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. 1998 Nov;112(5):593-609.
doi: 10.1085/jgp.112.5.593.

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals

K Narita  1 , T Akita, M Osanai, T Shirasaki, H Kijima, K Kuba
Affiliations

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals

K Narita et al. J Gen Physiol. 1998 Nov.

Abstract

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.

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Figures

Figure 1
Figure 1
A scheme to illustrate Ca2+ dynamics involving CICR in frog motor nerve terminals. Ca2+ enters into the motor nerve terminals through voltage-dependent Ca2+ channels (JCa*), and is extruded out of the terminal by Ca2+ pumps (p) at the cell membrane. Cytoplasmic Ca2+ (x) activates Ca2+ release (γ) from Ca2+ stores and is taken into Ca2+ stores (z) by Ca2+ pumps (α) to resume Ca2+ concentration in Ca2+ stores.
Figure 6
Figure 6
Effects of thapsigargin and CN− on Ca2+-humps and effects of thapsigargin on MEPP-hump during continuous tetanic stimulation in a low Ca2+, high Mg2+ solution. (A) Effects of thapsigargin on a Ca2+-hump. 50 Hz tetanus was given to the nerve in the absence or presence of thapsigargin (1 μM). After recording the control, the preparation was left without stimuli for 30 min in a low Ca2+, high Mg2+ solution, and then superfused with a drug-containing solution for a period of 30 min before the beginning of stimulation. (B) Effects of thapsigargin (4 μM) on a MEPP-hump and post-pause rises in MEPP frequency. The time interval between the measurements of MEPPs in the absence and presence of the drug and the duration of drug application are the same as those in A. (C) Effects of CN− (2 mM). 50 Hz tetanus was given to the nerve in the absence or presence of CN−. After taking the control, the preparation was left for 30 min without stimuli in a low Ca2+, high Mg2+ solution and subsequently superfused with a CN−-containing solution for 9 min before the beginning of stimulation.
Figure 4
Figure 4
Quenching effects of Mn2+ on Indo-1 or Fura-2 fluorescence loaded in the terminals. (A) Quenching effects of Mn2+ on Indo-1 fluorescence. Mn2+ (1.8 mM) was applied after the generation of a Ca2+-hump. Note decreases in fluorescence at both wavelength ranges (F 412 and F 475). (The lack of an apparent increase in F 412 during a rise in F 412/F 475 can be explained by the shift of excitation spectrum of Indo-1 by an increase in [Ca2+]i to a shorter wavelength range; compare Kuba et al., 1994). (B) The lack of quenching effects of Mn2+ on Fura-2 fluorescence in the absence of nerve stimuli and the quenching effect during stimuli. Fluorescence was measured at a wavelength range peaked at 380 nm (F 380). Fluorescence intensity is shown in an arbitrary unit. (C) Quenching effects of Mn2+ on Fura-2 fluorescence during tetanic stimuli after the generation of a Ca2+-hump (seen as a transient decrease in fluorescence). Fluorescence was measured at F 380.
Figure 3
Figure 3
Tetanus-induced increases in [Ca2+]i (Ca2+-hump) and effects of TMB-8. Throughout the data points in graphs, tetanus (50 Hz) was continuously applied. All the recordings were made in a low Ca2+ (0.2 mM), high Mg2+ (10 mM) solution. Effects of TMB-8 (10 μM) were seen 30 min after treatment with the drug that began after a period of no stimuli for 30 min. Data were obtained from the terminals loaded with Indo-1. Ratios (F 412/F 475) of fluorescences peaking at 412 (F 412) and 475 (F 475) nm were converted to [Ca2+]i values. Experimental protocol is the same as that in Fig. 2 D.
Figure 10
Figure 10
The dependence of post-pause rises in [Ca2+]i and MEPP frequency on pause duration. (A) Ca2+-hump and increases in [Ca2+]i after brief pause in tetanus (50 Hz). The data points were plotted from the experiments shown in Figs. 5 and 9. (B) Relationships between the magnitude of post-pause rise in MEPP frequency or [Ca2+]i and the duration of pause. (○) MEPP frequency; (×) [Ca2+]i. Each data point is the average of those obtained from different terminals, whose number is shown above or below each point. The magnitudes of post-pause rises in MEPP frequency and [Ca2+]i for each duration of pause were normalized to those at 10 s pause, and then their average and SEM were taken. MEPPs were recorded from frog sartorius muscles, while changes in [Ca2+]i were from motor nerve terminals of frog cutaneous pectoris muscles.
Figure 2
Figure 2
