By immunoblot, there was an increase in Na(+)-K(+)-ATPase catalytic (alpha) subunit immunoreactive polypeptide in the epithelium of lenses exposed to amphotericin B. The increase stemmed from a marked increase of Na(+)-K(+)-ATPase alpha 2-immunoreactive polypeptide but little change in the amount of alpha 1-immunoreactive protein. As judged by immunoblot experiments, the amount of Na(+)-K(+)-ATPase beta 1-immunoreactive polypeptide also appeared to be higher in the epithelium of amphotericin B-treated lenses compared with control lenses. In summary, these results suggest that in response to a permeability challenge with amphotericin B, the porcine lens epithelium is able to increase the activity of Na(+)-K(+)-ATPase. The same permeability challenge also appears to stimulate the biosynthesis of Na(+)-K(+)-ATPase catalytic subunit as well as glycoprotein subunit polypeptides.

Amphotericin B↗
PubMed1996

Differential expression of sodium pump catalytic subunits in the lens epithelium and fibers.

Na,K-ATPase in lens epithelium plays a key role in conducting sodium-potassium transport. The purpose of this study was to test whether epithelium or fiber cells can synthesize new Na,K-ATPase protein in response to an increase of membrane permeability. Western blot methodology was used to identify Na,K-ATPase alpha subunit polypeptides in membrane material isolated from lens cells. As judged by immunoblot density, epithelial cell membrane material isolated from porcine lenses cultured 24 h with 1 microM amphotericin B contained more Na,K-ATPase alpha subunit polypeptide than epithelial material isolated from control lenses. This increase stemmed from the apparent synthesis of Na,K-ATPase alpha 2 isoform polypeptide by the epithelium; Na,K-ATPase alpha 1 isoform polypeptide abundance was not detectably altered. The apparent amphotericin B-induced expression of Na,K-ATPase alpha 2 was seen in lens epithelial cells but not fiber cells. This study suggests that the epithelium of the adult porcine lens may be capable of expressing additional sodium pump molecules of the alpha 2-subtype when membrane permeability is increased.

Amphotericin B↗