I. Moschen

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-β1 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-SGK1K127R 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. .

The Shrinkage-activated Na+ Conductance of Rat Hepatocytes and its Possible Correlation to rENaC

At moderate cell shrinkage, activation of Na+ channels is the most prominent mechanism of regulatory cell volume increase in rat hepatocytes. The amiloride sensitivity of these channels suggests a relation to the family of epithelial Na+ channels (ENaCs). The present study was performed to determine the pharmacological profile of shrinkage-activated Na+ channels and to test for ENaC expression in primary cultures of rat hepatocytes; in addition, the influence of the cell volume regulated serine/threonine kinase hSGK on activity and pharmacological profile of rENaC was examined in Xenopus oocytes. Conventional electrophysiology in hepatocytes reveals that the shrinkage-activated Na+ channel is inhibited by amiloride and EIPA with IC50 values of 6.0 and 0.12 μmol/l, respectively. Western blots and RT-PCR demonstrate that rat hepatocytes do express all three subunits (α, β, γ) of ENaC. Coexpression of hSGK with rENaC in Xenopus oocytes reveals that the kinase stimulates ENaC by a factor of 4. Moreover, hSGK decreases the affinity to amiloride (increase of IC50 from 0.12 to 0.26 μmol/l) and increases the affinity to EIPA (decrease of IC50 from 250 to 50 μmol/l). In conclusion, rat hepatocytes express ENaC, which is activated by the cell volume-sensitive kinase hSGK. ENaC may contribute to the Na+ channels activated by osmotic cell shrinkage in hepatocytes, whereby the relatively low amiloride and high EIPA sensitivity of the channel could at least be partially due to modification by SGK, which decreases the amiloride and increases the EIPA sensitivity of ENaC.

Salivary Anti-hsp65 Antibodies as a Diagnostic Marker for Gingivitis and a Possible Link to Atherosclerosis

Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.

Significance of Short Chain Fatty Acid Transport by Members of the Monocarboxylate Transporter Family (MCT)

Metabolism of short-chain fatty acids (SCFA) in the brain, particularly that of acetate, appears to occur mainly in astrocytes. The differential use has been attributed to transport, but the extent to which transmembrane movement of SCFA is mediated by transporters has not been investigated systematically. Here we tested the possible contribution of monocarboxylate transporters to SCFA uptake by measuring fluxes with labelled compounds and by following changes of the intracellular pH in Xenopus laevis oocytes expressing the isoforms MCT1, MCT2 or MCT4. All isoforms mediated significant transport of acetate. Formate, however, was transported only by MCT1. The contribution of MCT1 to SCFA transport was determined by using phloretin as a high-affinity inhibitor, which allowed a paired comparison of oocytes with and without active MCT1.

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.

The effect of sodium fluoride at prophylactic and toxic doses on renal structure and function in the isolated perfused rat kidney

To assess the renal effects of fluoride, isolated rat kidneys were perfused in single pass mode for 120 min. Five, 15 and 50, as well as 150, 500 and 1500 mumol NaF were administered 60, 80 and 100 min after starting the perfusion, respectively. Kidneys were perfused with constant pressure (100 mmHg). The perfusate consisted of a substrate supplemented Ringer solution containing hydroxy ethyl starch (HES) to produce isoncotic conditions. Concentrations below 500 mumol/l NaF did not induce major changes in the main parameters of renal function. Only upon admixture of the highest concentration of 1500 mumol/l NaF severe changes in renal function could be observed, resulting in complete anuria and a drastic reduction of renal perfusion to 5% of control, associated with a cessation of glomerular filtration. Due to the lack in tubular load, tubular reabsorptive processes inevitably declined to zero. The morphological analysis of kidneys exhibited to 500 mumol/l NaF revealed the occurrence of vesicular material within the urinary space. These vesicles could electron microscopically be identified as membrane enclosed material of podocytic origin. The interstitium was widened. Upon admixture of 1500 mumol/l NaF, kidneys responded with a decrease of the interstitial space. Moreover, epithelial cell swelling, hydropic degeneration of all proximal and distal tubular segments, bleb formation and intraluminal casts were observed frequently. Glomerular capillaries were filled with fine precipitates and their endothelium was severely damaged. The results of our studies in the isolated perfused rat kidney (IPRK) model clearly demonstrate a direct dose dependent acute nephrotoxic effect of NaF only for extremely high doses, which, however, may be reached in human cases of severe fluoride intoxication. On the contrary, for low fluoride doses, especially for those concentrations occurring in human plasma upon cariesprophylactic intake of fluorides, no signs of direct acute nephrotoxic action could be observed in the IPRK model.

