INTRODUCTION

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Ca²⁺ in presynaptic terminals activates the exocytosis of neurotransmitters, its plasticity, recycling of the synaptic vesicles and other functions (see Katz 1969; Schweizer et al. 1995; Zucker 1996). For these Ca²⁺ actions, the basal level of the intracellular free Ca²⁺ ([Ca²⁺]_i) must be maintained far below that of external Ca²⁺, while rises in [Ca²⁺]_i produced by physiological stimuli must be cleared out for the next stimulus. The low basal level of [Ca²⁺]_i is maintained by active Ca²⁺ extrusion via the activity of Ca²⁺ pumps and Na/Ca exchanger, which is in equilibrium with passive Ca²⁺ entry at the cell membrane (see Carafoli 1987). Rises in [Ca²⁺]_i by external stimuli or spontaneous cell activity are quickly buffered by binding to Ca²⁺-binding proteins (see Kasai 1993) and cleared out by extrusion at the cell membrane and uptake via Ca²⁺ pumps into the endoplasmic reticulum and/or other organelles and via Ca²⁺ uniporter into mitochondria (see Miller 1991; Pozzan et al. 1994).

It is not precisely known, however, how these Ca²⁺-buffering mechanisms operate in presynaptic terminals. Na/Ca exchange was reported to play a predominant role in the clearance of impulse-induced Ca²⁺ entry in presynaptic terminals of hippocampal neurons (Reuter and Poerzig 1995), while mitochondrial Ca²⁺ uptake was emphasized for Ca²⁺ clearance in lizard motor nerve terminals (David et al. 1998). We have suggested that Ca²⁺ clearance in frog motor nerve terminals occurs in a cytosolic Ca²⁺-dependent manner with the fastest component as fast as free diffusion (Suzuki et al. 2000). We report here that the not only mitochondrial Ca²⁺ uptake but also plasmalemmal Ca²⁺ extrusion clear out tetanus-induced rises in [Ca²⁺]_i in a [Ca²⁺]_i-dependent manner in frog motor nerve terminals, while thapsigargin-sensitive Ca²⁺ uptake boosts the clearance of a heavy Ca²⁺ load and that Ca²⁺ pump and Na/Ca exchanger at the cell membrane are complementary in operation to each other with the slight predominance of the latter.

	METHODS

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Preparations and Ca²⁺-imaging techniques are essentially similar to those of the previous study (Suzuki et al. 2000). Briefly, frogs (Rana nigromaculata) were decapitated, and cutaneous pectoris muscles were isolated with the nerve attached. The composition of normal Ringer solution was (in mM) 113 NaCl, 2.0 KCl, 1.76 CaCl₂, 2.3 NaHCO₃, 5 glucose, and 5 HEPES-Na (pH 7.4). To avoid muscle contraction, 5-10 µM D-tubocurarine (Sigma) was added to perfusate throughout experiments. All the experiments were performed at 22 ± 1°C (mean ± SE). The motor nerve terminals were loaded with Ca²⁺ indicators [Indo-1/K, 19 mM, or Oregon green 488 BAPTA-1/K (OGB-1), 30 mM] from the cut end of the nerve bundle. Fluorescence of Indo-1 loaded in the nerve terminals was measured by line-scanning at 250 Hz with a UV confocal laser-scanning microscope (UV-CLSM; BioRad MRC-500 attached to Nikon TMD-300 with an objective, Nikon, Fluor ×40 water, NA 1.15; Argon laser, 351 nm) (Kuba et al. 1994) or by horizontal scanning at 60 Hz with a fast-scanning UV-CLSM (Noran Odessey System; Argon laser, 363 nm) with the same objective. Indo-1 fluorescence was used to observe effects of a mitochondrial uncoupler on tetanus-induced rises in [Ca²⁺]_i and changes in the basal [Ca²⁺]_i. Indo-1 fluorescence was separated into two wavelength ranges peaked at 406 and 475 nm, their ratio (F₄₀₆/F₄₇₅) was taken and converted to [Ca²⁺]_i values as described previously (Suzuki et al. 2000). In other experiments, the fluorescence of OGB-1 was recorded with a cooled CCD camera (ARGUS/HiSca, Hamamatsu Photonics, Hamamatsu, Japan) with an image intensifier (Stardancer 2, Videoscope International, Sterling, VA; at 33 Hz) or an intensified CCD camera (Argus 50, Hamamatsu Photonics; at 30 Hz). The ratios of fluorescence changes during and after tetanic nerve stimulation to that before the tetanus were taken and converted to [Ca²⁺]_i values as described previously (Suzuki et al. 2000).

