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Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee
Received December 16, 2002; accepted March 11, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). The major calcium stores in frog motor nerve
terminals include smooth endoplasmic reticulum (SER)
(Pezzati et al., 2001),
mitochondria (Calupca et al.,
2001), and secretory vesicles
(Pezzati et al., 2001). Second
messengers can activate or mobilize these stores. For example, injection of
IP3, cyclic ADP ribose (cADPR), and NAADP into the presynaptic site
of Aplysia californica motoneurons enhances transmitter output
(Chameau et al., 2001).
Similarly, liposomal delivery of IP3, cADPR, or NAADP into frog
motor nerve terminals increases spontaneous transmitter release
(Brailoiu and Miyamoto, 2000;
Brailoiu et al., 2001). In the
case of IP3, a similar enhancement in transmitter release was
reported at the crayfish glutamatergic motor synapse
(Dixon and Atwood, 1989).
Sphingomyelin is a major constituent of synaptic plasma membranes
(Cotman et al., 1969),
endoplasmic reticulum (Vale,
1980), and synaptic vesicles
(Breckenridge et al., 1973;
Deutsch and Kelly, 1981).
Similar to phosphatidylinositol bisphosphate, sphingomyelin breakdown
generates several active lipid messengers, such as sphingosine 1-phosphate
(S1-P) (Le Stunff et al.,
2002; Pyne and Pyne,
2002), sphingosylphosphorylcholine (SPC) (Meyer zu Heringdorf et
al., 1997,
2002), and ceramide
1-phosphate (Le Stunff et al.,
2002). These lipids can act as extracellular messengers to
activate G-protein-coupled receptors (Kluk
and Hla, 2002; Meyer zu
Heringdorf et al., 2002; Xu,
2002) or as intracellular second messengers to release
Ca2+ from internal stores
(Ghosh et al., 1990;
Meyer zu Heringdorf et al.,
2002), including brain microsomes
(Dettbarn et al., 1995;
Furuya et al., 1996;
Huang and Chueh, 1996).
Aside from the fact that sphingomyelin metabolism occurs in neurons
including motoneurons (Irie and
Hirabayashi, 1999), little is known regarding the possible
involvement of sphingomyelin-derived messengers in neurosecretion in general
and transmitter release in motoneurons in particular. In our previous study,
we show that intracellularly but not extracellularly applied S1-P enhanced
spontaneous transmitter release from frog motor nerve terminals, a process
that is subject to receptor desensitization
(Brailoiu et al., 2002). In
view of a role for S1-P in modulating neurosecretion in motor nerve endings
(Brailoiu et al., 2002), we
were interested to know whether or not SPC might also affect transmitter
release. If positive, does SPC act extracellularly or intracellularly or
both?
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). For other
series of experiments, D-erythro-SPC or
L-threo-SPC/methanol aliquots were dried under a
low-pressure concentrator and dissolved in 1 mg/ml fatty acid-free bovine
serum albumin (Meyer zu Heringdorf et al.,
1996). For intracellular delivery, SPC mixture,
D-erythro-SPC and L-threo-SPC were
dissolved in methanol (10–2 M stock solution). In
another set of experiments, D-erythro-SPC/BSA or
L-threo-SPC/BSA complex was entrapped in liposomes.
For intracellular administration, reverse-phase evaporation vesicles (REV
liposomes) (Szoka and Papahadjopoulos,
1978) were prepared from 60 mg/ml egg yolk phosphatidylcholine.
Drugs were entrapped into liposomes as described previously
(Brailoiu et al., 2002).
Liposome batches were dialyzed (Sigma dialysis sacs) against control Ringer's
solution [1/600 (v/v), 150 min] to remove nonincorporated agent, and the
Ringer's solution was changed every 30 min. Liposome suspensions were
administered by continuous perfusion (1.5 ml/min) after 1/20 (v/v) dilution in
control Ringer's solution.
