Human Na+-myo-inositol cotransporter gene: alternate splicing generates diverse transcripts

Francesca Porcellati1, Tommy Hlaing2, Masaki Togawa1, Martin J. Stevens1, Dennis D. Larkin1, Yoshiyuki Hosaka1, Thomas W. Glover3, Douglas N. Henry4, Douglas A. Greene1, and Paul D. Killen2

Departments of 1 Internal Medicine, 2 Pathology, 3 Human Genetics, and 4 Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan 48109

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Na+-myo-inositol cotransport activity generally maintains millimolar intracellular concentrations of myo-inositol and specifically promotes transepithelial myo-inositol transport in kidney, intestine, retina, and choroid plexus. Glucose-induced, tissue-specific myo-inositol depletion and impaired Na+-myo-inositol cotransport activity are implicated in the pathogenesis of diabetic complications, a process modeled in vitro in cultured human retinal pigment epithelium (RPE) cells. To explore this process at the molecular level, a human RPE cDNA library was screened with a canine Na+-dependent myo-inositol cotransporter (SMIT) cDNA. Overlapping cDNAs spanning 3569 nt were cloned. The resulting cDNA sequence contained a 2154-nt open reading frame, 97% identical to the canine SMIT amino acid sequence. Genomic clones containing SMIT exons suggested that the cDNA is derived from at least five exons. Hypertonic stress induced a time-dependent increase, initially in a 16-kb transcript and subsequently in 11.5-, 9.8-, 8.5-, 3.8-, and ~1.2-kb SMIT transcripts, that was ascribed to alternate exon splicing using exon-specific probes and direct cDNA sequencing. The human SMIT gene is a complex multiexon transcriptional unit that by alternate exon splicing generates multiple SMIT transcripts that accumulate differentially in response to hypertonic stress.

human retinal pigment epithelial cells; hypertonic stress; exon splicing; osmoregulation; diabetes mellitus

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE WATER-SOLUBLE CYCLIC hexitol myo-inositol is a constituent of virtually every living cell and an essential nutrient for most mammalian cells in culture. Intracellular concentrations 50- to 1,000-fold greater than that of extracellular fluid are with few exceptions (25) ascribed to the action of membrane-associated, Na+-dependent myo-inositol cotransporters (SMITs) (2). At the cellular level, myo-inositol is an obligate and sometimes rate-limiting (19) substrate for phosphoinositide synthesis. Along with sorbitol, taurine, and betaine, myo-inositol belongs to a family of alternative nonionic organic intracellular osmolytes whose regulated accumulation and efflux in response to hypertonic stress preserve intracellular volume and tonicity without perturbing the ionic milieu (2, 5). Organic osmolyte accumulation is coordinated by the regulated expression and/or activity of osmotically responsive, osmolyte-specific genes or gene products: distinct Na+-cotransporters for myo-inositol, taurine, and betaine and aldose reductase (AR) for the synthesis of sorbitol from glucose. Gradient-dependent organic osmolyte efflux is thought to be regulated primarily but not exclusively by a nonselective, ATP-dependent, volume-sensitive organic anion channel (2, 14, 27). Thus, at any given tonicity, the relative abundance of each osmolyte depends in part on substrate availability, the abundance and activity of their transporter or biosynthetic enzyme, and their efflux or degradation. The ready availability of one osmolyte may lead to reciprocal decreases in the abundance of others. For example, hyperglycemia is associated with increased flux through AR such that sorbitol accumulates with attendant depletion of other osmolytes including myo-inositol.

Despite the ubiquitous role of myo-inositol as an osmolyte and metabolic precursor, the metabolism and transport of myo-inositol are highly cell and tissue specific. The kidney synthesizes, concentrates, excretes, and reabsorbs myo-inositol and uniquely expresses the specific mammalian cytoplasmic oxygenase for myo-inositol degradation. Brisk Na+-myo-inositol cotransport activity in the small intestine, the choroid plexus, the ocular ciliary body, and the retinal pigment epithelium (RPE) (8) accounts for rapid intestinal absorption and the higher myo-inositol levels in cerebrospinal fluid and vitreous humor (14). Human RPE cells (8) and bovine lens epithelial cells (4) demonstrate Na+-myo-inositol cotransport activities with distinct kinetics. Whether this is due to genetically diverse SMITs or due to posttranslational modification is not clear, but Na+-myo-inositol cotransport activity can be regulated posttranscriptionally in RPE cells by intermediary metabolites such as pyruvate and in RPE and in Madin-Darby canine kidney (MDCK) cells through the action of protein kinases (14, 24). Thus Na+-myo-inositol cotransport is implicated in the regulation of both cellular and systemic myo-inositol homeostasis.

Despite its physiological importance, widespread distribution, and functional complexity, relatively little is known about the molecular regulation of Na+-myo-inositol cotransport. The functional cloning of a canine SMIT cDNA in Xenopus oocytes (16) by assaying Na+-dependent myo-[3H]inositol uptake led to the first elucidation of a SMIT primary structure. The deduced 718-amino acid polypeptide was predicted to have multiple potential protein kinase A/C (PKA/PKC) phosphorylation sites (16) and multiple hydrophobic "membrane-spanning" domains, homologous with the mammalian Na+-dependent glucose transporter (SGLT) family (12). More significantly, voltage-clamp studies of Na+-myo-inositol cotransport in Xenopus oocytes expressing the recombinant canine SMIT cDNA confirm the functional identity of the recombinant transporter through its substrate specificity and kinetic parameters (11). With the use of this cDNA as a probe, SMIT transcripts ranging from ~1.0 to >13.5 kb have been identified following hypertonic stress in Northern blots of RNA from kidney (16), lens (33), neural (21), and vascular (28) endothelial cells. The increased abundance of these transcripts in hypertonically stressed MDCK cells is at least in part due to increased transcription as detected by nuclear run-on analysis (31), but it is not clear whether these diverse transcripts are the products of a single gene or a gene family. Human genomic DNA containing a full-length open reading frame (ORF), potentially encoding human SMIT (SLC5A3), has been cloned using the canine cDNA as a probe (1). It was speculated that this intronless sequence gives rise to diverse SMIT transcripts through alternate transcript termination and polyadenylation sites. We now demonstrate that the human SMIT gene is a complex transcriptional unit comprising at least five exons, which are alternately spliced into a family of SMIT transcripts. The SMIT coding sequence is contained entirely within exon 2. The resulting transcripts differ, as a function of their exon composition, size, the presence or absence of the SMIT coding sequence, and the sequence of their 3' untranslated regions (UTR).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

RNA isolation. Primary cultures of human RPE cells were established and passaged (8) in minimal essential medium (MEM) with 20% calf serum at 37°C in a humidified 5% CO2 atmosphere. Confluent cells were exposed to isotonic (295 mosmol/kgH2O) MEM (IMEM) or 300 mM mannitol-supplemented hypertonic (595 mosmol/kgH2O) MEM (HMEM) with 20% calf serum for various times, after which RNA was purified (13).

