Regulation of uptake of inositol by glucose in cultured retinal pigment epithelial cells

Long term and acute effects of glucose on myo-inositol (MI) uptake were studied in primary cultures of bovine retinal pigment epithelial (RPE) cells. RPE cells were grown under low (5 mM) or high (20, 40, or 50 mM) glucose levels in the growth medium for up to 18 days. The concentrative capacity of confluent RPE cells to accululate [3H]MI (10 microM) was reduced up to 41% as the glucose concentration in the growth medium increased. When the growth medium glucose was switched from 5 to 40 mM, or vice versa, the capacity of cells to accumulate MI was reversed. Treatment of cells grown in 40 or 50 mM glucose with 0.1 mM Sorbinil (an aldose reductase inhibitor) minimally reversed the ability of cells to accumulate MI. RPE cells, grown in 5 mM glucose, were incubated with 10-60 mM D-glucose or its nonmetabolizable analogues (acute effect). Kinetics of MI uptake inhibition by alpha-methyl glucose according to Dixon plots were characteristic of competitive inhibition (Ki = 28 mM). MI uptake was strongly inhibited by phlorizin. The ability of RPE cells to bind 5 microM [3H]phlorizin also was reduced by increased glucose levels in the growth medium. These studies indicated that MI and glucose shared the same transporter system. Glucose in the incubation medium directly interfered with MI binding to the transporter. High glucose feeding of the cells also down-regulated the transporter density. The uptake and function of solutes such as MI in tissues that operate on the glucose carrier system may be severely impaired in diabetes.     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

Non-competitive inhibition of myo-inositol transport in cultured bovine retinal capillary pericytes by glucose and reversal by Sorbinil

myo-Inositol transport by retinal capillary pericytes in culture was characterized. The major myo-inositol transport process was sodium-dependent, ouabain-sensitive, and saturable at 40 mM, indicating a carrier-mediated process. The sodium ion concentration required to produce one-half the maximal rate of myo-inositol uptake ([Na+]0.5) did not show dependence on the external myo-inositol concentration (22.3 mM sodium for 0.005 mM myo-inositol; 18.2 mM sodium for 0.05 mM myo-inositol). myo-Inositol transport was an energy-dependent, active process functioning against a myo-inositol concentration gradient. The kinetics of the sodium-dependent system fitted a 'velocity type' co-transport model where binding of sodium ion to the carrier increased the velocity (Vmax 28 to 313 pmol myo-inositol/micrograms DNA per 20 min when [Na+] varied from 9 to 150 mM) but not the affinity for myo-inositol (Km 0.92 to 0.83 mM when [Na+] varied from 9 to 150 mM). Metabolizable hexoses (D-glucose or D-galactose; greater than 5 mM) inhibited myo-inositol uptake. Dixon-plot analysis indicated that the inhibition was non-competitive with a Ki of 22.7 mM for D-glucose and 72.6 mM for D-galactose. The inhibition was significantly reversed by Sorbinil (0.1 mM), an aldose reductase inhibitor. In contrast, high concentrations of non-metabolizable hexoses (L-glucose, 3-O-methyl-D-glucose), or partially metabolizable 2-deoxy-D-glucose, did not significantly inhibit myo-inositol uptake. The inhibitory effect of D-glucose or D-galactose on myo-inositol transport appeared to be related to glucose or galactose metabolism via the polyol pathway.     

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: Comparison with neuronal types of ion channels

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na⁺ and Ca²⁺ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na⁺ channels and Na⁺ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca²⁺ channel-expressing cells and Ca²⁺ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na⁺ current in the RPE cells was similar to that in neurons. The voltage dependence of Na⁺ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca²⁺ current expressed in cultured RPE cells was too small to detect without replacement of external Ca²⁺ with Ba²⁺. The Ba²⁺ current, like Ca²⁺ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca²⁺ channels in culture. The possibility that the development of both Na²⁺ and Ca²⁺ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

Inwardlyrectifying K⁺ current(I_Kir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, I_Kir randown completely in <10 min [half time(t_1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustainedI_Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used,I_Kir ran down by~71%. Mg²⁺ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg²⁺(t_1/2 = 1.8 min).I_Kir also randown when the pipette solution contained 4 mMMg²⁺ + 4 mM5'-adenylylimidodiphosphate(t_1/2 = 2.7 min)or 4 mM adenosine 5'-O-(3-thiotriphosphate)(t_1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity ofI_Kirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis.

