ryanodine and 2-tert-butylhydroquinone

The relation between Ca2+-induced Ca2+ release (CICR) elicited by action potentials (APs) and a Ca2+-dependent slow post-spike hyperpolarization (AHPslow) in acutely dissociated adult rabbit nodose neurons was studied using microfluorimetric calcium measurements in conjunction with standard intracellular current- and voltage-clamp recording techniques. The magnitude of the AP-induced transient increase in [Ca2+]i (DeltaCat) was used to monitor CICR. There was a close correlation between the magnitude of the DeltaCat and the AHPslow current over the range of 1-16 APs (r = 0.985). Functional CICR blockers, ryanodine (10 muM), thapsigargin (100 nM), 2,5-di(t-butyl)hydroquinone (10 muM) or cyclopiazonic acid (10 muM), selectively reduced the peak amplitude of the AHPslow >/=91%. In five neurons, simultaneous recordings of the DeltaCat and the AHPslow revealed that both responses were blocked in parallel. These findings indicate that CICR is necessary for the generation of the AHPslow in rabbit nodose neurons. The DeltaCat rises and decays significantly faster than the AHPslow. This temporal disparity suggests that activation of the AHPslow by Ca2+ may require additional signal transduction steps.

Topics: Action Potentials; Animals; Calcium; Calcium Channel Blockers; Chelating Agents; Egtazic Acid; Enzyme Inhibitors; Female; Hydroquinones; Indoles; Male; Membrane Potentials; Neurons, Afferent; Nodose Ganglion; Patch-Clamp Techniques; Rabbits; Ryanodine; Thapsigargin; Vagus Nerve

We investigated the effect of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) of leech P neurones by using the fluorescent indicator Fura-2. Caffeine induced a [Ca2+]i increase that was strongly reduced, but not abolished, in Ca(2+)-free solution. The effect of caffeine on [Ca2+]i was dose-dependent: while 5 mM caffeine evoked a persistent [Ca2+]i increase that could be elicited repetitively, 10 mM caffeine or more induced a transient [Ca2+]i increase that was strongly reduced upon subsequent applications at the same concentration. Surprisingly, the cells remained fully responsive to a moderately increased caffeine concentration. The caffeine-induced [Ca2+]i increase was not blocked by millimolar concentrations of La3+, Mg2+, Cd2+, Zn2+, Co2+, Ni2+, or Mn2+. While La3+ and Mg2+ had no effect on the caffeine response, the other cations caused irreversible changes in the Fura-2 fluorescence. The inhibitors of intracellular Ca2+ pumps-thapsigargin, cyclopiazonic acid (CPA), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ)--had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, but they reduced it in Ca(2+)-free solution. Ryanodine had no effect on the caffeine-induced [Ca2+]i increase at normal extracellular Ca2+ concentration, and also in Ca(2+)-free solution it seemed to be largely ineffective. Caffeine evoked complete fluctuations of the membrane potential. The effect in Ca2+ free and in Na(+)-free solution suggests that the depolarizing response components were mainly due to Na+ influx, while Ca2+ reduced the Na+ influx and/or activated mechanisms which re- or hyperpolarize the cells. It is concluded that leech P neurones possess caffeine-sensitive intracellular Ca2+ stores, as well as caffeine-sensitive ion channels, in the plasma membrane that are activated by a voltage-independent mechanism. The plasma membrane channels are permeable to various divalent cations including Ca2+, and possibly also to Na+.

Topics: Animals; Caffeine; Calcium; Calcium-Transporting ATPases; Cations; Dinucleoside Phosphates; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroquinones; Intracellular Fluid; Leeches; Membrane Potentials; Neurons; Ryanodine; Sodium; Solutions; Thapsigargin