Renal handling of taurine,l-alanine,l-glutamate andd-glucose inOpsanus tau: studies on isolated brush border membrane vesicles

Summary Renal brush border membrane vesicles (bbmv) from the aglomerular toadfish (Opsanus tau), isolated by differential precipitation, were tested for their ability to actively translocate (i) taurine, known to be secreted by the kidney of several marine teleosts, and (ii)l-alanine,l-glutamic acid, andd-glucose, solutes that are normally reabsorbed in the filtering nephron. Vesicular taurine uptake displayed a Na⁺ dependence. Transport was greatest under conditions of an inward-directed Na⁺ gradient, but a significant stimulation by Na⁺ over K⁺ could also be observed in the absence of a salt gradient. At high extravesicular K⁺, the addition of valinomycin reduced taurine uptake. Na⁺-dependent³H-taurine flux was almost completely inhibited by non-labeled taurine (tracer replacement) or -alanine, but was unaffected byl-alanine. Replacement of medium chloride by SCN^– or NO ₃ ^– in the presence of Na⁺ resulted in significantly lower uptake rates under both anion gradient and anion equilibrium conditions, whereas Br^– could almost fully substitute for the stimulatory Cl^– action. These results indicate the presence of an electrogenic Na⁺-cotransport mechanism with specificity for -amino acids in the toadfish renal brush border. Whether the system under physiological conditions mediates reabsorption or secretion of taurine remains to be determined. Toadfish bbmv also translocatedl-alanine andl-glutamic acid in a Na⁺-dependent manner. Possible roles for these most likely reabsorptive transport systems in a non-filtering kidney are discussed.d-glucose uptake, however, appeared to occur via Na⁺-independent pathways, since it was not affected by phlorizin in the presence of Na⁺, or by Na⁺ replacement.Abbreviation bbmv brush border membrane vesicles     

Regulation of intracellular pH in LLC-PK1 cells studied using 31P-NMR spectroscopy

31P-NMR spectroscopy was used to monitor intracellular pH (pHi) in a suspension of LLC-PK1 cells, a renal epithelial cell line. The regulation of intracellular pH (pHi) was studied during intracellular acidification with 20% CO2 or intracellular alkalinization with 30 mM NH4Cl. The steady-state pHi in bicarbonate-containing Ringer's solution (pHo 7.40) was 7.14 +/- 0.04 and in bicarbonate-free Ringer's solution (pHo 7.40) 7.24 +/- 0.04. When pHo was altered in nominally HCO3(-)-free Ringer's, the intracellular pHi changed to only a small extent between pHo 6.6 and pHo 7.6; beyond this range pHi was linearly related to pHo. Below pHo 6.6 the cell was capable of maintaining a delta pH of 0.2 pH unit (inside more alkaline), above pH 7.6 a delta pH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO2 in HCO3(-)-free Ringer's solution, pHi dropped initially to 6.9 +/- 0.05, the rate of realkalinisation was found to be 0.071 pH unit X min-1. After removal of CO2 the pHi increased by 0.65 and the rate of reacidification was 0.056 pH unit X min-1. Exposure to 30 mM NH4Cl caused a raise of pHi by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit X min-1, after removal of NH4Cl the pHi fell by 0.58 pH unit below the steady-state pHi, followed by a subsequent re-alkalinization of 0.083 pH unit X min-1. Under both experimental conditions, the pHi recovery after an intracellular acidification, introduced by exposure to 20% CO2 and by removal of NH4+, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pHi transients in suspensions of epithelial cells. The results support the view that LLC-PK1 cells use an Na+-H+ exchange system to readjust their internal pH after acid loading of the cell.     

Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish,Squalus acanthias

Summary A membrane fraction, rich in brush border membranes, was prepared from renal proximal tubules of the spiny dogfish,Squalus acanthias, and the sodium-proton exchange mechanism in these membrane vesicles was investigated by both a rapid filtration technique and the fluorescence quenching of acridine organe.²²Na⁺ uptake was stimulated by an outwardly directed H⁺ gradient, and was inhibited by amiloride at a single inhibitory site with an apparentK _i of approximately 1.7×10^–5 M. In the presence of an H _i ⁺ >H _o ⁺ gradient, the of the Na⁺/H⁺ exchanger were 9.7±0.8 mM and 48.0±12.0 nmol·mg protein^–1·min^–1, respectively. The uptake of Na⁺ was electroneutral in the presence of a H⁺ gradient, indicating a stoichiometry of 1. In the fluorescence studies, quenching of acridine orange occurred in the presence of an outwardly directed Na⁺ gradient which was inhibited by amiloride. Thus, an electroneutral Na⁺/H⁺ exchanger with properties similar to those found in the mammalian kidney is also present in the spiny dogfish and may contribute to the urinary acidification of this marine animal.     

