Decreased Urinary Sodium-to-urinary Creatinine Ratio Identifies Sodium Depletion in Pediatric Acute Gastroenteritis.

UNLABELLED: In acute gastroenteritis (AG) fecal losses may cause depletion of sodium (NaD) which may not be recognized because of normal plasma Na (pNa) concentrations. We studied the incidence of this state of normonatremic sodium depletion (NNaD) and the suitability of the urinary Na/urinary creatinine ratio (uNa/uCr) for diagnosing NNaD. PATIENTS: 16 AG- and 16 healthy control children aged 0.8-15.0 years. METHODS: Prospective cross sectional pilot study. Measurements of Na, K and creatinine in plasma (p) and urine (u). Calculation of uNa/uCr Ratio, fractional excretion of Na (FENa) and uNa/uK ratio as the hitherto best known parameters of prerenal Na depletion, respectively. RESULTS: pNa concentrations were normal in 15/16 AG patients (93.8%) with only one subnormal value of 133 mmol/L, and a mean value of 137.9±2.3 mmol/L not different from the normal control group (139.4±2.2 mmol/L). Also, mean uNa concentrations and uNa/uK ratios did not differ between both groups. However, uNa/uCr ratios were below normal in 13/16 AG children (81.3%) but normal in all healthy controls with a significantly lower mean value in the AG group (12.6±8.8 vs. 31.2±8.3 mmol/mmol; p<0.0001). Similarly, 14/16 AG patients (87.5%) had a decreased FENa<0.5% with a mean FENa value significantly lower than in controls (0.36±0.28% vs. 0.95±0.26%, p<0.0001). The good agreement between FENa and uNa/uCr results was also reflected by a high correlation coefficient of r=0.9333. CONCLUSIONS: The majority of AG patients was found to have NNaD as determined by uNa/uCr and FENa. Calculation of uNa/uCr may be useful for diagnosing NNaD in AG.

KCNQ2 and KCNQ3 mutations contribute to different idiopathic epilepsy syndromes.

OBJECTIVE: To explore the involvement of M-type potassium channels KCNQ2, Q3, and Q5 in the pathogenesis of common idiopathic epilepsies. METHODS: Sequence analysis of the KCNQ2, Q3, and Q5 coding regions was performed in a screening sample consisting of 58 nuclear families with rolandic epilepsy. Subsequently, an association study was conducted for all discovered variants in a case-control sample comprising 459 German patients with idiopathic generalized epilepsy (IGE) and 462 population controls. RESULTS: An in-frame deletion of codon 116 in KCNQ2 (p.Lys116del) and a missense mutation in KCNQ3 (p.Glu299Lys) were detected in two index cases exhibiting rolandic epilepsy and benign neonatal convulsions. Both mutations resulted in reduced potassium current amplitude in Xenopus oocytes. Mutation analysis of families with rolandic epilepsy without neonatal seizures discovered three novel missense variations (KCNQ2 p.Ile592Met, KCNQ3 p.Ala381Val, KCNQ3 p.Pro574Ser). The KCNQ2 p.Ile592Met variant displayed a significant reduction of potassium current amplitude in Xenopus oocytes and was present only once in 552 controls. Both missense variants identified in KCNQ3 (p.Ala381Val and p.Pro574Ser) were present in all affected family members and did not occur in controls, but did not show obvious functional abnormalities. The KCNQ3 missense variant p.Pro574Ser was also detected in 8 of 455 IGE patients but not in 454 controls (p = 0.008). In KCNQ2, a silent single nucleotide polymorphism (rs1801545) was found overrepresented in both epilepsy samples (IGE, p = 0.004). CONCLUSION: Sequence variations of the KCNQ2 and KCNQ3 genes may contribute to the etiology of common idiopathic epilepsy syndromes.

Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1.

The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability.

Colocalization of KCNQ1/KCNE channel subunits in the mouse gastrointestinal tract.

