SUMMARY1. The mechanism of spontaneous and rhythmic hyperpolarizations which occur in bullfrog sympathetic ganglion cells under the effect of caffeine (2-10 mM) were further analysed.2. Intracellular injection ofEGTA blocked generation of caffeine hyperpolarizations (C-hyperpolarizations): this confirmed the previous conclusion (Kuba & Nishi, 1976) that these hyperpolarizations are caused by rhythmic increases in the K+ conductance (GK) of the membrane as a result of rises in free intracellular Ca2+.3. The amplitude and duration of a C-hyperpolarization induced by an action potential was a function of the time since the previous one; the longer the interval, greater the area.4. The relationship between the product of the amplitude and duration of a Chyperpolarization and the preceding interval depended on external Ca2+; when this was low the relationship shifted, so as to indicate an involvement of a Ca2+ accumulating process in the generation mechanism of C-hyperpolarizations.5. A rapid lowering of temperature triggered the generation of a C-hyperpolarization before appearance of a rhythmic one. There seemed to be no threshold temperature for the effect of such a cold shock; cooling from any temperature within a certain range (10-25 0C) by more than a few degrees was effective.6. The rapid cooling effect was observed even in a Ca2+-free Mg2+ solution. 7. Dantrolene Na increased the interval between rhythmic C-hyperpolarizations or blocked them, but affected less those triggered by an action potential or cold shock.8. Intracellular injection of Ca2+ triggered the generation of a C-hyperpolarization before the appearance of a rhythmic one.9. The latency of the generation of an action potential-evoked C-hyperpolarization was dependent on the preceding interval; the shorter the interval, the longer the latency. There was a refractory period for induction of an action potential-induced C-hyperpolarization.10. The interval between rhythmic C-hyperpolarizations was increased by a small or moderate membrane hyperpolarization (5-20 mV) and decreased by a larger * Present address.

Superfusion of the isolated sympathetic ganglion of the bullfrog with a caffeine-containing (1-6 mM) solution caused in many cells an initial slow hyperpolarization which was followed by a subliminal depolarization interruped by rhythmic hyperpolarizations. A hyperpolarization, similar to one of the rhythmic hyperpolarizations, could be triggered by an action potential in the presence of caffeine. The action potential itself was not markedly affected by caffeine except for its afterhyperpolarization which was prolonged. All these caffeine-induced hyperpolarizations were associated with a marked reduction of the membrane resistance, their amplitude was increased in a K+-free solution and decreased in a high-K+ solution, and their polarity was reversed at the same level at which the afterhyperpolarization was also inverted. This reversal level was not altered by omission of Na+ or C1- from the external medium. These hyperpolarizations were reversibly abolished by depletion of external Ca2+ or replacement of external Ca2+ by Mg2+. Excess of external Ca2+ caused a shortening of the interval between rhythmic hyperpolarizations. Furthermore, iontophoretic injection of EDTA into the cytoplasm markedly depressed the initial caffeine hyperpolarizatin and abolished both the rhythmic and evoked caffeine hyperpolarizations. The caffeine-induced depolarization was not affected by omission of external Cl-. It was decreased in a Na+-free medium, but completely eliminated by omission of both Na+ and Ca2+ from the external medium. Tetrodotoxin did not impair the production of the initial and the rhythmic hyperpolarizations. A strong depolarizing pulse could evoke a typical hyperpolarizing response in the presence of this compound. Dibutyryl cyclic AMP, d-tubocurarine, atropine, and phenoxybenzamine were without effect on the caffeine-induced hyperpolarizations and depolarization. It was concluded that each caffeine-induced hyperpolarization is the result of an increased K+ permeability, which is probably caused by a rise in the internal Ca2+ concentration. It was also concluded that the caffeine-induced depolarization is due to an increased membrane permeability to Ca2+ and Na+.

Ca2+-induced Ca2+ release (CICR) enhances a variety of cellular Ca2+ signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca2+ entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca2+ entry inactivates the CICR mechanism and the absence of Ca2+ entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10–20 Hz, 2–10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca2+ in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca2+ during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca2+ channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca2+-dependent priming.

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20–50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05–0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN−. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.