Construction and screening of the cDNA library. Total RNA was isolated 24 h after exposure of RPE cells to HMEM. An oligo(dT)-primed, size-selected (>1 kb) cDNA library was commercially prepared from the RNA using a unidirectional strategy in pCDNA1 (Invitrogen, San Diego, CA) and transformed in Escherichia coli strain MC 1061/P3 by electroporation. The library was screened at reduced stringency using an EcoR I-BamH I fragment of the canine SMIT cDNA (generously provided by H. Moo Kwon, Johns Hopkins University, Baltimore, MD; GenBank accession no. M85068; nt 724-2870, containing most of the coding sequence and 236 nt of 3' UTR) (16) labeled as described in Probes. Recombinants (6 × 105) were plated at high density and lifted on nitrocellulose filters, colonies were lysed in 0.5% SDS, and the DNA was denatured before fixation in vacuo for 2 h at 80°C. Filters were prehybridized as previously described (13) for 4-6 h before addition of denatured probe (2 × 106 dpm/ml). After 18 h at 55°C, filters were washed at 55°C before exposure to preflashed photographic film (X-Omat AR, Eastman-Kodak, Rochester, NY) at -70°C. Three SMIT clones [clones 1-1, 1-2 (Fig. 1) and 1-3] were obtained. The library was rescreened at high stringency using a Hind III-Sph I fragment from the 5' end of the most 5' extended clone and clone 2-1 was obtained (Fig. 1). Additional 5' sequence was obtained by amplification of 1 µg of purified plasmid DNA representing the entire library, using nested primers 3' flanking an EcoR I site in clone 2-1 and a T7 primer in the pCDNA1 vector (5' flanking an EcoR I site in the vector). The amplification products were digested with EcoR I and subcloned in pBluescript (Stratagene, La Jolla, CA), and recombinant plasmids containing the 5' end of the cDNA were identified by colony hybridization with a 5' fragment of cDNA clone 2-1. The most 5' extended cDNAs were identified by EcoR I digestion, and the largest was designated clone 3-1 (Fig. 1). Complementary DNA clone 4-1 (Fig. 1) was obtained by specific primer extension and DNA amplification. After reverse transcription of RNA annealed to an oligonucleotide complementary to sequences in clone 3-1, the cDNA was tailed using terminal deoxytransferase and dATP. This served as a template for amplification with an oligo(dT) primer and the original primer. Appropriately sized PCR products (see below) were excised from a gel, subcloned in pBluescript, and identified by nucleotide sequencing.


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Fig. 1.   Partial restriction map of overlapping cDNA clones encoding human retinal pigment epithelium (RPE) Na+-dependent myo-inositol cotransporter (SMIT). Three clones [clones 1-1, 1-2 (shown), and 1-3] were obtained by screening a human RPE cell library prepared from RNA isolated 24 h after hypertonic stress at reduced stringency with a canine SMIT cDNA clone. The most 5' sequence was used to rescreen library, obtaining clone 2-1. Clone 3-1 was obtained by PCR amplification of library as described in METHODS. Clone 4-1 was obtained by 5' rapid amplification of cDNA ends as described in METHODS. Overlapping cDNAs span 3569 bp and contain a 2154-bp open reading frame (ORF; shaded bar).

Primer extension. Analytic primer extension was performed with synthetic oligonucleotides derived from sequences in clone 3-1 (5'-CTTAATCCACTCTCCACAAGACCATCAG-3' complementary to nt 241-268 and 5'-CTAAGTCTAGTGAAGCCTACTGTACCAAC-3' complementary to nt 173-201; see Molecular cloning of the full-length human SMIT cDNA). HPLC-purified oligonucleotides were end labeled with [gamma -32P]ATP (6,000 Ci/mmol, Amersham Life Science, Arlington Heights, IL) and annealed either to RNA from RPE cells exposed to HMEM for 24 h or to yeast tRNA as previously described (15). After reverse transcription, the end-labeled products were analyzed on a 6% sequencing gel and their size compared with the nucleotide sequence obtained by sequencing cDNA clone 3-1 with the same primers.

Cloning human SMIT exons. A human peripheral blood leukocyte genomic DNA library prepared in lambda -FIX was screened. Recombinant phage (1.2 × 106) were plated at high density, and filter lifts were prepared. In an attempt to clone the full length of the gene, filters were hybridized at high stringency (13) with cDNA clone 3-1 and a Pst I-Xho I fragment from cDNA clone 1-2 (corresponding to nt 3306-3570). Two distinct subsets of nonoverlapping phage were obtained, each hybridizing to a single probe. After plaque purification, large-scale phage DNA isolation, and partial restriction mapping, restriction fragments hybridizing with the original probe were subcloned, and the exons were sequenced. To obtain the intervening exon(s), the library was rescreened with a Pst I fragment from cDNA clone 1-2 (nt 2330-3309). A single clone, lambda 2-24, which did not overlap the sequences contained within the other phage, was obtained. A 3.0-kb Hind III-Xba I restriction fragment was subcloned, and the nucleotide sequence of the contained exon was determined. The putative first exon was identified by Southern hybridization of an EcoR I-Xba I digest of yeast artificial chromosome (YAC) DNA 860G11 with a fragment of cDNA clone 4-1 (nt 36-169). Appropriately sized EcoR I-Xba I fragments were excised and subcloned in pBluescript, and recombinant plasmids were identified by colony hybridization. The nucleotide sequence of the putative exon 1 was determined.

                              
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Table 1.   Exon-intron boundaries in the SMIT gene

DNA sequencing. The nucleotide sequence of both strands of the overlapping cDNA clones 1-2, 2-1, 3-1, and 4-1 were determined an average of 4.5 times/base by the dideoxy chain-termination method (15). Clone 2-1 was sequenced using a modified shotgun strategy (15). The cDNA insert was purified free of vector sequences, concatenated by ligation, and randomly sheared by sonication. The resulting DNA fragments were digested with S1 nuclease and polished with the Klenow fragment of DNA polymerase before cloning into the Sma I site of pUC19. After transformation, recombinants were identified by colony hybridization and sequenced with M13 universal primers and Sequenase (United States Biochemical, Cleveland, OH) following alkaline denaturation. The nucleotide sequence was analyzed using Genetics Computer Group software. Gaps in the sequence were determined with synthetic oligonucleotide primers.

Chromosomal assignment and fluorescence in situ hybridization. PCR amplification was performed using 20 pmol of each synthetic oligonucleotide primer [5'-ACCACCACCTTTTGGTCTAAG-3' (nt 2132-2152) and 5'-TATCCTTAAGTTCATAAGGAGAAA-3' (complementary to nt 2650-2673)] and 200 ng of genomic DNA in 50 µl of 15 mM (NH4)2SO4, 2 mM MgCl2, 60 mM Tris, pH 10.0, containing 1 U AmpliTaq (Perkin-Elmer, Norwalk, CT). A single DNA fragment of appropriate size was amplified from human genomic DNA but not from murine or hamster genomic DNA. DNA (200 ng) isolated from 24 human-rodent somatic cell hybrids, each retaining one intact human chromosome (mapping panel no. 2, Coriell Institute for Medical Research, Camden, NJ) were used in 50-µl reactions. After denaturation at 95°C for 3.5 min, the DNA was amplified 30 times as follows: denaturation at 94°C for 1.5 min, annealing at 56°C for 1.5 min, and extension at 72°C for 2 min. A final elongation reaction was conducted at 72°C for 8 min. Aliquots of reactions were electrophoretically separated on a 2% agarose gel, and DNA was visualized by staining with ethidium bromide. A full-length SMIT cDNA was used for fluorescence in situ hybridization, as previously described (22).

