Role of wild-type estrogen receptor-beta in mitochondrial cytoprotection of cultured normal male and female human lens epithelial cells.

The influence of sexual category as a modifier of cellular function is underinvestigated. Whether sex differences affect estrogen-mediated mitochondrial cytoprotection was determined using cell cultures of normal human lens epithelia (nHLE) from postmortem male and female donors. Experimental indicators assessed included differences in estrogen receptor-beta (ERbeta) isoform expression, receptor localization in mitochondria, and estrogen-mediated prevention of loss of mitochondrial membrane potential using the potentiometric fluorescent compound JC-1 after nHLE were exposed to peroxide. The impact of wild-type ERbeta (wtERbeta1) was also assessed using wtERbeta1 siRNA to suppress expression. A triple-primer PCR assay was employed to determine the proportional distribution of the receptor isoforms (wtERbeta1, -beta2, and -beta5) from the total ERbeta message pool in male and female cell cultures. Irrespective of sex, nHLE express wtERbeta1 and the ERbeta2 and ERbeta5 splice variants in similar ratios. Confocal microscopy and immunofluorescence revealed localization of the wild-type receptor in peripheral mitochondrial arrays and perinuclear mitochondria as well as nuclear staining in both cell populations. The ERbeta2 and ERbeta5 isoforms were distributed primarily in the nucleus and cytosol, respectively; no association with the mitochondria was detected. Both male and female nHLE treated with E(2) (1 muM) displayed similar levels of protection against peroxide-induced oxidative stress. In conjunction with acute oxidative insult, RNA suppression of wtERbeta1 elicited the collapse of mitochondrial membrane potential and markedly diminished the otherwise protective effects of E(2). Thus, whereas the estrogen-mediated prevention of mitochondrial membrane permeability transition is sex independent, the mechanism of estrogen-induced mitochondrial cytoprotection is wtERbeta1 dependent.

Overexpression of Na(+)-dependent myo-inositol transporter gene in mouse lens led to congenital cataract.

PURPOSE: Maintaining appropriate osmotic pressure is essential for maintaining lens transparency. This study was performed to investigate whether high levels of myo-inositol, one of the major organic osmolytes in the lens, would lead to cataract development. METHODS: Transgenic mouse lines carrying the bovine Na(+)-dependent myo-inositol transporter (bSMIT) cDNA under the control of the mouse alphaA-crystallin promoter were generated. RESULTS. Increased bSMIT expression was accompanied by increased myo-inositol level in the lens and increased uptake of (3H) myo-inositol by the lens in culture. The transgenic mice developed observable cataract under normal rearing conditions beginning at 2 to 8 weeks of age, and the severity of cataract development was correlated to the level of bSMIT gene expression and lens myo-inositol accumulation. For transgenic mouse line 3352, heterozygous mice did not develop cataract, whereas homozygous ones did. Prenatal feeding of heterozygous 3352 mice with high myo-inositol diet led to cataract development, indicating that cataract development was not merely due to a nonspecific effect of SMIT overexpression. Introducing aldose reductase overexpressing transgene into heterozygous 3352 mice also led to cataract development, indicating that this type of cataract is primarily due to osmotic stress. CONCLUSIONS: The present results indicate that high levels of myo-inositol and sorbitol in the lens contribute to cataract development. This is a useful model to study the role of osmotic stress in cataractogenesis during lens development.

A transgenic animal model of osmotic cataract. Part 1: over-expression of bovine Na+/myo-inositol cotransporter in lens fibers.