Tetanus-induced transient increases (MEPP-hump) and late slow rises in MEPP frequency in a low Ca2+, high Mg2+ solution. Tetanus at 50 Hz (except for one of the data in C) was continuously applied throughout each experiment. (A) MEPPs recorded at 1 (at the beginning of MEPP-hump shown in B), 2 (at the peak of MEPP-hump), 3 (at the end of MEPP-hump), and 4 (during a late slow rise) min after the beginning of a continuous tetanus (50 Hz) in a low Ca2+ (0.2 mM), high Mg2+ (10 mM) solution. (B) Time courses of MEPP-hump and the late slow rise in MEPP frequency produced by a continuous tetanus (50 Hz) and effects of lowering [Ca2+]o. [Ca2+]o was reduced from 0.2 mM (○; control) to 0.1 (Δ) or 0.05 (•) mM. (C) Effects of decreasing tetanus frequency on MEPP-humps. (D) Effects of TMB-8 (8 μM) on MEPP-humps and late slow rises in MEPP frequency. After recording the control response, the preparation was left for 30 min without nerve stimulation. Then, a solution containing TMB-8 was superfused to the preparation for 30 min and a tetanus (50 Hz) was given. MEPPs were again recorded 1 h after the removal of TMB-8.
Figure 5
Figure 5
Tetanus-induced increases in the fluorescence of Oregon Green BAPTA-1 in the motor nerve terminals. 50 Hz tetanus was given to the nerve throughout the experiments. All the images are shown by the ratio to that before tetanus. The image in the top is the control, which is the ratio of an image to another before the beginning of stimulation. The second to sixth images are those taken at 1, 2, 3, 4, and 5 min after the beginning of tetanus. The ratio values averaged over each image are used in the initial part of the graph in Fig. 10 A.
Figure 9
Figure 9
Increases in the fluorescence of Oregon Green BAPTA-1 in the motor nerve terminals after pauses of different duration during a 50-Hz tetanus. All the images are shown in ratios of fluorescence to that before the beginning of tetanus. The first, third, and fifth images from the top were taken at the end of each pause, while the second, fourth, and sixth images were taken at the peak of increases after pauses of 10, 30, and 60 s. The images were taken immediately after recording the images shown in Fig. 5 from the same cell. The ratio values averaged over each image fulfilled the data points in the later part of the graph in Fig. 10 A.
Figure 7
Figure 7
The time course of CICR activation: effects of brief pauses in tetanus on MEPP frequency during and after a MEPP-hump. (A) Changes in MEPP frequency caused by a pause in tetanus (50 Hz) applied during and after a MEPP-hump. Pauses of 5 or 9 s were applied during a period indicated by a horizontal bar. Inset is the expansions of the rising phases of MEPP frequency after a short pause (5 s) given at 1 (a), 1.5 (b), 2 (c), 2.5 (d), and 3 (e) min after the beginning of a tetanus (50 Hz). Abscissa is the time after resumption of tetanus, while ordinate represents a net increase in MEPP frequency. (B) Changes in the initial rate of rise of MEPP frequency after a pause during the course of a series of tetani (50 Hz). The rate of rise of MEPP frequency was measured from the slope of the regression line fitted to the initial three data points. Note that the time for the peak of the rate of rise roughly corresponds to the peak of MEPP-hump.
Figure 8
Figure 8
The dependence of post-pause increases in MEPP frequency on pause duration and effects of TMB-8. (A) Rises in MEPP frequency after brief pauses of different duration in tetanus (50 Hz) applied after a MEPP-hump. (B) Effects of TMB-8 (8 μM) on MEPP-hump and post-pause rises in MEPP frequency. Experimental protocol is the same as that in Fig. 2 D.
Figure 11
Figure 11
Ca2+ dependence of the priming effect of tetanus. (A) The lack of the facilitatory effect of a tetanus (the first of horizontal bars) in a Ca2+ free (1 mM EGTA), Mg2+(1 mM) solution on the induction of MEPP-hump. After tetanus (50 Hz) was given for ∼7 min in a Ca2+ free (1 mM EGTA), Mg2+ (1 mM) solution, the preparation was superfused with a low Ca2+ (0.2 mM), high Mg2+ (10 mM) solution for 5 min. This was followed by several tetani (50 Hz; horizontal bars) of 7 min at an interval of 5 or 79 min. (B) Transient rises in MEPP frequency after resumption of tetanus (a, b, c, d) replotted in the expanded time scale. Abscissa represents the time after resumption of tetanus.
Figure 12
Figure 12
Evidence for depriming process: effects of long pauses in tetanus on MEPP frequency after a MEPP-hump. (A) Rises in MEPP frequency produced by the resumption of tetanus (50 Hz) after pauses of different duration. Time scale in abscissa represents the time after resumption of tetanus. (B) The relationship between the initial rate of post-pause rise in MEPP frequency and the duration of pause. The initial rate of rise was defined as the rate of rise from the MEPP frequency at the beginning of a resumed tetanus to 10% of the peak. (○ and •) The data points obtained from two different end-plates. The data points shown by open circles were normalized to that shown by the closed circle at 1 min, and then rescaled to values in MEPPs/s2. (inset) The expansion of the initial part of the graph on a faster time scale.
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