calcimycin and methylglucoside

Aminoglycoside-induced renal cell injury was investigated using the LLC-PK1 pig kidney epithelial cell line. The development of aminopeptidase and gamma-glutamyltransferase, marker enzymes for apical membranes, was inhibited in the LLC-PK1 cells cultured with gentamicin. The inhibitory effect of gentamicin on the enzyme activities was dose dependent and was related to its accumulation within the cells. The development of Na+ -dependent active transport of alpha-methyl-D-glucoside, a nonmetabolizable hexose, was also inhibited by treatment with gentamicin. Inhibitions in apical membrane enzyme activities and Na+ -dependent transport of alpha-methyl-D-glucoside were associated with the elevation of cytosolic free calcium, determined with a fluorescent indicator fura-2. The correlation between the alterations in apical membrane functions and the increase in cytosolic free calcium concentration was also supported by the study using the calcium ionophore A23187. The present data suggest that aminoglycoside-induced alterations in apical membranes of the LLC-PK1 cells are related to the increase in cytosolic free calcium levels.

Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Cell Line; Cell Membrane; Cytosol; Gentamicins; Kidney; Methylglucosides; Sodium

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.

Topics: Animals; Antimycin A; Calcimycin; Calcium; Calcium-Transporting ATPases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Membrane Permeability; Edetic Acid; Glycolysis; Hyaluronoglucosaminidase; Intestinal Absorption; Intestine, Small; Methylglucosides; Nigericin; Oligomycins; Rats; Vanadates; Vanadium

Active transport of amino acids and the sugar, alpha-methyl-D-glucoside (alpha-MG) caused an increase in the rate of K efflux from isolated rabbit enterocytes. These effects were inhibited by apamin (5 X 10(-7) M), quinidine (10(-3) M), Ba (5 X 10(-3) M) and trifluoperazine (5 X 10(-5) M) but not by the loop diuretic furosemide (10(-4) M). None of these drugs affected the basal rate of K efflux. The stimulatory effects of amino acids or alpha-MG on K efflux are too great to be explained in terms of an increase in the electrical driving force across the plasma membrane of these cells and a change in membrane permeability is envisaged. An apparent Ca-dependent K permeability in isolated enterocytes can be demonstrated using the Ca ionophore A23187. The effect of the ionophore on K efflux is abolished by apamin or Ba. It is proposed that Ca-dependent K channels mediate the sugar and amino acid induced increases of K efflux. Under control conditions there is a decrease in intracellular K concentration during accumulation of alanine or alpha-MG. Ba by itself does not alter K concentration but it did produce a marked increase when used in conjunction with alanine or alpha-MG. The accumulation of alpha-MG was inhibited in the presence of Ba. This is consistent with an interference with the driving force for sugar accumulation. It is suggested that the increase in K permeability described has a role in both maintaining ion homoeostasis during Na-coupled transport and contributing to the driving force for sugar and amino acid absorption.

Topics: Amino Acids; Animals; Apamin; Barium; Biological Transport, Active; Calcimycin; Cell Membrane Permeability; Female; Furosemide; In Vitro Techniques; Jejunum; Male; Methylglucosides; Methylglycosides; Potassium; Quinidine; Rabbits; Trifluoperazine

We examined the effects of acute changes in extracellular and intracellular calcium on transport processes in primary culture of proximal rabbit renal cells. A change in extracellular calcium from 0 to 3 mM inhibited amiloride-sensitive sodium uptake by 30%, and this effect was maximal at 1 mM calcium. Other polyvalent cations (Mn2+, Mg2+, La3+, and Ba2+) produced quantitatively similar inhibition of amiloride-sensitive sodium uptake compared with calcium. An increase in cytosolic calcium produced by calcium loading (20 mM) or by A23187 (20 microM) resulted in an inhibition of 25-40% of amiloride-sensitive sodium uptake. Moreover, quinidine (10(-4)M) and ruthenium red (3 microM), agents presumed to increase cytosolic calcium, inhibited amiloride-sensitive sodium uptake by 20-60%. Both these agents also inhibited sodium-dependent phosphate uptake by 20% but had no effect on ouabain-sensitive 86Rb+ uptake or on sodium-dependent alpha-methylglucoside uptake. Our data indicate that increases in extracellular calcium inhibit amiloride-sensitive sodium uptake and increases in cytosolic calcium inhibit sodium-dependent phosphate and amiloride-sensitive sodium uptakes. The effect of extracellular calcium may be due to charge screening and/or binding to the negatively charged plasma membrane or due to alterations in membrane fluidity.

Topics: Amiloride; Animals; Biological Transport; Calcimycin; Calcium; Cells, Cultured; Cytosol; Kidney Tubules, Proximal; Kinetics; Male; Methylglucosides; Ouabain; Phosphates; Rabbits; Rubidium; Sodium