Instituto de Investigaciones Médicas A. Lanari, Universidad de Buenos Aires, 1427 Buenos Aires, Argentina
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ABSTRACT |
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Spontaneous secretion of the neurotransmitter acetylcholine in
mammalian neuromuscular synapsis depends on the
Ca2+ content of nerve terminals.
The Ca2+ electrochemical gradient
favors the entry of this cation. We investigated the possible
involvement of three voltage-dependent Ca2+ channels (VDCC) (L-, N-, and
P/Q-types) on spontaneous transmitter release at the rat neuromuscular
junction. Miniature end-plate potential (MEPP) frequency was clearly
reduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to a
lesser extent by the N-type VDCC blocker, -conotoxin GVIA (
-CgTx,
5 µM). On the other hand, nifedipine and
-CgTx had no effect on
K+-induced transmitter secretion.
-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, prevents
acetylcholine release induced by
K+ depolarization but failed to
affect MEPP frequency in basal conditions. These results suggest that
in the mammalian neuromuscular junction Ca2+ enters nerve terminals
through at least three different channels, two of them (L- and N-types)
mainly related to spontaneous acetylcholine release and the other
(P/Q-type) mostly involved in depolarization-induced neurotransmitter
release. Ca2+-binding
molecule-related spontaneous release apparently binds Ca2+ very rapidly and would
probably be located very close to
Ca2+ channels, since the fast
Ca2+ chelator (BAPTA-AM)
significantly reduced MEPP frequency, whereas EGTA-AM, exhibiting
slower kinetics, had a lower effect. The increase in MEPP frequency
induced by exposing the preparation to hypertonic solutions was
affected by neither external Ca2+
concentration nor L-, N-, and P/Q-type VDCC blockers, indicating that
extracellular Ca2+ is not
necessary to produce hyperosmotic neurosecretion. On the other hand,
MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the same
extent, supporting the view that hypertonic response is promoted by
"bulk" intracellular Ca2+
concentration increases.
spontaneous transmitter release; L-type voltage-dependent calcium
channel; nifedipine; -agatoxin IVA;
-conotoxin GVIA; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester; ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester; hypertonic response
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INTRODUCTION |
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THERE IS EVIDENCE THAT BOTH spontaneous and evoked quantal release of acetylcholine at the neuromuscular junction obey different mechanisms, although to some extent both are dependent on external Ca2+ concentration ([Ca2+]o) (4, 11, 21, 24, 29, 33), linked to ion influx through voltage-dependent Ca2+ channels (VDCCs) (11, 46).
Several VDCCs have been identified using molecular cloning and
selective Ca2+ channel toxins; at
least five distinct types are known to exist in nerve cells, namely, L,
N, T, P, and Q (2, 22, 41, 42). Available data indicate that multiple
types of channels may coexist at a given individual synapse. Synapses
differ in their channel types, and the same kind of synapse uses
different VDCCs in different species. In this regard, -conotoxin
GVIA (
-CgTx), an N-type VDCC blocker, was shown to inhibit
neuromuscular transmission in the frog (10, 13, 16, 34) but was
reported to be ineffective in mammals (34, 48). In contrast, it has
been shown that mammalian-evoked neuromuscular transmission is blocked
by P/Q-type VDCC antagonists such as funnel-web spider toxin,
-conotoxin MVIIC, and
-agatoxin IVA (
-Aga) (5, 18, 28, 46).
Intracellular free Ca2+ concentration ([Ca2+]i) seems to play a relevant role in spontaneous neurotransmitter secretion (3, 23, 30), particularly under hypertonic conditions (36). However, Tanabe and Kijima (40) have questioned the putative relationship between miniature end-plate potential (MEPP) frequency and [Ca2+]i in hypertonicity (40).
The aim of the present work was to identify the VDCCs responsible for
spontaneous quantal secretion in mammalian synapse and its relationship
with
[Ca2+]o
and
[Ca2+]i.
Experiments were performed in isotonic and hypertonic conditions and in
the presence of -Aga,
-CgTx, or nifedipine (P/Q-, N-, and L-type
VDCC blockers, respectively). To study the effect of [Ca2+]i
on the kinetics of spontaneous acetylcholine release, nerve terminals
were loaded with buffers having similar equilibrium Ca2+ affinities but different
binding kinetics. The effect of the slowly binding
Ca2+ buffer ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA)-acetoxymethyl ester (AM) was compared with the rapidly binding homologue
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (1, 44, 45).
