Biomedical subjects

M B Burg

Publications and source records attributed to M B Burg.

At least 37 records · Page 2Linked to original sources

PubMed1996

ORE, a eukaryotic minimal essential osmotic response element. The aldose reductase gene in hyperosmotic stress.

Organisms, almost universally, adapt to hyperosmotic stress through increased accumulation of organic osmolytes but the molecular mechanisms have only begun to be addressed. Among mammalian tissues, renal medullary cells are uniquely exposed to extreme hyperosmotic stress. Sorbitol, synthesized through aldose reductase, is a predominant osmolyte induced under hyperosmotic conditions in renal cells. Using a rabbit renal cell line, we originally demonstrated that hyperosmotic stress induces transcription of the aldose reductase gene. Recently, we cloned the rabbit aldose reductase gene, characterized its structure, and found the first evidence of an osmotic response region in a eukaryotic gene. Now, we have progressively subdivided this 3221-base pair (bp) region into discrete fragments in reporter gene constructs. Thereby, we have functionally defined the smallest sequence able to confer hyperosmotic response on a downstream gene independent of other putative cis-elements, that is, a minimal essential osmotic response element (ORE). The sequence of the ORE is CGGAAAATCAC(C) (bp -1105/-1094). A 17-bp fragment (-1108/-1092) containing the ORE used as a probe in electrophoretic mobility shift assays suggests hyperosmotic induction of a slowly migrating band. Isolation of trans-acting factor(s) and characterization of their interaction with the ORE should elucidate the basic mechanisms for regulation of gene expression by hyperosmotic stress.

Aldehyde Reductase↗

PubMed1996

Coordinate regulation of organic osmolytes in renal cells.

Adaptation of cells to prolonged hypertonicity generally involves accumulation of compatible organic osmolytes. Renal medullary cells in vivo and in tissue culture accumulate several different organic osmolytes, including sorbitol, inositol, betaine, and glycerophosphocholine (GPC) in response to hypertonicity. For the total concentration of these organic osmolytes to be appropriate for the ambient tonicity, an increase in one should cause the others to fall, minimizing changes in their total concentration. The experiments presented here demonstrate this in tissue culture and investigate the mechanisms involved. Sorbitol is synthesized from glucose, catalyzed by aldose reductase. Betaine is transported into the cells. Hypertonicity increases transcription of the aldose reductase and betaine transporter genes, ultimately elevating cell sorbitol and betaine. If aldose reductase is inhibited, which prevents accumulation of sorbitol, betaine transporter gene expression increases, resulting in a higher cell betaine that compensates for the lower sorbitol. Conversely, when cell betaine is altered by changing its concentration in the medium, aldose reductase transcription changes reciprocally, resulting in compensating changes in cell sorbitol. Hypertonicity increases GPC by inhibiting GPC:choline phosphodiesterase (GPC:PDE), an enzyme that degrades GPC. When cell betaine or inositol is increased by raising its concentration in the medium, GPC:PDE activity rises, reducing cell GPC. Thus, the total of the osmolytes, rather than the level of any individual one, is maintained.

Animals↗

PubMed1996

Osmotic regulation of gene expression.

Cells react to increased osmolality with numerous changes in gene expression. The specific genes affected differ between species, but the known osmoprotective effects of the gene products are remarkably similar, particularly with regard to cellular accumulation of compatible organic osmolytes. Here we concentrate on the molecular basis for osmotic regulation of gene expression, emphasizing certain genes expressed in bacteria, yeast, and the mammalian renal medulla because their expression is best understood. Thus, we emphasize 1) bacterial and yeast two-component histidine kinase systems, each consisting of a membrane osmolality sensor and a separate cytoplasmic response regulator that, when phosphorylated, alters transcription, 2) volume regulatory increases in cellular K+ salts that can prompt increased gene transcription in bacteria through direct effects on DNA and that in mammalian renal cells increase transcription, seemingly via trans-activating proteins, 3) a yeast kinase cascade that transmits an osmotic signal to the gene regulating the level of glycerol, and 4) in mammalian cells, several homologous cascades that are activated by hypertonicity, but whose osmoregulatory targets are not yet known.

Animals↗

PubMed1996

Betaine and inositol reduce MDCK cell glycerophosphocholine by stimulating its degradation.

