Committee on Neurobiology and Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Fox, Lyle E. and Philip E. Lloyd. Role of cAMP in the Short-Term Modulation of a Neuromuscular System in Aplysia. J. Neurophysiol. 83: 1567-1579, 2000. Neuromuscular synapses in buccal muscle I3a of Aplysia are modulated by the small cardioactive peptide (SCP), a peptide cotransmitter that is intrinsic to the motor neurons, and by serotonin (5-HT) released from modulatory neurons that are extrinsic to the motor circuit. Although the modulation of excitatory junction potentials (EJPs) and contractions by 5-HT and SCP has been studied extensively in this muscle, little is known about the mechanisms that underlie the modulation. 5-HT and SCP, at 1 µM, were found to potently increase the level of cAMP in I3a. Therefore we investigated whether the activation of the cAMP pathway was sufficient to modulate EJPs and contractions. The direct activation of adenylyl cyclase with forskolin increased the level of cAMP, facilitated EJPs, and potentiated contractions. Indeed, the short-term effects of forskolin were very similar to all aspects of the short-term effects of 5-HT and SCP. Membrane-permeable cAMP analogues also mimicked the effects of 5-HT and SCP on EJPs and contractions. However, it seems likely that some effects of 5-HT are also mediated through other second-messenger pathways because low concentrations of 5-HT modulate EJPs and contractions but do not significantly increase cAMP levels in I3a. It is possible that lower concentrations of 5-HT function through receptors linked to protein kinase C (PKC) because phorbol, an activator of PKC, modulated EJPs and contractions without increasing the levels of cAMP. In conclusion, we provide evidence that pharmacological agents that activate the cAMP pathway mimicked most of the effects of 5-HT or SCP and that more than one second-messenger system appears to be involved in the modulation of the I3a neuromuscular system.
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INTRODUCTION |
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One mechanism utilized by the nervous system to
modify behavior is the modulation of synaptic transmission. There is
increasing evidence that major sites for this modulation in both
vertebrates and invertebrates are peripheral at neuromuscular synapses
(Calabrese 1989; Worden 1998
). Peripheral
modulation has been examined extensively in the muscles that control
feeding movements in Aplysia (Weiss et al.
1992
, 1993
; Whim et al. 1993
).
Many of the motor neurons that drive ingestive and egestive movements
have been identified, and all of them express modulatory peptide
cotransmitters (Church and Lloyd 1991
,
1994
; Cohen et al. 1978
; Gardner
1971
). In addition, buccal muscle is innervated by purely
modulatory serotonergic neurons termed the metacerebral cells (MCCs)
(Weiss et al. 1978
). Therefore these muscles are
modulated by neuropeptides intrinsic to the motor neurons and by
serotonin (5-HT) released from modulatory neurons that are extrinsic to
the motor circuit (Church and Lloyd 1994
; Church
et al. 1993
; Cropper et al. 1987
,
1990
; Lloyd et al. 1984
,
1987
; Weiss et al. 1978
; Whim and
Lloyd 1989
, 1990
).
The preparation we have chosen to study consists of a muscle (termed
the intrinsic anterior muscle 3, I3a) that participates in the closing
of the jaws. It is innervated by two identified excitatory motor
neurons B3 and B38, which use glutamate as their fast transmitter and
also express modulatory peptide cotransmitters; B3 expresses FMRFamide,
and B38 expresses the small cardioactive peptides (SCP). All the muscle
fibers in I3a are functionally innervated by both motor neurons;
however, we do not know whether individual fibers are anatomically
innervated by both neurons because the muscle fibers are electrically
coupled (Church and Lloyd 1991; Church et al.
1993
; Fox and Lloyd 1999
; Lotshaw and Lloyd 1990
). I3a is also innervated by the two MCCs, a
bilateral pair of large serotonergic neurons that have extensive
central and peripheral synaptic outputs (Eisenstadt et al.
1973
; Weinreich et al. 1973
; Weiss and
Kupfermann 1976
). Release of peptides from the motor neurons
and 5-HT from the MCCs acts peripherally to modulate excitatory
junction potentials (EJPs) and contractions of I3 (Church et al.
1993
; Fox and Lloyd 1998
; Keating and
Lloyd 1999
; Lotshaw and Lloyd 1990
).
Specifically, these modulatory transmitters enhance the speed and
strength of biting responses. EJPs and contractions of I3a muscle
fibers are also modulated by application of exogenous neuropeptide
cotransmitters and 5-HT (Church et al. 1993
; Fox
and Lloyd 1997
; Lotshaw and Lloyd 1990
). In
previous studies, we have concentrated on the actions of 5-HT and SCP.
Both modulators produce similar short-term effects including the
following: the selective facilitation of B38-evoked EJPs; an increase
in the amplitude and relaxation rate of contractions evoked by either
B3 or B38; and a decrease in the latency between the onset of a motor
neuron burst and the onset of the resulting contraction, which is
larger for B38 than for B3.