The KCNQI potassium channel alpha-subunit can associate with various KCNE beta-subunits that drastically influence channel gating. Here we show that in the mouse gastrointestinal tract KCNQ1 is prominently expressed in stomach, small intestine and colon, while KCNE3 is expressed in the colon and to a lesser extent in small intestine. Immunostaining revealed that KCNQ1 colocalizes with KCNE3 in the basolateral membranes of crypt cells of the colon and small intestine. Together with the previously shown electrophysiological properties of KCNQ1/KCNE3 channels, this strongly suggests that they form the basolateral potassium conductance that is required for transepithelial cAMP-stimulated chloride secretion. In the stomach, KCNQ1 is expressed together with the H+/K+-ATPase in the luminal membrane of acid-secreting parietal cells of gastric glands. KCNE2, but neither KCNE1 nor KCNE3 was detected in the stomach by Northern analysis. Similar to KCNQ1, KCNE2 was present in gastric glands in only a subset of cells that probably represent parietal cells. The coexpression of KCNQ1 and KCNE2 in HEK293 cells yielded potassium currents that were open at resting voltages, suggesting that these heteromeric channels may underlie the apical potassium conductance in acid-secreting parietal cells that is necessary for the recycling of potassium ions during acid secretion via the H+/K+-ATPase.

Effects of the serine/threonine kinase SGK1 on the epithelial Na(+) channel (ENaC) and CFTR: implications for cystic fibrosis.

Cystic fibrosis (CF) is characterized by impaired Cl(-) secretion and increased Na(+) reabsorption in several tissues including respiratory epithelium. Many CFTR mutations have been identified over the past years. However, only a poor correlation between the genotype and lung phenotype was found suggesting additional factors influencing the phenotype and course of the disease. The serine/threonine kinase SGK1 has recently been shown to stimulate the activity of the epithelial Na(+) channel ENaC. A variety of stimuli such as aldosterone, cell shrinkage, insulin or TGF-beta1 stimulate transcription and activate the SGK1 kinase. Here we further examined the effects of SGK1 on ENaC and CFTR which have mutual interactions and we analyzed sgk1 mRNA abundance in lung tissue from CF patients. Coexpression of CFTR and h-SGK1 in Xenopus oocytes increased ENaC currents as previously described. In addition CFTR mediated currents were also stimulated. h-SGK1 accelerated the expression of the amiloride sensitive Na(+)- current in Xenopus oocytes paralleled by increased ENaC-protein abundance in the oocyte membrane, an effect which was reversed by a h-SGK1(K127R) mutation lacking the ATP-binding site. The cation selectivity or Na(+) affinity were not affected. However, coexpression of h-SGK1 with ENaC altered the sensitivity of the Na(+)-channel to the inhibitors amiloride and triamterene. The inhibitory effect of CFTR expression on ENaC current was not affected by coexpression of h-SGK1 in Xenopus oocytes. Lung tissue from CF patients strongly expressed the serine/threonine kinase h-sgk1 which was not the case for non-CF lung tissue. Loss of CFTR function itself in a CF lung epithelial cell line did not increase SGK1 expression. In summary, enhanced expression of h-SGK1 in epithelial cells of CF-lung tissue may be a novel pathophysiological factor contributing to increased Na(+) channel activity and thus to increased Na(+) transport in CF.

Intracellular anions as the voltage sensor of prestin, the outer hair cell motor protein.

Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement ("gating currents") driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.

Cloning and characterization of SLC26A6, a novel member of the solute carrier 26 gene family.

The SLC26 gene family (solute carrier family 26) comprises five mammalian genes that encode anion transporter-related proteins. In addition to sat-1 and prestin, which were cloned from rat and gerbil, respectively, three human members have been identified and associated with specific genetic diseases (DTD, diastrophic dysplasia; CLD, congenital chloride diarrhea; PDS, Pendred syndrome). In this study we used a homology approach combined with RACE PCR to identify human SLC26A6, the sixth member of this gene family. Northern blot analysis showed the highest SLC26A6 transcript levels in kidney and pancreas. Expression in MDCK cells and in Xenopus oocytes demonstrated trafficking of the SLC26A6 protein to the cell membrane but did not reveal anion transport activity with tracer uptake or intracellular pH measurements. We determined the genomic structure of the SLC26A6 gene and excluded mutations in the 21 coding exons as the cause of DFNB6 and USH2B, which closely map to the SLC26A6 chromosomal locus (3p21).

