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.

Excretion profile of boldenone and its metabolites after oral administration to veal calves.

The residue profiles of boldenone (17beta-Bol), its epimer (17alpha-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17alpha-Bol decreased rapidly after end of treatment; detectable amounts of 17alpha-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17beta-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.

Accelerated solvent extraction and liquid chromatography-tandem mass spectrometry quantitation of corticosteroid residues in bovine liver.

A new method for the rapid extraction and unequivocal confirmation of two highly potent fluorinated synthetic corticosteroids, dexamethasone and its beta-epimer betamethasone, in bovine liver was developed. Flumethasone was used as internal standard. An extraction procedure using an accelerated solvent extraction system was employed for the isolation of the analytes in liver samples. The procedure was highly automated, including defatting and extraction steps, sequentially carried out under 1.0 x 10(4) kPa in about 35 min. The extracts were then directly analysed by tandem mass spectrometry with on-line liquid chromatography. The analytes were ionised in a heated nebulizer interface operating in the negative ion mode where the molecular related ions [M-H-CH2O]- were generated for each analyte, at m/z 361 for betamethasone and dexamethasone and at m/z 379 for flumethasone. They served as precursor ions for collision-induced dissociation and three diagnostic product ions for the drugs were identified to carry out analyte confirmation by selected reaction monitoring. Assessment of recovery, specificity and precision for betamethasone, dexamethasone and flumethasone proved the method suitable for confirmatory purposes. The limit of quantification of betamethasone and dexamethasone in liver tissue was 1.0 microg/kg.

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.

Quantitation of anabolic hormones and their metabolites in bovine serum and urine by liquid chromatography-tandem mass spectrometry.

A specific and sensitive method based on tandem mass spectrometry with on-line high-performance liquid chromatography using atmospheric pressure chemical ionisation (LC-APCI-MS-MS) for the quantitation of anabolic hormone residues (17beta-19-nortestosterone, 17beta-testosterone and progesterone) and their major metabolites (17alpha-19-nortestosterone and 17alpha-testosterone) in bovine serum and urine is reported. [2H2]17Beta-testosterone was used as internal standard. The analytes were extracted from urine (following enzymatic hydrolysis) and serum samples by liquid-liquid extraction and purified by C18 solid-phase extraction. Ionisation was performed in a heated nebulizer interface operating in the positive ion mode, where only the protonated molecule, [M+H]+, was generated for each analyte. This served as precursor ion for collision-induced dissociation and two diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring LC-MS-MS. The overall inter-day precision (relative standard deviation) ranged from 6.37 to 2.10% and from 6.25 to 2.01%, for the bovine serum and urine samples, respectively, while the inter-day accuracy (relative error) ranged from -5.90 to -3.18% and from -6.40 to -2.97%, for the bovine serum and urine samples, respectively. The limit of quantitation of the method was 0.1 ng/ml for all the hormones in bovine serum and urine. On account of its high sensitivity and specificity the method has been successfully used to confirm illegal hormone administration for regulatory purposes.

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.

Quantification of 17 beta-estradiol residues in bovine serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

A method for the quantification of the natural hormone 17 beta-estradiol (17 beta-E2) in bovine serum by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS) was developed. Ethinylestradiol (EE2) was used as internal standard. Analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a polymeric reversed-phase (PLRP-S) LC column. They were ionized in a heated nebulizer (HN) interface operating in the negative ion mode, where only the intact deprotonated molecules, [M - H]-, were generated at m/z 271 and 295 for 17 beta-E2 and EE2, respectively. These served as precursor ions for collision-induced dissociation (CID) and diagnostic product ions were identified for the unambiguous hormone confirmation by selected reaction monitoring (SRM) LC-APCI-MS-MS. The method was validated on bovine serum and the limit of quantification (LOQ) was 30 pg ml-1 for 17 beta-E2. The inter-day precision (relative standard deviation, RSD) and accuracy (relative error, RE) derived from the analyses of validation samples at three concentrations ranged from 1.76 to 3.76 and from -4.18 to -2.01%, respectively. This method is currently being successfully applied to measure the bovine serum concentration of 17 beta-E2 in order to discriminate between the physiological concentrations of 17 beta/E2 and the hormone levels resulting from illegal administration.

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.

Determination of thyreostatic residues in cattle plasma by high-performance liquid chromatography with ultraviolet detection.

A procedure is described for the concurrent assay of thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil and methimazole in bovine plasma. In this procedure, reversed-phase high-performance liquid chromatography is performed after liquid-liquid extraction of plasma with ethyl acetate. Compounds are quantified by ultraviolet detection using a wavelength of 276 nm for thiouracil, methylthiouracil, propylthiouracil and phenylthiouracil and 258 nm for methimazole. The linearity range, precision, recovery and detection limits were determined and the method was shown to be applicable to samples of plasma from young bulls experimentally treated with methylthiouracil.

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.