PURPOSE: Intracellular osmotic stress is believed to be linked to the advancement of diabetic cataract. Although the accumulation of organic osmolytes (myo-inositol, sorbitol, taurine) is thought to protect the lens by maintaining osmotic homeostasis, the physiologic implication of osmotic imbalance (i.e., hyperosmotic stress caused by intracellular over-accumulation of organic osmolytes) on diabetic cataract formation is not clearly understood. Studies from this laboratory have identified several osmotic compensatory mechanisms thought to afford the lens epithelium, but not the lens fibers, protection from water stress during intervals of osmotic crisis. This model is founded on the supposition that the fibers of the lens are comparatively more susceptible to damage by osmotic insult than is the lens epithelium. To test this premise, several transgenic mouse lines were developed that over-express the bovine sodium/myo-inositol cotransporter (bSMIT) gene in lens fiber cells. METHODS: Of the several transgenic mouse lines generated, two, MLR14 and MLR21, were analyzed in detail. Transgenic mRNA expression was analyzed in adult and embryonic transgenic mice by a coupled reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization on embryonic tissue sections, respectively. Intralenticular myo-inositol content from individual mouse lenses was quantified by anion exchange chromatography and pulsed electrochemical detection. Ocular histology of embryonic day 15.5 (E15.5) embryos from both transgenic (TG) families was analyzed and compared to their respective nontransgenic (NTG) littermates. RESULTS: Both RT-PCR and in situ hybridization determined that transgene expression was higher in line MLR21 than in line MLR14. Consistent with this, intralenticular myo-inositol from MLR21 TG mice was markedly higher compared with NTG littermates or MLR14 TG mice. Histologic analysis of E15.5 MLR21 TG embryos disclosed a marked swelling in the differentiating fibers of the bow region and subcapsular fibers of the central zone, whereas the lens epithelium appeared morphologically normal. The lenticular changes, initiated early during lens development in TG MLR21 embryos, result in severe bilateral nuclear cataracts readily observable in neonates under normal rearing and dietary conditions. In contrast, TG MLR14 pups reared under standard conditions produced no lens opacity. CONCLUSIONS: Lens fiber swelling and related cataractous outgrowth positively correlated to the degree of lens bSMIT gene expression and intralenticular myo-inositol content. The affected (i.e., swollen) lens fibers appeared to be unable to cope with the water stress generated by the transgene-induced over-accumulation of myo-inositol and, as a result of this inability to osmoregulate, suffered osmotic damage due to water influx.

Expression of pI(Cln) mRNA in cultured bovine lens epithelial cells: response to changes in cell volume.

PURPOSE: The authors recently established a link between swelling-activated myo-inositol efflux and chloride movement via anion channels in cultured bovine lens epithelial cells (BLECs). To further define this pathway, the relationship between cell volume, myo-inositol movement and mRNA expression of pI(Cln), a proposed chloride channel regulatory protein was investigated. METHODS: To demonstrate the effect of cell volume changes on pIcln transcription, BLECs were exposed to either hypertonic or hypotonic medium conditions. For rapid cellular shrinkage, BLECs were maintained at confluence in physiologic medium (257+/-2 mosm) then transferred to sodium hypertonic medium (473+/-6 mosm) or raffinose hypertonic medium (452+/-2 mosm). For rapid cellular swelling, cells were switched from sodium hypertonic medium to physiologic medium+/-tamoxifen. The expression of pI(Cln) mRNA was determined by Northern blot analysis. RESULTS: Upon cell volume reduction (increasing intracellular osmolality), BLECs upregulate the expression of pI(Cln) mRNA. Contrastly, when cell volume rapidly increases (decreasing intracellular osmolality), BLECs moderately downregulate pIcln mRNA, with expression levels reaching near physiologic control by 24 h. Blockage of swelling-activated chloride movement and osmolyte efflux with either tamoxifen or niflumic acid enhances the downregulation of pIcln mRNA expression. CONCLUSIONS: In cultured BLECs, pI(Cln) transcriptional regulation appears to be responsive to cell volume fluctuations. These data suggest a converse relationship exists between pIcln mRNA expression and changes in cell volume.

Effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells.

