Transcriptional regulation of the betaine/gamma-aminobutyric acid transporter by hypertonicity.
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Animals↗
Biomedical subjects
H M Kwon
Publications and source records attributed to H M Kwon.
The MAP kinase cascade is not essential for transcriptional stimulation of osmolyte transporter genes.
Kidney derived MDCK cells are protected from the stress of hypertonicity by accumulating compatible osmolytes. Accumulation of the compatible osmolytes myo-inositol and betaine is driven by hypertonicity-induced stimulation of transcription of the genes coding for the myo-inositol cotransporter and the betaine cotransporter. We tested the importance of the mitogen-activated protein kinase pathway in transcriptional activation of the genes for the two osmolytes cotransporters because this kinase pathway is rapidly activated when cells are exposed to hypertonicity and a mitogen-activated protein kinase pathway is essential for the osmo-protective transcriptional response of yeast to hypertonicity. Eliminating the activation of mitogen-activated protein kinase did not block the hypertonicity induced increase in accumulation of osmolyte transporter mRNA.
Animals↗
The canine betaine gamma-amino-n-butyric acid transporter gene: diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.
The Na(+)- and Cl(-)-coupled betaine transporter, designated BGT1, a member of the neurotransmitter transporter gene family, is responsible for accumulation of betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. The canine gene for the betaine gamma-amino-n-butyric acid transporter has been cloned and analyzed. The gene extends over 28 kb and consists of 18 exons. The 5' end of the gene has three alternative first exons (1A, 1B, and 1C+D). Analysis of BGT1 mRNA revealed that there is considerable divergence in the 5' untranslated sequence resulting from three different 5' end motifs (A, B, and C) followed by an alternative motif (D) as well as two internal acceptor sites for splicing. Eight kinds of BGT1 mRNA were classified into three types (A, B, and C) according to the 5' end sequence. Northern blot analysis using probes specific for the A, B, or C motif revealed that hypertonicity induces all three types in MDCK cells. Reverse transcription and polymerase chain reaction showed that each type was expressed in a tissue-specific manner. Primer extension and/or RNase protection assays as well as transfection assays into MDCK cells demonstrated that exons 1A, 1B, and 1C+D have independent transcription initiation sites under control of independent promoters. Diverse mRNA isoforms are regulated by hypertonicity and are expressed in a tissue-specific manner.
Animals↗
The human osmoregulatory Na+/myo-inositol cotransporter gene (SLC5A3): molecular cloning and localization to chromosome 21.
A human Na+/myo-inositol cotransporter (SLC5A3) gene was cloned; sequencing revealed a single intron-free open reading frame of 2157 nucleotides. Containing 718 amino acid residues, the predicted protein is highly homologous to the product of the canine osmoregulatory SLC5A3 gene. The SLC5A3 protein is number 3 of the solute carrier family 5 and was previously designated SMIT. Using fluorescence in situ hybridization, the human SLC5A3 gene was localized to band q22 on chromosome 21. Many tissues including brain demonstrate gene expression. The inability of a trisomic 21 cell to downregulate expression of three copies of this osmoregulatory gene could result in increased flux of both myo-inositol and Na+ across the plasma membrane. The potential consequences include perturbations in the cell membrane potential and tissue osmolyte levels. The SLC5A3 gene may play a role in the pathogenesis of Down syndrome.
Amino Acid Sequence↗
Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains. Correlation with antigenicity and pathogenicity.
Transmissible gastroenteritis virus (TGEV) causes an economically important enteric disease of swine. Differences in the pathogenicity, antigenicity and tissue tropism have been observed among porcine coronaviruses. Although porcine respiratory coronavirus (PRCV) is antigenically similar but not identical to TGEV isolates, these respiratory coronaviruses differ markedly in pathogenicity and tissue tropism compared to TGEV isolates. Using a reverse transcriptase/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) assay, TGEV and PRCV isolates were assigned to several distinct groups. By RFLP analysis of the 5' region of the S gene, TGEV strains were differentiated into 4 groups using the restriction enzyme Sau3AI. A fifth Sau3AI group contained the PRCV isolates. These 5 groups correlated with antigenic groups previously defined using monoclonal antibodies in our laboratory. Several restriction enzymes could be used to differentiate the TGEV strains into Miller and Purdue types. Analysis of a PCR amplified product in the 3 and 3-1 genes indicated the RT/PCR-RFLP assay results for TGEV Miller strains could be correlated with lower virulence created by passage in cell culture.
Animals↗
Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes.