The decay time course of an increase in [Ca²⁺]_i produced by tetanic stimulation was fitted 5 s after the beginning of decay with a double-exponential function plus a constant value, which represents the slowest decay component with the time constants of several tens of second (Suzuki et al. 2000). The relationship between the rate of the decay of [Ca²⁺]_i and different levels of [Ca²⁺]_i was measured as follows. The digitized decay phases of tetanus-induced rises in [Ca²⁺]_i obtained from different experiments, which showed the [Ca²⁺]_i value of plateau between 1 and 2 µM, were averaged. The slopes of the decline of [Ca²⁺]_i at individual [Ca²⁺]_i values were then measured between two digitized points and plotted to each [Ca²⁺]_i values. The rate of [Ca²⁺]_i decay indicates the sum of Ca²⁺ effluxes out of the cytosol, which include both Ca²⁺ extrusion at the cell membrane and Ca²⁺ uptake into mitochondria and Ca²⁺ storing organelles. Thus the rate of [Ca²⁺]_i decay is simply defined as Ca²⁺ efflux out of the cytosol (J_Ca) (see Colegrove et al. 2000). The relationships between Ca²⁺ efflux and [Ca²⁺]_i were fitted by the equation

(1)

where J_max and K are the maximum Ca²⁺ efflux and the apparent dissociation constant, respectively (see Gunter and Gunter 1994). Equation 1 is based on the assumption that a Ca²⁺ transporter translocates Ca²⁺ across the membrane after the cooperative binding of two Ca²⁺ to the translocation sites. The subtraction of the component of Ca²⁺ efflux via mitochondrial Ca²⁺ uptake or Ca²⁺ extrusion was made by first fitting the control data points and those after the blockade of Ca²⁺ clearance by Eq. 1 and then taking the differences between the two asymptotic curves at each [Ca²⁺]_i value.

Data for each parameter of tetanus-induced Ca²⁺ transient in various conditions were expressed as means ± SE. Their statistical significance was examined by Student's t-test. Statistical significance for differences between the relationships of Ca²⁺ efflux to [Ca²⁺]_i in different conditions were examined as follows. Equation 1 was linearized by ignoring the first term of the denominator in the right side and taking the logarithm because all the K_d values (>7 µM) in fitting the data to the equation (Figs. 1D and 2, C and D) were greater than the range of [Ca²⁺]_i fitted (<1.2 µM). Thus

(2)

where Y_i is log (J_Ca) at each [Ca²⁺]_i, X_i = log ([Ca²⁺]_i), ₁ = log (A/K), and ₂ = 2, which is an assumed value. The null hypothesis was applied to pairs of the relationships of J_Ca to [Ca²⁺]_i obtained before and after the blockade of Ca²⁺ extrusion or mitochondrial Ca²⁺ uptake. For instance, it was assumed that the value for ₁ before the application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was not different from that after the application and vice versa. t-test was made as to the sample regression coefficient (^*₁) for ₁ by calculating the standard error of ^*₁ and then a t-value for ^*₁.

Indo-1/K and OGB-1/K were obtained from Molecular Probes (Eugene, OR). Thapsigargin, CCCP, and dinitrophenol (DNP) were from Sigma.