Serum- and glucocorticoid-dependent kinase, cell volume, and the regulation of epithelial transport.

Ample pharmacological evidence points to a role of kinases in the regulation of cell volume. Given the limited selectivity of most inhibitors, however, the specific molecules involved have remained largely elusive. The search for cell volume regulated genes in liver HepG2 cells led to the discovery of the human serum- and glucocorticoid-dependent serine/threonine kinase hsgk1. Transcription and expression of hsgk1 is markedly and rapidly upregulated by osmotic and isotonic cell shrinkage. The effect of osmotic cell shrinkage on hsgk1 is mediated by p38 kinase. Further stimuli of hsgk1 transcription include glucocorticoids, aldosterone, TGF-beta1, serum, increase of intracellular Ca2+ and phorbolesters, whereas cAMP downregulates hsgk1 transcription. The hsgk1 protein is expressed in several epithelial tissues including human pancreas, intestine, kidney, and shark rectal gland. Co-expression of hsgk1 with the renal epithelial Na+-channel ENaC or the Na+/K+/2Cl(-)-cotransporter NKCC2 (BSC1) in Xenopus oocytes, accelerates insertion of the transport proteins into the cell membrane and thus, stimulates channel or transport activity. Thus, hsgk1 participates in the regulation of transport by steroids and secretagogues increasing intracellular Ca2+-activity. The stimulation of hsgk1 transcription by TGF-beta1 may further bear pathophysiological relevance.

h-sgk serine-threonine protein kinase as transcriptional target of p38/MAP kinase pathway in HepG2 human hepatoma cells.

The human serum and glucocorticoid dependent serine/threonine kinase h-sgk has previously been discovered as cell volume regulated gene. The present study has been performed to elucidate the involvement of p38-kinase in the transcriptional control of h-sgk by osmotic cell shrinkage. The p38-kinase has previously been cloned as the mammalian homologue of HOG1 kinase, which constitutes a part of the osmosensor in the yeast Saccharomyces cerevisiae. Phosphorylated (active) p38-kinase has been estimated with Western blotting, transcription of hsgk using Northern blotting. Both, increase of extracellular NaCl concentration by 50 mmol/l and addition of 10 micromol/l anisomycin increase phosphorylation of the p38-kinase within 5 to 10 minutes. h-sgk transcription is upregulated by addition of 50 mmol/l NaCl and by anisomycin (10 micromol/l), effects completely inhibited by the specific p38-kinase inhibitor, SB 203580 (10 micromol/l). In conclusion, the stimulation of h-sgk transcription by osmotic cell shrinkage is mediated by p38-kinase.

Expression of cell volume-regulated kinase h-sgk in pancreatic tissue.

Transcript levels of the human serine/threonine kinase h-sgk have been found to be highest in pancreas. In the present study, localization and regulation of h-sgk transcription in pancreatic tissue were elucidated. As was apparent from radioactive in situ hybridization, most pancreatic acinar cells expressed high levels of h-sgk mRNA. h-sgk mRNA-positive cells were also found in ductal epithelia but not in pancreatic islets. In biopsy specimens from patients with pancreatitis, h-sgk mRNA levels were decreased in acinar cells but abundant in numerous mononuclear interstitial cells within areas of pancreatic necrosis and fibrosis. As shown by Northern blotting, h-sgk transcription in DAN-G pancreatic tumor cells is upregulated by osmotic cell shrinkage, serum, phorbol esters (phorbol 12,13-didecanoate), and Ca(2+) ionophore A-23187 and decreased by staurosporine and cAMP. In conclusion, h-sgk transcription is regulated not only by cell volume but also by serum, protein kinase C stimulation, cAMP, and increase of intracellular Ca(2+) activity. The kinase may participate not only in normal function of exocrine pancreas but also in fibrosing pancreatitis.