YAC localization. To more precisely localize the human SMIT gene, YAC DNA containing segments of genomic DNA from the region of 21q22.13 to proximal q22.3 were screened by Southern hybridization. YAC DNAs obtained from the University of Michigan Genomic Resources Core facility and from Research Genetics (Huntsville, AL) were hybridized with exon-specific probes as described below (Probes). YACs spanning the region included YAC clones 860G11, 872B5, 874C1, 191D3, and 73D10. After determination that all of the probes hybridized to the YAC DNA 860G11, Southern blots were prepared from human genomic DNA, YAC 860G11 DNA, and lambda 1-36 and lambda 1-38 phage DNA after restriction endonuclease digestion with Hind III or Pst I.

Northern blots. Denatured total RNA (10 µg) from cells exposed to IMEM or HMEM for various times, or poly(A)+ RNA (3 µg) from cells exposed to IMEM, and a 0.24- to 9.5-kb RNA ladder (Life Technologies, Gaithersburg, MD) were resolved on 2.2 M formaldehyde-1% agarose gels, transferred by capillary blotting to ZetaBind filters (CUNO, Meriden, CT), stained with methylene blue to confirm the integrity, uniformity of loading, and completeness of transfer, and fixed in vacuo at 80°C for 2 h. Filters were prehybridized (13) at 65°C for 4-6 h before adding denatured probe (1 × 106 dpm/ml). After 18 h, the filters were washed at 65°C, before exposure to preflashed film at -70°C. Exposure was adjusted from 3 to 15 days, depending on the probe.

Probes. Northern and Southern blots were hybridized with the 2.3-kb clone 1-2 cDNA insert or a 1.0-kb Pst I fragment that spanned exons 2-5. Probes for exons 2 and 5 were derived from a 1.3-kb Pst I fragment (nt 1064-2334) from cDNA clone 2-1 and a Xho I fragment (nt 2976-3569) from cDNA clone 1-2, respectively. Probes for exons 1, 3, and 4 were prepared by amplification from cDNA clones using synthetic oligonucleotides as follows: exon 1, a 134-nt fragment from cDNA clone 4-1 (corresponding to nt 36-169); exon 3, a 93-nt fragment from cDNA clone 1-2 (nt 2680-2772); and exon 4, a 131-nt fragment from cDNA clone 1-2 (nt 2793-2923). All amplification products were subcloned in pBluescript and sequenced to confirm their identity. All fragments were excised from the vector, purified from agarose or acrylamide gels, and labeled to a specific activity of ~109 dpm/µg using random primers (24) and [alpha -32P]dCTP (3,000 Ci/mmol, Amersham, Arlington Heights, IL). Labeled fragments were separated from unincorporated label by gel filtration, denatured by boiling, and added at a final amount of 2 × 106 dpm/ml. Northern blots were subsequently stripped and rehybridized with human AR (24) and chicken beta -actin (7) cDNA probes.

Cloning the "1.2-kb SMIT transcript." The smallest ~1.0-kb transcripts from canine kidney cortex, medulla, and MDCK cells (16) and human RPE cells (see Northern analysis) that hybridize, respectively, with canine and human SMIT cDNAs are too small to encode an ~718-amino acid SMIT protein. Exon-specific probes suggested that the "1.2-kb SMIT transcript" from human RPE cells contains sequences from putative exons 1, 4, and 5 but not exon 2 (which contains the SMIT ORF; see Cloning exons of the human SMIT gene). To clone cDNAs derived from the 1.2-kb SMIT transcript, the osmotically induced RPE cDNA library (4 × 105 transformants) was screened at high stringency with the exon 5 probe. More than 700 colonies hybridized strongly to the probe. Eighteen of these were screened by PCR using a 5' primer from exon 1 (corresponding to nt 36-169) and a 3' primer spanning exons 4 and 5 (complementary to nt 2902-2923). cDNAs derived from longer SMIT transcripts (i.e., those containing exon 2 sequence) would not be expected to amplify efficiently under the conditions employed and should yield products >2 kb in size. Six clones yielded products consistent with the predicted composition of the 1.2-kb SMIT transcript. Three of these were purified and sequenced in their entirety on both strands. To more accurately determine the size of the 1.2-kb SMIT transcript, RNA (5 µg) was resolved as described above on 2% agarose gels containing a 0.16- to 1.77-kb RNA ladder (Life Technologies), and Northern blots were probed with an exon 5 probe.

Synthetic oligonucleotide synthesis. All synthetic oligonucleotides were synthesized by the DNA Synthesis Core Facility, a part of the Michigan Diabetes Research and Training Center and the University of Michigan's Biomedical Research Core Facilities, on automated DNA synthesizers (Applied Biosystems, Foster City, CA) employing beta -cyanoethyl phosphoramidite chemistry on controlled pore glass supports.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Molecular cloning of the full-length human SMIT cDNA. Human SMIT cDNAs were cloned from a human RPE cell library prepared from RNA isolated after 24 h exposure to HMEM. The library was initially screened at reduced stringency with a fragment of the canine SMIT cDNA containing coding and 3' untranslated sequence (16). Three unique overlapping cDNAs [clone 1-1, 1-2 (Fig. 1), and 1-3] with similar restriction maps spanned some 3 kb. Limited nucleotide sequencing from the 5' end demonstrated considerable homology with the canine sequence. The nucleotide sequence of clones 1-2 and 1-3 terminated in a 3' poly(A) tail, but otherwise there was no similarity to the canine 3' sequence. Because none of the cDNAs extended to the translation initiation site, the 5' end of the most 5' extended clone was used to rescreen the library at high stringency. This resulted in a single additional clone (clone 2-1, Fig. 1), which was 100% identical in a 136-nt overlap with clone 1-2 but which failed to extend to the 5' end of the transcript. To clone more 5' sequence, PCR was performed using an anchored primer 5' flanking the polylinker in pCDNA1 and nested primers, complementary to sequences in clone 2-1, 3' to an EcoR I site. Total library DNA was amplified, digested with EcoR I, and subcloned. Nucleotide sequence analysis of the largest clone (Fig. 1, clone 3-1) was identical to clone 2-1 in a 268-nt overlap, but primer extension (see below) experiments suggested that the 5' end of the transcript was ~200 nt upstream. Because PCR amplification of the library failed to yield this sequence, a strategy of 5' rapid amplification of cDNA ends (9) was employed, using specific primer cDNA synthesis from RPE cell RNA and poly(A) tailing. Subsequent amplification, purification, and subcloning of appropriately sized PCR products yielded clone 4-1 (Fig. 1), spanning 268 nt with 100% sequence identity with clone 3-1 in a 72-nt overlap. Analytic primer extension was performed with different primers complementary to sequences in cDNA clones 3-1 and 4-1 (Fig. 2). Resolution of the primer extension products on a denaturing sequencing gel revealed little heterogeneity, and the size obtained with each primer was consistent with a single transcription initiation site only 5 nt 5' to the end of clone 4-1. No difference in the mobility of the primer extension products was noted when RNA from RPE cells in IMEM or HMEM was utilized (data not shown), suggesting that a single promoter transcribes the gene under both conditions.