We continue our study of the I3a neuromuscular system in an attempt to
determine the second-messenger pathways that are involved in mediating
the effects of 5-HT or SCP. Because they have similar short-term
effects on EJPs and contractions, it is possible that they act through
the same second-messenger system. We concentrated on the cAMP pathway
because cAMP levels in I3a are increased by the exogenous application
of 5-HT or SCP as well as by release of endogenous SCP from motor
neurons when they are stimulated in a manner similar to their activity
recorded during feeding-like motor patterns (Church and Lloyd
1994; Church et al. 1993
; Keating and
Lloyd 1999
; Lotshaw and Lloyd 1990
). In
addition, cAMP working through protein kinase A (PKA) has been shown to
mediate many, but not all, of the effects of 5-HT and SCP in another
Aplysia buccal muscle preparation (Brezina et al.
1994a
,b
; Probst et al. 1994
). Here we provide
evidence that the cAMP pathway mediates most of the effects of SCP and
some of the effects of 5-HT in I3a. However, it seems likely that some
of the effects of 5-HT are also mediated through other pathways.
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METHODS |
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Animals
Aplysia californica (60-300 g) were obtained from Marinus (Long Beach, CA), maintained in circulating artificial sea water (ASW) at 16°C, and fed dried seaweed every 3 days.
Neuron stimulation
Detailed experimental methods have been described previously
(Fox and Lloyd 1997). Briefly, animals were immobilized
with an injection of isotonic MgCl2 and the
dissection carried out in high Ca2+ (33 mM; 3 times normal), high Mg2+ (165 mM; 3 times normal)
ASW (termed high Ca, Mg ASW). The buccal mass and buccal ganglia were
removed and the mass bisected along the midline. Buccal nerve 2, which
contains the peripheral axons of B3 and B38, was left intact (nerve
designations from Gardner 1971
; muscle nomenclature from
Howells 1942
; also see Lloyd 1988
). The
ganglia were desheathed and superfused with low
Ca2+ (0.5 mM; 0.05 times normal) high
Mg2+ (110 mM; 2 times normal) ASW (termed low Ca
ASW). Neurons were normally impaled with two microelectrodes (2-4
M
; filled with 3 M K acetate), one to inject current and one to
monitor membrane potential. B3 and B38 were identified by their
position, size, and muscle innervation patterns (Church et al.
1993
). All experiments were performed at room temperature
(~22°C). Individual spikes in motor neurons were driven by brief
(10-20 ms) depolarizing current pulses. The frequency of action
potentials within a burst was usually 16 Hz. The burst durations were
adjusted so that the compound EJPs or contractions evoked by B3 or B38
were similar in amplitude. Many experiments were carried out by
alternatively stimulating bursts in B3 and B38 at 50-s intervals (100-s
intervals for each neuron). These long interburst intervals were used
to minimize release of endogenous peptide cotransmitters and
posttetanic potentiation (Church et al. 1993
;
Lotshaw and Lloyd 1990
; Whim and Lloyd
1990
).
Measurement of I3a contractions
The bath containing the I3a muscle was superfused with ASW and was separated from the ganglia by a partition through which ran the intact nerve containing the axons of the motor neurons. Transmitters and/or pharmacological agents were dissolved in ASW and applied via the superfusion. Typical application periods were 20 min to ensure adequate penetration into the muscle. Note that the partition prevented the ganglia from being exposed to these substances. Contraction amplitudes were monitored with an isotonic transducer (Harvard Apparatus), and submaximal contractions were evoked by stimulating B3 or B38. Burst durations were adjusted (from 0.5 to 4 s) so that contractions evoked by B3 or B38 were similar in amplitude.
Measurement of I3a EJPs
EJPs were recorded with a perfusion electrode (Church et
al. 1993; Fox and Lloyd 1997
). The perfusion
electrode consisted of a small chamber (100 µl) and aperture (~1.5
mm) that was positioned to press firmly down on a portion of the muscle
(see Fig. 1 in Church et al. 1993
). The inside of this
electrode was perfused with ASW, whereas the rest of the preparation
was superfused with low Ca ASW to suppress synaptic transmission and
muscle contractions. This procedure confined the contractions to the
small area of the muscle covered by the recording chamber and thus
markedly reduced movement artifacts in the recordings. The earliest
evoked muscle contractions occur after the sixth EJP so the early EJPs in a burst are recorded in the absence of any movement. EJPs were recorded by extracellular electrodes placed inside and just outside the
wall of the perfusion apparatus. Signals were amplified using a Grass
P15D AC amplifier. Transmitters and/or pharmacological agents were
applied in ASW to the inner chamber of the perfusion electrode so the
ganglia and the remainder of the muscle were not exposed to these
substances. Typical application periods were also 20 min. This
procedure permits us to simultaneously record from a population of
muscle fibers thereby reducing sampling bias. Burst durations were
adjusted (from 0.15 to 2 s) so that the compound EJPs evoked by B3
or B38 were similar in amplitude.
Measurement of cAMP
Muscle segments were dissected in high Ca, Mg ASW, weighed (~3
mg each), and washed in several changes of ASW for 1 h. Segments were incubated in ASW, transmitter, or pharmacological agents for 20 min (the same period used in most physiological experiments), extracted
immediately in 2% 2N HCl in ethanol at 30° C, homogenized, and
centrifuged at 10,000 × g. The supernatants were used
for duplicate cAMP determinations using a commercial radioimmunoassay (Biomedical Technologies, Norwood, MA).