The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (Vm), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: KMI0.5, KNa0.5, and the Hill coefficient n. At 100 mM NaCl, KMI0.5 was about 50 microM and was independent of Vm. At 0.5 mM myo-inositol, KNa0.5 ranged from 76 mM at Vm = -50 mV to 40 mM at Vm = -150 mV. n was voltage independent with a value of 1.9 +/- 0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent KI of 64 microM at Vm = -50 mV and 130 microM at Vm = -150 mV. To examine sugar specificity, sugar-induced steady-state currents (at Vm = -150 mV) were recorded for a series of sugars, each at an external concentration of 50 mM. The substrate selectivity series was myo-inositol, scylloinositol > L-fucose > L-xylose > L-glucose, D-glucose, alpha-methyl-D-glucopyranoside > D-galactose, D-fucose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose > D-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at Vm = -150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: D-glucose, alpha-methyl-D-glucopyranoside, D-galactose, D-fucose, 3-O-methyl-D-glucose > D-xylose, L-xylose, 2-deoxy-D-glucose > myo-inositol, L-glucose, L-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na(+)-dependent uptake of 3H-D-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mM Na+ that was abolished by phlorizin (0.5 mM). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in Vm. The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.
Animals↗
Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus.
The nucleotide sequences of S1 glycoprotein genes of the Gray and JMK strains of avian infectious bronchitis virus (IBV) were determined and compared with published sequences for IBV. The IBV Gray and JMK strains had 99% nucleotide sequence similarity. The overall nucleotide sequence similarity of the Gray and JMK strains compared with other IBV strains was between 82.0% and 87.4%. The similarity of the predicted amino acid sequence for the S1 glycoproteins of the Gray and JMK strains was 98.8%. Six of the 10 differences in the amino acid sequence were found between residues 99 and 127, suggesting a possible role for that region in the tissue trophisms of the viruses. The S1 glycoprotein of the Gray and JMK strains had 79.5%-84.6% amino acid similarity with the published sequence of other IBV strains. Serine instead of phenylalanine was observed in the protease cleavage site between the S1 and S2 glycoprotein subunits for the Gray and JMK strains, which was similar to the published sequence for the Ark99 and SE17 strains. The significance of that amino acid change is not known. Based on the nucleotide sequence of the Gray and JMK strains, the BsmAI restriction enzyme was selected by computer analysis and was used in restriction fragment length polymorphism analysis to differentiate the two strains.
Amino Acid Sequence↗
Cell volume regulated transporters of compatible osmolytes.
Virtually all cells respond to hypertonicity by accumulating certain small organic solutes (compatible osmolytes) that, in contrast to intracellular ions, do not perturb macromolecular function. Several important compatible osmolytes are accumulated by coupled transport. Transcription of genes encoding these cotransporters is increased by hypertonicity and a tonicity-responsive enhancer element has been identified. When cells return to an iso-osmotic environment, osmolytes are rapidly lost through a pathway that current evidence indicates may be a volume-sensitive chloride channel.
Animals↗
Activators of protein kinase A and of protein kinase C inhibit MDCK cell myo-inositol and betaine uptake.
Amino acid sequences of the myo-inositol and betaine cotransporters that are induced in MDCK cells by hypertonicity include consensus sequences for phosphorylation by protein kinase A and by protein kinase C. To test for the effect of activation of protein kinases A and C on the activity of those cotransporters, MDCK cells were exposed to activators of each kinase and the activity of both cotransporters was assayed. Incubation with 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP) or 3-isobutyl-1-methylxanthine (IBMX), activators of protein kinase A, and incubation with an active phorbol ester or with an active diacylglycerol, activators of protein kinase C, inhibited the activity of both cotransporters by about 30%. The relative effect of the activation of protein kinase A and of protein kinase C was similar in hypertonic and isotonic cells. The effects of activators of protein kinase A and of protein kinase C were not additive. The two cotransporters behaved differently when protein kinase C activity was down-regulated by prolonged incubation with a higher concentration of phorbol 12-myristate 13-acetate. There was a doubling of activity of the myo-inositol cotransporter and no change in the activity of the betaine cotransporter in hypertonic and isotonic cells. Although the mechanisms of the effects of activation of the two kinases remain to be established, it is clear that the kinases can mediate post-translational regulation of the uptake of compatible osmolytes.
1-Methyl-3-isobutylxanthine↗
Clinical significance of serum TSH in euthyroid patients with paroxysmal atrial fibrillation.