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Fig. 2.   Analytic primer extension indicates a single major transcript initiation site. Specific primer extension was performed to determine whether SMIT clone 4-1 extended to 5' end of SMIT transcripts. Primers complementary to cDNA sequences between nt 241 and 268 (lanes 1 and 2) and between nt 173 and 201 (lanes 3 and 4) (see Fig. 3) were end labeled, annealed to 10 µg of RNA, and reverse transcribed as described in METHODS. Primer extension products were resolved after denaturation on 6 M urea-6% polyacrylamide gels, and size (in nt; scale at left) was determined by comparison with DNA sequence determined with same primers. Specific primer extension products were only noted with annealing to RPE cell RNA (lanes 1 and 3) and not with annealing to yeast tRNA (lanes 2 and 4). Results suggest that SMIT clone 4-1 extends to within 5 nt of 5' end of transcripts and that there is little heterogeneity at 5' end, consistent with a single major transcript initiation site.

The nucleotide and deduced amino acid sequence of SMIT is shown in Fig. 3. The 3569-nt sequence from clones 1-2, 2-1, 3-1, and 4-1 was analyzed using the Wisconsin Genetics Computer Group software, revealing a single ORF of 2154 nt, a 505-nt 5' UTR, and a 910-nt 3' UTR that terminated in a poly(A) tail. The coding nucleotide sequence was highly conserved (94.5% identity over 2154 nt) compared with the canine SMIT cDNA sequence (16). The deduced amino acid sequence was 97% identical with the 718-residue canine polypeptide. Comparison with the nucleotide sequence of an ORF on chromosome 21, thought to encode the human SMIT (SLC5A3) (1), differed in only three conservative amino acid substitutions. It is unclear whether these are polymorphisms or sequencing errors. The deduced amino acid sequence was analyzed with Kyte-Doolittle plots, which revealed at least 12 hydrophobic domains that on further analysis based on homologies with the SGLT family (12) appeared to define 14 membrane-spanning domains (Fig. 3). Analysis with the PROSITE Dictionary of Protein Sites and Patterns identified a single conserved possible N-linked glycosylation site, 10 potential PKC phosphorylation sites, a single potential PKA phosphorylation site, and a potential tyrosine kinase phosphorylation site (Fig. 3). The 505-nt 5' UTR demonstrated 94.1% identity in a 457-nt overlap with the canine 5' UTR. Comparison of the 910-nt 3' UTR with the canine sequence revealed 100% identity at the stop codon and 15 nt adjacent to it, but [with the exception of the poly(A) tails] no significant homology with the remaining 193 nt of canine 3' UTR.


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Fig. 3.   Nucleotide and deduced amino acid sequence of human RPE SMIT cDNA. Nucleotide sequence of RPE SMIT transcript was determined from cDNA clones 1-2, 2-1, 3-1, and 4-1. The 3569-nt sequence (1st row) contained a single major ORF of 2154 residues, a 505-nt 5' untranslated region (UTR) and a 910-nt 3' UTR that terminates in a consensus polyadenylation sequence (double underline) and a poly(A) tail. Deduced amino acid sequence (2nd row) is highly conserved when compared with canine SMIT amino acid sequence (3rd row). It contains 14 putative transmembrane domains (shaded bars), a single conserved N-linked glycosylation site (open rectangle), 10 potential PKC phosphorylation sites (open circles), and single potential PKA (shaded square) and tyrosine phosphorylation (shaded circle) sites.

Northern analysis. Northern blots of total RNA isolated from RPE cells maintained for 24 h in IMEM or HMEM were hybridized under high-stringency conditions with the 2.3-kb SMIT clone 1-2 and the size of the SMIT transcripts determined from an RNA ladder. Six transcripts were evident in RNA from RPE cells exposed to HMEM (16, 11.5, 9.8, 8.5, 3.8, and 1.2 kb), all of which were significantly increased compared with the RNA from IMEM cells. Indeed, only the 1.2-kb transcript was readily detectable in cells in IMEM, with an 11.5-kb transcript evident only after prolonged (2 wk) exposure (Northern not shown). After transfer to HMEM, there was a time-dependent increase in SMIT transcripts (Fig. 4), with the 16-kb transcript appearing earliest and declining more rapidly than the other SMIT transcripts. SMIT transcripts <16 kb in apparent size reached a maximum abundance (40-fold increase) after 24 h in HMEM and returned to baseline after 48 h in HMEM. In contrast, AR mRNA increased at 8 h, reached a maximum at 24 h, but persisted for at least 48 h in HMEM. When a 1.3-kb Pst I fragment from SMIT clone 2-1 containing only coding sequence was used as a hybridization probe, all of the transcripts except the 1.2-kb mRNA hybridized (see Differential exon utilization and SMIT transcript diversity).


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Fig. 4.   Hypertonic exposure induces differential expression of SMIT transcripts in human RPE cells. Northern blots were prepared from 10 µg RNA isolated from RPE cells exposed to hypertonic (300 mM mannitol) minimal essential medium (MEM) for times indicated. Filter was sequentially hybridized with a 1-kb Pst I fragment from SMIT cDNA clone 1-2, a partial human aldose reductase (AR) cDNA, or a chicken beta -actin cDNA. A 1.2-kb SMIT mRNA was detectable in RPE cells before hypertonic exposure (an 11.5-kb transcript was evident after prolonged exposure; not shown). After hypertonic exposure, there was little change in beta -actin mRNA. As previously reported, AR mRNA was first increased at 8 h and then peaked at 24 h before slowly declining. In contrast, SMIT mRNA increased earlier than AR mRNA and declined more rapidly to baseline after hypertonic exposure. The 16-kb SMIT transcript increased earlier and declined more rapidly than smaller SMIT transcripts, whereas smaller SMIT transcripts were maximally induced at 24 h.

Cloning exons of the human SMIT gene. Different transcription initiation sites (alternate promoters) could not readily explain the presence of diverse SMIT transcripts, since primer extension studies with several different primers all suggested a single initiation site. Heterogeneity at the 3' end of the transcripts, either through the use of alternate polyadenylation sites in the same (1) or different exons, was explored as an alternative basis for multiple SMIT transcripts. A human genomic library screened with fragments of the cDNA from the 5' and 3' ends yielded two nonoverlapping clones, lambda 1-36 and lambda 1-38 (Fig. 5). The restriction fragments containing the putative exons were subcloned and sequenced using synthetic oligonucleotide primers. A single exon identified in lambda 1-36 was 100% identical to the sequence between nt 172 and 2675 (Fig. 3) and flanked by appropriate splice donor-acceptor sequences (Table 1). The map of this clone was similar to that described by Berry et al. (1), and this exon contained the entire ORF, as reported. Clone lambda 1-38 contained a single exon encoding a 3' UTR sequence distal to nt 2913. Rescreening the library yielded one additional clone, lambda 2-24 (Fig. 5), which contained a 140-nt exon encoding sequences between nt 2773 and 2912. Thus only 171 nt of 5' UTR sequence and 96 nt of 3' UTR were unaccounted for, suggesting that the SMIT gene is encoded by a minimum of five exons. The putative first exon was subsequently subcloned from YAC DNA 860G11 (see below).