The amount of glycerophosphocholine (GPC) in renal medullary cells in vivo and in cultured renal [Madin-Darby canine kidney (MDCK)] cells varies with extracellular NaCl and urea. We previously showed that this is largely due to modulation of GPC degradation catalyzed by GPC:choline phosphodiesterase (GPC: PDE). GPC also varies inversely with the levels of other compatible osmolytes, the accumulation of which is induced by high tonicity. We tested whether GPC:PDE activity and GPC degradation are affected by accumulation of compatible osmolytes other than GPC. We find that MDCK cell GPC content decreases when the cells take up betaine and/or inositol from the medium. The effect is considerably greater for cells in isosmotic or high-NaCl medium than in high-urea medium. This difference is associated with suppression of betaine and inositol accumulation with high urea. We then measured GPC:PDE activity with a novel chemiluminescent assay. Addition of inositol and/or betaine to the medium greatly increases GPC:PDE activity in cells in isosmotic or high-NaCl media, but the increase is much less in high-urea medium. The increases in GPC:PDE activity, associated with the presence of betaine, are accompanied by commensurate increases in absolute rates of endogenous GPC degradation by cells in isosmotic or high-NaCl medium. We found previously that, in MDCK cells incubated for 2 days in high-NaCl medium, the rate of GPC synthesis from phosphatidylcholine is increased, correlated with an increase in phospholipase activity. However, in the present experiments, betaine accumulation has no effect on phospholipase activity under those conditions and, thus, presumably does not affect GPC synthesis. Collectively, these data support the conclusion that betaine and/or inositol reduces GPC by increasing GPC degradation catalyzed by GPC:PDE. This mechanism enables GPC to be reciprocally regulated relative to other compatible osmolytes, thus maintaining an appropriate total osmolyte content.

Animals↗

PubMed1996

CD9 antigen mRNA is induced by hypertonicity in two renal epithelial cell lines.

In diverse organisms, cells adapt to hyperosmotic stress by accumulating organic osmolytes. Mammalian renal medullary cells are routinely under osmotic stress. Two renal cell lines, Madin-Darby canine kidney (MDCK) and PAP-HT25, have been widely used to study mammalian osmotic regulation. In these epithelial cells, extracellular hypertonicity induces gene transcription of proteins directly involved in the metabolism and transport of organic osmolytes. This induction is relatively specific and not part of a generalized stress response. Little is known about the signal transduction pathway between cellular detection of extracellular osmolality and increased specific gene transcription. Here, using differential mRNA display polymerase chain reaction on MDCK cells in isotonic vs. hypertonic medium, we identify a cDNA product corresponding to CD9 antigen mRNA. CD9 antigen is a cell surface glycoprotein originally found in cells of the immune system. Although CD9 antigen has been structurally characterized, its function is unclear. We further demonstrate that CD9 antigen mRNA is present in MDCK and PAP-HT25 cells and that its mRNA abundance is induced by extracellular hypertonicity, but not by heat stress. Also, we show that accumulation of organic osmolytes markedly attenuates the CD9 mRNA induction, as only recently demonstrated with genes involved in the hyperosmotic stress response. This suggests a role for CD9 antigen in this response.

Animals↗

PubMed1996

Betaine transporter cDNA cloning and effect of osmolytes on its mRNA induction.

Cells generally adapt to long-term hyperosmolality by accumulating compatible organic osmolytes, thereby helping to normalize both volume and intracellular inorganic ion concentration. When organic osmolytes are accumulated, as in renal inner medullary cells, it is the sum of their concentrations that is theoretically important. In effect, when one organic osmolyte rises, the others generally fall to maintain their sum approximately constant. The present study addresses the mechanism controlling betaine accumulation. Hypertonicity induces accumulation of betaine, sorbitol, inositol, and other organic osmolytes in PAP-HT25 cells, a line derived from rabbit renal papilla. Hypertonicity increases the betaine transporter expression in these cells. To obtain a specific probe for betaine transporter mRNA, we cloned from PAP-HT25 cells a cDNA that encodes the full protein. We then examined the effect of betaine, sorbitol, and inositol on betaine transporter mRNA abundance. Increased accumulation of any of these three organic osmolytes reduces betaine transporter mRNA. We previously observed similar results for aldose reductase, the enzyme responsible for osmotically regulated sorbitol accumulation. We conclude that the accumulation of organic osmolytes regulates betaine transporter gene expression. Because the aldose reductase gene is controlled in a similar fashion, we surmise that the two genes share a common signal for induction.

