Effects of membrane depolarization on nicotinamide nucleotide fluorescence in brain slices.
1. Simultaneous measurement of tissue NAD(P)H fluorescence and respiration was used to elucidate some of the early chemical changes after depolarization of the membranes of cerebral-cortical cells. Depolarization was effected by the application of a train of short-duration voltage pulses across a slice of guinea-pig cerebral cortex. The pulses cause a biphasic change in fluorescence corresponding to an early (within 1s) oxidation and later (about 10s) reduction of nicotinamide nucleotide. The major portions of both these redox changes are unrelated to the accompanying increase in slice respiration of about 75%. 2. The early oxidation requires Ca(2+) and phosphate in the bathing medium and appears to be largely due to an early mitochondrial uptake of these ions. 3. The ensuing reduction occurs in the cytosol, as judged by its almost complete elimination in the presence of exogenous pyruvate. It becomes markedly attenuated with increasing time of incubation. This and the ability of exogenous 6-N-2'-O-dibutyryl cyclic AMP to cause a reduction in the absence of electrical pulses suggest that it results from a cyclic AMP-mediated activation of glycogenolysis. 4. Further results, obtained in the presence of exogenous pyruvate, indicate that in addition to the above effects a net transfer of NADH from the mitochondrial to cytosolic space (presumably via a shuttle mechanism) is activated by the electrical pulses. 5. The lack of any sizeable (more than 2% of basal fluorescence) fluorescence change associated with the respiratory increase caused by the pulses, and the fact that any change which may be associated with it corresponds to a small reduction of mitochondrial nicotinamide nucleotide, show that a simple state 4 --> state 3 mitochondrial transition cannot account for the increased respiration. The results suggest, rather, a co-ordinated control of respiration, at more than one site.
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004558:Electric Stimulation; D004594:Electrophysiology; D005453:Fluorescence; D005965:Glucuronates / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010710:Phosphates / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D013997:Time Factors
D005965:Glucuronates; D010710:Phosphates; D011773:Pyruvates; D009243:NAD; D009249:NADP; D006003:Glycogen; D000242:Cyclic AMP; D002118:Calcium