	RESULTS

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Characteristics of tetanus-induced rises in [Ca²+]_i

As in the previous study (Suzuki et al. 2000), a high-frequency tetanus (100 Hz, 100 or 200 stimuli) caused a rise in [Ca²⁺]_i in the nerve terminals (Ca²⁺ transient: measured by OGB-1 fluorescence), which rose quickly for the initial 6-20 stimuli, reached a plateau, and decayed in three phases after the end of tetanus (Figs. 1 and 2). The time constant and fraction of the initial, fast and second decay components of 100 stimuli-induced Ca²⁺ transients were 87 ms and 85.2% and 2.2 s and 11.0%, respectively (Fig. 3A). The slowest component had the time constant much longer than several tens of second and therefore handled as a constant value (3.8% of the peak) in analysis (Fig. 3A: see METHODS). The time constant and fraction of the initial and second decay components of 200 stimuli-induced Ca²⁺ transient (OGB-1 fluorescence) were 82 ms and 80.5 ± 1.1% (n = 20) and 1.14 s and 12.1 ± 1.3%, respectively, with the slowest component of 4.9 ± 0.5% (Fig. 3B for the time constants). In the previous study (Suzuki et al. 2000), the time constant of the initial decay component was found to decrease with the elevation of the plateau phase of tetanus-induced Ca²⁺ transients produced by tetani of different frequencies, indicating the [Ca²⁺]_i dependence of the initial component. The [Ca²⁺]_i dependence of Ca²⁺ clearance can be more clearly demonstrated by measuring the slope of the decay phase at different levels of [Ca²⁺]_i along the course of the decay and plotting them against [Ca²⁺]_i (see METHODS). The rate of the decay of the increased [Ca²⁺]_i reflects the magnitude of Ca²⁺ efflux out of the cytoplasmic space at each [Ca²⁺]_i value (see Colegrove et al. 2000). Ca²⁺ efflux out of the cytosol after the end of a 100-stimuli-induced Ca²⁺ transient clearly decreased with the reduction of [Ca²⁺]_i level (Figs. 1D and 2C). A similar [Ca²⁺]_i-dependence of Ca²⁺ efflux was seen after a 200-stimuli-induced Ca²⁺ transient (not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 1. Effects of blocking Ca2+ pumps and/or Na/Ca exchangers at the cell membrane on tetanus-induced Ca2+ transients. A: effects of raising external pH to 9.0. B: effects of replacement of most external Na+ with Li+. C: effects of combination of treatments with Li+ and high external pH. Ca2+ transients were induced by a tetanus of 100 stimuli at 100 Hz to the nerve and recorded by measuring the fluorescence of OGB-1. Thin traces are the control records, while thick traces are those after treatment with high pH (A), Li+ (B), or both (C). Insets: the falling phase of Ca2+ transients on semi-log coordinates. D: the [Ca2+]i dependence of the rate of the decay of tetanus-induced Ca2+ transient and effects of blocking Ca2+ pumps and Na/Ca exchangers. The slope of the decline of [Ca2+]i, Ca2+ efflux, along the decay time course of Ca2+ transient was plotted against the corresponding [Ca2+]i value. Open triangles, the control Ca2+ efflux; closed triangles, those after treatment with external Li+ at pH 9. Data points were the averages of the relationships obtained from 5 terminals and fitted by Eq. 1 (see METHODS), where values for K were 7.6 and 3.5 µM for the control relationship (a) and that in the presence of Li+ at pH 9 (b), respectively, and Jmax were 514.0 and 84.3 µM/s, respectively. The difference between the control curve and that after blocking Ca2+ extrusion represents the component sensitive to Li+ and high pH and is shown by a thick curve (a  b: JCa(pm)). The difference is statistically significant with P < 0.05 (see METHODS). It is to be noted that fitting the data points with Eq. 1 was only for approximation to take the difference between the 2 relationships.