Previously we have shown that hyperosmolarity increases Na(+)-myo-inositol cotransporter (SMIT) activity and mRNA levels in cultured endothelial cells. Because hyperosmolarity and cytokines, such as tumor necrosis factor-alpha (TNF-alpha), activate similar signal transduction pathways, we examined the effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation. In contrast to the effect of hyperosmolarity, TNF-alpha caused a time- and concentration-dependent decrease in SMIT mRNA levels and myo-inositol accumulation. The effect of TNF-alpha on myo-inositol accumulation was found in large-vessel endothelial cells (derived from the aorta and pulmonary artery) and cerebral microvessel endothelial cells. In bovine aorta and bovine pulmonary artery endothelial cells, TNF-alpha activated nuclear factor (NF)-kappa B. TNF-alpha also increased ceramide levels, and C2-ceramide mimicked the effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in bovine aorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and 7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibit NF-kappa B activation, partially prevented the TNF-alpha-induced decrease in myo-inositol accumulation. The effect of TNF-alpha on myo-inositol accumulation was also partially prevented by the protein kinase C inhibitor calphostin C but not by staurosporine. These studies demonstrate that TNF-alpha causes a decrease in SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells, which may be related to the activation of NF-kappa B.

Cloning the bovine Na+/myo-inositol cotransporter gene and characterization of an osmotic responsive promoter.

Hyperosmotic-induced enhancement of myo-inositol accumulation in cultured bovine lens epithelial cells stems from increased uptake activity due to upregulation of Na+/myo-inositol cotransporter mRNA and de novo synthesis in myo-inositol carrier protein. The molecular mechanism of osmoregulation of Na+/myo-inositol cotransporter gene transcription was further investigated. The effect of hypertonicity on transcription initiation of the Na+/myo-inositol cotransporter gene was examined by use of the rapid amplification of cDNA ends (RACE) technique. 5"-RACE analysis revealed that one hypertonic and two isotonic (i.e. physiologic) transcription start sites are present in the Na+/myo-inositol cotransporter gene of cultured bovine lens epithelial cells. Moreover, the bovine Na+/myo-inositol cotransporter gene was cloned and its promoters were characterized by transient transfection assay using luciferase reporter constructs of various fragments of the 5"-flanking regions upstream of the individual transcription start sites. Among its promoters, only one was osmotically responsive and showed approximately a 3-fold induction of activity subsequent to hypertonic insult. Transient transfection assays revealed that the region between -398 and -331 bp, upstream of this hypertonic transcription start site, contains the putative osmotic response element. Preferential utilization of this osmotically responsive promoter contributes to the elevation of Na+/MI cotransporter mRNA abundance and increased myo-inositol uptake activity as initiated by osmotic stress.

Inducible expression of Na+/myo-inositol cotransporter mRNA in anterior epithelium of bovine lens: affiliation with hypertonicity and cell proliferation.

Bovine lenses were pretreated with physiological (314 +/- 6 mosm) or hypertonic medium (> 450 mosm) for 20 hours prior to introduction of the lenses into modified Ussing chambers (providing a leakproof seal at the lens equator). The anterior (epithelial-containing) and posterior (cell-free) aspects of the mounted intact lens could thus be independently bathed with myo-[3H]inositol. Myo-inositol uptake as a function of concentration across the anterior aspect was indicative of an active carrier-mediated component and at high concentration, significant myo-inositol influx due to passive diffusion. Myo-inositol uptake was markedly stimulated across the anterior aspect and inhibitable by ouabain but not stimulated across the posterior aspect, when lenses were exposed to hyperosmotic medium by the addition of raffinose or sodium chloride. Myo-inositol rapidly desaturates across both aspects of the lens consistent with its efflux being largely a function of passive diffusional leakout from the extracellular space. In order to corroborate the apparent hypertonic-induced upregulation of gene expression inferred by kinetic analysts, the level of Na+/myo-inositol cotransporter mRNA was determined by reverse transcription and quantitative PCR using harvested anterior epithelial cells from intact lenses exposed to physiologic or hypertonic medium for 20 hours. RNA samples were microextracted from both the central and equatorial regions of the lenticular epithelium of the anterior lens capsule. The abundance of Na+/myo-inositol cotransporter mRNA was similar from epithelium of the central zone irrespective of medium tonicity but was markedly elevated from the equatorial zone of osmotically-stressed lenses. While both the quiescent epithelial cells of the central region and actively dividing epithelial cells of the equatorial zone of the intact lens express Na+/myo-inositol contransporter mRNA, only the equatorial epithelium responds to hypertonic insult with enhanced uptake of myo-inositol due to increased transcription of the Na+/myo-inositol cotransporter gene, suggesting a possible role for the state of cell proliferation.