Potent stimulation and inhibition of the CFTR Cl(-) current by phloxine B.

The effects of the fluoresceine derivative, phloxine B, on the Cl(-) current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR. In whole oocytes, the CFTR Cl(-) current (I(CFTR)) was activated by superfusion with isobutylmethylxanthine and forskolin. I(CFTR) was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited I(CFTR) weakly. In excised inside-out macropatches, I(CFTR) was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01 - 3 microM), applied after activation, increased I(CFTR) within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations. The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 45 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC(50) value of approximately 1 microM. In the absence of cPKA, phloxine B did not stimulate I(CFTR). In the presence of cPKA and MgATP, the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM). The data show that phloxine B modulates I(CFTR) by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell.

Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy.

Transforming growth factor beta (TGF-beta) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-beta stimulates the expression of a distinct serine/threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-beta (2 microgram/liter), phorbol 12,13-didecanoate (1 microM), or the Ca(2+) ionophore ionomycin (1 microM) and blunted by high concentrations of nifedipine (10 and 100 microM). In situ hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in Xenopus oocytes expressing the renal epithelial Na(+) channel ENaC or the mouse thick ascending limb Na(+),K(+),2Cl(-) cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 microM) and chelerythrine (1 microM) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na(+) transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.

Effect of urea and osmotic cell shrinkage on Ca2+ entry and contraction of vascular smooth muscle cells.

The present study was performed to elucidate the effects of urea on vascular smooth muscle cells (SMC). Addition of urea (20, 50, 100 mM) to physiological salt solution blunted the vasoconstrictory effect of phenylephrine (by 17, 25 and 30%, respectively) and of an increased extracellular K+ concentration (by 7, 14 and 19%, respectively) without affecting the basal tone of rabbit arterial rings. According to Fura-2 fluorescence in cultured SMC (A7r5), urea had no effect on basal intracellular calcium activity ([Ca2+]i), but significantly blunted the increase of [Ca2+]i following an increase of extracellular K+. Whole-cell patch-clamp studies revealed that the Ca2+ current through voltage-sensitive Ca2+ channels is significantly inhibited in the presence of urea. As evident from calcein fluorescence, addition of urea leads to sustained cell shrinkage. The effects of urea on vascular tone, [Ca2+]i activity, voltage-gated Ca2+ channels and cell volume are mimicked by addition of raffinose or NaCl. However, the cell shrinkage induced by urea is sustained, whereas the addition of equiosmolar NaCl is only transient and followed by a regulatory cell volume increase. Moreover, hypertonic NaCl increases, whereas urea decreases, the transcription of cell-volume-regulated kinase hsgk. In conclusion, urea leads to sustained shrinkage of vascular smooth muscle cells, which is followed by inhibition of voltage-gated Ca2+ channels, a decrease of [Ca2+]i and thus blunts the vasoconstrictory action of phenylephrine and increased extracellular K+ concentration.

Functional and structural analysis of ClC-K chloride channels involved in renal disease.

ClC-K channels belong to the CLC family of chloride channels and are predominantly expressed in the kidney. Genetic evidence suggests their involvement in transepithelial transport of chloride in distal nephron segments; ClC-K1 gene deletion leads to nephrogenic diabetes insipidus in mice, and mutations of the hClC-Kb gene cause Bartter's syndrome type III in humans. Expression of rClC-K1 in Xenopus oocytes yielded voltage-independent currents that were pH-sensitive, had a Br(-) > NO(3)(-) = Cl(-) > I(-) conductance sequence, and were activated by extracellular calcium. A glutamate for valine exchange at amino acid position 166 induced strong voltage dependence and altered the conductance sequence of ClC-K1. This demonstrates that rClC-K1 indeed functions as an anion channel. By contrast, we did not detect currents upon hClC-Kb expression in Xenopus oocytes. Using a chimeric approach, we defined a protein domain that, when replaced by that of rClC-K1, allowed the functional expression of a chimera consisting predominantly of hClC-Kb. Its currents were linear and were inhibited by extracellular acidification. Contrasting with rClC-K1, they displayed a Cl(-) > Br(-)> I(-) > NO(3)(-) conductance sequence and were not augmented by extracellular calcium. Insertion of point mutations associated with Bartter's syndrome type III destroyed channel activity. We conclude that ClC-K proteins form constitutively open chloride channels with distinct physiological characteristics.