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Effects of blocking Ca2+ uptake into thapsigargin-sensitive Ca2+ stores and mitochondrial Ca2+ uptake on tetanus-induced Ca2+ transients. A: effects of thapsigargin. B: effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Ca2+ transients were induced by a tetanus of 100 stimuli at 100 Hz to the nerve and recorded by changes in fluorescence of OGB-1 (A) or Indo-1 (B). Thin traces are the control records, while thick traces are those after treatment with thapsigargin (2 µM: A) or CCCP (1 µM: B). Insets: the falling phase of Ca2+ transients on semi-log coordinates. C: the [Ca2+]i dependence of the rate of the decay of tetanus-induced Ca2+ transient and effects of blocking mitochondrial Ca2+ uptake. The slope of the decline of [Ca2+]i, Ca2+ efflux, along the decay time course of Ca2+ transient was plotted against the corresponding [Ca2+]i value. Open circles, the control Ca2+ efflux; closed circles, those after treatment with CCCP. Data points were the average of the relationships obtained from 10 terminals and fitted by Eq. 1, where K values were 7.8 and 12.0 µM for the control curve and that after treatment with CCCP and Jmax values were 423.1 and 204.7 µM/s, respectively. The difference between the control curve and that in the presence of CCCP represents the component sensitive to CCCP and is shown by a thick curve (a  b: JCa(mito)). The difference is statistically significant with P < 0.05 (see METHODS). It is to be noted that fitting the control data points with Eq. 1 was only for approximation to take the difference between the 2 relationships. D: the summary and comparison of the components of [Ca2+]i-dependent Ca2+ efflux under different conditions. The data points with the asymptotic curve relating Ca2+ efflux to [Ca2+]i in the presence of Li+ at high pH, JCa(mito), the data points with the asymptotic curve relating Ca2+ efflux to [Ca2+]i in the presence of CCCP and JCa(pm) were replotted for comparison. JCa(mito) and JCa(pm) were nicely fitted by Eq. 1 with K values of 6.8 and 9.4 µM, respectively, and Jmax values of 254 and 197.1 µM/s, respectively. The curve fitted to JCa(mito) is not seen because it is overlapped with JCa(mito), while the curve fitted to JCa(pm) is shown by a thin interrupted curve. Note the similarity of the curve in the presence of Li+ at high pH to JCa(mito) and that of the curve in the presence of CCCP to JCa(mito).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Summary of the effects of blocking Ca2+ extrusion and uptake on various parameters of tetanus-induced Ca2+ transients. The decay phase of Ca2+ transients induced by a tetanus (100 Hz, 100 stimuli) was fitted by the equation, [Ca2+]i = Cainitial exp(t/initial) + Casecond exp(t/second) + Cathird. A: effects of blocking Ca2+ extrusion and Ca2+ uptake on Ca2+ transients induced by 100 stimuli. a: effects on the resting [Ca2+]i. b: effects on the amplitude of the initial decay component (Cainitial). c: effects on the amplitude of the 2nd decay component (Casecond). d: effects on the magnitude of the slowest decay component (Cathird). e: effects on the time constant of the initial decay component (initial). f: effects on the time constant of the 2nd decay component (second). All the values for each parameter after blocking 1 or 2 of Ca2+ clearance mechanisms were normalized to the control value in individual terminals and rescaled to the absolute value by multiplying the control value for each parameter. Vertical interrupted lines indicate the control value for each parameter. n, the number of terminals experimented. *, significantly different from the control with P < 0.05; **, significant with P < 0.025. B: effects of blocking Ca2+ extrusion and thapsigargin-sensitive Ca2+ uptake on Ca2+ transients induced by a tetanus (100 Hz) of 200 stimuli. a: effects on the time constant of the initial decay component (initial). b: effects on the time constant of the 2nd decay component (second). *, significantly different from the control with P < 0.05; **, significant with P < 0.025. It is to be noted that other parameters of Ca2+ transient were not changed by these treatments.

