Kenji Kuba

Cyclic ADP-ribose modulates Ca2+ release channels for activation by physiological Ca2+ entry in bullfrog sympathetic neurons

Although Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors has been found to occur in intact neurons, little is known about the physiological processes that regulate it. We studied the effects of cyclic ADP-ribose (cADPR) on CICR in cultured bullfrog sympathetic neurons by fura-2 fluorescence recording and patch-clamp techniques. cADPR applied through a patch pipette augmented action potential- or depolarizing pulse-induced rises in intracellular Ca2+ without a change in Ca2+ entry initiating the responses, but not in the presence of ryanodine. Likewise, cADPR enhanced a single or oscillatory rise(s) in intracellular Ca2+ induced by caffeine. These results strongly suggest that cADPR can be an endogenous modulator of ryanodine receptors in neurons.

Long-term potentiations in vertebrate synapses: a variety of cascades with common subprocesses

Article de synthese sur la potentialisation a long terme de la transmission synaptique dans des synapses des systemes nerveux central et peripherique chez les vertebres

Characteristics of Ca2+ release induced by Ca2+ influx in cultured bullfrog sympathetic neurones.

1. A rise in intracellular Ca2+ ([Ca2+]i) and a Ca2+ current (ICa) induced by a depolarizing pulse were simultaneously recorded by fura‐2 or indo‐1 fluorescence and whole‐cell patch clamp techniques in cultured bullfrog sympathetic ganglion cells. 2. [Ca2+]i (calculated from the ratio of fura‐2 fluorescences excited at 380 and 340 nm and recorded with a photomultiplier at > 492 nm) rose regeneratively (in most cells) during a command pulse (from ‐60 to 0 mV, 100 ms), continued to rise thereafter, peaked at 666 ms (on average) and decayed slowly with a half‐decay time of 22.8 s. 3. Scanning a single horizontal line across the cytoplasm with an ultraviolet argon ion laser (351 nm) and recording indo‐1 fluorescences at two wavelengths (peaked at 410 and 475 nm) with a confocal microscope demonstrated that [Ca2+]i beneath the cell membrane rose much faster than that in the deeper cytoplasm. The time course of the spatial integral of [Ca2+]i, however, corresponded well with that recorded with fura‐2 fluorescence using a photomultiplier. 4. [Ca2+]i measured by fura‐2 fluorescence ratio using a photomultiplier did not increase during a strong depolarizing pulse (‐60 to +80 mV), but sometimes rose after the pulse. A depolarization‐induced rise in [Ca2+]i ([Ca2+]i transient) was blocked in a Ca(2+)‐free, EGTA solution, reduced by lowering the extracellular Ca2+ concentration ([Ca2+]o) to 0.45 or 0.9 mM and enhanced by raising [Ca2+]o to 7.2 or 14.4 nM. 5. The extracellular Ca2+ dependence was non‐linear when long depolarizing pulses (up to 500 ms) were applied; the amplitude of [Ca2+]i transient/Ca2+ entry (unit [Ca2+]i transient) increased with an increase in Ca2+ entry. 6. Increasing the duration of depolarization (‐50 or ‐60 to 0 mV) from 20 to 500 ms enhanced asymptotically the integral of ICa (due to inactivation), and progressively the magnitude of [Ca2+]i transients, leading to the apparent non‐linear dependence of unit [Ca2+]i transient on Ca2+ entry as well as on the duration of membrane depolarization. The peak time of [Ca2+]i transient was unchanged for pulse durations up to 300 ms, but prolonged with an increase in pulse duration to 500 ms. 7. Inhibitors of Ca2+ release from intracellular Ca2+ reservoirs, dantrolene (10 microM) and ryanodine (50 microM), blocked the [Ca2+]i transient to 56 and 30%, respectively, of the control. 8. The higher the basal [Ca2+]i level, the greater was the magnitude of the [Ca2+]i transients.(ABSTRACT TRUNCATED AT 400 WORDS)

Simulation of intracellular Ca2+ oscillation in a sympathetic neurone.

Abstract Three different theoretical models were considered for the mechanism of the oscillation of the intracellular free Ca 2+ ([Ca 2+ ] i ) linked to the K + conductance of the plasma membrane ( G K ) observed in bullfrog sympathetic ganglion cells. The models assumed a Ca 2+ -induced Ca 2+ release mechanism, an active Ca 2+ uptake mechanism at a Ca 2+ reservoir site in the ganglion cell, and a Michaelis—Menten type relationship between [Ca 2+ ] i and G K . Including both active and passive Ca 2+ transport mechanisms at the plasma membrane, either a one-compartment model or a two-compartment model for the intracellular Ca 2+ store reconstructed successfully the [Ca 2+ ] i oscillation and rhythmic membrane hyperpolarizations observed in the ganglion cell, and simulated most of their characteristics. On the other hand, a two-compartment model disregarding of Ca 2+ transport at the plasma membrane failed to reproduce the oscillations of [Ca 2+ ] i and membrane potential.