Atrial fibrillation may occur in patients with a variety of cardiovascular or chronic disease as well as in normal subjects. Many authors reported that atrial fibrillation occurs in patients with thyrotoxicosis. It is reported that a low serum thyrotrophin concentration in an asymptomatic person with normal serum thyroid hormone concentrations can be a independent risk factor for developing atrial fibrillation. But we focused on the significance of serum thyroid stimulating hormone (TSH) in the euthyroid patient with atrial fibrillation whose serum level of T3, T4, fT4, and even TSH were absolutely within normal range. On our results, there was no significant differences in age, sexual distribution, and left ventricular ejection fraction between the patients group of paroxysmal and chronic persistent atrial fibrillation (p > 0.05), but there was larger left atrial dimension (LAD) and more cases of rheumatic heart disease in the chronic persistent atrial fibrillation group and there was more cases of lone atrial fibrillation in the paroxysmal atrial fibrillation group (p < 0.05). There was no significant differences in serum levels of T3, T4, fT4 between paroxysmal and chronic persistent atrial fibrillation, but significantly lower serum TSH was found in patients with paroxysmal atrial fibrillation (p < 0.001), and these findings were more significant after the control of hemodynamic change (p < 0.001 vs p < 0.05). The discriminant value in serum TSH between the paroxysmal and chronic atrial fibrillation group was 1.568U/mL with about 76% of predictive power. There was significantly lower serum TSH in paroxysmal atrial fibrillation in all age groups (p < 0.05). There was a significantly higher prevalence of cerebral thromboembolic events in chronic persistent (27.7%) and disease-associated (15.0% atrial fibrillation than in the paroxysmal (3.3%) and lone (4.5%) atrial fibrillation group (p < 0.001). Therefore, we suggest that serum TSH below the serum concentration of 1.5U/mL can be a risk factor for developing atrial fibrillation when the serum level of T3, T4, fT4, and even TSH were within absolutely normal range.
Adult↗
The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress.
BGT1, the Na(+)-and Cl- coupled betaine transporter, is responsible for the accumulation of high concentrations of the non-perturbing osmolyte betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. In MDCK cells, the increase in activity of the betaine transporter is preceded by an increase in transcription of BGT1 and in the abundance of BGT1 mRNA. To investigate the molecular mechanism of transcriptional regulation by tonicity, we have characterized the 5'-flanking region of the gene. Transient transfection assays in MDCK cells cultured in isotonic or hypertonic medium using luciferase reporter constructs containing various fragments of the 5'-flanking region revealed that the region spanning base pairs -69 to -50 5' to the transcription initiation site (-69/-50) has hypertonicity-responsive enhancer activity. A double-stranded -69/-50 concatemer cloned 5' to an SV40 basal promoter and luciferase reporter gene in hypertonic cells exhibited more than 11-fold the activity in isotonic cells. Expression assays and electrophoretic mobility shift assays of mutants of -69/-50 identified a smaller region that is required for hypertonicity to induce increased expression and a slowly migrating band on mobility shift assays.
Animals↗
Acute myocardial infarction in the young adults.
A review was done on 631 patients with acute myocardial infarction who underwent coronary angiography within 30 days after onset of myocardial infarction at Yonsei University Severance Hospital from January, 1985, to August, 1993. The incidence of acute myocardial infarction in patients under 40 years of age was 10.3% (65/631). Acute myocardial infarction below the fourth decades was the predominant disease of men. Risk factor analysis revealed a history of cigarette smoking and hypercholesterolemia were more frequently found in the young patients, but a history of hypertension and diabetes were more frequently found in the elderly patients. Angiographically, the incidence of one vessel disease and normal or minimal lesion coronary anatomy were more frequent in the young patients and incidence of multi-vessel disease were more frequent in the elderly patients. Of the 65 patients under 40 with acute myocardial infarction, the patients with multi-vessel disease tended to have a history of diabetes mellitus in comparison with those with normal coronary anatomy or one vessel disease.
Adult↗
Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.
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Amino Acid Sequence↗
Sodium- and chloride-dependent transporters in brain, kidney, and gut: lessons from complementary DNA cloning and structure-function studies.
The family of Na(+)- and Cl(-)-dependent, 12 transmembrane domain transporter proteins now includes transporters for neurotransmitter molecules in the brain and for substances important in extraneuronal tissues, including adrenal, kidney, and gut. Transported substrates include monoamine and amino acid neurotransmitters and nonperturbing osmolytes. A common protein topology is predicted and features intracellular N- and C-termini possessing phosphorylation sites and at least one large extramembranous loop with N-linked glycosylation. Using the rat dopamine transporter as a template, molecular modeling of putative transmembrane domains coupled with amino acid sequence conservation analysis indicates amino acid residues potentially involved in substrate and/or ion recognition. Targeting such residues with site-directed mutagenesis will help clarify substrate and ion binding sites and should facilitate rational design of therapeutics to combat depression, locomotor disorders, and substance abuse.
Amino Acid Sequence↗
Regulation of renal cell organic osmolyte transport by tonicity.