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Fig. 5.   Partial restriction maps of genomic clones containing putative exons 2, 4, and 5. A human genomic DNA library in lambda -FIX was screened with SMIT cDNA clone 1-2, and 2 nonoverlapping clones (lambda 1-36 and lambda 1-38) were obtained. Library was rescreened with an ~1-kb Pst I fragment from SMIT cDNA clone 1-2, and clone lambda 2-24 was obtained. Restriction fragments containing putative exons were subcloned and sequenced. There was 100% identity between genomic DNA sequence and cDNA, and exons appropriately conformed to AG/GT rule. Exon boundaries and flanking sequences are given in Table 1.

Chromosomal localization. Because the description of the SLC5A3 locus (1) did not include unique flanking sequences that could be used to confirm the identity of clone lambda 1-36, chromosomal assignment studies were performed. A mapping panel of human-rodent somatic cell hybrid DNAs (panel 2, Coriell Institute for Medical Research) was screened by PCR using sequences derived from exon 2. These primers amplified an appropriately sized product only from GM/NA10323 (data not shown), consistent with SMIT assignment to chromosome 21. Southern hybridization of the PCR products failed to reveal additional bands. High-resolution fluorescence in situ hybridization studies (data not shown) were concordant with the mapping panel results and the localization of the gene to 21q22.1 to q22.2 (1). Refinement of this localization was performed using YAC DNAs from this region. Southern blots of YAC DNA were hybridized to fragments of the cDNA. YAC clone 860G11 but not flanking YAC clones 872B5, 874C1, 191D3, and 73D10 hybridized with the cDNA probes. To confirm that this hybridization was not spurious, Southern blots of human genomic DNA, YAC 860G11, and lambda 1-36 or lambda 1-38 restricted with Hind III or Pst I were hybridized with fragments derived from exon 2 or 5, respectively (Fig. 6). In each case, the restriction fragments were similar in size and number. Because the most 5' untranslated sequence contained within clone 4-1 hybridized to an appropriately sized 4.0-kb EcoR I-Xba I fragment from YAC clone 860G11, appropriately sized fragments of YAC clone 860G11 DNA were subcloned and identified by colony hybridization and nucleotide sequencing. The nucleotide sequence was 100% identical to clone 4-1 in a 173-nt overlap, and the 3' end was flanked by appropriate splice donor sequences (Table 1).


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Fig. 6.   Putative exons 2 and 5 colocalize to yeast artificial chromosome (YAC) clone 860G11. Human genomic DNA, YAC clone 860G11 DNA, and lambda 1-36 or lambda 1-38 DNA were digested with Hind III (HIII) or Pst I (P), and Southern blots were prepared. A 1.3-kb Pst I fragment from putative exon 2 hybridized to similar and appropriately sized fragments in human genomic and YAC clone 860G11 DNA. Similarly, an Xho I fragment of cDNA clone 1-2 containing sequence derived from putative exon 5 hybridized to similarly sized fragments in human genomic DNA and YAC 860G11 DNA.

Differential exon utilization and SMIT transcript diversity. Because little heterogeneity of SMIT transcripts was identified at the 5' end by primer extension, exon-specific probes were hybridized to Northern blots of RNA obtained from RPE cells maintained in IMEM or HMEM for 24 h (Fig. 7). As noted above, six transcripts were evident when hybridized using the 2.3-kb clone 1-2 cDNA insert that spanned exons 2-5. Only the 1.2-kb transcript was readily identified in RNA from cells in IMEM with this probe. A probe from exon 1 hybridized to all six transcripts, consistent with the lack of heterogeneity at the 5' end. However, as described above, the exon 2 probe failed to hybridize to the 1.2-kb transcript. Exon 3 sequences were preferentially represented in the 11.5-kb transcripts, whereas exon 4 and 5 sequences were preferentially represented in the 3.8- and 1.2-kb transcripts. These results demonstrate that the diverse SMIT transcripts noted by filter hybridization were due at least in part to differential exon utilization.


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Fig. 7.   Differential exon utilization results in diverse SMIT RNA transcripts. A: Northern blots of total RNA (10 µg) from RPE cells maintained in isotonic (I) or hypertonic (H) MEM were hybridized with exon-specific probes or SMIT cDNA clone 1-2, as described in METHODS. cDNA clone 1-2 hybridized with 6 distinct transcripts, as described above (Fig. 4). The 1.2- and 11.5-kb bands hybridized most strongly with exon 1 probe; however, prolonged exposure (not shown) demonstrated exon 1 sequences in all SMIT transcripts. Exon 2 probe hybridized to all transcripts except 1.2-kb band. Exon 3 probe hybridized preferentially to 11.5-kb transcripts. Exon 4 and 5 probes hybridized to similar transcripts, including 1.2-, 3.8-, and 16-kb bands. B: poly(A)+ RNA (6 µg) from RPE cells in IMEM was hybridized with exon-specific probes. Exon composition of 3.8-kb transcript in cells unexposed to hypertonic conditions is shown to be identical to that in A in cells exposed to hypertonic stress, indicating that alternate SMIT exon utilization is not confined to hypertonicity-stressed cells.

Filter hybridization demonstrated that 3.8-kb transcript(s) included sequences contained in exons 1, 2, 4, and 5. The overlapping cDNAs could account for the existence of a hypothetical transcript of this electrophoretic mobility derived from the combined sequences of exons 1-5. However, the exon 3 probe failed to hybridize with the 3.8-kb band, suggesting that this hypothetical transcript, if present, was not abundant and that other SMIT sequences must contribute to this band. Filter hybridization with exon-specific probes suggests a complex explanation for the electrophoretic mobilities of transcripts >3.8 kb. These larger bands appear to include sequences derived from various permutations of exons 1-5, but these sequences alone cannot account for transcripts of this electrophoretic mobility. That these results were not an artifact of hypertonic stress is demonstrated by the presence of the 1.2- and 3.8-kb SMIT transcripts with similar exon composition in RNA isolated from RPE cells exposed to IMEM (Fig. 7, A and B, respectively).