Long-term potentiation of transmitter release induced by repetitive presynaptic activities in bull-frog sympathetic ganglia.

Long‐lasting potentiation of transmitter release induced by repetitive presynaptic activities in bull‐frog sympathetic ganglia was studied by recording intracellularly fast excitatory post‐synaptic potentials (fast e.p.s.p.s.). Following a brief period of post‐tetanic potentiation or depression (less than 10 min), the amplitude of the fast e.p.s.p. was potentiated for a period between several tens of minutes and more than 2 h in response to tetanic stimulation of the preganglionic nerve in twenty‐one out of twenty‐eight cells. Quantal analysis revealed that this long‐term potentiation of the fast e.p.s.p. (l.t.p.) was accompanied by an increase in quantal content m (in nine out of twenty‐one cells), quantal size (four cells) or both (eight cells). The increased quantal content (presynaptic l.t.p.) declined exponentially (ten cells) or decayed gradually to a certain enhanced level which lasted several hours. In contrast, the increased quantal size grew with a relatively long latency (10‐25 min) and remained relatively constant for at least 2 h. The magnitude of presynaptic l.t.p. increased with increased duration of the presynaptic tetanus (33 Hz) from 2 to 5 s. No l.t.p. was elicited by a 1‐s tetanus, whereas the time course appears to be independent of the tetanus duration and the magnitude of l.t.p. There was a positive correlation between the magnitude of presynaptic l.t.p. and the pre‐tetanic quantal content up to m = 3, but the former deviated from linear regression when the value of the latter exceeded 3. No l.t.p. occurred when quantal content was less than 0.5. A tetanus (33 Hz, 10 s) applied in Ca2+‐free solution elicited no presynaptic l.t.p., while the same tetanus in normal Ringer solution produced a large presynaptic l.t.p. Presynaptic l.t.p. was enhanced in magnitude at low temperature (8‐10 degrees C). These results demonstrate the existence of a use‐dependent, long‐term potentiation of transmitter release in bull‐frog sympathetic ganglia. Several possible mechanisms are discussed in terms of Ca2+‐buffering mechanisms of the presynaptic nerve terminals.

Calcium localization in the sympathetic ganglion of the bullfrog and effects of caffeine.

The localization of Ca2+ in the bullfrog sympathetic ganglion was studied using electron microscopy with Oschman and Walls technique. When the ganglion was incubated and processed in an extremely high Ca2+ solution (20 mM) for electron microscopy, electron-dense deposits (EDD) were found at or in the plasma membranes, subsurface cisterns and mitochondria of the postganglionic neurons. These EDD were proved to contain calcium by X-ray microprobe analysis. On the other hand, they were not significant in the preganglionic terminals except those in the synaptic vesicles. Addition of caffeine (10 mM) to the incubation media and fixatives caused a drastic decrease in number of EDD of the subsynaptic membranes and the subsurface cisterns. Caffeine also reduced, but less markedly, the size and number of EDD in mitochondria. Caffeine (10 mM) prolonged the afterhyperpolarization of an action potential, reduced the amplitude of the ACh (nicotinic) potential and induced slow rhythmic hyperpolarizations in a 20 mM Ca2+ solution. These effects of caffeine which were presumably the result of an increase in the intracellular free Ca2+ were discussed in relation to the morphological data.

(+)-Tubocurarine blocks the Ca2+-dependent K+-channel of the bullfrog sympathetic ganglion cell.

(+)-Tubocurarine [+)-Tc: 10-100 microM) reduced the duration of the afterhyperpolarization, which was induced by the activation of Ca2+-dependent K+-conductance (GK,Ca) following an action potential in the bullfrog sympathetic ganglion cell, but did not affect the maximum rates of rise and fall of Na+- and Ca2+-dependent action potentials. The amplitudes of slow rhythmic membrane hyperpolarizations produced by rhythmic rises in the GK,Ca were also decreased by (+)-Tc without a change in their intervals. Thus, (+)-Tc appears to block the Ca2+-dependent K+-channel of the bullfrog sympathetic ganglion cell.

Long-term potentiation induced by a sustained rise in the intraterminal Ca2+ in bull-frog sympathetic ganglia.