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 5. Mechanism of the myo-inositol efflux pathway.

PURPOSE: Cultured bovine lens epithelial cells (BLECs) exposed to sodium hypertonicity respond with an accumulation of intracellular myo-inositol. Using BLECs initially maintained at hypertonicity and reacting to a decrease in medium osmolality, a mechanism for the tonicity-activated release of myo-inositol was recognized. Alternatively, BLECs acclimated to sodium hypertonicity and subsequently transferred to high sodium osmolality plus hypergalactosemia rapidly accumulate intracellular galactitol, an experimental manipulation that permitted characterization of the role of sugar alcohols in polyol-activated myo-inositol efflux. The authors identify a communal transport route for tonicity-activated and polyol- activated myo-inositol release from cell to medium and demonstrate an association for myo-inositol efflux with chloride movement. METHODS: Two distinct experimental approaches were designed to delineate the physiological circumstances that initiate myo-inositol efflux. For tonicity-induced inositol efflux, BLECs were maintained at confluence in sodium hypertonic medium (473+/-6 mOsm) for 48 hours; afterward, the medium was replaced with isotonic medium (285+/-4 mOsm) containing 40 mM galactose +/- Sorbinil. For polyol-induced inositol release, hypertonically adapted BLECs were transferred to fresh sodium hypertonic medium containing 40 mM galactose (513+/- 10 mOsm). RESULTS: On reduction in medium osmolality, intracellular myo-inositol was lost because of a rapid, transient efflux during the first 30 minutes, which was followed by a slow, sustained decrease in efflux during the next 12 hours. Inhibition of aldose reductase activity substantially diminished myo-inositol efflux from cell to galactose-containing, isotonic medium. Administration of phloretin significantly inhibited both tonicity-activated and polyol-activated myo-inositol release, as did the chloride channel blocker, niflumic acid. CONCLUSIONS: In cultured bovine lens epithelial cells, tonicity-activated movement of myo-inositol from cell to medium and myo-inositol efflux as induced by intracellular polyol accumulation appear to be interactively associated with chloride movement and moderated by a common anionic (chloride) channel, carrier-mediated transport protein, or both.

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 4: Induction pattern of Na(+)-myo-inositol cotransporter mRNA under hypertonic conditions denoting an early-onset, interactive, protective mechanism against water stress.

PURPOSE: To examine the effect of hypertonicity on the induction of the Na(+)-myo-inositol (Na(+)-MI) cotransporter(s) in cultured bovine lens epithelial cells (BLECs). METHODS: Na(+)-MI cotransporter 626-bp reverse transcription-polymerase chain reaction product amplified from lens cell RNA and aldose reductase (AR) cDNA probes were used to measure respective mRNA content by Northern blot analysis. RESULTS: Northern blot analysis of BLEC mRNA hybridized to Na(+)-MI cotransporter cDNA showed that Na(+)-MI cotransporter mRNA increased when secondary cultures of BLECs were exposed to physiological medium supplemented with 116 mmol/l NaCl. A time course further revealed a maximal increase in Na(+)-MI cotransporter mRNA by 8 hours. Thereafter, the level of Na(+)-MI cotransporter mRNA steadily declined for the duration of the 72-hour incubation period despite continuous exposure of BLECs to hypertonicity. AR mRNA levels maximally increased by 24 h of cell exposure to hypertonic condition. Unlike Na(+)-MI cotransporter mRNA, AR mRNA remained elevated throughout the duration of the experiment. Hypertonic exposure resulted in a steady state accumulation of myo-inositol and sorbitol for 6 days. Inhibition of sorbitol formation prompted the intracellular myo-inositol content to a higher level. CONCLUSIONS: These data suggest that enhanced MI transport and accumulation, as an adaptive osmoregulatory response to hypertonicity in cultured BLECs, is a primary, early-onset, protective mechanism against water stress, succeeded by, enhanced sorbitol formation and accumulation, a secondary, late-onset protective mechanism. The lens appears to respond to the preliminary stages of hyperosmotic stress by induction of Na(+)-MI cotransporter mRNA, indicating that the myo-inositol carrier protein(s) play an initial responsive role in the management of osmotic stress. Lens water stress management is interactive because myo-inositol and sorbitol levels are regulated in concert.

