The Test of Everyday Attention for Children (TEA-Ch): patterns of performance in children with ADHD and clinical controls.

The present study explores the utility of the Test of Everyday Attention for Children (TEA-Ch) as a measure of the attentional impairments displayed by children with Attention Deficit Hyperactivity Disorder (ADHD). Sixty-three children with ADHD and 23 non-ADHD Clinical Control children were compared on subtests of the TEA-Ch reflecting three attentional domains: sustained, selective, and attentional control. Results show that children with ADHD performed significantly worse than clinical controls on subtests of sustained attention and attentional control. The groups did not differ, however, on subtests of selective attention. These findings suggest that the TEA-Ch is sensitive to attentional deficits unique to ADHD and holds promise as a useful tool in the assessment of ADHD. Performance patterns and future directions are discussed.

Representational momentum for rotations in depth: effects of shadings and axis.

Representational momentum is a positive memory distortion for an object's final position following the presentation of an implied event (J.J. Freyd, 1987). Positive memory distortions occur when observers accept test positions beyond the final presented position, or forward along the implied trajectory, as the same more readily than positions behind the final position. Four experiments explored implied events depicting rotations about various depth axes in shaded and silhouette conditions. Positive memory distortions were observed for all depth rotations under certain shading conditions, with some differences in the size of the distortion between axes. No directional effects (e.g., right vs. left) were observed. The overall positive memory distortions observed for depth rotations contrasted with the negative distortions previously observed for translation motion in depth (T.L. Hubbard, 1995 ).

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.

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.

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.

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.

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.

Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity.

Cells in the hypertonic renal medulla maintain their intracellular ion concentration at isotonic levels, despite much higher concentrations of extracellular electrolytes, by accumulating high concentrations of nonperturbing small organic solutes termed osmolytes. Taurine has been identified as a nonperturbing osmolyte in the renal medulla and Madin-Darby canine kidney (MDCK) cells. In hypertonic medium, the increased accumulation of taurine in MDCK cells is the result of increased activity of a Na(+)- and Cl(-)-dependent taurine transporter. We have isolated a cDNA encoding a Na(+)- and Cl(-)-dependent taurine transporter, whose sequence corresponds to a protein of 655 amino acids with significant amino acid sequence similarity to previously cloned Na(+)- and Cl(-)-dependent transporters, including the MDCK cell betaine/gamma-aminobutyric acid transporter and several brain neurotransmitter transporters. Northern hybridization indicates that mRNA for the taurine transporter is present in renal cortex and medulla, ileal mucosa, brain, liver, and heart. The abundance of mRNA for the taurine transporter is increased in MDCK cells cultured in hypertonic medium, suggesting that regulation of transport activity by medium hypertonicity occurs at the level of mRNA accumulation.

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.

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.

Taurine behaves as an osmolyte in Madin-Darby canine kidney cells. Protection by polarized, regulated transport of taurine.

Using a clonal growth assay, we demonstrated that taurine, a nonperturbing osmolyte accumulated in kidney medulla, brain, and some other tissues of hypertonic experimental animals can function as a nonperturbing osmolyte in Madin-Darby canine kidney (MDCK) cells. The taurine content of hypertonic MDCK cells is twice that of isotonic MDCK cells (isotonic 160 nmol/mg protein; hypertonic 320 nmol/mg protein). Therefore we studied taurine transport in MDCK cells grown on porous supports and then studied the effect of hypertonicity which is known to elicit increased uptake of some other nonperturbing osmolytes by MDCK cells. Basal uptake exceeded apical uptake, with Km and Vmax of 56 microM and 933 pmol/min.mg protein on the basal surface and 10 microM and 50 pmol/min.mg protein on the apical surface. On both surfaces, virtually all taurine uptake was Na+ and Cl- dependent. 24 h after cells were shifted to hypertonic medium (500 mosmol/kg), taurine uptake doubled on the basolateral surface without change on the apical surface. The response to hypertonicity was the result of an increase in Vmax without change in Km. There was no change in taurine efflux when cells were shifted from isotonic to hypertonic medium. When cells adapted to hypertonic medium were shifted to isotonic medium, a large transient basolateral efflux of taurine occurred within 10 min. We conclude that taurine can function as a nonperturbing osmolyte in MDCK cells and that tonicity-regulated taurine transport is a basolateral function in MDCK cells.

