monensin and gluconic-acid

The amount of neurotransmitter stored in a single synaptic vesicle can determine the size of the postsynaptic response, but the factors that regulate vesicle filling are poorly understood. A proton electrochemical gradient (Δμ(H+)) generated by the vacuolar H(+)-ATPase drives the accumulation of classical transmitters into synaptic vesicles. The chemical component of Δμ(H+) (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters as well as in protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. However, synaptic vesicle uptake of the principal excitatory transmitter glutamate depends on the electrical component of Δμ(H+) (Δψ). We found that rat brain synaptic vesicles express monovalent cation/H(+) exchange activity that converts ΔpH into Δψ, and that this promotes synaptic vesicle filling with glutamate. Manipulating presynaptic K(+) at a glutamatergic synapse influenced quantal size, indicating that synaptic vesicle K(+)/H(+) exchange regulates glutamate release and synaptic transmission.

Topics: Amiloride; Analysis of Variance; Animals; Animals, Newborn; Arthropod Proteins; Aspartic Acid; Biological Transport; Biophysical Phenomena; Brain; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Postsynaptic Potentials; Gluconates; Glutamic Acid; Hydrogen-Ion Concentration; In Vitro Techniques; Ionophores; Membrane Potential, Mitochondrial; Monensin; Oligopeptides; Potassium; Presynaptic Terminals; Radionuclide Imaging; Rats; Rats, Wistar; Sodium Isotopes; Synapses; Synaptic Vesicles; Synaptosomes; Tritium; Vesicular Glutamate Transport Proteins

The relationships between extracellular pH (pHo), intracellular pH (pHi), and loss of cell viability were evaluated in cultured rat hepatocytes after ATP depletion by metabolic inhibition with KCN and iodoacetate (chemical hypoxia). pHi was measured in single cells by ratio imaging of 2',7'-biscarboxy-ethyl-5,6-carboxyfluorescein (BCECF) fluorescence using multiparameter digitized video microscopy. During chemical hypoxia at pHo of 7.4, pHi decreased from 7.36 to 6.33 within 10 min. pHi remained at 6.1-6.5 for 30-40 min (plateau phase). Thereafter, pHi began to rise and cell death ensued within minutes, as evidenced by nuclear staining with propidium iodide and coincident leakage of BCECF from the cytoplasm. An acidic pHo produced a slightly greater drop in pHi, prolonged the plateau phase of intracellular acidosis, and delayed the onset of cell death. Inhibition of Na+/H+ exchange also prolonged the plateau phase and delayed cell death. In contrast, monensin or substitution of gluconate for Cl- in buffer containing HCO3- abolished the pH gradient across the plasma membrane and shortened cell survival. The results indicate that intracellular acidosis after ATP depletion delays the onset of cell death, whereas reduction of the degree of acidosis accelerates cell killing. We conclude that intracellular acidosis protects against hepatocellular death from ATP depletion, a phenomenon that may represent a protective adaptation against hypoxic and ischemic stress.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acidosis; Amiloride; Animals; Bicarbonates; Carrier Proteins; Cell Survival; Cells, Cultured; Chlorides; Fluoresceins; Gluconates; Hydrogen-Ion Concentration; Liver; Male; Monensin; Oxygen; Rats; Rats, Inbred Strains; Sodium-Hydrogen Exchangers