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. III. Effects of cycloheximide and colchicine on Na(+)-myo-inositol cotransporter activity under hypertonic conditions, inhibition of a plasma membrane osmotic stress protein.

Cultured bovine lens epithelial cells adapt to hypertonic sodium stress via an increase in Na(+)-myo-inositol cotransporter activity and accumulate myo-inositol. At least 12 hr of hypertonic exposure was necessary to enhance myo-inositol accumulation; and thereafter, uptake activity continued to increase throughout the duration of a 72-hr exposure period. Switching from hypertonic to isotonic medium for 24 hr reversed the otherwise elevated accumulation activity. The protein synthesis inhibitor, cycloheximide, did not affect myo-inositol uptake in isotonic medium but markedly decreased myo-inositol uptake in hypertonic medium. Cells exposed to hypertonic conditions and the microtubule disrupter, colchicine, similarly showed marked impairment of the otherwise enhanced myo-inositol uptake. These studies indicated that hypertonicity-induced elevation of Na(+)-myo-inositol cotransporter activity in cultured bovine lens epithelial cells is not solely attributed to the increased sodium gradient alone, but rather involves increased de novo synthesis of the Na(+)-myo-inositol cotransporter protein(s).

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 1: A hypertonicity-induced protein enhances myo-inositol transport.

PURPOSE: The nature of the association between attenuated myo-inositol-concentrating capability, intracellular polyol accumulation, and hypertonicity-enhanced myo-inositol uptake was investigated in cultured bovine lens epithelial cells (BLECs) exposed to high ambient galactose. METHODS: The kinetic characteristics of myo-inositol accumulation based on the measurement of in vitro myo-[3H]inositol (3H-MI) uptake was determined with cultured BLECs incubated in either high ambient galactose or galactose-free, physiological medium under experimental conditions that included both aldose reductase inhibition and elevation of extracellular osmotonicity. RESULTS: The uptake of 3H-MI was lowered after chronic (20 hour) preincubation of cultured BLECs in 40 mmol/l galactose (i.e., conditions that would favor galactitol synthesis) compared with control cells in galactose-free, physiological medium. Acute exposure (3 hours) of cultured BLECs to a range of 10 to 40 mmol/l galactitol or 5.5 to 44 mmol/l galactose plus the aldose reductase inhibitor (ARI), sorbinil, established by Dixon plot that galactitol, but not galactose, inhibited both the high- and the low-affinity MI transport sites. MI uptake was markedly stimulated in cultured BLECs exposed to galactose-free, hyperosmotic medium by the addition of extracellular raffinose, mannitol, or sorbitol for 20 hours. The enhanced uptake involved increase in the maximal velocity without significant change in Km of both the high- and low-affinity MI transport sites, as indicated by Lineweaver-Burk analysis. However, a similar coadministration of 150 mmol/l sorbitol to the 40 mmol/l galactose (Gal) medium significantly increased, but failed to normalize, the MI uptake relative to that observed with galactose-free, physiological medium. The combined administration of 150 mmol/l sorbitol and the ARI, zopolrestat, to Gal significantly exceeded the MI uptake observed with physiological medium. Exposure of BLECs to cycloheximide for 20 hours did not affect MI uptake in cells maintained in 40 mmol/l galactose but inhibited the otherwise enhanced MI uptake in cells maintained in Gal plus 150 mmol/l sorbitol and zopolrestat in the omission versus the inclusion of cycloheximide. CONCLUSIONS: These results suggest that bovine lens epithelial cells respond to hypertonic stress by elevating myo-inositol transport activity. The increase in MI uptake is due to an increase in the number (or, possibly, a change in the transporter turnover rate) of high- and low-affinity, sodium-dependent MI transporters expressed as a result of the osmotic shock stemming from exposure to hypertonic medium.

Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 2: Cloning of a 626 bp cDNA portion of a Na+/myo-inositol cotransporter, an osmotic shock protein.