Frogs (Rana pipiens) were decapitated and rapidly double-pithed, and sciatic-sartorius nerve-muscle preparations were isolated. Every effort was made to use the minimum number of animals required for valid statistical analyses. Procedures were reviewed and approved by the University Committee for Animal Care. Muscles were mounted in a 3-ml Sylgard-lined Petri dish bath that was continuously perfused with Ringer's solution using a dual-chambered roller pump. The Ringer's solution contained 110 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 2.0 mM Tris, pH 7.2, and 5.6 mM glucose. Ca2+-free Ringer's solution contained 108 mM NaCl, 2.5 mM KCl, 1.8 mM MgCl2, 2.5 mM EGTA, 2.0 mM Tris, pH 7.2, and 5.6 mM glucose.
MEPPs were recorded using conventional microelectrode (3 M KCl, 5–15
M
) techniques similar to those described previously
(Brailoiu and Miyamoto, 2000).
Selection of recordings was made from impalements that showed large MEPP size
(>0.3 mV), good signal-to-noise ratio (baseline peak-to-peak noise <0.1
mV), and high and stable muscle resting membrane potential (>–80 mV,
with <3 mV decline during the control period). Resting potentials ranged
between –80 and –90 mV in different fibers. Data from muscle
fibers that showed more than 10% drop in the resting membrane potential during
an experiment were not used. Experiments were conducted at the ambient room
temperature (21–22°C), and only one trial was carried out on each
muscle. Preparations were equilibrated for at least 30 min before use. Signals
were fed into a high-impedance preamplifier (A-M Systems, Carlsberg, WA) and
viewed on a R5103N oscilloscope (Tektronix, Beaverton, OR). Signal-to-noise
ratio was increased with a band-pass filter (1 kHz) and boosted for
interfacing with a data acquisition unit with 1 MHz digitization frequency (RC
Electronics, Goleta, CA). MEPPs were recorded with a modified videocassette
recorder (AM Vetter, Rebersburg, PA) for off-line analysis.
MEPP frequency represents the number of miniature endplate potentials per 60 s. MEPP amplitudes (100 samples for each time point) were measured using stored digitized data and a grid template on a flat screen monitor. To minimize the effects of junction-to-junction variation, data for each experiment were expressed as percentage of values at time 0, and results from six single experiments were averaged (plots show mean ± S.E.M.). Analysis of statistical differences was made by comparing each point with points obtained in control Ringer's solution, with p < 0.05 indicating significant differences (Student's t test followed by analysis of variance and Bonferroni's test). Relative amplitude-frequency histograms were compared using the Kolmogorov-Smirnov test for significant difference (p < 0.05).
Occasionally, MEPPs of much larger amplitude, referred to herein as giant
MEPPs, were recorded. Giant MEPPs are spontaneous potentials with amplitudes
of more than twice that of the regular MEPPs and with a slower, smoother
rising phase (Brailoiu et al.,
2002).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Discrimination between D-erythro-SPC and L-threo-SPC. Local administration of 10 µM D-erythro-SPC dissolved in DMSO (0.1% final concentration in the bath) induced an increase in MEPP frequency of 143% (Fig. 2A), with a peak effect at 5 to 6 min. This effect was stronger and more sustained than that observed with 10 µM SPC mixture. There was no significant change in median amplitude or frequency-amplitude distribution (data not shown). On the other hand, administration of 10 µM D-erythro-SPC/BSA complex had no significant effect on the frequency (Fig. 2B) and amplitude of MEPPs or the muscle resting potential. At the concentration as high as 50 µM, D-erythro-SPC/BSA complex had no significant effect on the MEPP frequency or amplitude (n = 3; data not shown).
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Local application of 10 µM L-threo-SPC dissolved in DMSO or L-threo-SPC/BSA complex had no effects on the MEPP frequency (Fig. 2, C and D), amplitude, or the muscle resting potential.
Intracellular Administration of SPC Mixture. Perfusion with
liposomes containing 10, 100, or 1000 µM SPC mixture in aqueous phase
induced a concentration-dependent increase in MEPP frequency of 45, 91, and
100%, respectively (Fig. 1B).
The final concentration within the nerve terminal was estimated to be 100-fold
lower than that in the liposome vesicle for molecules of the size of SPC
(Brailoiu et al., 2001). The
enhancing effect was phasic, with a peak after 3 min of perfusion. There was
no significant change in MEPP amplitude or the muscle resting potential. The
effect induced by SPC-filled liposomes had a faster onset compared with that
obtained by extracellular SPC application (3 min versus 5 min).