Myo-inositol and betaine transporters regulated by tonicity are basolateral in MDCK cells.

Myo-inositol and glycinebetaine are compatible osmolytes accumulated in the renal medulla and in MDCK cells cultured in hypertonic media. Both osmolytes are taken up by MDCK cells on Na-coupled transporters. The maximal velocity (Vmax) of both cotransporters is increased by culture in hypertonic medium. When hypertonic MDCK cells are shifted to isotonic medium there is a large transient efflux of osmolytes. To determine the polarity of the cotransporters and the transient efflux, we grew MDCK cells on a porous support to assay transport separately at their apical and basolateral surfaces. In hypertonic cells, basolateral uptake of both osmolytes was 1) more than 10-fold apical uptake, 2) greater than 96% Na dependent, 3) 25- (myo-inositol) and 16-fold (glycinebetaine) uptake in isotonic cells, reaching a maximum 24 h after the switch to hypertonic medium. When medium osmolarity was decreased from hypertonic to isotonic, myo-inositol uptake reversed to the isotonic level within 1 day; glycinebetaine uptake decreased more slowly. When medium osmolarity was decreased from hypertonic to isotonic, there was a large transient increase in basolateral efflux of both osmolytes.

Expression of Madin-Darby canine kidney cell Na(+)-and Cl(-)-dependent taurine transporter in Xenopus laevis oocytes.

Expression of a Madin-Darby canine kidney (MDCK) cell taurine transporter was examined in Xenopus oocytes that had been injected with poly(A)+ RNA extracted from MDCK cells. Compared with water-injected oocytes, injection of total poly(A)+ RNA resulted in an increase in Na(+)-dependent taurine uptake which was directly related to the amount of RNA injected. The magnitude of expression in poly(A)+ RNA-injected oocytes was 5-10-fold higher than that of water-injected oocytes. Since the Vmax of taurine uptake in MDCK cells is increased by culture in hypertonic medium, we compared oocyte taurine uptake after injection with poly(A)+ RNA from MDCK cells cultured in hypertonic medium with uptake in oocytes injected with poly(A)+ RNA from hypertonic cells elicited twice the taurine uptake elicited by poly(A)+ RNA from isotonic cells. The transporter expressed in oocytes was like that in MDCK cells: it was completely dependent on external sodium and was also anion dependent (Cl- greater than or equal to Br- greater than SCN- much greater than gluconate-). Other beta-amino acids, beta-alanine and hypotaurine, inhibited taurine uptake, but L-alanine and 2-(methylamino) isobutyric acid did not. The apparent Km of the transporter was 7.0 microM. After size fractionation on a sucrose density gradient, poly(A)+ RNA encoding for the MDCK taurine transporter was found in the fraction whose average size was 4.4 kilobases.

Dexamethasone accelerates differentiation of A6 epithelia and increases response to vasopressin.

When seeded heavily on a porous tissue culture dish, A6 cells, derived from the kidney of Xenopus laevis, form a highly differentiated epithelium within 4-6 days. When dexamethasone is added to the culture medium, morphological differentiation is completed by day 2, a time at which the control (untreated) is still a disorganized multilayer of cells. In addition to the morphologically evident monolayer of cuboidal cells, the accelerated differentiation is expressed as high transepithelial electrical resistance, short-circuit current, and adenylate cyclase response to vasopressin. When grown on impermeable plastic tissue culture dishes, A6 epithelia are less differentiated and do not respond to vasopressin. With the addition of dexamethasone at the time of seeding on a plastic tissue culture dish, vasopressin responsive adenylate cyclase activity is expressed, albeit at a slower rate than when grown on a porous surface. In addition, dexamethasone treatment of mature epithelia grown on a porous surface results, in hours, in an increase in the adenylate cyclase response to vasopressin. These results reveal two previously unrecognized interactions between adrenal steroid hormones and vasopressin, namely, accelerated differentiation and increased responsiveness of adenylate cyclase.

Porous-bottom dishes for culture of polarized cells.

Porous-bottom dishes offer several advantages for growing and studying epithelia in culture. Many epithelia differentiate more on porous surfaces than on plastic tissue culture dishes. In addition, separate solutions can be maintained on each side of the epithelium and can be sampled easily for studies of transport and other polarized functions. We describe the fabrication of dishes with a cellulose ester filter, a collagen-coated polycarbonate filter, or a collagen membrane forming the surface for cell attachment at the bottom of the dish.