PURPOSE: Bovine lens epithelial cells (BLECs) accumulate osmotically active organic solutes (i.e., osmolytes) including myo-inositol when exposed to hypertonic stress (osmotic shock). In hypertonic medium, the increase in myo-inositol accumulation is attributed to an elevation in activity of Na+/myo-inositol cotransporter(s). The authors report the nature of the hypertonicity-induced enhancement of myo-inositol uptake in cultured BLECs by amplifying a 626 bp cDNA from lens cell RNA. METHODS: A portion of cDNA encoding a Na+/myo-inositol cotransporter was isolated from cultured BLECs using PCR primers designed from an established myo-inositol transporter from Madin-Darby canine kidney (MDCK) cells. Using the reverse transcription-polymerase chain reaction, a 626 bp PCR product was amplified. Its nucleic acid sequence was determined by the dideoxynucleotide method using Sequenase kit. Na+/Myo-inositol cotransporter mRNA expression in the cultured cells was demonstrated under physiological and hypertonic conditions by northern analysis of poly(A)+ RNA using the lens cell 626 bp cDNA as probe. RESULTS: The BLEC cDNA sequence was 92% identical with the Na+/myo-inositol cotransporter of MDCK cells. Myo-inositol transporter mRNA was demonstrated in cultured BLECs and was significantly induced by hypertonic stress. CONCLUSIONS: These data suggest that cultured bovine lens epithelial cell adaptation to hypertonicity involves intracellular accumulation of small organic osmolytes (i.e., myo-inositol) through elevation of myo-inositol uptake activity resulting from the upregulation of transporter mRNA.

Protein kinase C activity and its relationship to myo-inositol uptake during hyperglycemic conditions in cultured bovine lens epithelial cells.

Incubation of cultured bovine lens epithelial cells (BLECs) in minimal essential medium (MEM) containing 40 mM galactose for 20 hr results in an attenuation of 3H-myo-inositol (3H-MI) concentrating ability. Decreased MI uptake could negatively impact on normal phosphoinositide turnover and diacylglycerol production, and presumably, protein kinase C (PKC) activation. The present report examines the relationship between PKC activity, myo-inositol transport and hyperglycemic conditions. PKC activities in the cytosol and particulate fractions of bovine lens epithelial cells in culture were quantitated using a mixed micelle assay following DEAE-cellulose (DE52) and Sephadex G-25 chromatography. Protein kinase C activity was assessed as Ca2+ and phospholipid-dependent Ac-myelin basic protein substrate peptide phosphorylation and confirmed using a PKC pseudosubstrate inhibitor peptide (PKC 19-36). Total PKC activity was similar in galactose-incubated cells (871 +/- 64 pmol/mg total protein/min) and control cells (881 +/- 8 pmol/mg total protein/min) after 20 hr. In unstimulated cells, approximately 90% of the total cellular PKC activity was recovered in the cytosolic fraction. Enzyme translocation was induced with the tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), resulting in a 6-fold increase in membrane-associated PKC activity. A similar PMA-induced translocation was observed in BLECs incubated with 40 mM galactose MEM-maintained cells briefly treated with PMA or the non-phorbol PKC activators, SC-10 and mezerein, displayed a rate of 3H-MI uptake similar to the untreated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of myo-[3H]inositol uptake by glucose and sorbitol in cultured bovine lens epithelial cells. I. Restoration of myo-inositol uptake by aldose reductase inhibition.