Pathways for organic osmolyte synthesis in rabbit renal papillary tissue, a metabolic study using 13C-labeled substrates

Renal papillary collecting duct cells have been postulated to adapt their intracellular osmolality to the large changes in interstitial osmolality by changing their content of 'non-perturbing' organic osmolytes such as sorbitol and myo-inositol. 13C-NMR was used in this study to elucidate the metabolic pathways leading to a synthesis of those compounds. Incubation of rabbit renal papillary tissue with [1-13C]glucose showed label scrambling mainly into sorbitol (C-1) and lactate (C-3). This result confirms activity of aldose reductase and glycolytic enzymes in renal papillary cells. Using [3-13C]alanine or [2-13C]pyruvate as carbon source, 13C-labeling of sorbitol and myo-inositol was observed, indicating that renal papillary tissue possesses, in addition, gluconeogenic activity. The latter assumption is supported by the result that in enzyme assays rabbit kidney papilla and isolated rat kidney papillary collecting duct cells show significant fructose-1,6-bisphosphatase activity.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

Buchbesprechungen

Ohne Zusammenfassung

---

Book reviews

---

Analyses

     

Growth and reproduction as a function of temperature and salinity inClava multicornis (Cnidaria,Hydrozoa)

Summary 1. Rates of growth (length increase of stolons) and of asexual reproduction (increase in number of polyps) were determined in secondaryClava multicornis colonies of a clone exposed to 12 different combinations of water temperature and salinity (12°, 17°, 22° C; 16 , 24 , 32 , 40 S). Sexual reproduction (via gonophores) has been observed only at 12° and 17° C; temperature and salinity ranges are narrower for sexual than for asexual reproduction.2. The data obtained are insufficient for a detailed analysis; they provide, however, interesting insights into the variability of growth and reproduction ofC. multicornis caused by different intensities of temperature and salinity.3. It appears that temperature requirements for maximum colony increase are reduced as the colony grows older.4. One feeding period per 24 hours seems insufficient for maximum growth and reproduction at the higher temperature levels, especially at 22° C.5. The different degrees of environmental stress endured during the initial period of transfer into the test combinations of temperature and salinity have affected the resulting colony size at least up to an age of 39 days. More appropriate criteria for assessment of rates of growth and reproduction are therefore the doubling times (number of days within which stolon length and polyp numbers taken 20 days after initiation of experiments have doubled).6. On the basis of doubling time values, increase in stolon length is progressively reduced with increasing water temperature (12°, 17°, 22° C). At 12° and 17° C stolons grow fastest in 32 , followed by 24 , 16 and 40 S; at 22° C stolon growth rates are identical in 32 and 24 S.7. Doubling times of polyp numbers per colony show a less obvious trend. In 56-day-old colonies, however, stolon length and polyp number are modified to similar degrees by the various temperatures and salinities offered. The sequence of temperatures causing fastest increase in polyp number is 12°>17°>22° C; the respective sequence of salinities reads: 24 , 32 , 16 , 40 S.8. Stolon length and polyp number per colony increase exponentially; most curves obtained exhibit undulations indicating endogenous growth rhythms.9. During the initial period of transfer into the final test media, asexual reproduction via budding seems to have been stimulated by a reduction in salinity.10. The doubling times obtained forC. multicornis are considerably longer than those found forCordylophora caspia and indicate that our culture conditions may have been suboptimal.

---

Wachstum und Reproduktion als Funktion von Temperatur und Salzgehalt beiClava multicornis (Cnidaria, Hydrozoa)

Kurzfassung Einzelpolypen eines Klons vonC. multicornis Forskål wurden schrittweise in 12 verschiedene Temperatur-Salzgehalts-Kombinationen überführt und — während sie zu neuen Kolonien heranwuchsen — das Längenwachstum ihrer Stolonen, die Geschwindigkeit ihrer asexuellen Vermehrung durch Knospung neuer Hydranthen sowie die Gonophorenausbildung (sexuelle Fortpflanzung) registriert. Die erhaltenen Daten sind unzureichend für eine detaillierte Analyse, gewähren jedoch interessante Einblicke in die Bedeutung der verschiedenen Temperatur- und Salzgehaltsbedingungen für Wachstum und Vermehrung. Die anfängliche, schrittweise Überführung in die Testmedien verursacht per se Leistungsunterschiede, deren Auswirkungen sich mindestens bis zu einem Alter von 39 Tagen verfolgen lassen. Doubling times stellen daher objektivere Kriterien dar als absolute Zuwachswerte. Die doubling times von Kolonien, welche länger als 20 Tage in den Testmedien gewachsen waren, zeigen eine Verringerung der Stolonenzuwachsrate mit steigender Temperatur (12°, 17°, 22° C). Die Reihenfolge der fördernden Wirkung der einzelnen Salzgehaltsstufen ergibt sich zu 32 , 24 , 16 , 40 S. Im Prinzip ähnliche Verhältnisse liegen hinsichtlich der asexuellen Vermehrungsrate vor. Bemessen an den getesteten Kriterien scheinen die Temperaturansprüche mit zunehmendem Koloniealter abzunehmen. Die errechneten doubling times sind wesentlich länger als beiCordylophora; möglicherweise deutet dieser Unterschied auf inadäquate Kulturbedingungen (Fütterung, Wasserbewegung) hin.