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MATERIALS AND METHODS |
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Wistar rat diaphragm muscles were used. Rats (180-220 g) were
anesthetized with thiopental sodium (50 mg/kg), and the left hemidiaphragm was excised and transferred to a chamber filled with
Krebs-Ringer [(in mM) 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 11 glucose, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, pH 7.3-7.4] and bubbled with
O2. Hyperosmotic media were
freshly prepared by adding sucrose to the Ringer solution, and their
osmolarity was checked with a Fiske osmometer before each experiment.
MEPP frequency was recorded intracellularly at the end-plate region of
the muscle fiber with glass microelectrodes filled with 3 M KCl
(resistance of 5-10 M). Muscle fibers with a resting membrane potential less negative than
60 mV or MEPP with a rise time >1 ms were rejected. To study the time course of hyperosmotic response, 10 junctions were sampled in the control solutions (isotonic) and their
values were averaged (see Figs. 3 and 5). Immediately after the change in osmolarity, at least 11 synapses were sampled repeatedly from the same small area of diaphragm over brief intervals for 40 min at the most (see Figs. 3 and 5). An effort was made to keep
the intervals between sampling as short as possible. In these
experiments, tetrodotoxin
(10
6 M; Sigma) was added to
external medium to prevent the muscle from twitching violently, which
otherwise occurred when preparations were suddenly exposed to
hypertonic solutions. To render comparable frequency values from
different experiments, mean MEPP frequency of the peak hypertonic
response was normalized with respect to mean MEPP frequency obtained in
isotonic solution (9, 12). The comparison between hypertonic responses
in control and test solutions was expressed as the ratio of the areas
under their respective curves measured by numerical integration. When
addition of drugs to the saline significantly increased osmolarity
(Co2+, 13.5 mM), the control
solution was corrected to the same tonicity as the test solution. To
eliminate the inward Ca2+
gradient, a Ringer solution containing 0 Ca2+, 2 mM
Mg2+, and 1 mM EGTA was employed.
Other details are described when required in
RESULTS. To decrease
[Ca2+]i,
nerve terminals were loaded with BAPTA-AM (Molecular Probes) as
follows: the preparation was immersed in a
Ca2+-free Ringer solution
containing BAPTA-AM dissolved in dimethyl sulfoxide (3 × 10
7 mol/30 mg of muscle).
Preparations were incubated, under these conditions, for 2 h at room
temperature (14, 44, 45) and then rinsed during a 40-min period with
Ca2+-free Ringer solution. In some
experiments, an equal concentration of EGTA-AM (Molecular Probes), a
buffer with similar equilibrium Ca2+ affinity but slower binding
kinetics, was used, following a similar procedure. Both buffers are
cell permeant.
Nifedipine (Sigma; 5 µM, in darkness), -CgTx (Alomone
Laboratories; 5 µM), and
-Aga (Peptide Institute; 100 nM) were
used as L-, N-, and P/Q-type VDCC blockers, respectively.
In results, n represents number of fibers per number of muscles.
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RESULTS |
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Effect of -Aga on spontaneous acetylcholine release.
It is known that, in mice neuromuscular junctions, 100 nM
-Aga
abolishes the presynaptic Ca2+
currents and acetylcholine release induced by electrical stimulation or
by K+ depolarization (28). In
isotonic conditions (Fig. 1),
MEPP frequency was unaltered by 100 nM
-Aga [results (means ± SD) are 1.9 ± 0.6 for control,
n = 38/4, and 2.0 ± 0.5 for
-Aga, n = 42/4]. However,
when nerve terminals were depolarized by increasing extracellular
K+ concentration to 10 and 15 mM
(Fig.
2A),
-Aga exerted a clear drop in the
K+-induced increase in spontaneous
release [results (means ± SD) are 8.4 ± 0.1 (n = 38/4) with 10 mM
K+ and 23.2 ± 1.3 (n = 37/4) with 15 mM
K+ for control and 2.9 ± 0.4 (n = 40/4) with 10 mM
K+ and 8.4 ± 0.4 (n = 40/4) with 15 mM
K+ for
-Aga;
P < 0.0001 and
P < 0.0001, respectively].
These results suggest that the P/Q-type VDCCs at the mammalian
neuromuscular junction are not involved in spontaneous acetylcholine
release but are related to the
K+-evoked MEPPs. When Ringer
solution tonicity was raised 35%, measurements of the hypertonic
response area remained similar to control values (ratio of areas in
control and
-Aga = 1.0 ± 0.2; n = 4). Figure 3A shows a
typical experiment.
