Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium.

Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H(2)O(2). The inhibitory effects of H(2)O(2) were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.

Creatine kinase in human retinal pigment epithelium.

Retinal pigment epithelial ion transport activity, and consequent ATP consumption vary significantly as a function of photoreceptor activity. In a variety of cell types, ATP levels are maintained during high-energy usage by phosphocreatine hydrolysis, catalysed by the enzyme creatine kinase. The present work was designed to assess the importance of creatine kinase in retinal pigment epithelial cell metabolism. To this end, activity measurements, non-denaturing gel electrophoresis, Western blot analysis and immunohistochemistry were used to characterize creatine kinase in retinal pigment epithelium. Total creatine kinase activity in the retinal pigment epithelium is approximately 0.05 micromol ATP mg protein(-1) min(-1). The bulk of this activity was mediated by the B-CK isoform. However, by immunoblotting, non-denaturing gel electrophoresis and immunohistochemistry, the presence of the M-CK isoform of creatine kinase was also detected. The M-CK isoform was plasma membrane associated and predominately localized to the apical surface. Creatine kinase in the retinal pigment epithelium could function in a spatial energy shuttle that helps to sustain apical plasma membrane ion transport activity.

Transthyretin localization in cultured and native human retinal pigment epithelium.

The aim of this study was to determine transthyretin subcellular localization in cultured and native human retinal pigment epithelium. Monoclonal and polyclonal antibodies directed against human plasma transthyretin were used to detect transthyretin-specific immunoreactivity in cultured human retinal pigment epithelium. At the light microscopic level, transthyretin-specific immunoreactivity was observed throughout the cytosol with intense perinuclear staining. Nuclear staining was faint, but detectable. Both monoclonal and polyclonal antibodies exhibited similar staining patterns. An electron microscopic immunogold labeling protocol detected transthyretin-specific immunoreactivity in cultured and native human retinal pigment epithelium. Transthyretin-specific immunogold labeling within mitochondrial, apical, basal, nuclear and cytosolic subcellular compartments was quantitated and statistically analyzed. The pattern of transthyretin labeling was similar for each antibody, and comparable between cultured and native human retinal pigment epithelium. Transthyretin labeling was observed in mitochondrial and nuclear compartments, and in close apposition to both apical and basal membranes. Transthyretin labeling density was highest in the mitochondrial compartment and was significantly greater than labeling in all other compartments. Detection of transthyretin labeling in mitochondrial and nuclear compartments suggests an intracellular role for transthyretin in human retinal pigment epithelium, possibly as a cytoplasmic carrier protein for thyroxine.

Sodium-calcium exchanger in cultured human retinal pigment epithelium.

Regulation of intracellular free Ca2+ concentration ([Ca2+]i) by an Na+/Ca2+ exchanger was studied in cultures of human retinal pigment epithelial cells using Ca(2+)-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]i of cultured RPE in a control Ringer solution was 189 +/- 16 nM. Replacing extracellular Na+ with N-methyl-D-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]i varied as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+ channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+ stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]i was significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+ exchange inhibition. These findings indicate that removal of extracellular Na+ promotes net [Ca2+]i gain via Na+/Ca2+ exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+ exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at approximately 126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+ exchanger showed that the major exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-exchanger-related transcript was approximately 6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+ exchanger in cultures of human RPE.

Plasma membrane calcium-ATPase in cultured human retinal pigment epithelium.

The present work demonstrates the presence of plasma membrane (Ca(2+) + Mg2+)-ATPase (PMCA) activity in cultured human retinal pigment epithelium (HRPE). Whole-cell HRPE homogenates exhibited Ca(2+)-stimulated ATPase activity that was inhibited with high affinity (IC50 = 60 nM) by eosin, a potent inhibitor of the erythrocyte PMCA. This activity was not inhibited by thapsigargin, a selective inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). A PMCA-specific monoclonal antibody exhibited staining in attached HRPE monolayers. By Western blot analysis of SDS-PAGE separations of whole cell lysates of HRPE, this antibody identified a single band at approximately 145 kD. Finally, a Ca(2+)-dependent, La(3+)-augmented phosphoprotein, which comigrated with anti-PMCA immunoreactivity, was also detected. Taken together, these results show that cultured HRPE express PMCA activity.

Immunodetection of an arrestin-like protein in human retinal pigment epithelium.

The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity.

Molecular analysis of CD2 gene expression in acute myeloblastic leukemia expressing T-lineage associated surface antigens.