Discrimination between D-erythro-SPC and L-threo-SPC. Perfusion with liposomes containing 1 mg/ml BSA dissolved in 140 mM KCl significantly decreased the MEPP frequency by 20% (Fig. 3, B and D). Administration of liposomes containing 100 µM D-erythro-SPC increased the MEPP frequency by 117% (Fig. 3A), the effects were more sustained than that of the SPC mixture. Again, there was no significant change in the median MEPP amplitude. However, the relative frequency-histogram indicates an increase in the number of giant MEPPs (data not shown). In contrast with extracellular application of D-erythro-SPC/BSA complex, perfusion of liposomes containing 100 µM D-erythro-SPC/ BSA transiently enhanced the MEPP frequency by 91% (Fig. 3B); the MEPP amplitude or muscle resting potential was not changed by D-erythro-SPC/BSA.
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Perfusion with 100 µM L-threo-SPC or L-threo-SPC/BSA entrapped into liposomes enhanced the MEPP frequency by 67 and 61% (Fig. 3, C and D). As shown above, these compounds when applied extracellularly had no significant effects on MEPP frequency.
Extra- and Intracellular D-erythro-SPC Induced
Effect Partly Insensitive to Thapsigargin. In this series of experiments,
we used thapsigargin to produce a functional deletion of SER in the nerve
terminals. Thapsigargin inhibits Ca2+-ATPase in SER
(Takemura et al., 1989),
thereby abolishing Ca2+ release in response to
IP3 or cADPR (Berridge,
1993; Lee, 2001).
Muscles were incubated in Ca2+-free Ringer's solution
plus 2.5 mM EGTA and 1 µM thapsigargin for 30 min, followed by restoration
of muscles to normal Ca2+-containing Ringer's solution
and 1 µM thapsigargin for another 30 min.
After thapsigargin treatment, extracellular administration of 10 µM D-erythro-SPC dissolved in DMSO enhanced the transmitter release by only 74%, which is significantly smaller compared with the effect on nontreated preparations (Fig. 4A). Perfusion of liposomes containing 100 µM D-erythro-SPC on preparations pretreated with thapsigargin still induced a phasic increase in MEPP frequency of 76% (Fig. 4B). This effect was smaller, but statistically significant, compared with that observed on untreated muscles.
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Effects of Liposomes Containing D-erythro-SPC in Ca2+-Free and after Depletion of Intracellular Ca2+ Stores. After depletion of thapsigargin-sensitive Ca2+ stores (procedure described above), muscles were perfused 30 min with Ca2+-free Ringer's solution, which totally abolished neurotransmitter release. Administration of liposomes containing 100 µM D-erythro-SPC had no effect (n = 3; data not shown).
Interferences between IP3 and D-erythro-SPC. Administration of 100 µM IP3-filled liposomes induced an increase in MEPP frequency of 105% (Fig. 5), without affecting the muscle resting potential. Perfusion of liposomes containing both 100 µM IP3 and D-erythro-SPC enhanced the MEP frequency by only 70% (Fig. 5), a value that is smaller, but statistically significant, compared with that caused by IP3 or D-erythro-SPC alone. This increase was followed by a decrease (30%) below the control level in MEPP frequency after 10 min of liposome perfusion (n = 6).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Contrary to HEK-293 cells, in which
D-erythro-SPC/BSA is active
(Meyer zu Heringdorf et al.,
1998), the D-erythro-SPC/BSA complex is found
to have no significant effect on MEPP frequency at the frog nerve-muscle
junction. One possible explanation is that the D-erythro-
SPC/BSA complex is too large to diffuse into the intact synaptic cleft. This
is in contrast with cell suspension, where the molecule is likely to be
interacting directly with receptors exposed from the surface of plasmalemmal
membrane. This suggests that D-erythro-SPC might act via
plasmalemmal receptors and/or by diffusing into cytoplasm. Because both the
D- and L-isoforms act intracellularly, it is possible
that extracellularly applied D-erythro-SPC may diffuse
into cytoplasm and act on intracellular receptors. Because a stereospecific
transporter for lipids has yet to be documented, our observation that the
D- but not the L-isoform is effective when applied
extracellularly argues against the idea that SPC, irrespective of the isoform,
acts only intracellularly. As a corollary, D-erythro-SPC
probably acts via a stereospecific plasmalemmal receptor situated at the
presynaptic site. Similar to other models (for review, see
Meyer zu Heringdorf et al.,
2002), activation of these receptors induces a response partly by
mobilizing Ca2+ from thapsigargin-sensitive
Ca2+ pool.