Amino Acid Sequence↗

PubMed1996

Organic osmolyte distribution and levels in the mammalian urinary bladder in diuresis and antidiuresis.

Inositol, sorbitol, glycerophosphocholine (GPC), and betaine are organic osmolytes that are accumulated by renal medullary cells in response to hyperosmotic stress. Previous screening studies, using nuclear magnetic resonance spectroscopy, have shown some of these same compounds to be present in extracts of whole urinary bladder from rabbits and rats. In the present study, we used high-performance liquid chromatography to quantify levels of these compounds in the separated epithelium and muscle of bladders taken from normal rabbits as well as diuretic and thirsted rats. We find that 1) high concentrations of organic osmolytes, namely inositol, GPC, and sorbitol, are present in urinary bladder; 2) levels of these osmolytes in the bladder epithelium are higher than in the muscle; 3) increased urinary osmolality due to antidiuresis is associated with a 2.4-fold increase in total osmolyte levels in rat bladder epithelium and a lesser (1.5-fold) increase in the muscle, compared with corresponding levels in tissues from diuretic animals; and 4) these increases in total osmolyte amounts in the epithelium are due to increased levels of GPC, sorbitol, and perhaps inositol (P = 0.07), whereas only GPC increases in the bladder muscle.

Animals↗

PubMed1996

Glycerophosphocholine and betaine counteract the effect of urea on pyruvate kinase.

Renal medullary cells contain large quantities of organic osmolytes when the levels of salt and urea in renal medullary interstitial fluid are high. Two of these osmolytes, betaine and glycerophosphocholine (GPC), are methylamines. Methylamines generally counteract the perturbing effects of urea on enzymes and other macromolecules. Betaine was previously shown to counteract the effect of urea on enzymes in vitro and to protect renal cells in tissue culture from harmful effects of high urea. Nevertheless, renal medullary cells in vivo and in tissue culture specifically accumulate GPC rather than betaine, in response to high urea. In the present studies we tested directly whether GPC counteracts the effect of urea on the Km of pyruvate kinase (PK) for ADP and compared the effectiveness in this regard of GPC to that of betaine. We find that urea increases the Km (as previously observed), that betaine and GPC decrease it, and that the increase caused by urea is counteracted by betaine or by GPC. The effects of GPC are slightly less than those of betaine. In addition, other renal medullary organic osmolytes (namely sorbitol, inositol and taurine) were already known to be compatible osmolytes whose accumulation protects renal medullary cells from hypertonicity because they have little effect on enzyme function. In agreement with this generalization we find that high sorbitol or inositol has little or no effect on PK activity, but surprisingly that taurine reduces Vmax and greatly elevates Km. In conclusion, the main finding is direct evidence that GPC is a counteracting osmolyte, which explains its accumulation in response to high urea. However, we do not find that GPC is a more effective counteracting osmolyte than betaine, which leaves unexplained the preference of renal cells for GPC over betaine for counteracting the perturbing effect of urea.

Animals↗

PubMed1995

Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells.