CD2 is a surface marker of T cells and NK cells, and is not normally expressed on human myeloid cells, but is found on a significant minority of cases of acute myeloid leukemia (AML). Molecular studies were performed on bone marrow myeloblasts from two patients with CD2 surface positive AML. No abnormality of the CD2 gene was detected on Southern blot analysis. On Northern blots, CD2 mRNA of normal size was present. The CD2 gene contained a site which was unmethylated, consistent with active transcription, in a CD2 positive AML case, and in a CD2 positive T cell line, but methylated in CD2 negative AML cells. The evidence does not support the hypothesis that inappropriate surface expression of lineage markers is due to leukemia-related genetic changes, such as amplification or rearrangement, of the CD2 gene itself. Rather, the results are consistent with the hypothesis that mixed lineage leukaemia arises from rare normal bone marrow progenitors with multilineage phenotypes.

Cloning and DNA sequence analysis of the region containing attP of the temperate phage phi AR29 of Prevotella ruminicola AR29.

Phage phi AR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29. By DNA hybridization studies, the point of integrative recombination on the phage genome (attP) was located on a 4.5 kb EcoRV fragment. After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRV/HindIII fragment was isolated, cloned in Escherichia coli and sequenced. DNA hybridization localized the attP site to the vicinity of an internal DraI site. Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage lambda. Two open reading frames are present adjacent to attP (ORF1 and ORF2). The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases. Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role. However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to attP. The proposed phi AR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria.

Volume regulation in cultured cells derived from human retinal pigment epithelium.

To characterize volume regulatory mechanisms, unidirectional Rb+ efflux and influx, unidirectional Cl- influx, and cell volume were measured in cultured human retinal pigment epithelium (HRPE). The HRPE was found to be capable of both regulatory volume increase (RVI), in response to a hypertonic challenge, and regulatory volume decrease (RVD), in response to a hypotonic challenge. Bumetanide-sensitive Rb+ influx increased almost threefold on incubation in a hypertonic (390 mosmol/kgH2O) medium. Bumetanide-insensitive Rb+ influx was activated by hypotonic (190 mosmol/kgH2O) challenge as well as by treatment with N-ethylmaleimide (NEM). Exposure to hypotonic media also activated unidirectional Cl- influx and unidirectional Rb+ efflux. Both the RVD and hypotonically activated Rb+ efflux were inhibited by the K(+)-channel blocker barium. On the other hand, hypotonically activated Rb+ influx was increased by barium treatment. In sum, the HRPE exhibits volume-sensitive transport mechanisms over a range of volumes from 190 to 390 mosmol/kgH2O. Cultured HRPE possess hypertonically activated Na-K-Cl cotransport, hypotonically activated K-Cl cotransport, and a barium-inhibitable hypotonically activated K+ efflux pathway.

Increased expression of interferon-gamma in hyperplastic lymph nodes from HIV-infected patients.

Polyclonal B cell activation is characteristic of HIV infection and occurs in the presence of severe CD4+ lymphocyte depletion. In contrast, CD4+ lymphocytes are the dominant T cell in the reactive lymphoid tissues of patients not infected with HIV. In this study, lymph node biopsies from eight HIV-infected patients with persistent generalized lymphadenopathy syndrome (PGL) were assessed for IL-1 beta, IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) gene expression using the polymerase chain reaction (PCR). The cytokine gene expression of two cases of reactive adenopathy in patients not infected with HIV was assessed for comparison. IFN-gamma was expressed much more strongly in the PGL samples than in control reactive lymphoid tissues, whereas the other cytokines were expressed to a similar extent in both types of tissues. IFN-gamma may have an important role in maintaining the adenopathy of HIV-infected patients. Expression of cytokines such as IL-2, IL-4 and IL-10 in HIV nodes may be adequate to allow the recruitment of naive B cells to the reactive process.

Rubidium transport in cultured monkey retinal pigment epithelium.

Rb+ influx was used to assess Na-K-Cl cotransport and Na,K-ATPase activities in cultured monkey retinal pigment epithelium. Bumetanide-sensitive (Na-K-Cl cotransport-mediated) Rb+ influx exceeds ouabain-sensitive (Na,K-ATPase-mediated) Rb+ influx, with these two transporters accounting for approximately 95% of total Rb+ uptake. Half-maximal inhibition of Rb+ influx by bumetanide is attained at 75 nM bumetanide. The bumetanide-sensitive Rb+ influx depends on both extracellular Na+ and Cl-, and is activated by extracellular Rb+ with a relatively high affinity. Na-K-Cl cotransport activity is stimulated (2.5-fold) by increased extracellular osmolarity. Elevated cAMP content and glycolytic inhibition both depress cotransport activity. Cyanide application, however, had very little effect on Na-K-Cl cotransport activity. Monkey retinal pigment epithelial cells, maintained in culture, provide a system in which the activity and regulation of cation transport mechanisms can be examined.

Na(+)-K(4)-Cl- cotransport in cultured cells derived from human retinal pigment epithelium.