The promoter for constitutive expression of the human ICln gene CLNS1A.

The ICln protein is expressed ubiquitously in mammals. Experiments designed to knock down the ICln protein in NIH 3T3 fibroblasts as well as in epithelial cells led to the conclusion that this protein is crucially involved in volume regulation after cytoplasmic swelling. Reconstitution of the ICln protein in lipid bilayers revealed the ion channel nature of ICln. Here we describe a new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be effected at multiple sites. In addition to the starting sites, upstream sequence elements are mandatory for an efficient transcription of the ICln gene (CLNS1A). These new nucleotide elements were defined by site-directed mutagenesis.

A constitutively open potassium channel formed by KCNQ1 and KCNE3.

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.

Effect of verapamil enantiomers and metabolites on cardiac K+ channels expressed in Xenopus oocytes.

The effect of verapamil and its enantiomers and metabolites on cardiac action potential repolarizing potassium channels was tested. For this purpose, the potassium channels Kv1.1, Kv1.5, Kir2.1, and HERG, and the IsK subunit of the IKs-channel complex were expressed in Xenopus oocytes and two-electrode voltage-clamp experiments were performed. Verapamil induced a concentration-dependent block of Kv1. 1-, Kv1.5-, IKs-, and HERG-induced currents with IC50 values of 14.0 +/- 2.7 microM (n = 4), 5.1 +/- 0.5 microM (n = 6), 161.0 +/- 26.3 microM (n = 4), and 3.8 +/- 0.2 microM (n = 5), respectively. The same potency of HERG channel inhibition was observed for the optical enantiomers (+)-verapamil (IC50 = 3.5 +/- 0.4 microM, n = 5) and (-)-verapamil (IC50 = 4.0 +/- 0.7 microM, n = 4), as well as the derivatives norverapamil (D591; IC50 = 3.8 +/- 0.3 microM, n = 4) and D703 (IC50 = 2.2 +/- 0.4 microM, n = 4). The verapamil metabolites D620 and D617 did not block HERG-induced currents at concentrations of up to 30 microM (n = 3). These results demonstrate that cardiac delayed rectifier potassium currents are sensitive targets to calcium channel blockers.

Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.

h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor beta in human intestine.

BACKGROUND & AIMS: Recently, the immediate early gene h-sgk was cloned as a hypertonicity-induced gene from human hepatoma cells. The aim of this study was to localize h-sgk messenger RNA (mRNA) expression in normal and inflamed intestinal mucosa and to identify potential transcriptional regulators. METHODS: h-sgk mRNA in small intestinal mucosa from healthy persons and patients with Crohn's disease was determined by in situ hybridization. Transcriptional regulation was studied by Northern blot analysis of total RNA isolated from cultured human Intestine 407, U937, and HepG2 cells. RESULTS: In normal ileum, h-sgk mRNA was selectively localized to the apical villus enterocytes, whereas no staining was detected in crypt cells. In Crohn's disease, enterocytes of the crypts expressed h-sgk and abundant h-sgk positive inflammatory cells appeared in the lamina propria. Combined h-sgk in situ hybridization and immunohistochemical analysis of CD68 antigen expression identified a part of these cells as macrophages. In addition to spatial correlation of transforming growth factor (TGF)-beta1 protein and h-sgk mRNA expression, h-sgk transcription in human Intestine 407 and HepG2 cells as well as in U937 monocytes/macrophages was strongly induced by TGF-beta1 in vitro. CONCLUSIONS: h-sgk expression in normal and inflamed intestinal mucosa may be regulated by TGF-beta1 and may contribute to the pleiotropic actions of TGF-beta1 in mucosal cell populations.

Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.