Effects of blocking Ca²+ extrusion at the plasma membrane

We first tested the effect of inhibition of Ca²⁺ pumps at the cell membrane on tetanus-induced Ca²⁺ transients by raising external pH (Benham et al. 1992; Milanick 1990; Niggli et al. 1982). Increasing external pH to 9.0 affected neither the resting [Ca²⁺]_i nor the amplitude and decay phases of Ca²⁺ transients induced by a tetanus (100 Hz) of 100 stimuli with a tendency of an increase in the amplitude of the second decay component (but, not significant: Figs. 1A and 3A). The initial and second decay time constant of Ca²⁺ transients induced by 200 stimuli, however, tended to increase with no change in other parameters. Although the increases in the initial and second decay constants averaged over all the terminals examined were not statistically significant (Fig. 3B), some of the terminals showed significant increases to 130% (n = 3) and 165% (n = 5), respectively, with no change in other parameters.

We next examined the effect of blockade of Na/Ca exchanger at the cell membrane on tetanus-induced Ca²⁺ transients. This was achieved by replacing external Na⁺ with Li⁺, which is known to be incapable of substituting the role of Na⁺ in Na/Ca exchange (see Reuter and Porzig 1995). External Li⁺ slowly but only slightly elevated the basal level of [Ca²⁺]_i over several minutes (Fig. 3A), which was measured with changes in the ratio of indo-1 fluorescence. Under this condition, the amplitude and decay phase of Ca²⁺ transients induced by 100 stimuli (100 Hz) remained unchanged except for a small increase in the time constant of the initial decay component (Figs. 1B, e, and 3A). Both the initial and second decay time constants of Ca²⁺ transients produced by 200 stimuli (100 Hz), however, were prolonged to 140 and 135%, respectively, with no change in other parameters (Fig. 3B). These effects are apparently stronger than those of high alkaline, which were not consistent among all the terminals.

In contrast to the effect of blocking Na/Ca exchangers or Ca²⁺ pumps alone, the combined application of high external pH and Li⁺ significantly increased the time constants of the initial and second decay components of Ca²⁺ transients induced even by 100 stimuli (100 Hz: Figs. 1C and 3A). Under this condition, there was little change in the magnitude of each component of the decay although the second component tended to increase.

The results shown in the preceding text indicate that both the initial and second decay components are caused by Ca²⁺ extrusion operating in different modes depending on [Ca²⁺]_i. Effects of blocking Ca²⁺ extrusion on the [Ca²⁺]_i dependence of Ca²⁺ clearance are more clearly shown by the effects on the [Ca²⁺]_i dependence of the rate of the decay of Ca²⁺ transient. The rates of the decay of 100 stimuli-induced Ca²⁺ transient at different [Ca²⁺]_i values in the presence of external Li⁺ at high external pH were plotted against the corresponding [Ca²⁺]_i value and the relationship was fitted by Eq. 1 (Fig. 1D, b). This asymptotic curve was subtracted from the asymptotic curve for the control relationship (curve a in Fig. 1D, a). The net difference (Fig. 1D, a  b) yielded the fraction of Ca²⁺ clearance achieved by Ca²⁺ extrusion at the cell membrane. The rate of Ca²⁺ extrusion was thus clearly [Ca²⁺]_i-dependent and amounted to be ~30% of the total flux.

Effects of blockade of Ca²+ uptake into thapsigargin-sensitive Ca²⁺ stores

Next, we tested the effect of blocking Ca²⁺ uptake into Ca²⁺-storing organelles. Thapsigargin (2 µM), a blocker of Ca²⁺ pump at Ca²⁺-storing organelles, did not affect Ca²⁺ transients caused by 100 stimuli (100 Hz), although there was a tendency of a slight increase in the time constant of the initial component of the decay (Figs. 2A and 3A). The initial decay time constant of Ca²⁺ transients induced by 200 stimuli (100 Hz), however, was significantly increased to 134% by thapsigargin (2 µM: Fig. 3B) with no change in other parameters. This suggests that Ca²⁺ uptake into thapsigargin-sensitive Ca²⁺ stores plays minor roles in Ca²⁺ clearance of a small Ca²⁺ load but operates as a Ca²⁺ sink only for a greater Ca²⁺ load. This may conform to the previous suggestion that this Ca²⁺ store involved in Ca²⁺-induced Ca²⁺ release is normally filled with Ca²⁺ (Narita et al. 1998).

Effects of blockade of mitochondrial Ca²+ uptake

Blocking Ca²⁺ uptake into mitochondria had drastic effects on Ca²⁺ clearance in the nerve terminals. CCCP (1 µM), a mitochondrial uncoupler, markedly increased the plateau of tetanus-induced Ca²⁺ transients, the initial and second components of the decay phase, and the magnitude of the slowest component (Figs. 2B and 3). Under this condition, the basal level of [Ca²⁺]_i was tended to increase (Fig. 3A). In two terminals, a high concentration of CCCP (5 µM) caused an increase in the basal level of [Ca²⁺]_i by several tens of nM (unpublished observations; see also David 1999; Tsang et al. 2000; see Narita et al. 1998 for the action of CN, another mitochondrial poison). The increase in plateau must be mainly due to the decrease in the rate of Ca²⁺ clearance seen as increases in the time constants of the initial and second components of [Ca²⁺]_i decay and the magnitude of the latter because the plateau is determined by the apparent equilibrium of Ca²⁺ entry and clearance (see DISCUSSION).