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RESULTS |
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5-HT and SCP increase the level of cAMP in isolated I3a
Brief application (5.5 min) of both 5-HT and SCP potently increase
cAMP levels in the I3a muscle (Lotshaw and Lloyd 1990). However, in subsequent physiological experiments, we found that 20-min
applications of the modulators were necessary to maximally facilitate
EJPs or potentiate contractions (Fox and Lloyd 1997
, 1998
). Accordingly, we repeated the cAMP measurements
using 20-min applications. At 1 µM, both 5-HT and SCP dramatically
increased the level of cAMP in I3a muscle segments more than 250-fold
over control (Fig. 1). The cAMP levels
were ~10-fold higher measured after 20 min than those reported by
Lotshaw and Lloyd (1990)
for the shorter incubation
suggesting that the levels of cAMP continue to rise. Lower
concentrations of SCP (0.1 µM) were also effective at increasing cAMP
levels (46 ± 13-fold increase over control, this is equivalent to
a 4,500% increase, n = 8), but 0.1 µM 5-HT had no
significant effect on these levels (20 ± 11% increase, mean ± SE, n = 5; P > 0.05, t-test; Fig. 1). This steep dose response curve
for 5-HT, in which 0.1 µM essentially has no effect on cAMP levels
and 1 µM has very large effects, has also been observed in a previous
study (Lotshaw and Lloyd 1990
). Because 0.1 µM 5-HT had large effects on EJPs and contractions (Fox and Lloyd
1998
), this suggests that 5-HT may act through mechanisms other
than the cAMP pathway. This preparation contains both muscle fibers and
the axons and terminals of neurons that innervate them. The large
increases in cAMP levels are of such a magnitude that they must occur
in the muscle fibers because they constitute nearly the total volume of
the muscle. It is unlikely that an increase in cAMP exclusively in the
axons and terminals of neurons could account for the measured increase
in cAMP levels because of their small volume. This conclusion is
supported by work on another buccal muscle in Aplysia
(termed I5 or ARC). 5-HT and SCP increase the level of cAMP in
dissociated I5 muscle fibers and modulate their whole cell currents
recorded by voltage clamp (Brezina et al. 1994a
,b
).
Although most of the increase in cAMP must be in the muscle fibers, it
is possible that the cAMP levels are also increased in the axons or
terminals of the motor neurons.
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Forskolin selectively facilitates B38-evoked EJPs and modulates contractions of both neurons
Superfusion with 5-HT or SCP has a number of effects including
facilitation of EJPs, potentiation of contractions, increased rate of
muscle relaxation, and a decrease in the latency between the onset of a
burst and the onset of the evoked contraction (Fox and Lloyd
1997). Although 5-HT and SCP had similar effects on the
amplitude of contractions evoked by both motor neurons, they facilitated B38-evoked EJPs much more than those of B3. If cAMP mediates all of the actions of 5-HT and SCP, then activators of the
cAMP pathway should also mimic these effects. We focused on the
facilitation of EJPs to screen the effects of activators of the cAMP
pathway. Because B38-evoked EJPs are potently facilitated by the 5-HT
and SCP, they served as a sensitive assay for the effects of the
reagents tested, whereas B3-evoked EJPs, which are only slightly
facilitated, served as a control for possible nonspecific effects.
Elevation of cAMP levels by activation of adenylyl cyclase with
forskolin selectively facilitated B38-evoked EJPs in a
concentration-dependent manner. At 10 µM, forskolin slightly
facilitated B3-evoked EJPs and potently facilitated those of B38 (Figs.
2 and 3).
We believe that this selective facilitation was caused by the
activation of adenylyl cyclase because 10 µM forskolin increased the
levels of cAMP in I3a muscle segments by 191 ± 46%
(n = 8) and a forskolin analogue that does not activate
adenylyl cyclase but has many of forskolin's nonspecific effects (10 µM 1,9-dideoxy-forskolin) (Laurenza et al. 1989) had a
much smaller effect on EJPs (Figs. 2 and 3). Lower concentrations of
forskolin (1 µM) also facilitated the B38-evoked EJPs more than those
of B3 (Fig. 3). Two other active forskolin derivatives were tested;
7-deacetyl-6-(N-acetylglycyl)-forskolin (DAAG; 75 µM)
because it has fewer nonspecific effects than forskolin (Baxter
and Byrne 1990b
) and
7-deacetyl-7-(O-(N-methylpiperazino)-
-butyryl)-forskolin (DMPB; 100 µM) because it is more soluble and more stable in water (Laurenza et al. 1987
). As reported elsewhere, it was
necessary to use higher concentrations of these forskolin derivatives
because they are less effective than forskolin at activating adenylyl cyclase (Baxter and Byrne 1990b
; Laurenza et al.
1987
). Both DAAG and DMPB facilitated B38-evoked EJPs much more
than those of B3 (Fig. 3). Therefore forskolin and its analogues that
activate adenylyl cyclase selectively facilitated B38-evoked EJPs,
presumably by increasing cAMP levels.