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Effect of nifedipine on spontaneous acetylcholine release. L-type VDCCs are sensitive to 1,4-dihydropyridine compounds (8, 26, 27), some of which, like nifedipine, act as blockers and whose effect on spontaneous neurotransmitter release was studied. As shown in Fig. 1, in isotonic conditions, 5 µM nifedipine reduced MEPP frequency by roughly 53% (3.6 ± 1.8 for control, n = 34/5, 1.7 ± 0.9 for nifedipine, n = 32/5; P < 0.005). The effect was not evident under hyperosmotic conditions (ratio of areas in control and nifedipine = 1.0 ± 0.1; n = 3; Fig. 3B). These results indicate that nifedipine-blockable channels play a major role in resting spontaneous acetylcholine release and, furthermore, that they do not participate to any appreciable extent in osmotic response.
Figure 2B shows the effect of the same nifedipine concentration on nerve terminals depolarized by increasing external K+ concentration to 10 and 15 mM. Interestingly enough, nifedipine appeared to exert a selective inhibitory effect on spontaneous acetylcholine release only in basal conditions (5 mM K+), without interfering with the Ca2+ current associated with nerve terminal depolarization (10 and 15 mM K+). A nifedipine effect independent of Ca2+ channel blockade was ruled out, since MEPP frequency in the presence of nifedipine + zero Ca2+-EGTA was similar to values obtained in zero Ca2+-EGTA solution (percent of control values: 35.1 ± 2.6% for zero Ca2+-EGTA, n = 40/4, and 37.8 ± 4.9% for 5 µM nifedipine + zero Ca2+-EGTA, n = 42/4).Effect of -CgTx on spontaneous acetylcholine
release.
To determine whether L-type VDCCs were the only channels involved in
spontaneous secretion in mammals, MEPP frequency was recorded in zero
Ca2+-EGTA solution. In Fig. 1, the
decrease in spontaneous transmitter release produced by 5 µM
nifedipine (47 ± 12%, n = 32/5)
can be compared with that observed in zero
Ca2+-EGTA solution (33.5 ± 3.8%, n = 60/6). Values obtained in
zero Ca2+-EGTA were significantly
lower than those observed after addition of nifedipine
(P < 0.01). These results suggest
that more than one type of VDCC is involved in spontaneous release.
Effect of BAPTA-AM and EGTA-AM on spontaneous acetylcholine release. It is known that the slowly binding Ca2+ buffer EGTA fails to block evoked neurotransmitter release, whereas a rapidly binding homologue, BAPTA, efficiently blocks release (1, 35, 47). However, scarce information is available concerning the kinetics of spontaneous transmitter release. Figure 4 shows that nerve terminals loaded with BAPTA-AM display a reduction in MEPP frequency to 49.2 ± 3.6% (n = 40/4) and to 16.7 ± 3.2% (n = 58/6) when the recordings were performed in muscles exposed to normal Ringer (2 mM Ca2+) and to zero Ca2+ Ringer solutions, respectively. Control values obtained in normal Ringer solution before BAPTA-AM loading are expressed as 100%. It is evident that the efficacy of the chelator decreases when Ca2+ is reentering the terminal during the recording period. The effect of BAPTA-AM was also demonstrated when the MEPP frequency recorded in zero Ca2+ solution from loaded end plates was compared with that obtained in zero Ca2+ solution from not loaded preparations [16.7 ± 3.2% for loaded (n = 58/6) and 43.4 ± 8.6% for not loaded (n = 38/4), P < 0.0002]. These results are different from those found in frog (40).