The effect of blocking mitochondrial Ca²⁺ uptake on the decay phase of Ca²⁺ transient can be shown more relevantly by the effect on the [Ca²⁺]_i dependence of the decay rate of the increased [Ca²⁺]_i. The [Ca²⁺]_i-dependent Ca²⁺ efflux from the cytosol was considerably decreased under the blockade of mitochondrial Ca²⁺ uptake (Fig. 2C). The CCCP-sensitive component of Ca²⁺ efflux obtained by subtraction [Fig. 2C, thick curve (a  b)] was also [Ca²⁺]_i dependent and amounted to be ~70% of the total Ca²⁺ efflux. This [Ca²⁺]_i-dependence of the CCCP-sensitive Ca²⁺ efflux (J_Ca(pm) replotted in Fig. 2D), reflecting that of Ca²⁺ efflux via mitochondrial Ca²⁺ uniporter (see DISCUSSION), is very similar to that in the presence of Li⁺ at high pH (thin curve with triangles replotted in Fig. 2D). On the other hand, the relationship between the Ca²⁺ efflux remaining in the presence of CCCP and [Ca²⁺]_i (thin curve with circles replotted in Fig. 2D) fairly resembles that of the Ca²⁺ efflux sensitive to Li⁺ at pH 9 (J_Ca(pm) replotted in Fig. 2D). This remaining component must be caused by Ca²⁺ extrusion at the cell membrane.

It is to be added that DNP (20 or 50 µM), an uncoupler, and NaCN (2 mM), a blocker of an electron transfer system, had effects similar to those of CCCP on the decay phases of tetanus-induced Ca²⁺ transients (unpublished observations).

	DISCUSSION

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The present study demonstrates the primary role of mitochondrial Ca²⁺ uptake and the secondary role of plasmalemmal Ca²⁺ extrusion in the [Ca²⁺]_i-dependent Ca²⁺ clearance of a small Ca²⁺ load caused by a relatively short repetitive activity in frog motor nerve terminals and the boosting role of thapsigargin-sensitive Ca²⁺ uptake in the clearance of a heavier Ca²⁺ load. How Ca²⁺ extrusion and mitochondrial Ca²⁺ uptake depend on [Ca²⁺]_i and how much each of them contributes to the total Ca²⁺ clearance are discussed more in detail within the limitation of understanding of the time course of Ca²⁺ transients averaged over the whole terminal as in the following text.

Nonlinear dynamics of Ca²+ and its reactions with Ca²⁺ indicators in the nerve terminals

Ca²⁺ entry caused by a nerve impulse first produces a large increase in [Ca²⁺]_i to >10-100 µM in a localized region (Ca²⁺ domain) around Ca²⁺ channels (Bollmann et al. 2000; Heidelberger et al. 1994; Llinás et al. 1992; Schneggenburger and Neher 2000; Schweizer et al. 1995), which dissipates within a few milliseconds, by binding, diffusion, and uptake (DiGregorio et al. 1999; Sala and Hernández-Cruz 1990; Sinha et al. 1997; Suzuki et al. 2000). On the other hand, high-affinity Ca²⁺ indicators in the Ca²⁺ domain quickly bind with Ca²⁺ and diffuse out immediately after each nerve impulse, while free Ca²⁺ indicators in neighboring regions would diffuse into the domain and bind Ca²⁺ (Naraghi and Neher 1997; Suzuki et al. 2000; Tank et al. 1995). Thus during the plateau of Ca²⁺ transient, Ca²⁺ entry at a constant rate would be in pseudo-equilibrium with Ca²⁺ extrusion at the cell membrane and mitochondrial Ca²⁺ uptake (David 1999), producing standing gradients in [Ca²⁺]_i, free and bound forms of Ca²⁺ indicators from the Ca²⁺ microdomain to the global space. The high [Ca²⁺]_i in the Ca²⁺ domain in a small volume and a short life time would have escaped detection by high-affinity Ca²⁺ indicators. The measured changes in [Ca²⁺]_i during the plateau are therefore largely underestimated, while the changes in [Ca²⁺]_i after the dissipation of the Ca²⁺ domains would faithfully reflect the time course of the averaged change in [Ca²⁺]_i in the terminals. Simulation indeed revealed that the fluorescence changes averaged over the whole terminal represent the true impulse-induced changes in the [Ca²⁺]_i only 20 ms after the end of stimuli (Suzuki et al. 2000).