On the basis of the hybridization of exon-specific probes, it was surmised that the 1.2-kb cDNA transcript derived in part from exons 1, 4, and 5 (Fig. 8). This was confirmed by two approaches. RNA isolated from cells exposed to HMEM was reverse transcribed and amplified using synthetic oligonucleotide primers corresponding to a sequence in exon 1 and complementary to sequences flanking the junction of exons 4 and 5. The resulting PCR product was of the size predicted by a transcript comprised of these exons, and its composition was further established by cloning and sequencing. Because PCR can amplify very rare sequences, a more direct approach was taken. The hypertonically induced RPE cell library was screened at high stringency with an exon 5 probe, and >700 positive recombinant clones were identified. Thus the sequence was well represented in the library. Eighteen clones were screened by PCR for sequences intervening between exon 1 and exon 4. Fully one-third of these yielded product consistent with the hypothetical structure shown in Fig. 8. Three of these were randomly chosen for nucleotide sequencing. Each was essentially identical (1 base difference in >950-base overlap) with the hypothetical sequence in Fig. 8. They differed only in their extension to the 5' end, length of poly(A) tail, and, in one clone, in the position downstream from the polyadenylation sequence from which the poly(A) tail was synthesized. The 50-kb difference between the size determined by electrophoretic mobility and that based on the cDNAs is most likely due to the length of the poly(A) tail. This divergence is consistent with the known infidelity of polyadenylation and established these clones as independent isolates. These cDNA clones were ~950 bp [depending on the size of the poly(A) tail and the 5' end of the clones], rather than the expected 1.2 kb (Fig. 8). This size difference was attributed to the relatively poor resolution of small RNAs in a 1% gel. Indeed, a Northern blot prepared from RNA run on a 2% agarose gel demonstrated a mobility consistent with a 1-kb transcript. Although devoid of exon 2 coding sequence, this small SMIT transcript contained an ORF that could encode a 14-kDa peptide. Searches of protein and nucleic acid databases failed to identify other homologous proteins or sequences.


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Fig. 8.   Nucleotide and amino acid sequence for "1.2-kb SMIT transcript" devoid of exon 2 coding sequence. Hypothetical structure of 1.2-kb SMIT transcript is depicted, based on Northern blot hybridization. Exon junctions are depicted by vertical lines. Confirmation of hypothetical sequence was conclusively established by direct cDNA sequencing. Three clones were isolated from hypertonicity-stressed RPE cell library. The 3 cDNA clones began at positions 19, 16, and 16, respectively, and, with exception of a single base (position 706, Fig. 8; position 3322, Fig. 3), were identical to depicted sequence between their 5' ends and polyadenylation signal (double underline). An ORF that lies between boxed translation initiation site and double-underlined polyadenylation signal would encode a 14-kDa peptide whose predicted amino acid sequence is shown in 2nd line. Putative PKC phosphorylation sites are indicated by open circles. Sequence identity of this transcript with that of exons 1, 4, and 5 in full-length cDNA indicates that it arises by alternate splicing from same SMIT gene. Its presence in RNA from cells unexposed to hypertonic stress further supports contention that alternate splicing of SMIT transcripts is not a product of hypertonic stress.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Intracellular concentrations of myo-inositol and the kinetic and biochemical characteristics of myo-inositol transport vary widely among and within mammalian cell types and tissues (3, 4, 6, 8, 14, 20, 21, 25, 29; Porcellati, Hosaka, Hlaing, Togawa, Larkin, Stevens, Killen, and Greene, unpublished observations), with some cells expressing more than one class of Na+-myo-inositol cotransport activity (4, 8). The effects of diabetes or hyperglycemia on SMIT regulation are equally complex (20). The maximal velocity of Na+-myo-inositol cotransport activity has been reported to be increased by experimental diabetes or hyperglycemic concentrations of glucose in renal (6) and endothelial (20) cells. The same manipulations have been reported to diminish Na+-myo-inositol cotransport activity in a variety of experimental systems including incubated endoneural preparations from streptozotocin-diabetic rats (10) and various cell culture models (3, 4, 14, 18, 32). AR inhibitors that block the formation of the intracellular osmolyte sorbitol prevent both the reported increases (6) and decreases (3, 10, 18, 32) in Na+-myo-inositol cotransport activity produced by glucose, suggesting that the regulation of Na+-myo-inositol cotransport by alternative intracellular osmolytes may be complex.

The heterogeneity of Na+-myo-inositol cotransport function and regulation and SMIT expression is poorly understood in molecular terms. A SMIT cDNA was functionally cloned in Xenopus oocytes (16) from a hypertonic MDCK cDNA library prepared from poly(A)+ RNA with a median size of 4 kb (17). Despite its origin in a 4-kb RNA pool, the "full-length" canine SMIT cDNA comprising 5' UTR, ORF, and 3' UTR hybridized to multiple transcripts ranging from 1 to 13.5 kb but most strongly to a 10.5-kb transcript (16). Subsequent studies using the canine probe (21) or probes amplified from the coding sequences of rat (29) or bovine (33) SMIT mRNA demonstrated hybridization to a similarly broad diversity of SMIT transcripts in hypertonically stressed cells in vitro (21, 28, 33) or tissues in vivo (29, 30). Although SMIT steady-state mRNA is generally induced by hypertonicity (31), the multiple SMIT transcripts consistently noted by Northern analysis (16, 21) are rapidly up- and downregulated by osmotic stress (21) in a cell-specific fashion not fully explained by tonicity alone (29). Both transcriptional regulation (31) and alterations in mRNA stability (21) have been invoked in this process.

It has been suggested that this heterogeneity may derive from distinct members of a SMIT gene family (1), yet only a single genomic sequence has been reported (1). Berry and co-workers obtained a genomic fragment localized to chromosome 21q22.1-22.2 (1) containing an intronless ORF potentially encoding a full-length SMIT polypeptide. Northern blots performed with a 1.3-kb Pst I fragment of the ORF hybridized to transcripts in a variety of tissues. SMIT mRNA was most abundant in the kidney, with multiple transcripts similar in mobility to those described by others (16, 21, 29, 30, 33). It was speculated that the diverse SMIT transcripts derived from the use of alternate polyadenylation sites in the 3' UTR. A similar intronless ORF has been noted in the canine gene (1).

Here we describe the isolation and characterization of overlapping SMIT cDNAs derived from a size-selected, tonicity-induced human RPE cell library. The 5' UTR and coding sequences are highly conserved compared with the canine sequence and the coding sequence is virtually identical with the sequence reported for the chromosome 21 ORF (1). The 3' UTR, however, diverges significantly from the canine sequence, suggesting a possible cloning artifact. However, the 3' UTR sequence was similar in two independent isolates (clones 1-2 and 1-3), and Northern blots using probes derived from the 3' UTR hybridized (with the exception of the 1.2-kb transcript) to transcripts with the same mobility as detected by probes containing the coding sequence. Isolation of genomic DNA fragments containing putative SMIT exons 1, 2, 4, and 5 suggests that the SMIT gene is more complex than previously envisioned (1) (Fig. 9). The genomic DNA clones did not overlap, but all of the sequences were contained in YAC 860G11, making it unlikely that they derive from distinct transcriptional units. Indeed, all SMIT RNA transcripts hybridized with a putative exon 1 probe and primer extension studies using multiple primers were consistent with a single transcription initiation site for the SMIT gene. These results were the same with RNA isolated from cells in IMEM or HMEM, suggesting that there is a single promoter. The ~900 bp sequence 5' flanking the putative first exon functioned to initiate transcription using chimeric reporter constructs, but its activity was not altered by hypertonic stress (Hlaing, Killen, and Greene, unpublished observations). Similar results have been obtained with the canine SMIT gene (Ref. 26; personal communication, H. Moo Kwon, Johns Hopkins University).