SPC acts intracellularly to release Ca2+ from
internal stores in different types of permeabilized cell (Ghosh et al.,
1990,
1994;
Yule et al., 1993;
Kindman et al., 1994), via a
nonstereospecific receptor (Meyer zu
Heringdorf et al., 1998). SPC is also able to elicit a rapid
Ca2+ release from cerebral and cerebellar microsomes
(Dettbarn et al., 1995;
Furuya et al., 1996;
Huang and Chueh, 1996). In the
frog nerve-muscle preparations, perfusion of liposomes containing the SPC
mixture induced a concentration-dependent enhancement in MEPP frequency,
without affecting the median amplitude. The onset of MEPP frequency increase
seems to be faster (3 versus 5 min) in the case of intracellular application
compared with that caused by extracellular application. Similar to HEK-293
cells (Meyer zu Heringdorf et al.,
1998), both D-erythro-SPC and
L-threo-SPC administered intracellularly enhance the
spontaneous transmitter release. In contrast to HEK-293 cells, in which these
compounds elicit a release of Ca2+ with the same
magnitude and time course (Meyer zu
Heringdorf et al., 1998), D-erythro-SPC is
more potent than L-threo-SPC in our preparations, where
comparison was made with one single concentration. It should also be noted
that both D-erythro-SPC/BSA and
L-threo-SPC/BSA complex administered intracellularly are
active in enhancing the MEPP frequency, suggesting that SPC is coupled to
albumin. This is in contrast to extracellular application, in which only the
D-erythro-SPC is active. The presence of internal
Ca2+ stores in frog motor nerve terminals
(Pezzati et al., 2001),
together with the ability of SPC to release Ca2+ from
brain microsomes (Dettbarn et al.,
1995; Furuya et al.,
1996; Huang and Chueh,
1996) suggest that SPC enhances transmitter release by mobilizing
Ca2+ from internal stores.
Unlike other models, in which where SPC was ineffective in eliciting an
effect after depletion of intracellular Ca2+ stores
(Yule et al., 1993;
Meyer zu Heringdorf et al.,
1998), the effect of SPC is only partly inhibited by thapsigargin
pretreatment. Our observation that administration of IP3 or cADPR
induced no facilitatory effect on MEPP frequency at frog motor nerve terminals
after thapsigargin pretreatment (Brailoiu
et al., 2001) indicates that SPC may act on
thapsigargin-insensitive Ca2+ stores represented in
nerve terminals by synaptic vesicles
(Brailoiu et al., 2001) or
cause Ca2+ sensitization
(Shirao et al., 2002). It is
less likely for SPC to release Ca2+ from mitochondrial
stores because among several lipid metabolites, only arachidonic acid in lower
concentrations has been shown to be effective
(Huang and Chueh, 1996).
Moreover, SPC at high concentration (100 µM) damages the mitochondrial
functions (Strasberg and Callahan,
1988), resulting in a decrease of MEPP frequency. Because the
intracellular SPC-induced effect is abolished in the absence of extra- and
intracellular Ca2+, the possibility that the
thapsigargin-insensitive effect is caused by a heretofore-unknown
Ca2+ sensitive intracellular pathway activated by SPC
cannot be excluded.
Future studies are needed to determine whether or not SPC acts on
`sphingolipid Ca2+-release-mediating protein of the
endoplasmic reticulum' (SCaMPER), which is proposed to be an endoplasmic
reticulum Ca2+ channel
(Mao et al., 1996).
Alternatively, there is some evidence that SPC may bind to SCaMPER and
function as a modulatory protein that opens the ryanodine
Ca2+ channel (Betto
et al., 1997; Schnurbus et
al., 2002) in a manner similar to that proposed for cADPR. In the
later case, cADPR binds to FK-506 binding- or 100-kDa protein and activates
ryanodine receptors (for review, see Lee,
2001).
