An NMR spectroscopic characterization of a new epithelial cell line, TALH-SVE, with properties of the renal medullary thick ascending limb of Henle's loop.

NMR spectroscopy has been used to characterize a new renal cell line, TALH-SVE.1, which is derived from the medullary thick ascending limb of Henle's loop. From the 31P-NMR spectrum of a suspension of TALH-SVE cells using the chemical shift of the intracellular inorganic phosphate a value of 7.24 +/- 0.04 for the steady-state intracellular pH (pHi) was determined at pHo = 7.40. In addition, the 31P-NMR spectrum indicated rather high levels of UDPG, a finding confirmed by 1H-NMR spectra of perchloric acid extracts. The 1H-NMR data also demonstrate the presence of 'organic osmolytes' such as inositol, sorbitol, choline and glycerophosphoryl choline (GPC). 13C-NMR spectra of perchloric acid extracts of TALH-SVE cells incubated with [2-13C]- and [3-13 C]alanine were used to determine the relative influx in the Krebs cycle via pyruvate carboxylase (PCB) versus the influx via pyruvate dehydrogenase (PDH). The ratio was 0.41, while about 52% of all acetyl-CoA entering the Krebs cycle was unlabeled. 13C-NMR experiments also indicated that TALH-SVE cells lack gluconeogenic activity. The NMR study presented indicates that TALH-SVE cells possess metabolic pathways similar to those of the parental cells.

Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR.

The aim of this study was to investigate the effect of NH4+ on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH4+ transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pHi) under standard conditions was 7.24 +/- 0.04 (n = 46). Exposure to NH4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pHi value. The NH4(+)-induced acidification followed saturation kinetics up to 20 mM NH4Cl (delta pHmax = 0.2 pHunits). Half-maximal acidification was observed at 0.6 mmol/l. The intracellular acidification due to NH4Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter. The effect of bumetanide was dose-dependent and a Ki value of 8.10(-7) M was calculated. NH4+ influx via K+ channels or the (Na+ + K+)ATPase could not be detected. pHi recovery to the initial value was caused mainly by amiloride-sensitive Na+/H+ exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (beta i) in TALH SVE.1 cells of 26 +/- 3 mM x pH-1 (pHi = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal 'functional buffer capability' between pHout 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na+/K+/2Cl- cotransporter is the main site of NH4+ entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH4+ influx into the cell.

Characterization of three novel members of the Arabidopsis SHAGGY-related protein kinase (ASK) multigene family.

In this paper we report the characterization of three novel members of the Arabidopsis shaggy-related protein kinase (ASK) multigene family, named ASKdzeta (ASKzeta), ASKetha (ASKeta) and ASKiota (ASKiota). The proteins encoded by the ASK genes share a highly conserved catalytic protein kinase domain and show about 70% identity to SHAGGY (SGG) and glycogen synthase kinase-3 (GSK-3) from Drosophila and rat respectively. SGG is an ubiquitous intracellular component of the wingless signalling pathway that establishes cell fate and/or pattern formation in Drosophila. At least ten different ASK genes are expected to be present per haploid genome of A. thaliana. Different amino- and carboxy-terminal extensions distinguish different ASK family members. Five ASK gene sequences were analysed and shown to be present as single-copy genes in the Arabidopsis genome. A comparison based on the highly conserved catalytic domain sequences of all known sequences of the GSK-3 subfamily of protein kinases demonstrated a clear distinction between the plant and the animal kinases. Furthermore, we established the presence of at least three distinct groups of plant homologues of SGG/GSK-3. These different groups probably reflect biochemical and/or biological properties of these kinases. The differential expression patterns of five ASK genes were accessed by northern and in situ hybridization experiments using gene-specific probes. While ASKzeta is expressed in the whole embryo during its development, ASKeta expression is limited to the suspensor cells. No signal was detected for ASKalpha, ASKgamma and ASKiota in developing embryos.