[Ca²+]_i-dependent mitochondrial Ca²⁺ uptake

The [Ca²⁺]_i dependence of mitochondrial Ca²⁺ uptake, seen as CCCP-sensitive component of Ca²⁺ efflux (J_Ca(mito)) induced by 100 impulses or that of the Ca²⁺ efflux remaining in the presence of Li⁺ at high pH, is nicely fitted by the Hill's equation (Eq. 1) assuming the cooperative binding of two Ca²⁺ ions to a Ca²⁺ transporter. This is consistent with the recent findings in bullfrog sympathetic ganglion cells (Colegrove et al. 2000) and also with the known property of mitochondrial Ca²⁺ uniporters binding two Ca²⁺ for translocation (Bygrave et al. 1971; Scarpa and Grazziotti 1973; see Gunter and Pfeiffer 1990). The threshold level of [Ca²⁺]_i for mitochondrial Ca²⁺ uptake to occur, however, was found to be 0.2 µM in the present study (see also Colegrove et al. 2000), which is smaller than the known value of 0.5 µM (see Carafoli 1987; Gunter and Pffeifer 1990). The discrepancy was explained by the ignorance of Ca²⁺ release at [Ca²⁺]_i <0.5 µM in the previous measurement of mitochondrial Ca²⁺ uptake (see Colegrove et al. 2000).

The increase in the third, slowest component by blocking mitochondrial Ca²⁺ uptake is unexpected because the blockade of Ca²⁺ uptake would have also reduced mitochondrial Ca²⁺ release that occurs on restoration of [Ca²⁺]_i close to the resting level (Colegrove et al. 2000; David et al. 1998; Duchen et al. 1990; Friel and Tsien 1994). The increase in the slowest component could be explained by a large [Ca²⁺]_i load exceeding the capacity of Ca²⁺ extrusion as a result of the blockade of mitochondrial Ca²⁺ uptake. Presumably, mitochondrial Ca²⁺ release may have occurred after the end of 100 stimuli at 100 Hz but must have been too small to apparently affect the slowest decay component.

[Ca²+]_i-dependent Ca²+ extrusion at the cell membrane

The component of Ca²⁺ efflux via Ca²⁺ extrusion (J_(ca(pm)) after 100 impulse-induced Ca²⁺ load was dependent on [Ca²⁺]_i and amounted to be ~30% of the total Ca²⁺ efflux caused by 100 impulses. The J_Ca(pm), seen as the Li⁺ and high-alkaline-sensitive component or the Ca²⁺ efflux remaining in the presence of CCCP, was well fitted by Eq. 1 assuming the Hill number of two (Fig. 2D). This is consistent with the known property of the Ca²⁺-ATPase having two Ca²⁺-binding sites at the cell membrane (Ferreira and Lew 1976; Lew et al. 1982) but not with the binding of a single Ca²⁺ to a Na/Ca exchanger molecule (see Carafoli 1987). Therefore there must be several other possible mechanisms for the apparent cooperative [Ca²⁺]_i dependence of J_Ca(pm).

First, the Ca²⁺ affinity of Ca²⁺ pumps at the cell membrane is markedly enhanced by the binding of calmodulin (see Carafoli 1991). Thus rises in [Ca²⁺]_i would promote the action of calmodulin and amplify the [Ca²⁺]_i dependence of Ca²⁺ pump. Second, a rise in [Ca²⁺]_i should increase the driving force for Na/Ca exchangers. For instances, 10-fold increase in [Ca²⁺]_i would shift the equilibrium potential for Na/Ca exchange (E_Na/Ca) to a more positive value by 56 mV [using the equation, E_Na/Ca = 3E_Na  2E_Ca (Mullins 1977)]. These two effects would have changed the [Ca²⁺]_i-dependent property of J_Ca(pm) to that more closely expected from Eq. 1. The deviation of the relationship of Ca²⁺ efflux to [Ca²⁺]_i in the presence of CCCP from the equation in the range of [Ca²⁺]_i > 1.5 µM (see Fig. 2C) might presumably be explained by the recruitment of another Ca²⁺ clearance mechanism unidentified yet.