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Because forskolin mimicked the effects of 5-HT and SCP on EJPs, we next examined whether it also modulated evoked contractions. At 10 µM, forskolin potentiated both B3- and B38-evoked contractions (by 850 ± 88% for B3, n = 4; by 1,455 ± 420% for B38, n = 5; Fig. 4). It also increased the relaxation rate of the contractions and reduced the latency between the onset of the motor neuron burst and the onset of the evoked contraction. The relaxation of the contractions was well fitted to a single exponential, and the time constant was calculated only for regions in which the amplitude of control and potentiated contractions overlapped. Forskolin reduced the relaxation time constant by 32 ± 8% (n = 4) for contractions evoked by B3 and by 29 ± 14% for those evoked by B38 (n = 5). As for the effects of 5-HT and SCP, it is difficult to measure small changes in the relaxation rate because of the large change in the contraction amplitude. The effects of forskolin on the latency were similar to the application of 5-HT or SCP in that the latency was reduced more for B38-evoked contractions than those of B3. The latency of B3-evoked contractions were reduced by 20 ± 7% (n = 4), whereas the B38-evoked contractions were reduced by 54 ± 5% (n = 5; Fig. 4). These effects of forskolin appeared to be caused by activation of adenylyl cyclase because 1,9-dideoxy-forskolin (10 µM) had little effect on the amplitude, latency, or relaxation rate of contractions (Fig. 4). Thus the effects of forskolin were similar to the effects of 5-HT and SCP: it increased the level of cAMP, it selectively facilitated B38-evoked EJPs, it potentiated B38-evoked contractions somewhat more than those evoked by B3, it reduced the latency more for B38 than B3, and it increased the relaxation rate of contractions for both neurons. These observations, combined with the effects of 5-HT and SCP on cAMP levels, suggest that these modulators act, at least in part, through the cAMP pathway.
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Membrane-permeable cAMP analogues selectively facilitate B38-evoked EJPs and modulate contractions of both neurons
We next investigated whether membrane-permeable cAMP analogues
selectively facilitated B38-evoked EJPs. We used a cAMP analogue, cpt-cAMP, that has been used extensively in Aplysia and
other animals (Baxter and Byrne 1990a; Goldsmith
and Abrams 1992
; Rydel and Greene 1988
;
Weiss et al. 1979
). Two concentrations (100 and 500 µM) were tested and found to facilitate EJPs evoked by B38 more than
those evoked by B3 (Figs. 5 and
6). The facilitation does not appear to
be mediated by the extracellular effects of cpt-cAMP because cAMP (500 µM), which poorly diffuses across the cell membrane (Sandberg
et al. 1991
), had no effect on EJPs. Because cpt-cAMP is
hydrolyzed by phosphodiesterases (Sandberg et al. 1991
)
and high concentrations of it have nonspecific effects on ion channels
(Brezina et al. 1994a
,b
; Lambert and Harrison
1990
; Sugita et al. 1994a
), we tried to reduce
these effects by using the lower concentration while simultaneously
slowing its degradation with phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX). The facilitation of EJPs produced
by 100 µM cpt-cAMP was significantly enhanced by IBMX. Simultaneous
application of IBMX (100 µM) with cpt-cAMP selectively facilitated
B38-evoked EJPs much more effectively than either cpt-cAMP or IBMX
alone (Fig. 6). We believe that this facilitation was caused by the
activation of PKA and not the extracellular actions of cpt-cAMP or IBMX
because the coapplication of structurally related molecules, cAMP and
adenosine (each at 100 µM), had no effect on EJPs. In addition, the
purinergic receptors agonists ATP and adenosine (each at 100 µM) did
not facilitate EJPs.
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Our working hypothesis was that cpt-cAMP and IBMX were activating
PKA. However, it was possible that they were acting through the cGMP
pathway because both cpt-cAMP and IBMX inhibit the cGMP-specific phosphodiesterases leading to an elevation of cGMP levels, and cpt-cAMP
directly activates cGMP-dependent protein kinase (PKG) (Beavo
and Reifsnyder 1990; Connolly et al. 1992
;
Gillespie and Beavo 1989
; Sandberg et al.
1991
). We tested for this by using a second more specific
activator of PKA and by comparing the effects of PKA and PKG activators
on the same preparations.
