Electrophysiological and metabolic effects of a convulsant barbiturate on dissociated mouse primary sensory neurons.
1. The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) depolarizes dorsal root ganglion (DRG) neurons. We have applied microfluorimetric and whole-cell patch clamp techniques to investigate the mechanisms underlying this response in freshly dissociated mouse DRG cells. 2. Application of CHEB (2-200 microM) raised cytosolic calcium concentration ([Ca2+]i) rapidly and reversibly in 55% of eighty-three neurons tested. This population did not correlate with other classifications of sensory neurons based on either cell size or the expression of membrane currents. 3. The response was dependent on external calcium and was reduced by 81 +/- 22% by Ruthenium Red. A rise in [Ca2+]i was still seen with the membrane potential clamped at -70 mV, excluding membrane depolarization and activation of voltage-dependent Ca2+ channels as the principal mechanism for the response. 4. The rise in [Ca2+]i was associated with an increase in membrane conductance and a current, ICHEB, which was inward at -70 mV. Both the rise in [Ca2+]i and the current showed 'run-down' under whole-cell recording conditions. When K+ conductances were blocked, the reversal potential of ICHEB was close to 0 mV. This was independent of the Cl- reversal potential, suggesting that ICHEB is carried as a non-specific cation current. 5. In contrast to the change in [Ca2+]i, ICHEB was not dependent on external Ca2+ and the current was still seen when [Ca2+]i as strongly buffered by the pipette filling solution. These data suggest that CHEB opens a non-selective cation channel permeant to Ca2+, raising [Ca2+]i and further depolarizing the cell membrane potential. The exact nature of this conductance remains unknown. These actions could readily account for the convulsant actions of the drug, depolarizing neurons and increasing transmitter release. 6. It was also noted that CHEB increases autofluorescence derived from mitochondrial NAD(P)H. Further examination of this phenomenon using the dye rhodamine 123 to follow changes in mitochondrial potential (psi m) suggested that CHEB is a potent inhibitor of mitochondrial respiration, probably acting at complex I. These effects appeared to be quite distinct from the action of CHEB at the level of the plasma membrane.
Pearce
R J
RJ
Department of Physiology, University College London, UK.
Duchen
M R
MR
D000818:Animals; D001463:Barbiturates / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003292:Convulsants / Q000494:pharmacology*; D004594:Electrophysiology; D005456:Fluorescent Dyes; D005727:Ganglia, Spinal / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology; D009249:NADP / Q000187:drug effects; D009475:Neurons, Afferent / Q000187:drug effects*; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D012235:Rhodamines; D012430:Ruthenium Red / Q000494:pharmacology; D012680:Sensitivity and Specificity
D001463:Barbiturates; D003292:Convulsants; D005456:Fluorescent Dyes; D012235:Rhodamines; D012430:Ruthenium Red; C024321:5-(2-cyclohexylidene-ethyl)-5-ethylbarbiturate; D009249:NADP; D002118:Calcium