The association between high-ambient glucose, the polyol pathway, and aldose reductase inhibition on in vitro myo-[3H]inositol uptake was examined in cultured bovine lens epithelial cells (BLECs). Myo-[3H]inositol accumulation in the presence of 5.5 mmol/l D-glucose was rapid and linear for 8 hr. When Na+ was replaced on an equal molar basis with N-methyl-D-glucamine chloride, myo-[3H]inositol uptake was reduced by more than 95%. The myo-inositol transport system appear to be distinct from glucose transport, based upon three criteria: (1) 2-deoxy-D-[3H]glucose uptake, unlike myo-[3H]inositol uptake, was largely sodium independent; (2) L-glucose was a competitive inhibitor of myo-[3H]inositol uptake but had no effect on 2-deoxy-D-[3H]glucose uptake; and (3) 2-deoxy-D-[3H]glucose uptake appeared independent of myo-inositol concentration. Sodium-dependent myo-[3H]inositol uptake was substantially inhibited after chronic (20 hr) exposure of cultured cells to 40 mmol/l glucose. Inhibition of aldose reductase activity partially prevented the inhibitory effect of glucose on myo-[3H]inositol accumulation. No significant difference in the rates of passive efflux of myo-[3H]inositol from preloaded high glucose-treated and control cultures was observed. Although the coadministration of sorbinil to the high-glucose medium partially protected against the attendant decrease in transport activity, the failure to normalize myo-[3H]inositol uptake suggested that glucose-sensitive and sorbitol-sensitive processes were involved in the uptake of myo-inositol.

Modulation of myo-[3H]inositol uptake by glucose and sorbitol in cultured bovine lens epithelial cells. II. Characterization of high- and low-affinity myo-inositol transport sites.

Myo-[3H]inositol accumulation in cultured bovine lens epithelial cells (BLECs) occurred by high- and low-affinity, Na(+)-dependent transport sites. The high- and low-affinity transport systems had a Km of 27 +/- 4 and 157 +/- 22 mumol/l, respectively, and Vmax of 652 +/- 35 and 2952 +/- 308 pmol/mg protein/hr, respectively. The uptake of myo-[3H]inositol was lowered after chronic (20 hr) incubation of cultured cells in 40 mmol/l glucose throughout the concentration range for 1.5-400 mumol/l myo-inositol. The coadministration of sorbinil (0.1 mmol/l) to 40 mmol/l glucose partially prevented the inhibitory effect of glucose on myo-[3H]inositol uptake. Although the aldose reductase inhibitor prevented the inhibitory effect of glucose on the low-affinity transport site, a glucose-sensitive process for myo-[3H]inositol uptake on the high-affinity transport site was uncovered by Lineweaver-Burk analysis. Acute exposure (3 hr) of cultured BLECs maintained in physiologic medium (Eagle's minimal essential medium, 5.5 mmol/l glucose) to a range of 5.5-44 mmol/l glucose plus sorbinil also caused a decrease in myo-[3H]inositol uptake. Dixon plot analysis confirmed that the acute effect of glucose was the result of competitive inhibition of the high-affinity myo-inositol transport site. Acute exposure of cultured cells to 10-40 mmol/l sorbitol also diminished the accumulation of myo-[3H]inositol. Dixon plot analysis established that the acute effect of exogenous sorbitol was the result of competitive inhibition of the low-affinity myo-inositol transport site.(ABSTRACT TRUNCATED AT 250 WORDS)

Uncoupling of attenuated myo-[3H]inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells.

Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-[3H]inositol was lowered after 20 h of incubation in galactose and remained significantly below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-[3H]inositol uptake. No significant difference in the rates of passive efflux of myo-[3H]inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

Depletion of glutathione by L-buthionine sulfoximine does not promote inactivation of myo-inositol transport in cultured bovine lens epithelial cells.

Exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) promotes a decrease in the cellular content of reduced glutathione (GSH) as galactitol increases. Incubation of BLECs with Gal also leads to a reduction in 3H-myo-inositol (3H-MI) concentrating capability. Studies were therefore initiated to determine the nature of the relationship between polyol accumulation, GSH content and the attenuation of the active transport of myo-inositol. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-maintained cells to a concentration below that characteristically observed in Gal-treated cells, under conditions whereby no galactitol accumulation could or had occurred. L-BSO (0.5 mM) was simultaneously administered to BLECs maintained in either MEM or Gal for up to five days. The cellular content of GSH after five days of continuous incubation was 3.3 micrograms GSH/micrograms PO4 in MEM alone and 0.45 microgram GSH/microgram PO4 in MEM + BSO. Moreover, the GSH content in BLECs exposed to Gal for five days was 1.9 micrograms GSH/micrograms PO4 and was not detectable in the Gal + BSO-treated cells. However, the ability to concentrate 3H-MI in MEM + BSO-treated BLECs was equivalent to that observed with MEM-maintained cells regardless of the significant difference in GSH content. Likewise, L-BSO addition to Gal-treated cells, while virtually depleting the intracellular GSH content, did not further decrease the ability of the cells to accumulate 3H-MI compared to that observed with BLECs in Gal alone. Indeed, supplementation of Gal-treated cells with exogenous GSH failed to correct the Gal-induced attenuation in myo-inositol concentrating ability. These studies demonstrate that the Gal-induced depletion of cellular GSH and the Gal-induced deficit in ability to concentrate myo-inositol are not associated and represent independent events. That is, depletion of lens cell GSH does not lead to the attenuation of myo-inositol uptake in cultured lens epithelial cells.