Sp-5,6-dichloro-1-
-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate (cBiMPS), is a potent and specific activator of PKA (Schultz et al. 1994
). It is not necessary to use IBMX in these experiments because cBiMPS is resistant to hydrolysis by phosphodiesterases (Sandberg et al. 1991
). cBiMPS (100 µM) facilitated
B3-evoked EJPs by 122 ± 51%, whereas it dramatically facilitated
B38-evoked EJPs by 1,442 ± 999% (n = 3;
B38-evoked EJPs were facilitated in all the experiments; however, the
facilitation was quite variable ranging from a minimum of a 4-fold to a
maximum of 35-fold; Fig. 5). Thus a second agent that directly
activates PKA mimicked the short-term effects of 5-HT and SCP by
selectively facilitating B38-evoked EJPs. Although cpt-cAMP can
activate the cGMP pathway by inhibiting cGMP-specific phosphodiesterase
and directly activating PKG, the effects of cpt-cGMP are more specific
in that it is a poor inhibitor of phosphodiesterases and a poor
activator of PKA (Connolly et al. 1992
; Francis
et al. 1988
; Sandberg et al. 1991
). Therefore
the contribution of the cGMP pathway to the facilitation produced by
cpt-cAMP can be determined by comparing the effects of cpt-cAMP to
those of cpt-cGMP. In four experiments, the effects of IBMX alone, a
cpt-cAMP/IBMX mixture, and a cpt-cGMP/IBMX mixture were compared on the
same preparation and found to selectively facilitate B38-evoked EJPs
(Fig. 6B). Whereas the cpt-cAMP/IBMX mixture was much
more effective at facilitating EJPs than IBMX alone, the effects of the
cpt-cGMP/IBMX mixture on B38-evoked EJPs was not significantly
different from IBMX alone. These results indicate that in this
preparation cpt-cAMP appears to act through the cAMP pathway.
The membrane-permeable cAMP analogues also potentiated contractions
evoked by B3 or B38. cpt-cAMP (500 µM) potentiated contractions evoked by both neurons to a similar degree (Figs.
7 and 8).
The effects of cpt-cAMP on contraction latency and relaxation rate were
inconsistent. The latency was significantly reduced only for the
B3-evoked contractions and the relaxation time constant only for
B38-evoked contractions (Fig. 8). The weak effects of cpt-cAMP on
contractions might be due to limited diffusion across the cell membrane
or its rapid degradation by phosphodiesterases (Sandberg et al.
1991). We believe that potentiation was not mediated by the
extracellular effects of cpt-cAMP because cAMP (500 µM), which poorly
diffuses across the cell membrane (Sandberg et al. 1991
), had no effect on contractions. Potentiation of
contractions by cpt-cAMP was enhanced and made more consistent by
co-application with IBMX. Simultaneous application of a lower
concentration of cpt-cAMP (100 µM) with IBMX (100 µM) increased
both B3- and B38-evoked contractions to a degree similar to the higher
concentration of cpt-cAMP alone. Surprisingly, contractions evoked by
stimulating B3 were potentiated more than those of B38 (Figs. 7 and 8).
The latency and the relaxation time constant were also reduced to a
similar degree for both neurons (Figs. 7 and 8). The potentiation caused by cpt-cAMP/IBMX application was not due to activation of
extracellular purinergic receptors because the application of cAMP and
adenosine (each at 100 µM) had no effect on contractions. Thus the
short-term effects of cpt-cAMP/IBMX, although not identical, were
qualitatively similar to the short-term effects of 5-HT and SCP. They
selectively facilitated B38-evoked EJPs and increased the relaxation
rate of contractions for both neurons. Although cpt-cAMP/IBMX
potentiated contractions and reduced the latency for both neurons, the
effects on B3-evoked contractions were larger than predicted based on
the actions of 5-HT and SCP in this preparation. These differences
could be due to some nonspecific effects of cpt-cAMP (see
DISCUSSION).
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In summary, there is good evidence that activation of the cAMP pathway can modulate EJPs and contractions in buccal muscle I3a. The short-term effects of 5-HT and SCP were mimicked well by procedures that increased cAMP levels or by cAMP analogues. Taken together with the observation that 5-HT and SCP (at 1 µM) potently elevate the cAMP concentration in the muscle, these results suggest that 5-HT and SCP modulate EJPs and contractions, at least in part, via the cAMP second-messenger pathway.
Activation of the protein kinase C (PKC) pathway facilitates EJPs, but is not selective for B38
Some of the effects of 5-HT appear to be mediated by a
second-messenger system other than cAMP pathway because low
concentrations of 5-HT (0.1 µM) effectively modulate EJPs and
contractions (Fox and Lloyd 1998
), whereas they have
little effect on cAMP in the muscle. Because the activation of both PKA
and PKC by 5-HT has been implicated in the facilitation of central
sensory neuron synapses in Aplysia (Byrne and Kandel
1996
; Ghirardi et al. 1992
; Sugita et al.
1992
, 1997a
), we examined whether the activation of PKC facilitated EJPs. Indeed, 4
-phorbol 12,13 diacetate
(
-PDA), used at concentrations necessary to reliably activate
PKC-mediated processes in intact ganglia (3 µM) (Sugita et al.
1992
, 1994b
), dramatically facilitated EJPs
evoked by both neurons to a similar degree (Figs.
9 and
10). This differs from 5-HT and SCP,
which selectively facilitated B38-evoked EJPs. Lower concentrations of
-PDA (0.3 µM) were also effective at facilitating EJPs evoked by
the two neurons (Fig. 10). This facilitation appears to be due to
activation of PKC because a phorbol isomer that does not activate PKC,
4
-phorbol 12,13 diacetate (
-PDA; 3 µM), did not affect EJPs
evoked by either neuron (Fig. 10). A second phorbol derivative that is
more selective in activating PKC, 12-deoxyphorbol 13-isobuterate (DPIB;
0.3 µM), also facilitated EJPs evoked by both neurons (Fig. 10). Thus
activation of PKC appears capable of facilitating EJPs, but was not
selective for B38.