Glycerophosphocholine (GPC) is osmotically regulated in renal medullary cells and in cultured Madin-Darby canine kidney (MDCK) cells. Previously, it was shown that a high extracellular concentration of urea or NaCl causes these cells to accumulate large amounts of GPC. GPC is known to be a product of phosphatidylcholine (PC) catabolism. The purpose of the present experiments was to examine the role of changes in the rate of GPC synthesis from PC in hyperosmotically induced GPC accumulation. When 1-palmitoyl-2-lysophosphatidyl-[methyl-3H]choline ([3H]LPC) is added to the medium, it is taken up by the cells and most of it is rapidly converted to PC. During a chase, 3H lost from PC appears almost exclusively in GPC and sphingomyelin. The rate of catabolism of PC is twofold greater in cells exposed to high NaCl (200 mosmol/kgH2O, added for 2 days) than in control or high-urea medium. Increased PC catabolism in NaCl-treated cells is associated with a 2.6-fold increase in GPC synthesis from PC; sphingomyelin synthesis decreases, and total cell PC does not change. Also, neither total mass nor specific radioactivity of lysophosphatidylcholine changes. PC catabolism is unaffected by short (2 h) exposure to high NaCl or urea. To investigate the enzymatic basis for the increased PC catabolism in response to high NaCl, phospholipase activity was measured in cell homogenates with 1-palmitoyl-2-[1-14C]palmitoyl-PC as a substrate. Exposure of cells to high NaCl for 2 days (but not 2 h) increases activity 2.8-fold compared with control or high-urea medium. Lysophospholipase activity (measured with [3H]LPC as the substrate) is unchanged. The increased phospholipase activity occurs with dipalmitoyl PC, but not sn-2-arachidonyl PC, as a substrate. Collectively, these data suggest a role for a phospholipase, unrelated to the arachidonyl-selective enzyme, in the regulation of PC catabolism during accumulation of GPC induced by prolonged exposure to high extracellular NaCl.

Animals↗

PubMed1995

Osmoregulation of GPC:choline phosphodiesterase in MDCK cells: different effects of urea and NaCl.

The organic osmolyte, glycerophosphocholine (GPC), accumulates in renal cells in response to high concentrations of either NaCl or urea, despite the very different effects of these solutes on cell function and volume. Together, high levels of these solutes increase GPC amount in Madin-Darby canine kidney cells by inhibiting its enzymatic degradation. The present study tests the effects of NaCl and urea, individually, on GPC accumulation and its degradation. A technique was developed to determine the absolute rate of GPC degradation by measuring the initial rate of disappearance of [3H]GPC (pulsed into the cells by hypotonic shock) and the specific activity of GPC in the cells. The mass of GPC in the cells was measured by another newly developed method, a sensitive chemiluminescent assay. We find that exposure to high NaCl or urea decreases the absolute rate of cellular GPC degradation by approximately one-half during the first 20.5 h. Reductions in GPC degradation are accompanied by commensurate decreases in the activity of GPC:choline phosphodiesterase (GPC:PDE; EC 3.1.4.2), an enzyme that catalyzes degradation of GPC. Activity of GPC:PDE falls > 50% in cells exposed for 2 h to high osmolality. Inhibition is sustained for 7 days with high urea alone. In contrast, with high NaCl alone, GPC:PDE activity reverts to control values by 7 days, by which time synthesis of GPC is increased, accounting for sustained GPC accumulation. Collectively, these data suggest that GPC accumulation in response to either high NaCl or urea occurs initially by inhibition of its degradation but that the effect of NaCl on degradation differs, in that it is transient, while that of urea is sustained.

Animals↗

PubMed1995

Molecular basis of osmotic regulation.

Cells almost universally respond to the stress of long-term hyperosmolality by accumulating compatible organic osmolytes. This allows them to maintain normal cell volume without a deleterious increase in intracellular inorganic ion concentration. Cells in the renal inner medulla are exposed to variable concentrations of salt and urea that may reach molal levels. The organic osmolytes that they accumulate include sorbitol, betaine, inositol, taurine, and glycerophosphocholine (GPC). This review considers recent advances in understanding osmotic regulation of these substances. Sorbitol is synthesized from glucose catalyzed by aldose reductase. Hypertonicity elevates the abundance of this enzyme by increasing transcription of its gene. Betaine is taken up via a specialized transporter. Hypertonicity raises the number of transporters by increasing their transcription. Current studies demonstrate that the 5' regions flanking the aldose reductase and betaine transporter genes contain osmotic response elements that increase transcription in response to hypertonicity. Osmotic regulation of inositol and taurine uptake also involves increased expression of specific transporter genes. GPC is unique in that its level rises in response to high urea, as well as hypertonicity. GPC accumulation is mainly regulated by changes in its degradation to choline, catalyzed by GPC:choline phosphodiesterase. Numerous other genes, including those for heat shock proteins, are also induced by hypertonicity. Their regulation and their role in osmotic regulation are the subject of considerable ongoing research.

Aldehyde Reductase↗

PubMed1994

Cloning, genomic organization, and osmotic response of the aldose reductase gene.

Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We have cloned and characterized the rabbit AR gene to determine how it is regulated by hyperosmolality. The length of the gene, not including 5' or 3' flanking regions, is approximately 14.7 kilobases (kb) organized into 10 exons and 9 introns. The transcription start site is 36 base pairs upstream of the initiator methionine codon. A 5-kb fragment containing approximately 3.5 kb of 5' flanking region was isolated. The 3.5-kb sequence was examined for basal promoter activity and hyperosmotic response in luciferase reporter gene constructs. A 235-base-pair fragment (base pairs -208 to +27) was able to drive the downstream reporter gene in transfected PAP-HT25 cells under isoosmotic conditions (300 mosmol/kg of H2O). When this fragment plus the remaining upstream sequence (from approximately base pair -3429 to base pair +27) was used, cells in hyperosmotic medium (500 mosmol/kg of H2O) showed about 40-fold induction of luciferase expression compared with cells in isoosmotic medium. The upstream fragment (from approximately base pair -3429 to base pair -192) also conferred osmotic response to a heterologous promoter (B19). This finding evidences putative osmotic response element(s) (OREs) within a specific DNA fragment in a eukaryotic genome. Identification and characterization of OREs within this fragment and their associated trans-acting factors should reveal the molecular mechanisms of gene regulation in osmotic stress.

Aldehyde Reductase↗

PubMed1994

Molecular basis for osmoregulation of organic osmolytes in renal medullary cells.

Renal medullary cells are naturally exposed to extremely high and variable interstitial concentrations of NaCl and urea, consequent to operation of the urinary concentrating mechanism. They respond by accumulating large and variable amounts of sorbitol, glycerophosphocholine (GPC), glycine betaine (betaine), myo-inositol (inositol), and taurine both in vivo and in cell cultures. Sorbitol is synthesized from glucose, catalyzed by aldose reductase. Hypertonicity increases aldose reductase activity by raising this enzyme's transcription, mRNA level, and translation, and thereby increases production of sorbitol. GPC is synthesized from choline via phosphatidylcholine. A combination of high NaCl plus urea does not increase GPC synthesis, but does reduce its degradation by inhibiting GPC:choline phosphodiesterase. Betaine, inositol and taurine are taken up into the cells, each by a different sodium-dependent transporter. Hypertonicity increases mRNAs of all three transporters. This is due to increased transcription (at least of the inositol and betaine transporters). The eventual result is greater betaine, inositol and taurine uptake and accumulation. Osmoregulation of net sorbitol and GPC synthesis and of betaine, inositol and taurine transport is slow, requiring hours to days. However, following an acute fall in tonicity, these organic osmolytes exit from the cells within minutes, via specialized efflux mechanisms. As demonstrated by cloning efficiency studies, renal cell survival and growth following hypertonicity depend on the sum of all organic osmolytes that are accumulated; altering one experimentally changes the others to maintain a nearly constant total. Methylamine accumulation protects these cells against high urea; the methylamine that is preferentially accumulated in response to high urea is GPC.

Animals↗

PubMed1994

Macromolecular crowding and confinement in cells exposed to hypertonicity.

The nonideal properties of solutions containing high concentrations of macromolecules can result in enormous increases in the activity of the individual macromolecules. It has been proposed that molecular crowding and confinement occur in cells and are major determinants of the activity of the proteins and other intracellular macromolecules. This concept has important implications for cell volume regulation because, under crowded conditions, relatively small changes in concentration, consequent to alterations of water content, lead to large changes in macromolecular activity. This review considers several aspects of macromolecular crowding and confinement, including: 1) the physical chemical principles involved; 2) in vitro demonstrations of the effects; 3) relation to water activity; 4) estimates of the actual intracellular activity of water and macromolecules; 5) relation to osmotic regulation in various types of cells, including bacteria, red blood cells, and complex nucleated cells; and 6) the relation to inorganic ions and organic osmolytes in cells stressed by hypertonicity. We conclude that, while there is compelling evidence for important effects of molecular crowding in vitro and in red blood cells, the role of macromolecular crowding and confinement in osmotic regulation of more complex cells is an open question that deserves the extensive attention it is currently receiving.

Animals↗

PubMed1994

Induction of gene expression by heat shock versus osmotic stress.