Effects of Gallium and Mercury Ions on Transport Systems

Mercury was previously shown to exert toxic effects by influencing ion channels and transporters in the kidney and brain. Gallium alloys were suggested as less toxic restorative materials. To compare the toxicity of gallium ions with those of mercury ions, we applied gallium nitrate Ga(NO3)3 (0.1-100 μM and mercuric chloride (HgCl2) (0.001-10 μM) to Xenopus oocytes expressing mammalian ion channels and transport proteins. Mercury (10 μM) inhibited the K+-channels ROMK and HERG, the phosphate transporter NaPi-3, the amino acid transporter rBAT, the cation transporter OCT-2, and the osmolyte transporter BGT. It activated the IKschannel but did not affect the Na+-channel ENaC, the anion channel NaPi-1, and the glucose transporter SGLT-1. Gallium was without significant effect on the channels and on SGLT1, NaPi-3, and rBAT, but inhibited BGT and OCT-2. In conclusion, both Hg2+ and Ga3+ may exert toxic effects on transport systems, which may partially explain their cytotoxic effects.

Effect of NaPi-mediated phosphate transport on intracellular pH.

Abstract. Extracellular pH has been shown previously to influence transport via type-II Na+/phosphate (NaPi) transporters by modifying the affinity of the carrier for Na+ and by altering the availability of divalent and monovalent phosphate. As the transport of monovalent phosphate would be expected to acidify, and that of divalent phosphate to alkalinize the cell interior, the effect of phosphate transport on cytosolic pH was studied using ion selective microelectrodes in Xenopus oocytes expressing NaPi-3 or NaPi-5. At an alkaline extracellular pH (pHe) of 8.0, addition of phosphate elicited a strong inward current, depolarization of the cell membrane and cytosolic alkalinization. At pHe 6.0 the phosphate-induced inward current and depolarization were reduced and the alkalinization completely abolished. In conclusion, at alkaline pHe phosphate transport is enhanced and the transport of divalent phosphate prevails. At pHe 6.0, phosphate transport is attenuated and is accomplished by transport of both divalent and monovalent phosphate.

Comparison of resin-bonded prosthesis groove parallelism with the use of four tooth preparation methods ☆ ☆☆ ★

STATEMENT OF PROBLEM A precise preparation is required to develop resistance form resulting in mechanical stability of the framework for resin-bonded prostheses (RBPs). PURPOSE The effects of 4 methods of tooth preparation (freehand, guiding pin, extraoral parallelometer, and intraoral parallelometer) on the deviation of proximal grooves from a preestablished path of insertion (guide planes) were investigated under clinical conditions. MATERIAL AND METHODS Tooth preparation of proximal grooves was performed by 32 dentists on resin substitutes of posterior segments intraorally with a single test patient. A Latin-square randomized cross-over design was selected as the experimental design. RESULTS The significant least angular deviation of proximal grooves from path of insertion was achieved with an intraoral parallelometer (mean +/- SD 3.15 +/- 1.67 degrees). Compared with freehand tooth preparations (4.37 +/- 2. 11 degrees), neither use of a guiding pin (4.10 +/- 1.62 degrees) nor an extraoral parallelometer (5.06 +/- 2.33 degrees) improved the results. CONCLUSION Divergence of guiding grooves from path of insertion was reduced with the use of an intra-oral parallelometer. This should improve mechanical stability of posterior RBPs.

Acute regulation of the Betaine/GABA transporter BGT-1 expressed in xenopus oocytes by extracellular pH

Besides uptake of Na+ and Cl–, mammalian cells counteract osmotic cell shrinkage also by Na+-coupled uptake of osmolytes, e.g., myo-inositol, taurine or betaine. The expression of the corresponding transporters is transcriptionally regulated by the ambient pH and osmolarity and is increased upon cell shrinkage, a process requiring hours. The present study has been performed to disclose rapid regulation by pH of osmolyte transport via BGT-1. Transport of GABA was investigated by using the two-electrode voltage-clamp technique with BGT-1 expressing Xenopus oocytes. GABA was used as a substrate, because of the low oocyte endogenous transport activity. Extracellular acidification to pH 5.5 reversibly decreased and extracellular alkalinization to pH 8.5 increased GABA-induced currents. Kinetic analysis revealed that extracellular alkalinization increases the affinity for Cl– as reflected by a decrease of the apparent Km-value for Cl– from >500 mM to 55.8 ± 4.7 mM upon an increase of the pH from 7.0 to 8.5. The apparent Km- values for Na+ and GABA remained unaltered in the pH range from 6.0 to 8.5. Instead, alkalinization increased the maximal current induced by saturating Na+ and GABA concentrations. The results are compatible with a model of interference of H+ ions with Cl– binding and a pH-dependent reduction of Vmax for Na+ and GABA.