gramicidin-a and thiocyanate

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Brain; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Chlorides; Dopamine; Energy Metabolism; gamma-Aminobutyric Acid; Glutamates; Glutamic Acid; Gramicidin; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membrane Potentials; Proteolipids; Rats; Reserpine; Synaptic Vesicles; Thiocyanates

A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90 degrees to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25 degrees C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43 +/- 0.044 sec-1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25 degrees C with an activation energy of 9.75 +/- 1.04 kcal/mole from 11 to 25 degrees C and 17.2 +/- 0.55 kcal/mole from 25 to 37 degrees C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51 +/- 0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 micron radius, an osmotic permeability coefficient of 1.2 X 10(-3) cm/sec can be estimated for the native brush-border membranes at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Body Water; Calorimetry; Cell Membrane Permeability; Gramicidin; Intestinal Mucosa; Intestine, Small; Kinetics; Male; Microvilli; Osmolar Concentration; Rats; Rats, Inbred Lew; Thiocyanates