Blocking either Na/Ca exchangers or Ca²⁺ pumps alone at the cell membrane affected only slightly or not obviously the decay rate of the clearance of a small Ca²⁺ load produced by a short tetanus (100 stimuli), while blocking both indeed slowed the decay rate. Presumably, a greater increase in [Ca²⁺]_i in the submembrane region as a result of the blockade of one mechanism would have enhanced another in compensation for the [Ca²⁺]_i dependence of their rate. Such a greater increase in [Ca²⁺]_i would have escaped detection by high-affinity Ca²⁺ indicators (see preceding text). Although the operations of Na/Ca exchanger and Ca²⁺ pumps are complementary to each other, the contribution of the former is stronger than the latter, as seen in the effect of Li⁺ on Ca²⁺ transients induced by 200 stimuli. This is consistent with the higher rate of Na/Ca exchanger than that of Ca²⁺ pumps at the cell membrane (see Carafoli 1987). A question remains, however, how such complementary operations of Na/Ca exchanger and Ca²⁺ pumps can be explained by their different Ca²⁺ affinity and transport rate (see Carafoli 1987) and the possible location of the latter close to Ca²⁺ channels as suggested for ciliary ganglion synapses (Juhaszova et al. 2000).

The apparent absence of changes in the plateau phase of Ca²⁺ transients induced by either 100 or 200 stimuli under the blockade of Ca²⁺ extrusion may be explained in part by the secondary role of Ca²⁺ extrusion in Ca²⁺ clearance and in part by the failure of detection of the high [Ca²⁺]_i in the submembrane regions with high-affinity Ca²⁺ indicators. This contrasts with the successful recording of an increase in the [Ca²⁺]_i in the bulk phase due to the blockade of mitochondrial Ca²⁺uptake. The little effect of blocking Ca²⁺ extrusion as well as mitochondrial Ca²⁺ uptake on the resting level of [Ca²⁺]_i could be accounted for presumably by small Ca²⁺ entry under the resting condition.

Comparison with Ca²+ clearance in other terminals and physiological significance

The predominant role of mitochondrial Ca²⁺ uptake in the clearance of the increased [Ca²⁺]_i found in the present study is consistent with the previous studies on the rat neurohypophysial nerve endings (Stuenkel 1994) and the motor nerve terminals of the crayfish (Ohnuma et al. 1999; Tang and Zucker 1997) and the lizard (David et al. 1998). On the other hand, the involvement of Na/Ca exchanger in Ca²⁺ clearance in frog motor nerve terminals conforms to its similar role in rat brain synaptosomes (Nachshen et al. 1986) and hippocampal presynaptic terminals (Reuter and Poerzig 1995). Furthermore, Na/Ca exchangers were shown to exist in the membrane of the rat motor nerve terminal (Luther et al. 1992).

The significance of the present study is twofold. First, the modes of clearance of Ca²⁺ load via both mitochondrial Ca²⁺ uptake and Ca²⁺ extrusion are Ca²⁺ dependent in frog motor nerve terminals. Second, the operations of Na/Ca exchangers and Ca²⁺ pumps at the cell membrane to a small Ca²⁺ load are complementary to each other with the slight predominance of the former. The effective operation of Ca²⁺ extrusion machinery, Na/Ca exchangers and Ca²⁺ pumps, is really physiological for the presynaptic terminals of small size whose surface/volume ratio is quite large and therefore effective for Ca²⁺ clearance. This contrasts to the negligible role of Ca²⁺ extrusion for the clearance of Ca²⁺ load caused by Ca²⁺ entry in the large-sized cell soma of bullfrog sympathetic neurons (Colegrove et al. 2000) and rat adrenal chromaffin cells (Park et al. 1996).