The observation that the increase in MEPP frequency occurs rapidly,
starting in the first minute after liposome perfusion, suggests that SPC may
activate the `recycling vesicular pool' rather than the `storage' pool.
Although the median amplitude of MEPPs is not changed by SPC, a
frequency-amplitude histogram indicates an increase in giant MEPPs, similar to
that observed in the presence of S1-P
(Brailoiu et al., 2002). This
suggests that sphingomyelin breakdown may be one of the intracellular pathways
generating giant MEPPs. More importantly, the finding that an increase in the
number of giant MEPPs is associated with intracellular application of SPC or
S1-P (Brailoiu et al., 2002),
but not extracellular application of SPC, provides additional evidence that
extracellular SPC activates a different intracellular pathway(s) than
sphingomyelin breakdown.
In other cells, the maximal Ca2+ release induced by
SPC was in the same range as that caused by IP3
(Yule et al., 1993; Meyer zu
Heringdorf et al., 1998,
2002). Similar effects were
observed in our model. Unexpectedly, the concomitant administration of SPC and
IP3 resulted in a significantly smaller increase in MEPP frequency
than these two compounds alone. This may be explained by the alteration of the
complex temporal and spatial interaction in Ca2+
signaling (Petersen and Cancela,
1999), which plays a critical role in exocytosis described by some
investigators (Hirose et al.,
1999). Concomitant application of IP3 and SPC may
inhibit Ca2+-induced Ca2+ release
mechanism, leading to an attenuated release of transmitters.
The estimated and expected concentration of S1-P and SPC in the blood is
reported to be 250 nM and 15 µM, respectively, with an approximate ratio of
2:1 (Liliom et al., 2001). Our
observation that extracellular SPC in micromolar concentrations activates
motoneuron secretion raises the possibility that circulating SPC may function
as a physiological regulator of acetylcholine release at the neuromuscular
junction.
In conclusion, our study shows that extra- as well as intracellular SPC can enhance spontaneous transmitter release at the frog neuromuscular junction, partly by activating internal Ca2+ stores. The extracellular effect is stereospecific, in that only the D-erythro-SPC is active, whereas both isoforms are active intracellularly.
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Footnotes |
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ABBREVIATIONS: SER, smooth endoplasmic reticulum; IP3, D-myo-inositol 1,4,5-trisphosphate; cADPR, cyclic adenosine diphosphate-ribose; S1-P, sphingosine 1-phosphate; SPC, sphingosylphosphorylcholine; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; MEPP, miniature end-plate potential; HEK, human embryonic kidney; SCaMPER, sphingolipid Ca2+-release-mediating protein of the endoplasmic reticulum.
Address correspondence to: Nae J. Dun, Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, PO Box 70577, Johnson City, TN 37614-1708. E-mail: dunnae{at}etsu.edu
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), 10 (
), and 25 µM(
) mixtures of SPC
administered extracellularly. The peak effect occurs 5 min after SPC
application. MEPP frequency 100% = 1.14 s–1 (1
µM SPC), 0.87 s–1 (10 µM SPC), and 1.26
s–1 (25 µM SPC). B, plots of effects of
liposomes containing 10 (
) versus 1 mg/ml BSA
entrapped liposomes (
). In contrast with extracellular application,
intracellular delivery of D-erythro-SPC/BSA complex
enhances the MEPP frequency. The maximal effect (97% increase) appears after 3
min of perfusion. Furthermore, the vehicle control (liposomes containing BSA)
induced a decrease in MEPP frequency. MEPP frequency 100% = 1.12
s–1 for liposomes containing 100 µM
D-erythro-SPC/BSA and 1.44 s–1
for liposomes containing only BSA. C, perfusion of liposomes containing 100
µM L-threo-SPC increased MEPP frequency by 67%, which
is statistically significant. Similar to D-erythro-SPC
filled liposomes, the maximal effect occurs after 3 min of perfusion. However,
note that the effect is significantly smaller than that induced by
D-erythro-SPC (67 versus 117%) and that the MEPP frequency
returns to control level before wash (e.g., at time = 8 min); n = 6.
D, plots of effects of liposomes containing 100 µM
L-threo-SPC/BSA complex (