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We also looked at the effects of kinase inhibitors on EJPs. The broad
spectrum kinase inhibitor H7 (Hidaka et al. 1984) had variable effects but clearly facilitated EJPs in some preparations (by
as much as 367% at 100 µM and 471% at 500 µM). Chelerythrine, a
relatively specific PKC inhibitor (Herbert et al. 1990
),
also facilitated EJPs (increased 29 ± 3% at 30 µM and 111 ± 10% at 100 µM; n = 2 for each). We did not
further pursue the use of these inhibitors because they caused
facilitation themselves.
Activation of the PKC pathway potentiates contractions, but does not increase relaxation rate
Next we examined the effects of phorbol on the properties of
contractions evoked by B3 and B38. -PDA (3 µM) dramatically changed the dynamics of contractions in that short bursts that were
barely above threshold for evoking a contraction before
-PDA application produced near maximal contractions that relaxed extremely slowly after its application. Therefore it was necessary to use lower
concentrations to study its effects on contractions. We found that
-PDA, at 0.03 and 0.3 µM, dramatically increased contraction amplitude and reduced the latency for both neurons (Figs.
11 and 12). However, unlike 5-HT and SCP,
phorbol did not increase the relaxation rate of the contractions.
Indeed, the relaxation rate actually decreased during the 0.03 and 0.3 µM
-PDA applications for both neurons (Fig. 12). We believe that
these effects were caused by the activation of PKC because
-PDA (3 µM), the inactive phorbol isomer, had no significant effects on the
amplitude, latency, or relaxation rate of contractions evoked by either
neuron (Figs. 11 and 12).
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Many adenylyl cyclases interact with and are regulated by other
second-messenger systems including the diacylglycerol (DAG)/PKC pathway
(Cooper et al. 1995; Pieroni et al.
1993
). Indeed, activation of PKC with phorbol increased the
level of cAMP in Aplysia sensory neurons (Sugita et
al. 1997b
). Because phorbol can activate the cAMP pathway, we
tested whether it increased the concentration of cAMP in I3a.
-PDA
does not appear to act through the cAMP pathway because it only
slightly increased the level of cAMP by 13 ± 9% at 0.3 µM
(n = 4; P > 0.05) and 24 ± 12%
at 3 µM (n = 4; P > 0.05). Thus
facilitation of EJPs by phorbol differs from the actions of 5-HT, SCP,
and activators of the cAMP pathway because phorbol facilitated the EJPs
and reduced the contraction latency of both motor neurons to a similar
degree. The nonselective facilitation of EJPs and the nonselective
reduction in latency caused by phorbol supports our hypothesis that
selective facilitation of B38-evoked EJPs leads to the selective
reduction in latency produced by 5-HT, SCP, and agents that activate
the cAMP pathway. Modulation by phorbol, without a concurrent increase
in the cAMP levels in the muscle, suggests that there are mechanisms
other than the cAMP pathway that can modulate EJPs and contractions.
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DISCUSSION |
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The effects of SCP appear to be mediated by the activation of the
cAMP pathway. SCP, at concentrations as low as 0.01 µM, increased the
level of cAMP in I3a (Lotshaw and Lloyd 1990), and activation of the cAMP pathway, with agents that bypass the SCP receptor and directly activate either adenylyl cyclase or PKA, mimicked
the short-term effects of SCP. Activation of the cAMP pathway also
mimicked the short-term effects of 1 µM 5-HT; however, it is unlikely
that the effects of lower concentrations of 5-HT (
0.1 µM) are
mediated by this pathway because, at these concentrations, 5-HT has
little effect on the cAMP levels. Because phorbol dramatically facilitated EJPs and potentiated contractions, it is possible that low
concentrations of 5-HT activate the PKC pathway.
Many of the effects of 5-HT or SCP are mediated postsynaptically in the
I3a muscle fibers. In previous studies, 5-HT and SCP were shown to
increase the relaxation rate of contractions, potentiate contractions
evoked by the bolus application of glutamate to isolated bundles of
muscle fibers, and modulate some step(s) in excitation-contraction coupling because they potentiate B3-evoked contractions even when the
B3-evoked EJPs were essentially unchanged (Church et al.
1993; Fox and Lloyd 1997
; Lotshaw and
Lloyd 1990
). In the present study we have shown that 1 µM
5-HT or SCP elevate cAMP levels in the I3a neuromuscular preparation
over 250-fold above control values. This preparation contains both
muscle fibers and the axons and terminals of neurons that innervate
them. Considering the magnitude of the increase in the cAMP levels, it
must occur primarily in muscle fibers because they represent nearly the
entire tissue volume. It is unlikely that an increase in cAMP
exclusively in the axons and terminals of neurons could account for the
measured increase in cAMP because of their small volume. It is still
possible that cAMP levels increased in the axons and terminals of
neurons; however, in this preparation, the concentration of cAMP in
neuronal terminals cannot be measured independently. Observations made in another buccal muscle in Aplysia (termed I5 or ARC)
support the conclusion that most of the increase in cAMP levels is
postsynaptic. 5-HT and SCP also potently increase the level of cAMP in
I5, and an increase in cAMP was also observed in dissociated muscle
fibers (Brezina et al. 1994a
,b
; Lloyd et al.