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Osmotic response dependence on [Ca2+]o and [Ca2+]i. With the assumption that nerve terminals behave in a similar way to muscle fibers, resting membrane potential was recorded from the latter during exposure to hypertonic solutions. No difference in resting membrane potential was found between isotonic and hypertonic conditions (72.5 ± 1.5 for isotonic and 73.2 ± 0.8 for hypertonic, n = 8); similar results were also reported by Hubbard et al. (12). To study osmotic response dependence on [Ca2+]o, MEPP frequency was evaluated in zero Ca2+-EGTA solution to preclude Ca2+ influx. In Fig. 5A, a single experiment is illustrated, showing that, by increasing tonicity from 280 to 420 mosM, MEPP frequency was raised both in control Ringer solution and in a Ringer made without Ca2+ and containing 1 mM EGTA. Although the magnitude of the hypertonic response in control solution was higher than in zero Ca2+-EGTA solution (ratio of areas in control and zero Ca2+-EGTA = 4.8 ± 0.3; n = 4), extracellular Ca2+ is hardly necessary to produce hyperosmotic neurosecretion, as shown by the similar ratios of peak osmotic response and mean isotonic MEPP frequencies in control and test solutions (8 ± 1.9 for control and 9.1 ± 3.5 for zero Ca2+-EGTA; n = 4). The relative decrease in MEPP frequency in zero Ca2+-EGTA may be due to a reduced [Ca2+]i in conditions in which the Ca2+ electrochemical gradient is reversed by the virtual absence of external Ca2+ (30, 49). Such an effect was not discernible when 13.5 mM Co2+ (a Ca2+ channel blocker) was added to the zero Ca2+-EGTA solutions (ratio of areas in control and zero Ca2+-EGTA + Co2+ = 1.1 ± 0.1; n = 4). To investigate further the role of free Ca2+ concentration within nerve terminals during hypertonic response, presynaptic terminals were loaded with BAPTA-AM or EGTA-AM, two membrane-permeant Ca2+ chelators. After chelator loading, MEPP frequency in isotonic and hypertonic conditions was recorded in Ca2+-free solution and compared with the response obtained in control Ringer solution (2 mM Ca2+) before BAPTA-AM or EGTA-AM loading. Figure 5B shows the effect of BAPTA-AM on hyperosmotic neurosecretion. The magnitude of such response, in terms of the area under the curve, was markedly reduced compared with the control response obtained in not loaded end plates (ratio of areas in control and BAPTA-AM = 8.1 ± 1.4; n = 4). When the early transient increase in MEPP frequency induced by exposing the preparation to hypertonic solution was related to the mean frequency obtained in isotonic condition, the ratio obtained in end plates loaded with BAPTA-AM was significantly lower than the ratio obtained in control Ringer solution before chelator treatment (ratio of peak osmotic response and mean isotonic MEPP frequency was 9.1 ± 0.8 for control and 5.6 ± 2.4 for BAPTA-AM; n = 4, P < 0.01), suggesting that the presence of Ca2+ within terminals is a basic requirement for hyperosmotic response. It is worthwhile pointing out that both BAPTA-AM and EGTA-AM (see Fig. 5C) had a similar effect on the response of MEPP frequency to hypertonicity (ratio of peak osmotic response and mean isotonic MEPP frequency was 7.6 ± 0.8 for control and 4.0 ± 1.4 for EGTA-AM; n = 4, P < 0.002), which contrasts with the greater potency of BAPTA-AM compared with EGTA-AM on spontaneous transmitter release observed in isotonic conditions (see DISCUSSION).
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DISCUSSION |
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This study investigates the control of spontaneous acetylcholine
release by Ca2+ influx into nerve
terminals through diverse VDCCs at mammalian neuromuscular junctions.
Our results in rat diaphragm strongly suggest the involvement of
nifedipine-blockable Ca2+
channels, since the addition of 5 µM of this drug to the saline reduced MEPP frequency by roughly 53%. A nifedipine effect other than
on VDCCs may be ruled out, since we obtained similar MEPP frequency
values in zero Ca2+-EGTA and in
nifedipine + zero Ca2+-EGTA
solutions. It may be assumed that most of the
Ca2+ current contributing to
resting MEPP frequency in mammalian neuromuscular junctions takes place
through L-type VDCCs. On the other hand, a different type of VDCC seems
to be involved, since the reduction in MEPP frequency induced by
nifedipine was significantly different from that recorded in zero
Ca2+-EGTA Ringer solutions. It was
found that -CgTx reduced MEPP frequency by ~29%, indicating that
N-type VDCCs also play a role in the regulation of spontaneous
secretion. The combined application of nifedipine and
-CgTx reduced
MEPP frequency to values lower than the sum of the individual effects
of each drug (see Fig. 1), suggesting that both VDCC types act
synergistically to control spontaneous release at individual release
sites. Remarkably, nifedipine and
-CgTx did not affect the high
K+-evoked increase in MEPP
frequency, suggesting that L- and N-type VDCCs do not interfere with
acetylcholine release associated with nerve terminal depolarization. On
the other hand, it has been shown that
-Aga (a P/Q-type VDCC
blocker) inhibits presynaptic Ca2+
current and acetylcholine release induced by either electrical or
K+ depolarization in mice
neuromuscular junction (5, 28). Our results in rat diaphragm muscles
are in agreement with those findings (see Fig.
2A). However, our experiments showed
that P/Q-type VDCCs were not involved in spontaneous acetylcholine
secretion in basal conditions (5 mM
K+). Coexistence of several
types of VDCCs at a single synapse has already been described in
mammalian central nervous system (6, 17, 32, 39). In the frog, it has
been shown that a significant fraction of
Ca2+ influx involved in the
maintenance of resting MEPP frequency occurs via
-CgTx-blockable
channels; however, in this species more than one type of channel may be
involved in spontaneous release, since the action of
-CgTx is not as
effective as zero Ca2+-EGTA Ringer
solution (10).