     

Na,K-ATPase inhibition alters tight junction structure and permeability in human retinal pigment epithelial cells

Na,K-ATPase regulates avariety of transport functions in epithelial cells. In cultures ofhuman retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPaseby ouabain and K⁺ depletion decreased transepithelialelectrical resistance (TER) and increased permeability of tightjunctions to mannitol and inulin. Electrophysiological studiesdemonstrated that the decrease in TER was due to an increase inparacellular shunt conductance. At the light microscopy level, thisincreased permeability was not accompanied by changes in thelocalization of the tight junction proteins ZO-1, occludin, andclaudin-3. At the ultrastructural level, increased tight junctionpermeability correlated with a decrease in tight junction membranecontact points. Decreased tight junction membrane contact points andincreased tight junction permeability were reversible inK⁺-repletion experiments. Confocal microscopy revealed thatin control cells, Na,K-ATPase was localized at both apical andbasolateral plasma membranes. K⁺ depletion resulted in alarge reduction of apical Na,K-ATPase, and after K⁺repletion the apical Na,K-ATPase recovered to control levels. Theseresults suggest a functional link exists between Na,K-ATPase and tightjunction function in human RPE cells.

     

Effects of chondroitin sulfate on cultured human retinal pigment epithelial cells

We investigated the effects of chondroitin sulfate on growth, morphology and ultrastructure of retinal pigment epithelial (RPE) cells in culture. When added to the medium, chondroitin sulfate reduced cell density in RPE cultures in a dose-dependent manner. Compared with the controls, the treated cells appeared to be larger and more granular. Electron microscopic examinations revealed accumulations of membrane-bound whorls. In addition, phagocytic activity in the treated cells was notably increased and the level of acid lipase was elevated. These data suggest that increased levels of chondroitin sulfate can induce alterations in both metabolism and activities of RPE cells.     

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Taurine is activelytransported at the retinal pigment epithelial (RPE) apical membrane inan Na⁺- and Cl-dependent manner. Diabetes mayalter the function of the taurine transporter. Because nitric oxide(NO) is a molecule implicated in the pathogenesis of diabetes, we askedwhether NO would alter the activity of the taurine transporter incultured ARPE-19 cells. The activity of the transporter was stimulatedin the presence of the NO donor 3-morpholinosydnonimine. Thestimulatory effects of 3-morpholinosydnonimine were not observed duringthe initial 16-h treatment; however, stimulation of taurine uptake waselevated dramatically above control values with 20- and 24-htreatments. Kinetic analysis revealed that the stimulation wasassociated with an increase in the maximal velocity of the transporterwith no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study providesthe first evidence of an increase in taurine transporter geneexpression in human RPE cells cultured under conditions of elevatedlevels of NO.

     

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na+-K+-ATPase and epithelial Na+ channels

Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O(2) on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 muM) for 3 days before exposure to hypoxia (1.5% O(2)). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (-30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na(+)-K(+)-ATPase and epithelial Na(+) channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of alpha- and gamma-ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of alpha(1)-Na(+)-K(+)-ATPase and alpha-ENaC increased in normoxia and hypoxia, and gamma-ENaC was increased in normoxia only. DEX stimulated the mRNA expression of alpha(1)-Na(+)-K(+)-ATPase and alpha-, beta-, and gamma-ENaC of A549 cells in normoxia and hypoxia (1.5% O(2)) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.     

Effect of glucose availability on glucose transport in bovine mammary epithelial cells

Primary bovine mammary epithelial cells (BMEC) were cultured in media containing varying concentrations of glucose, to determine the effects of glucose availability on glucose transport and its mechanism in bovine mammary gland. The BMEC incubated with 10 and 20 mM glucose had twofold greater glucose uptake than that with 2.5 mM glucose (P < 0.05). Increased glucose availability enhanced the cell proliferation (P < 0.05). As the glucose uptake is mediated by facilitative glucose transporters (GLUTs), the expression of GLUT mRNA was investigated. Compared with the control (2.5 mM), 5 and 10 mM glucose did not influence the abundance of GLUT1 mRNA (P < 0.05), whereas 20 mM glucose decreased the GLUT1 mRNA expression in the BMEC (P < 0.05). The expression of GLUT8 mRNA was not affected by any concentration of glucose (P > 0.05). As GLUTs are coupled with hexokinases (HKs) in regulating glucose uptake, the expression of HKs and their activities were also studied. The HK activity was greater in 5, 10 and 20 mM glucose than that in 2.5 mM glucose (P < 0.05). The expression of HK2 mRNA rather than HK1 mRNA was detected in the BMEC; however, the abundance of HK2 mRNA was not elevated by any concentrations of glucose compared with control (P > 0.05). Furthermore, addition of 3-bromopyruvate (30, 50 or 70 μM), an inhibitor of HK2, resulted in the decrease of glucose uptake and cell proliferation at both 2.5 and 10 mM glucose (P < 0.05). Therefore, the glucose concentrations may affect glucose uptake partly by altering the activity of HKs, and HK2 may play an important role in the regulation of glucose uptake in the BMEC.