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Fig. 9.   Deduced structure of multiple SMIT transcripts. Multiexon structure of SMIT gene gives rise to multiple SMIT transcripts through alternate splicing. The 1.2-kb transcript is most abundant and derives from exons 1, 4, and 5. Although several ORFs exist within its sequence, it is unclear whether 1.2-kb transcript encodes a polypeptide. The 3.8-kb transcripts would appear to be heterogeneous, with most abundant transcripts containing sequence derived from exons 1, 2, 4, and 5. A less abundant sequence deduced from overlapping cDNAs described above also contains sequence from exon 3, but this is not abundant enough to be identified by filter hybridization with a 92-nt probe. Transcripts >3.8 kb are heterogeneous in size and sequence. Some of these may derive from alternate transcript termination and polyadenylation sites, as previously suggested (2). Others may have a complex 3' UTR derived from exons 3, 4, and 5.

It seemed likely that the diverse SMIT transcripts observed by filter hybridization might derive in part from alternate splicing, as depicted in Fig. 9. Indeed, exon-specific probes selectively hybridized to transcripts of different size. Perhaps the most intriguing transcript is the 1.2-kb transcript (or, more appropriately, the "~1-kb transcript"), similar in mobility to transcripts noted in hypertonically stimulated MDCK and canine renal medulla; this transcript is too small to encode a SMIT-like molecule, and its function is unknown. This transcript was previously noted only when full-length canine SMIT probes were used, i.e., with probes containing sequence derived from the first exon (16). Others have failed to note this transcript, most likely because they employed probes derived from exon 2 coding sequence. In human RPE cells, this transcript is actually a 0.95-kb sequence derived from exons 1, 4, and 5, and it contains an ORF that shows little sequence homology with proteins within existing databases. It is, however, interesting to note that alternate splicing may not be limited to untranslated sequence. An alternate splice donor site within the SMIT ORF appears to give rise to a family of SMIT transcripts, which encode distinct SMIT isoforms different in their COOH-terminal sequence (Porcellati, Hosaka, Hlaing, Togawa, Larkin, Stevens, Killen, and Greene, unpublished observations).

The significance of the differing 3' UTR sequences derived from exons 3, 4, and 5 on the expression of the 718-amino acid SMIT is presently unknown. The largest SMIT transcript (16 kb) increases in abundance earlier and declines more rapidly than the smaller transcripts during exposure to HMEM. This is consistent with the hypothesis that alternate splicing is a regulated process and that the resulting distinct mRNA sequences could influence mRNA stability. The existence of these diverse 3' UTR sequences in RNA from RPE cells under isotonic conditions eliminates the possibility that these higher molecular weight transcripts are an artifact of osmotic stress. Preliminary studies suggest that the ~1-kb SMIT transcript devoid of exon 2 and exon 3 sequence appears to be considerably more stable than the 11.5-kb transcript, which contains these and other sequences, after inhibition of transcription (Hosaka, Larkin, Stevens, Killen, and Greene, unpublished observations). Exposure of RPE cells to isotonic hyperglycemic (i.e., 20 mM glucose) conditions simulating the diabetic milieu markedly decreases the apparent half-life of the 11.5-kb transcript (Hosaka, Larkin, Stevens, Killen, and Greene, unpublished observations). The extent to which variability in SMIT transcript stability contributes to tissue-specific differences in the response of Na+-myo-inositol cotransport activity to hyperglycemic or osmotic stress remains to be elucidated. Stability studies with specific reporter constructs will be required in order to establish the role of diverse 3' UTR sequences in these phenomena. At the very minimum, these results suggest that studies of the physiology of SMIT expression must address possible transcript-specific effects.

In summary, the studies reported in this communication confirm the reported chromosomal assignment of a human SMIT gene (1) but describe a complex multiexon transcriptional unit that gives rise to a series of transcripts by alternate splicing. The smallest SMIT transcript derives from exons 1, 4, and 5 and therefore lacks sequence from the ORF described by Berry et al. (1). Its functional significance, if any, is unclear. Exons distal to exon 2 encode a heterogeneous set of 3' UTRs that contribute to a diverse array of variously sized transcripts that may exhibit differential induction and half-life after osmotic stress. The complexity of the SMIT gene may have important but as yet unexplored implications for SMIT gene expression and the regulation of Na+-myo-inositol cotransport activity under physiological and pathophysiological conditions.

    ACKNOWLEDGEMENTS

We gratefully acknowledge helpful discussions of the data with David Kurnit (University of Michigan) and H. Moo Kwon (Johns Hopkins Medical School).

    FOOTNOTES

D. A. Greene, D. N. Henry, F. Porcellati, and M. Togawa were supported in part by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant P60-DK-20572 (Michigan Diabetes Research and Training Center). P. D. Killen was supported by NIDDK Grants RO1-DK-44848 and P50-DK-39255. D. A. Greene was supported by NIDDK Grant RO1-DK-38304. D. N. Henry was supported by National Institute of Child Health and Human Development Grant P30-HD-25520 and NIDDK Grant DK-02193 and by the Juvenile Diabetes Foundation.

Present addresses: F. Porcellati, Instituto di Medicina Interna e Scienza Endocrin e Metaboliche, Via Enrico Dal Pozzo, 06126 Perugia, Italy; M. Togawa, Shiga University of Medical Science, Third Department of Medicine, Seta, Otsu, Shiga 520-21, Japan; D. N. Henry, Michigan State University, Department of Physiology, 113 Giltner Hall, E. Lansing, MI 48814-1101.

Address for reprint requests: P. D. Killen, Dept. of Pathology, Box 0602, 1301 Catherine St., Ann Arbor, MI 48109-0602.

Received 23 January 1997; accepted in final form 16 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Berry, G. T., J. J. Mallee, H. M. Kwon, J. S. Rim, W. R. Mulla, M. Muenke, and N. B. Spinner. The human osmoregulatory Na+/myo-inositol cotransporter gene (SLC5A3): molecular cloning and localization to chromosome 21. Genomics 25: 507-513, 1995[Medline].

2.   Burg, M. B. Molecular basis of osmotic regulation. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F983-F996, 1995[Abstract/Free Full Text].

3.   Cammarata, P. R., H. Q. Chen, J. Yang, and T. Yorio. 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. Invest. Ophthalmol. Vis. Sci. 33: 3561-3571, 1992[Abstract].

4.   Cammarata, P. R., H. Q. Chen, J. Yang, and T. Yorio. 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. Invest. Ophthalmol. Vis. Sci. 33: 3572-3580, 1992[Abstract].

5.   Chamberlin, M. E., and K. Strange. Anisosmotic cell volume regulation: a comparative review. Am. J. Physiol. 257 (Cell Physiol. 26): C159-C173, 1989[Abstract/Free Full Text].

6.   Chatzilias, A. A., and C. I. Whiteside. Cellular mechanisms of glucose-induced myo-inositol transport upregulation in rat mesangial cells. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F459-F466, 1994[Abstract/Free Full Text].