Sorbinil prevents the hypergalactosemic-induced reduction in [3H]-myo-inositol uptake and decreased [3H]-myo-inositol incorporation into the phosphoinositide cycle in bovine lens epithelial cells in vitro.

The synthesis of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4-5-bisphosphate was studied using 3H-myo-inositol (3H-MI) as precursor in cultured bovine lens epithelial cells (BLECs) maintained in galactose-free, physiological medium or 40 mM galactose (Gal) +/- sorbinil for six days. The formation of inositol polyphosphates from phosphoinositides was also shown. Galactitol did not exceed 2mM in Gal-incubated cells after six days of exposure; no galactitol was observed in BLECs maintained in galactose-free, physiological medium or Gal supplemented with sorbinil. Uptake of 3H-myo-inositol(3H-MI) into BLECs was significantly reduced in cells exposed to Gal. A concomitant reduction in 3H-MI incorporation was observed in the phosphoinositides, as well as with the released inositol phosphates. The simultaneous addition of sorbinil to the Gal medium corrected the drop in 3H-MI uptake and normalized 3H-MI incorporation into the phosphoinositides and inositol phosphates. While an apparent decrease in the three inositol-containing lipids was observed with the Gal-incubated cells, based on 3H-MI incorporation, there was no change in total membrane phosphatidylinositol content when compared to cells maintained in physiological medium as determined by the microgram Pl PO4 per microgram total membrane PO4. The apparent loss of radiolabeled phosphoinositides was attributed to the decreased specific activity resulting from the lower internal pool of 3H-MI in the Gal-exposed cells available for incorporation into the phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

L-buthionine sulfoximine-induced loss of glutathione does not elicit PGH synthase inactivation in cultured bovine lens epithelial cells.

Previous studies from this laboratory have indicated that exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) for as short a duration as 20 h results in a reduction of microsomal prostaglandin biosynthesis as demonstrated by a decrease in PGH synthase activity (Invest. Ophthalmol. Vis. Sci. 29:1452-1460, 1988). The present study shows that upon brief exposure of BLECs to Gal, the cellular content of glutathione (GSH) decreases as galactitol increases. Studies were therefore undertaken to establish whether a positive correlation existed between polyol accumulation, GSH content and the activity of PGH synthase (an enzyme known to auto-oxidize) utilizing BLECs exposed to hypergalactosemic conditions. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-incubated cells to a level below that routinely observed in Gal-incubated cells, under conditions whereby no galactitol accumulation had occurred. The galactitol content was 92 nmol/micrograms PO4 in Gal-incubated cells after a 20 h exposure period; no detectable level of galactitol was observed in BLECs maintained in galactose-free MEM. L-(BSO) (1 mM) was co-administered to BLECs maintained in either MEM or Gal for 20 h. The cellular content of GSH was 1.70 +/- .02 micrograms GSH/micrograms PO4 in MEM alone and 0.540 +/- .02 micrograms GSH/micrograms PO4 in MEM + BSO. Furthermore, the GSH content in BLECs after 20 h of exposure to Gal was 0.960 +/- .01 microgram GSH/microgram PO4 but decreased to 0.110 +/- .01 microgram GSH/microgram PO4 in Gal + BSO.(ABSTRACT TRUNCATED AT 250 WORDS)