Madin-Darby canine kidney cells accumulate several nonperturbing organic osmolytes when cultured in a hypertonic medium. Myo-inositol, betaine, and taurine are accumulated secondary to an increase in uptake, the first coupled to sodium entry, the latter two coupled to sodium and chloride entry. The transport rates increase as the result of an increase in maximum velocity for each cotransporter, with peak activity 24 h after the increase in tonicity. The cDNA for each cotransporter has been cloned. Their sequences indicate that the myo-inositol cotransporter belongs to the gene family that includes the sodium-coupled glucose transporter (SGLT1); the betaine and taurine cotransporters belong to the gene family of sodium- and chloride-coupled transporters that are responsible for neuronal uptake of many neurotransmitters. Assays of mRNA abundance and nuclear run-on assays reveal that shifts in tonicity have a major effect on transcription of the genes for the sodium-myo-inositol (SMIT) and sodium-chloride-betaine (BGT1) cotransporters. The ensuing increase in mRNA abundance for the two cotransporters and presumed increase in synthesis of the cotransporter proteins can explain the increase in transport activity in response to changes in tonicity.
Amino Acid Sequence↗
Hypertonicity stimulates transcription of gene for Na(+)-myo-inositol cotransporter in MDCK cells.
Myo-inositol is a major compatible osmolyte accumulated in the hypertonic renal medulla and in Madin-Darby canine kidney (MDCK) cells cultured in hypertonic media. Myo-inositol is taken up by MDCK cells on a Na(+)-coupled transporter whose activity increases sixfold 24 h after cells are switched to hypertonic medium. To investigate the mechanism of regulation of the cotransporter by hypertonicity, we used the cDNA encoding the canine Na(+)-myo-inositol cotransporter that we recently cloned to measure the abundance of the mRNA for the cotransporter and its rate of transcription after changes in osmolality. When MDCK cells were switched from isotonic to hypertonic medium, cotransporter mRNA abundance rose 10-fold in 16 h. Transcription of the cotransporter gene also rose and 16 h after the switch reached a peak approximately 15-fold that in isotonic cells. When cells were switched back to isotonic medium, mRNA abundance and transcription of the gene returned to isotonic levels in 8 h and transport rate reached isotonic levels in 48 h. Thus transcription appears to be the primary step in regulation of myo-inositol transport by hypertonicity.
Animals↗
Expression of rat fibroblast growth factor receptor 1 as three splicing variants during kidney development.
Fibroblast growth factors (FGF) are known to participate in the processes of embryogenesis and angiogenesis. This study was undertaken to examine the transcriptional and posttranscriptional regulation of the FGF receptor 1 (FGFR-1) subclass in the embryonic rat kidney. Two full-length FGF receptor cDNAs were cloned using low-stringency screening of a neonatal rat kidney library with a chicken FGFR-1 cDNA probe. Sequencing revealed these cloned cDNAs to be rat homologues of the FGFR-1 subtype, with the two clones representing splicing variants beta and gamma of the FGFR-1. Evidence for renal expression of a third splicing variant (alpha) was obtained by use of the polymerase chain reaction. Splicing variants alpha and beta of FGFR-1 are predicted to produce cell-surface FGF receptors with three and two immunoglobulin-like domains, respectively, whereas the gamma-isoform may represent an intracellular form of the receptor. Although all three splicing variants were expressed in the developing kidney at days 14, 17, and 20 of gestation, at neonatal days 1 and 7 and in mature rats the beta-isoform was present in vastly larger abundance than alpha- and gamma-isoforms at all stages studied. Northern blot analysis revealed enhanced expression of FGFR-1 in the neonatal compared with the mature kidney. It is concluded that FGFR-1 is expressed in the kidney predominantly as the beta-isoform splicing variant and that expression of this receptor is enhanced during kidney development.
Aging↗
Medium tonicity regulates expression of the Na(+)- and Cl(-)-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene.
Betaine is one of the major compatible osmolytes accumulated by kidney derived Madin-Darby canine kidney cells cultured in hypertonic medium. Betaine is accumulated by Na(+)- and Cl(-)-dependent uptake from the medium. To gain insight into the mechanism by which hypertonicity evokes an increase in the Vmax of the betaine transporter in Madin-Darby canine kidney cells, we measured the relative abundance of mRNA for the transporter in cells shifted to a hypertonic medium and found parallel increases in mRNA abundance and cotransporter activity. The increase in mRNA levels preceded the increase in transporter activity slightly. Transcription of the gene for the transporter rose rapidly and to the same relative extent as mRNA abundance in cells shifted to hypertonic medium, indicating that transcription of the gene for the cotransporter plays a major role in regulating the accumulation of betaine in response to hypertonicity.
Animals↗