7.   Cleveland, D. W., M. A. Lopata, R. J. MacDonald, N. J. Cowan, W. J. Rutter, and M. W. Kirschner. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell 20: 95-105, 1980[Medline].

8.   Del Monte, M. A., R. Rabbani, T. C. Diaz, S. A. Lattimer, J. Nakamura, M. C. Brennan, and D. A. Greene. Sorbitol, myo-inositol, and rod outer segment phagocytosis in cultured hRPE cells exposed to glucose. In vitro model of myo-inositol depletion hypothesis of diabetic complications. Diabetes 40: 1335-1345, 1991[Abstract].

9.   Frohman, M. A., M. K. Dush, and G. R. Martin. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988[Abstract].

10.   Gillon, K. R. W., and J. N. Hawthorne. Transport of myo-inositol into endoneurial preparations of sciatic nerve from normal and streptozotocin-diabetic rats. Biochem. J. 210: 775-781, 1983[Medline].

11.   Hager, K., A. Hazama, H. M. Kwon, D. D. F. Loo, J. S. Handler, and E. M. Wright. Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes. J. Membr. Biol. 143: 103-113, 1995[Medline].

12.   Hediger, M. A., E. Turk, and E. M. Wright. Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters. Proc. Natl. Acad. Sci. USA 86: 5748-5752, 1989[Abstract].

13.   Henry, D. N., M. Del Monte, D. A. Greene, and P. D. Killen. Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells. J. Clin. Invest. 92: 617-623, 1993[Medline].

14.   Karihaloo, A., K. Kato, D. A. Greene, and T. P. Thomas. Protein kinase C and cytosolic Ca2+ modulation of myo-inositol transport in cultured retinal pigment epithelial cells. Am. J. Physiol. 273 (Cell Physiol. 42): C671-C678, 1997[Abstract/Free Full Text].

15.   Killen, P. D., P. D. Burbelo, G. R. Martin, and Y. Yamada. Characterization of the promoter for the alpha 1 (IV) collagen gene. DNA sequences within the first intron enhance transcription. J. Biol. Chem. 263: 12310-12314, 1988[Abstract/Free Full Text].

16.   Kwon, H. M., A. Yamauchi, S. Uchida, A. S. Preston, A. Garcia-Perez, M. B. Burg, and J. S. Handler. Cloning of the cDNA for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J. Biol. Chem. 267: 6297-6301, 1992[Abstract/Free Full Text].

17.   Kwon, H. M., A. Yamauchi, S. Uchida, R. B. Robey, A. Garcia-Perez, M. B. Burg, and J. S. Handler. Renal Na-myo-inositol cotransporter mRNA expression in Xenopus oocytes: regulation by hypertonicity. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F258-F263, 1991[Abstract/Free Full Text].

18.   Mistry, K. P., A. Beyer-Mears, and F. P. Diecke. Mechanisms for D-glucose inhibition of myo-inositol influx into rat lens. Diabetes 42: 1737-1744, 1993[Abstract].

19.   Nakamura, J., M. A. Del Monte, D. Shewach, S. A. Lattimer, and D. A. Greene. Inhibition of phosphatidylinositol synthase by glucose in human retinal pigment epithelial cells. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E417-E426, 1992[Abstract/Free Full Text].

20.   Olgemoller, B., S. Schwaabe, E. D. Schliecher, and K. D. Gerbitz. Upregulation of myo-inositol transport compensates for competitive inhibition by glucose. An explanation for the inositol paradox? Diabetes 42: 119-125, 1993.

21.   Paredes, A., M. McManus, H. M. Kwon, and K. Strange. Osmoregulation of Na+-inositol cotransporter activity and mRNA levels in brain glial cells. Am. J. Physiol. 263 (Cell Physiol. 32): C1282-C1288, 1992[Abstract/Free Full Text].

22.   Petty, E. M., D. E. Miller, A. L. Grant, E. E. Collins, T. W. Glover, and D. J. Law. FISH localization of the soluble thymidine kinase gene (TK1) to human 17q25, a region of chromosomal loss in sporadic breast tumors. Cytogenet. Cell Genet. 72: 319-321, 1996[Medline].

24.   Preston, A. S., A. Yamuchi, H. M. Kwon, and J. Handler. Activators of protein kinase A and protein kinase C inhibit MDCK cell myo-inositol and betaine uptake. J. Am. Soc. Nephrol. 6: 1559-1664, 1995[Abstract].

25.   Prpic, V., P. F. Blackmore, and J. H. Exton. myo-Inositol uptake and metabolism in isolated rat liver cells. J. Biol. Chem. 257: 11315-11322, 1982[Free Full Text].

26.   Rim, J. S., A. S. Preston, M. Takenaka, J. S. Handler, and H. M. Kwon. Cloning of the canine gene for the sodium/myo-inositol cotransporter (SMIT) (Abstract). J. Am. Soc. Nephrol. 6: 368, 1995.

27.   Strange, K., and R. Morrison. Volume regulation during recovery from chronic hypertonicity in brain glial cells. Am. J. Physiol. 263 (Cell Physiol. 32): C412-C419, 1992[Abstract/Free Full Text].

28.   Wiese, T. J., J. A. Dunlap, C. E. Conner, J. A. Grzybowski, W. L. Lowe, Jr., and M. A. Yorek. Osmotic regulation of Na-myo-inositol cotransporter mRNA level and activity in endothelial and neural cells. Am. J. Physiol. 270 (Cell Physiol. 39): C990-C997, 1996[Abstract/Free Full Text].

29.   Yamauchi, A., A. Miyai, S. Shimada, Y. Minami, M. Tohyama, E. Imai, T. Kamada, and N. Ueda. Localization and rapid regulation of the Na+/myo-inositol cotransporter in rat kidney. J. Clin. Invest. 96: 1195-1201, 1995[Medline].

30.   Yamauchi, A., T. Nakanishi, Y. Takamitsu, M. Sugita, E. Imai, T. Noguchi, Y. Fujiwara, T. Kamada, and N. Ueda. In vivo osmoregulation of Na/myo-inositol cotransporter mRNA in rat kidney medulla. J. Am. Soc. Nephrol. 5: 62-67, 1994[Abstract].

31.   Yamauchi, A., S. Uchida, A. S. Preston, H. M. Kwon, and J. S. Handler. Hypertonicity stimulates transcription of gene for Na+-myo-inositol cotransporter in MDCK cells. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F20-F23, 1993[Abstract/Free Full Text].

32.   Yorek, M. A., J. A. Dunlap, and B. H. Ginsberg. myo-Inositol metabolism in 41A3 neuroblastoma cells: effects of high glucose and sorbitol levels. J. Neurochem. 48: 53-61, 1987[Medline].

33.   Zhou, C., N. Agarwal, and P. R. Cammarata. Osmoregulatory alteration in myo-inositol uptake by bovine lens epithelial cells. II. Cloning of a 626 bp cDNA portion of a Na/myo-inositol cotransporter, an osmotic shock protein. Invest. Ophthalmol. Vis. Sci. 35: 1236-1242, 1994[Abstract].


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