1. The mechanism of a long‐term potentiation of transmitter release (pre‐LTP) induced by a tetanic stimulation (33 Hz for 1‐30 s) applied to the preganglionic nerve was examined by intracellularly recording the fast excitatory postsynaptic potentials (fast EPSPs) in bull‐frog sympathetic ganglia. 2. Short‐term facilitation induced by paired pulses was decreased during the course of pre‐LTP; the extent of reduction paralleled with the magnitude of pre‐LTP. 3. The frequency of miniature EPSPs increased after tetanic stimulation that produced the pre‐LTP. 4. The Ca2+ ionophore, A23187, increased both the amplitude and quantal content of fast EPSPs and frequency of miniature EPSPs while it decreased short‐term facilitation. 5. A Ca2+ chelating agent, Quin‐2, loaded as acetoxymethyl ester, reduced the amplitude and quantal content of fast EPSPs and short‐term facilitation, and blocked the generation of pre‐LTP. 6. Activators of protein kinase C, phorbol 12,13‐dibutyrate and 1‐oleoyl‐2‐acetyl‐rac‐glycerol, and its inhibitors, H‐7 and staurosporine, did not block the generation of pre‐LTP, while the activators enhanced transmitter release. 7. Inhibitors of calmodulin, trifluoperazine and W‐7, blocked the generation of pre‐LTP, whereas the amplitude and quantal content of fast EPSPs were not influenced. 8. These results suggest that the pre‐LTP results from a sustained rise in the basal level of intraterminal Ca2+ and an activation of the Ca(2+)‐calmodulin‐dependent process in the preganglionic nerve terminals.

Basal Ca2+ and the oscillation of Ca2+ in caffeine-treated bullfrog sympathetic neurones.

1. Effects of caffeine on the intracellular free Ca2+ concentration ([Ca2+]i) in single bullfrog sympathetic neurones in excised tissue were studied by recording Fura‐2 fluorescence excited at 340, 361 or 380 nm and taking their ratios (R340/380 or R361/380). 2. Caffeine (3‐10 mM) produced oscillation of [Ca2+]i and an ‘apparent’ decrease in the basal level of [Ca2+]i during a period between phasic rises. The mechanism of the latter effect was analysed in relation to the mechanism of the former. 3. Caffeine (3‐10 mM) increased Fura‐2 fluorescence in a range of excitation wavelength from 330 to 390 nm. The ratios of fluorescences, R340/380 and R361/380, however, were not significantly affected by caffeine. These results suggest that the ‘apparent’ reduction in the basal [Ca2+]i seen as a decrease in R340/380 or R361/380 results from a true decrease in [Ca2+]i. 4. Caffeine‐induced decrease in [Ca2+]i persisted for every period between phasic rises of [Ca2+]i during [Ca2+]i oscillation, and after the blockade of [Ca2+]i oscillation by ryanodine. The decrease in the latter condition lasted for more than 20 min. 5. The decrease in the basal [Ca2+]i depended on the external Ca2+ concentration and was not mimicked by the action of cyclic nucleotides. 6. Possible mechanisms underlying the decrease in the basal [Ca2+]i produced by caffeine (effects on Ca2+ transport at the cell or Ca(2+)‐storing organelle membrane) and their significance in relation to the [Ca2+]i oscillation were discussed.

Intracellular calcium dynamics in response to action potentials in bullfrog sympathetic ganglion cells

1. Dynamic changes in the intracellular free Ca2+ concentration ([Ca2+]i) following electrical membrane activity, were recorded from the neurone soma of the excised bullfrog sympathetic ganglion, using Fura‐2 fluorescence and compared with the accompanying Ca(2+)‐dependent electrical membrane responses. 2. The resting [Ca2+]i was about 100 nM, a value little changed by penetration with an intracellular electrode. 3. A net rise in fluorescence at a wavelength of 340 nm (Ca2+ transient) induced by a single action potential in Ringer solution rose almost in parallel with the initial decay phase of a slow Ca(2+)‐dependent after‐hyperpolarization; decayed in parallel with the late phase; and increased in amplitude and duration in the presence of tetraethylammonium (20 mM). 4. A Ca2+ transient induced by repetitive action potentials was increased asymptotically in amplitude and progressively in duration by increasing the number of spikes, and was slower in time course than the associated Ca(2+)‐dependent K+ current. 5. Scanning a single horizontal line across the cytoplasm with an ultraviolet argon ion laser (351 nm) and recording Indo‐1 fluorescence with a confocal microscope demonstrated an inward spread of a rise in [Ca2+]i following a tetanus. 6. Both single spike‐ and tetanus‐induced Ca2+ transients were abolished in a Ca(2+)‐free solution, while single or repetitive transient rises in [Ca2+]i induced by caffeine (5‐10 mM) were generated under the same conditions. 7. Ryanodine (10‐50 microM) did not affect tetanus‐induced Ca2+ transients, whereas it blocked completely the caffeine‐induced oscillation of [Ca2+]i. 8. Ca2+ transients induced by a tetanus in Ringer solution were independent of the interval from the preceding tetanus. The amplitude of Ca2+ transients induced by a tetanus in the presence of caffeine (5 mM) was equal to, or greater than, that generated in Ringer solution in any of the phases of [Ca2+]i oscillation. 9. It is suggested that under the physiological conditions here, the induction of action potentials does not cause the release of Ca2+ in the cells of the freshly excised bullfrog sympathetic ganglion, and that Ca(2+)‐buffering systems contribute not only to lowering a transient rise in [Ca2+]i but also to sustaining an increased [Ca2+]i after a large Ca2+ load into the cell.