Complete response to vasopressin requires epithelial organization in A6 cells in culture.

Arginine vasopressin (AVP) stimulates adenylate cyclase activity in A6 epithelia grown on filters but not in A6 epithelia grown on plastic culture dishes. When A6 cells are subcultured from culture dishes and seeded at high density on filters, stimulation of adenylate cyclase by AVP is not evident for approximately 3 days. Peak stimulation by AVP occurs between 5 and 10 days. The time course for development of responsiveness to AVP corresponds to the development of an ordered epithelium with polarized cells, tight junctions, and a high transepithelial resistance. A similar correlation is seen when mature filter-grown epithelia that respond to AVP are dissociated by chelation of calcium and subcultured as single cells onto another filter. Stimulation of adenylate cyclase is not evident until an epithelium with significant electrical resistance is reformed. Filters seeded at 1.6 X 10(6) cells/cm2 require 1-2 days to show AVP sensitivity and electrical resistance; filters seeded at 0.4 X 10(6) cells/cm2 require 4 days. Experiments to test the function of the subunits of adenylate cyclase with forskolin (catalytic subunit) and with cholera toxin (Ns, guanine nucleotide-binding subunit) indicate that they are functional during the entire time and that it is the receptor for AVP that is not functional until an ordered epithelium has been formed.

Adenosine stimulates sodium transport in kidney A6 epithelia in culture.

The effects of adenosine receptor agonists and antagonists were examined in epithelia formed in culture by A6 cells, a continuous cell line derived from Xenopus laevis kidney. A6 epithelia have a high electrical resistance and a short-circuit current that is equal to net sodium flux from mucosal to serosal surface. Adenosine, 2-chloroadenosine, 5'-(N-ethyl)carboxamidoadenosine, and N6-(L-2-phenylisopropyl) adenosine produced concentration-dependent increases in short-circuit current. Stimulation of short-circuit current by 2-chloroadenosine occurred at concentrations of 0.05 microM and above, with half-maximal stimulation occurring at 0.3 microM. 5'-(N-ethyl)carboxamidoadenosine was more potent than N6-(L-2-phenylisopropyl)adenosine, the usual order of potency for activation of stimulatory adenosine receptors. Theophylline (100 microM), an adenosine receptor antagonist, reduced the short-circuit current response to adenosine and 2-chloroadenosine by 85-90%. Amiloride, an agent that inhibits both basal and adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated short-circuit current in A6 epithelia, completely and reversibly inhibited short-circuit current stimulated by 2-chloroadenosine. Adenosine and 2-chloroadenosine stimulated adenylate cyclase activity in a crude membrane preparation from A6 cells. Stimulation by adenosine was blocked by adenosine deaminase. 2-Chloroadenosine increased cell cAMP accumulation in intact epithelia. The results provide evidence that adenosine and adenosine receptor agonists stimulate adenylate cyclase and active sodium transport in an epithelial cell line of renal origin.

Factors affecting the differentiation of epithelial transport and responsiveness to hormones.

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.

Epithelial organization and hormone sensitivity of toad urinary bladder cells in culture.

Two continuous cell lines (TB-M and TB-6c) derived from epithelial cells of the toad urinary bladder form epithelia in culture that manifest hormone-sensitive transepithelial transport. Development of transepithelial electrical resistance (R) and transport rate (ISC) are dependent on time and density of cells seeded, but steady-state ISC and R are characteristic for each cell line and independent of seeding density. Some responses of intact toad bladder are preserved in culture, whereas others are altered or absent. Neither cell line responds to vasopressin. Analogues of cAMP increase sodium transport and urea permeability in both cell lines but do not affect water permeability. The intramembrane particle aggregates associated with the vasopressin- and cAMP-induced increase in water permeability of the intact bladder could not be detected in the cell lines. Aldosterone increases sodium transport in both cell lines, and the time course and concentration dependence of the response to aldosterone are similar to those of the intact bladder. The relative effect of a series of steroids on ISC reveals corticosterone to be a more potent mineralocorticoid in cultured cells than in the intact bladder.