1984
; Weiss et al. 1979
; Whim and Lloyd
1989
). In addition, application of 5-HT and SCP also leads to
the phosphorylation of a contractile protein that is believed to
mediate the increase in relaxation rates (Probst et al.
1994
).
Two different procedures were used to bypass the 5-HT and SCP
receptors and stimulate the cAMP pathway, activation of adenylyl cyclase by forskolin and application of cAMP analogues. Both procedures yielded similar results. Individually, each of the agents used in this
study may have effects unrelated to their actions on the cAMP pathway.
Forskolin activates adenylyl cyclase but has also been shown to affect
both voltage-gated and ligand-gated currents in a cAMP-independent
manner (Baxter and Byrne 1990b; Brezina et al.
1994a
,b
; Laurenza et al. 1989
). We used
dideoxy-forskolin, which has many of the cAMP-independent effects of
forskolin but does not activate adenylyl cyclase as a control and found
it had little effect. Dideoxy-forskolin (100 µM) does significantly
reduce voltage-gated currents in another buccal muscle (Brezina
et al. 1994a
,b
); however, we used lower concentrations and
found that they had little effect on EJPs and contractions in I3a. In
addition, two hydrophilic derivatives of forskolin that activate
adenylyl cyclase but have fewer nonspecific effects were also found to mimic forskolin. cpt-cAMP also has effects unrelated to its actions on
the cAMP pathway. It has been shown to inhibit cGMP-specific phosphodiesterases (Connolly et al. 1992
), directly
activate PKG (Sandberg et al. 1991
), and affect both
voltage- and ligand-gated channels in a cAMP-independent manner
(Brezina et al. 1994a
,b
; Lambert and Harrison
1990
). Some of these PKA-independent effects appear to be
associated with the p-chlorophenylthio moiety because cpt-cGMP had
similar effects, whereas cAMP, 8-bromo-cAMP, or 8-bromo-cGMP do not
(Sugita et al. 1994a
). Although cpt-cAMP has many
nonspecific effects, we believe that its effects on EJPs and
contractions were mediated by the cAMP pathway for several reasons.
1) BiMPS, a different membrane-permeable cAMP analogue that
does not activate PKG and does not contain the p-chlorophenylthio
moiety (Sandberg et al. 1991
), also selectively
facilitated B38 evoked EJPs. 2) cpt-cGMP is much less
effective at facilitating EJPs than cpt-cAMP. 3) IBMX and
cpt-cAMP do not act extracellularly at purinergic receptors. Two native
purinergic agonists, adenosine and ATP, had no effect on EJPs even
though they were used at the same concentration as IBMX and cpt-cAMP
and are structurally similar to them. When the results are viewed
collectively, they provide good evidence in support of a prominent role
for the cAMP pathway in mediating many of the actions of 5-HT or SCP.
Cyclic nucleotides gated channels have been found in a variety of
sensory and nonsensory tissues including vertebrate muscle and
invertebrate neurons (Biel et al. 1994,
1999
; Kehoe 1990
; Price and
Goldberg 1993
; Sudlow et al. 1993
; Zufall
et al. 1997
). Therefore it is possible that cAMP modulates EJPs
and contractions by directly gating ion channels. We could not
determine whether cAMP acted directly on channels, if it acted through
PKA, or a combination of both mechanisms. However, we believe that PKA
has an important role in the modulation of I3a contractions based partially on results obtained in another buccal muscle (I5). In both
muscles, 5-HT and SCP increase the levels of cAMP, increase contraction
amplitude, and increase the relaxation rates of contractions (Fox and Lloyd 1997
; Kupfermann 1997
;
Lotshaw and Lloyd 1990
; Weiss et al.
1992
). In I5, SCP application leads to the activation of PKA,
and application of either SCP or 5-HT causes the phosphorylation of a
contractile protein termed twitchin (Hooper et al. 1994
; Probst et al. 1994
). The degree and time course of the
phosphorylation of twitchin correlates well with the increase in the
relaxation rates (Probst et al. 1994
). By contrast, the
role of PKA in the facilitation of EJPs is uncertain because the locus
of facilitation has not been identified in I3, and EJPs in I5 are only
slightly facilitated by 5-HT and SCP making it a poor model for
facilitation (Fox and Lloyd 1997
; Lloyd et al.
1984
; Weiss et al. 1978
).
Second-messenger systems other than the cAMP pathway may be involved in
the plasticity at I3a neuromuscular synapses. Phorbol isomers that
activate PKC modulated EJPs and contractions evoked by both B3 and B38.