Free Ca2+ concentration within the terminal depends on Ca2+ coming from the extracellular medium or released from an organelle into the cytosol (3, 31, 37, 43). After removal of all external Ca2+ and in conditions in which free Ca2+ is buffered by permeant Ca2+ chelators, residual secretion of transmitter quanta may depend on Ca2+-binding sites sensing a Ca2+ transient within a small subcellular volume near the organelle. The fact that MEPP frequency fell more sharply when nerve terminals were loaded with a rapid chelator (BAPTA-AM) than with similar concentrations of a slow one (EGTA-AM) indicates that spontaneous secretion seems to follow a rapid kinetic pattern. It may be speculated that the Ca2+ receptors that trigger spontaneous release bind Ca2+ within the L- and N-type VDCC nanodomains, so that nearby vesicles can promptly fuse (1, 25, 35, 38, 47). Strikingly enough, although BAPTA-AM has a greater potency compared with EGTA-AM on spontaneous secretion, this effect is more readily discernible on evoked neurotransmitter release (1, 25, 35, 38, 47).
To investigate further the contribution of [Ca2+]i to the release of acetylcholine in mammalian diaphragm muscle, we modified its concentration within nerve terminals by exposing the preparations to hypertonic solutions, which produced an associated increase in MEPP frequency (7, 9, 12). This osmotic response appears unrelated to any increase in intracellular K+ concentration, which tends to hyperpolarize nerve terminals, decreasing rather than increasing MEPP frequency (19). Although no direct measurements of resting membrane potential were performed in nerve terminals, our data obtained from muscle fibers in hypertonic and isotonic salines (see RESULTS) may be extrapolated to nerve terminals.
Increase in MEPP frequency promoted by hypertonicity varies widely, but
it has been suggested that a solution of a given tonicity increases the
frequency by a fixed factor with respect to the frequency recorded in
control solution, so that the ratio of the frequency in a hypertonic
solution to the control frequency should be roughly constant, at least
for the first 15 min following the change in tonicity (9, 12, 15).
Early experiments showed that the osmotic response seems to be
independent of
[Ca2+]o
(4, 12). In agreement with these findings, our results disclosed that
the ratio between the peak osmotic response and the mean isotonic MEPP
frequency was not significantly different in control and in zero
Ca2+-EGTA, although the absolute
values of both peak response and isotonic MEPP frequency were
considerably lower in the virtual absence of external
Ca2+ (See Fig.
5A). This behavior in zero
Ca2+-EGTA may be ascribed to
reduced
[Ca2+]i
when the electrochemical Ca2+
gradient is reversed (30, 49). Moreover, when a nonspecific Ca2+ blocker (13.5 mM
Co2+) was added to zero
Ca2+-EGTA solutions, osmotic
response was similar to that recorded in control solution. It is
worthwhile to mention that nifedipine-, -CgTx-, and
-Aga-blockable channels do not seem to be involved in the
hyperosmotic response.
Under hypertonic condition, a decrease in [Ca2+]i was then achieved by loading nerve terminals with Ca2+ chelators (BAPTA-AM or EGTA-AM). In frog neuromuscular junction, it has been shown that hypertonicity markedly increases MEPP frequency, even when cytosolic Ca2+ was buffered by BAPTA-AM, suggesting that factors other than [Ca2+]i regulate transmitter release (40). This effect seems to be a species-dependent phenomenon, since in mammalian muscle the present results showed a significant decrease in osmotic response when nerve terminals were loaded with BAPTA-AM (Fig. 5B). The drop in MEPP frequency in hypertonic conditions produced by this chelator was evident whether expressed in absolute values or as a ratio between peak osmotic response and mean MEPP frequency under isotonic conditions, implying that osmotic response may be dependent on [Ca2+]i. In such condition, both BAPTA-AM and EGTA-AM behave similarly, suggesting that in hypertonic conditions Ca2+ receptors sense the increase in bulk [Ca2+]i, thereby producing the observed increase in neurotransmitter release.
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ACKNOWLEDGEMENTS |
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We thank Dr. Alberto Venosa for valuable discussion and Raquel Almirón and Maria Fernanda Rodríguez for technical assistance.
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FOOTNOTES |
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This research was supported by grants from the Consejo Nacional de Investigaciones Científicas y Tecnológicas and University of Buenos Aires.
A preliminary report on some of these observations has been published elsewhere (20).
Address for reprint requests: A. Losavio, Instituto de Investigaciones Médicas A. Lanari, Donato Alvarez 3150, 1427 Buenos Aires, Argentina.
Received 25 November 1996; accepted in final form 14 August 1997.
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