Unidirectional fluxes of Rb+ and Cl- were measured in cultured human retinal pigment epithelium (HRPE) to characterize the Na(+)-K(+)-Cl-cotransport system in this epithelial tissue. In HRPE, the ouabain-sensitive fraction of Rb+ influx comprises 65% of the total Rb+ influx. Bumetanide [inhibition constant (KI) = 160 nM] inhibited the residual ouabain-insensitive fraction of Rb+ influx by 70%. The bumetanide-sensitive fraction of Rb+ influx was dependent on the presence of both extracellular Na+ and Cl-. Cl- influx was similarly inhibited by bumetanide and also dependent on extracellular Na+ and Rb+. The stoichiometry of bumetanide-sensitive Cl- influx to Rb+ influx was 2:1. Elevation of extracellular osmolarity (by 30%) caused a 2.5-fold increase in Na(+)-K(+)-Cl- cotransport activity in cultured HRPE. The present study provides direct evidence for the occurrence of Na(+)-K(+)-Cl- cotransport-mediated cation and anion transport in HRPE.

Calcium-stimulated sodium efflux from rabbit vascular smooth muscle.

1. The effects of the addition of Ca2+ on ouabain-resistant 22Na+ efflux from Na+-loaded strips of rabbit portal anterior mesenteric vein in Ca2+-free media have been studied. 2. Na+ efflux into Li+ media containing 5 mM-KCl is rapidly and transiently stimulated some 4- to 5-fold on the addition of Ca2+ (1.2 mM). No stimulation is observed if the Li+ medium is K+ free or if Na+ replaces Li+ ions. This Ca2+-activated Na+ efflux is not obligatorily coupled to Na+ influx. 3. The stimulation of Na+ efflux could also be triggered by the addition of 5 mM-K+ to a Ca2+-containing K+-free medium. The Ca2+-activated increase in Na+ efflux also occurred when K+ was the sole monovalent extracellular cation. Rb+ could substitute for the K+ requirement. Thus the Na+ efflux is not mediated by a system which has a specific requirement for counter-transport of Li+ or one in which Li+ but not K+ are counter-transported such as the familiar Na+-H+ exchange system. Acidification of the external medium reduced the Ca2+-stimulated Na+ efflux, in keeping with the conclusion that this efflux was not due to Na+-H+ exchange. 4. Progressive reduction of external [Ca2+] increased the time-lag to peak activation of Na+ efflux, suggesting that the effects of added Ca2+ were mediated by a rise in intracellular Ca2+. Under experimental conditions which did not result in activation of the Na+ efflux by the addition of extracellular Ca2+ alone (e.g. in Na+ media), addition of Ca2+ plus the Ca2+ ionophore, ionomycin, stimulated Na+ efflux. This further confirms that intracellular sites for Ca2+ are critical for the activation of Na+ efflux. In the absence of ionophore, in Na+ media, intracellular Ca2+ is not sufficiently increased when extracellular Ca2+ is added. A partial (40%) block of Ca2+-activated Na+ efflux by amiloride (2 X 10(-3) M) could also be overcome by the addition of ionomycin. 5. The lack of effect of a variety of inhibitors suggests that the Ca2+-stimulated Na+ efflux mechanism is not mediated via a Na+-K+-Cl- co-transport system or a Na+-H+ counter-transport system, or Na+-Ca2+ exchange. 6. The activation of Na+ efflux in smooth muscle by Ca2+ ions seems to involve Ca2+ entry partially via an extracellular Ca2+-intracellular Na+ exchange and also through other parallel pathway(s), followed by a rise in intracellular Ca2+ that activates Na+ efflux through a Ca2+-sensitive Na+ channel or other transport pathway.

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts.

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line.

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.

Amino acid transport in kidney epithelial cell line (MDCK): characteristics of Na+/amino acid symport in membrane vesicles and basolateral localization in cell monolayers.

Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.

Regulation of Na+,K+-ATPase activity in MDCK kidney epithelial cell cultures: role of growth state, cyclic AMP, and chemical inducers of dome formation and differentiation.

Na+,K+-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me2SO). Ouabain-sensitive Na+,K+-ATPase and 86Rb+ uptake activities, and the number of [3H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 X 10(7) pumps/cell was estimated for subconfluent cultures, declining to 9 X 10(5) pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt2cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me2SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na+ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na+ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me2SO partially inhibited ouabain-sensitive 86Rb+ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na+ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt2cAMP caused a furosemide-sensitive increase in intracellular Na+ content. This class of inducers stimulated ouabain-sensitive 86Rb+ uptake, presumably by substrate effects due to increased Na+ levels. The Na+ or ATP activation of Na+,K+-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb+ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na+ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.