Elevated temperature rapidly increases expression of genes for heat shock proteins (HSP), including HSP-70. The response is presumably triggered by denaturation of cell proteins and helps in their renaturation. Hypertonicity may also denature proteins, but the protective response, which is accumulation of compatible organic osmolytes [including betaine and inositol in Madin-Darby canine kidney (MDCK) cells], apparently differs and is slow. Recently, hypertonicity was found also to increase expression of HSP-70 in MDCK cells, a response proposed to provide protection until organic osmolytes can accumulate. Our purpose was to examine whether 1) a gene involved in accumulation of organic osmolytes also responds to heat stress and 2) whether accumulation of organic osmolytes affects expression of HSP-70. We find that 1) the betaine transporter mRNA, which is greatly increased by hypertonicity (515 vs. 315 mosmol), is unaffected by high temperature (42 degrees C vs. 37 degrees C); 2) hypertonicity-induced increases in HSP-70 and betaine transporter mRNA are much greater when the medium (and cell) contain no betaine and no inositol than when high concentrations of these are present; and 3) high betaine greatly inhibits the increase in HSP-70 mRNA at high temperature. We conclude the following. 1) Although heat shock and betaine transporter genes both respond to hypertonicity, the betaine transporter is not a HSP. 2) Accumulation of organic osmolytes attenuates the HSP-70 response to hypertonicity, as it might if the HSP-70 expression were a temporizing response. 3) Betaine inhibits HSP-70 response to elevated temperature, presumably by its known effect of stabilizing proteins.

Animals↗

PubMed1992

Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein.

Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.

Amino Acid Sequence↗

PubMed1992

Cloning of a Na(+)- and Cl(-)-dependent betaine transporter that is regulated by hypertonicity.

Many hypertonic bacteria, plants, marine animals, and the mammalian renal medulla are protected from the deleterious effects of high intracellular concentrations of electrolytes by accumulating high concentrations of the nonperturbing osmolyte betaine. When kidney-derived Madin-Darby canine kidney (MDCK) cells are cultured in hypertonic medium, they accumulate betaine to 1,000 times its medium concentration. This results from induction by hypertonicity of high rates of betaine transport into cells. We have isolated a cDNA (BGT-1) encoding a renal betaine transporter by screening an MDCK cell cDNA library for expression of a betaine transporter in Xenopus oocytes. The cDNA encodes a single protein of 614 amino acids, with an estimated molecular weight of 69 kDa. The deduced amino acid sequence exhibits highly significant sequence and topographic similarity to brain gamma-amino-n-butyric acid (GABA) and noradrenaline transporters, suggesting that the renal BGT-1 is a member of the brain GABA/noradrenaline transporter gene family. Expression in oocytes indicates that the BGT-1 protein has both betaine and GABA transport activities that are Cl(-)- as well as Na(+)-dependent and functionally similar to betaine and GABA transport in MDCK cells. Northern hybridization indicates that transporter mRNA is localized to the kidney medulla and is induced in MDCK cells by hypertonicity.

Amino Acid Sequence↗

PubMed1992

Kidney aldose reductase gene transcription is osmotically regulated.

Cells generally adapt to long-term hypertonic stress by accumulating organic osmolytes. PAP-HT25 renal medullary cells in hypertonic medium accumulate sorbitol through a reaction catalyzed by aldose reductase and betaine through osmotically regulated transport. Hypertonicity increases aldose reductase protein synthesis rate by elevating its mRNA abundance. To test whether the rise in aldose reductase mRNA is due to enhanced transcription, PAP-HT25 cells adapted to isotonic medium were switched to hypertonic medium, and transcription rate was measured by nuclear run-on. Aldose reductase transcription rate peaked at 17-fold the isotonic level after 12 h of hypertonicity. Then, transcription fell as sorbitol and betaine accumulated. Transcription stabilized at fivefold the isotonic level within days. Aldose reductase mRNA stability was not significantly different between the hypertonic and isotonic steady states. Thus aldose reductase mRNA is osmotically regulated through changes in its transcription. The osmotically induced rise in aldose reductase transcription is blunted by the accumulation of intracellular betaine and is exaggerated and prolonged by preventing the accumulation of both sorbitol (by aldose reductase inhibition) and betaine (by removal from the medium). This study presents the first description of osmoregulated gene transcription in animal cells.

Aldehyde Reductase↗