However, the effects of phorbol were different from those of 5-HT or
SCP because phorbol facilitated EJPs and reduced the latency of
contractions equally for both motor neurons. Phorbol also did not
increase the relaxation rate of contractions. The differences between
the actions of phorbol and 5-HT do not preclude PKC as a mediator of
the effects of 5-HT. It is possible that B3 synapses do not express
5-HT receptors linked to this pathway or that phorbol activates PKC
isoforms that are not normally activated by 5-HT. For example, sensory
neurons in Aplysia contain both Ca-dependent and
Ca-independent isoforms of PKC, and 5-HT activates only the
Ca-dependent isoform of PKC, but phorbol activates both isoforms
(Kruger et al. 1991; Sossin et al. 1993
;
Sossin and Schwartz 1992
). Our results also suggest that
5-HT could act through second-messenger pathways other than the cAMP
pathway. First, 0.1 µM 5-HT and SCP have large modulatory effects on
I3a (Fox and Lloyd 1998
, Lotshaw and Lloyd
1990
), but 5-HT does not significantly elevate the level of
cAMP, whereas 0.1 µM SCP causes a large increase in cAMP levels. In
addition, activation of the PKC pathway facilitated EJPs and
potentiated contractions without elevating cAMP levels. Both of these
second-messenger pathways probably participate in the modulation of
feeding by the MCCs because the maximal short-term effects of MCC
stimulation were similar to those of 0.1-0.3 µM 5-HT (Fox and
Lloyd 1998
); however, our data suggest that the
cAMP-independent pathway is more important at physiological
frequencies. Of course, the possibility remains that 5-HT might
function solely through cAMP if, for example, the increase in cAMP
caused by low concentrations of 5-HT was highly compartmentalized.
In many ways, these results are reminiscent of the facilitation
observed in the central ganglia of Aplysia. PKA and PKC are thought to be involved in mediating the 5-HT-induced facilitation of
excitatory postsynaptic potentials (EPSPs) between sensory and motor
neurons (Byrne and Kandel 1996). The two pathways were once thought to be activated serially with activation of the PKA leading to PKC activation (Goldsmith and Abrams 1991
).
Results from other work suggest that both pathways are activated in
parallel (Braha et al. 1993
; Ghirardi et al.
1992
; Sacktor and Schwartz 1990
). More recent
evidence suggests that both pathways function in parallel but that
there is considerable cross-talk between them. Indeed, activation of
PKC with phorbol attenuates subsequent responses to 5-HT and raises
cAMP concentrations in sensory neurons (Sugita et al.
1997b
). Activation of PKC has also been shown to increase
adenylyl cyclase activity and increase the cAMP concentration in other
systems (Cooper et al. 1995
; Pieroni et al.
1993
). However, this does not appear to be the case in the I3a
neuromuscular preparation because phorbol had little effect on cAMP
levels. Furthermore, the effects of phorbol on EJPs were different from
the effects of activators of the cAMP pathway. Phorbol equally
facilitated EJPs evoked by B3 and B38, whereas 5-HT, SCP, forskolin,
and cAMP analogues selectively facilitated B38-evoked EJPs.
Modulation of EJPs and contractions by both the cAMP and PKC
pathways has been observed in two other well-studied invertebrate neuromuscular preparations. In lobster, 5-HT facilitates EJPs, increases the tone of muscle, and increases the level of cAMP in both
motor neurons and muscle fibers (Goy and Kravitz 1989; Goy et al. 1984
; Hempel et al. 1996
).
Activation of the cAMP pathway with forskolin, IBMX, or 8-bromo-cAMP
only partially mimicked the actions of 5-HT. These pharmacological
agents had no effect on muscle tone and did not facilitate EJPs to the
same degree as 5-HT, suggesting that a component of the facilitation
was cAMP-independent (Goy and Kravitz 1989
). Similar
results have been obtained from the crayfish neuromuscular junction.
The facilitation induced by 5-HT in the crayfish has been divided into
two phases: early and late. Although the early phase of facilitation is
mimicked by activators of the PKC pathway, the late phase facilitation requires the activation of both the cAMP and PKC pathways (Dixon and Atwood 1989a
-c
). In addition, the cAMP pathway also
modulates vertebrate muscle preparations. Activation of the cAMP
pathway modulates electrical and contractile properties of the heart
and skeletal muscles (McDonald et al. 1994
;
Osterrieder et al. 1982
; Rodger and Bowman
1983
; Tsien 1977
).
In conclusion, we provided additional evidence that pharmacological agents that activate the cAMP pathway mimicked most of the effects of 5-HT or SCP. However, it seems likely that some effects of 5-HT are also mediated through another second-messenger pathway because low concentrations of 5-HT modulate EJPs and contractions but do not increase cAMP. It is possible that lower concentrations of 5-HT function through receptors linked to the PKC pathway because phorbol modulated EJPs and contractions without increasing the levels of cAMP. Therefore more than one second-messenger system appears to modulate synaptic efficacy in the I3a neuromuscular system.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Mental Health Grant 1-F31-MH-10656 to L. E. Fox and IBN-9728453 to P. E. Lloyd.
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FOOTNOTES |
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Address for reprint requests: P. E. Lloyd, Committee on Neurobiology, University of Chicago, 947 E. 58th St., Chicago, IL 60637.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 September 1999; accepted in final form 4 November 1999.
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REFERENCES |
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