1Ottawa-Carleton Institute of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada; and 2Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9QG, United Kingdom
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ABSTRACT |
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Brezden, B. L.,
M. S. Yeoman,
D. R. Gardner, and
P. R. Benjamin.
FMRFamide-activated Ca2+ channels in Lymnaea
heart cells are modulated by "SEEPLY," a neuropeptide encoded on
the same gene. The cell-attached, patch-clamp technique was
used to investigate the modulatory role of the neuropeptide
SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") on FMRFamide-activated
Ca2+ channels in isolated Lymnaea heart
ventricular cells. Both SEEPLY and FMRFamide are encoded on the same
neuropeptide gene and are coexpressed in a pair of excitatory motor
neurons that innervate the heart. FMRFamide applied alone was capable
of significantly increasing the P(open) time of
a Ca2+ channel in isolated heart muscle cells. However,
SEEPLY applied alone did not significantly alter the basal level of
Ca2+ channel activity in the same cells. Repeated
applications of FMRFamide (15 s every min) resulted in a progressive
reduction in the number of Ca2+ channel openings and the
overall P(open) time of the channel. The fifth
successive 15-s application of FMRFamide failed to cause the
Ca2+ channels to open in the majority of cells tested. When
FMRFamide and SEEPLY were repeatedly applied together (2-min
applications every 4 min) the FMRFamide-activated Ca2+
channels continued to respond after the fifth application of the two
peptides. Indeed channel activity was seen to continue after repeated
2-min applications of FMRFamide and SEEPLY for as long as the patch
lasted (60 min). As well as preventing the loss of response to
FMRFamide, SEEPLY was also capable of both up- and down-regulating the
response of the Ca2+ channel to FMRFamide. The direction of
the response depended on the P(open) time of the
channel before the application of SEEPLY. When the
P(open) time for the FMRFamide-activated channel
was initially 0.004 ± 0.002 (means ± SE), subsequent
perfusion with a mixture of FMRFamide and SEEPLY produced a
statistically significant increase in Ca2+ channel activity
(13 cells). In two cells where no channel activity was observed in
response to an initial application of FMRFamide, superfusing the heart
cells with a mixture of FMRFamide and SEEPLY induced openings of the
Ca2+ channel. When the P(open) time
of FMRFamide-induced Ca2+ channel openings was 0.058 ± 0.017 the subsequent application of a mixture of SEEPLY and
FMRFamide caused a statistically significant decrease in
Ca2+ channel activity (8 cells). As up- and down-regulation
of FMRFamide-activated Ca2+ channel openings by SEEPLY were
observed in the same cells (8 cells), this suggested that corelease of
the two peptides might act together to regulate the level of
Ca2+ channel activity within a defined range.
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INTRODUCTION |
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Neuropeptides, which form the largest and most structurally
diverse group of signaling molecules, mediate an extensive array of
central and peripheral effects in the nervous system of both vertebrates and invertebrates. Neuropeptide genes are often complex with multiple exons encoding diverse sets of neuropeptides (e.g., rat
calcitonin gene) (Amara et al. 1982). Alternative mRNA
splicing may occur so that different tissues express different mRNA
species originating from this same gene (Nawa et al.
1984
). The complexity of these patterns of expression and the
colocalization of classical low molecular weight transmitters makes the
study of multiple peptides in cell signaling difficult in the
vertebrate system. The number of neurons is very large, and molecular
and cellular analysis of neuropeptide function is difficult to study at
the level of identified neurons. This is not the case in the pond snail, Lymnaea, where individual peptidergic neurons can be
repeatedly identified for both molecular and electrophysiological
analysis (Benjamin and Burke 1994
).
Recent studies of the FMRFamide gene of Lymnaea showed that
it consists of five exons (Kellett et al. 1994) with
FMRFamide and the related FLRFamide encoded exclusively on exon II
(Linacre et al. 1990
). This exon alone is expressed in a
pair of heart motor neurons (Santama et al. 1995
) whose
excitatory actions on the myogenic heart can be mimicked by the
application of FMRFamide (Buckett et al. 1990c
).
FMRFamide is therefore a candidate transmitter for the excitatory
actions of the heart motor neurons in the snail heart. Two further
types of neuropeptides are also encoded on exon II of the
Lymnaea FMRFamide gene (Linacre et al. 1990
).
One of these, the 22-amino acid peptide SEQPDVDDYLRDVVLQSEEPLY
("SEEPLY") was recently sequenced from the snail CNS and shown to
be present in the FMRFamidergic heart motor neurons and the heart with
a specific antibody (Santama et al. 1993
). It is
therefore coexpressed with FMRFamide and may be a cotransmitter.
The objective of this investigation was to examine the function of
SEEPLY on its presumed target organ, the heart, with a previously
developed dissociated heart cell preparation (Brezden et al.
1986) that allowed patch-clamp techniques to be applied to
single Lymnaea heart ventricle cells maintained in temporary cell culture. Specifically, the effect of SEEPLY on a previously characterized Ca2+ channel was examined. This was shown to
be a non-voltage-gated, second-messenger-mediated channel activated
by the application of FMRFamide (Brezden et al. 1991
).
We will show that SEEPLY alone is unable to increase the
P(open) time of the Ca2+ channels
but acts by modulating the ability of FMRFamide to open these channels.
More specifically, SEEPLY can both up- and down-regulate FMRFamide-activated Ca2+ channel activity depending on the
P(open) time of the channel in response to an
initial application of FMRFamide.
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METHODS |
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The procedure for harvesting cells from Lymnaea heart
ventricles and patch-clamp recording from the isolated cells were
described in detail by Brezden et al. (1986) and Brezden and Gardner
(1990)
. Briefly, in a single dissociation, cells were harvested by
digestion of a minimum of five heart ventricles for 30 min in 0.25%
trypsin (SIGMA type XII-S) and subsequently for 2 h in 0.1%
collagenase (SIGMA, type II-S) in modified Leibovitz's (L-15) medium
containing 0.5 mM Ca2+. The harvested cells were then
divided into four equal aliquots, and each aliquot was plated onto a
glass coverslip placed in the bottom of a 35-mm plastic petri dish and
incubated at room temperature (20-25°C) in modified L-15 medium
containing 3.5 mM Ca2+. The modified L-15 medium was
prepared by diluting 7 parts of L-15 (stock prepared from powder,
Gibco) with 18 parts of saline stock. The salt concentration of the
saline stocks was adjusted to provide on dilution with the L-15 stock
59.0 mM Na+(dissociation) or 50.0 mM
Na+(incubation), 0.5 mM Ca2+ (dissociation) or
3.5 mM Ca2+ (incubation), 1.75 mM K+, 2.0 mM
Mg2+, 10.0 mM HEPES buffer (pH 7.7-7.8), 0.1 mg/ml
gentamycin (Gibco), and 32 mM glucose. In addition, the incubation
medium contained 2% vol/vol fetal calf serum (Gibco). For patch-clamp
recording the coverslips were transferred to normal saline containing
(in mM) 50.0 NaCl, 3.5 CaCl2, 1.6 KCl, 2.0 MgCl2, and 10.0 HEPES buffer (pH 7.7-7.8).
Patch pipettes were pulled from thick-walled Corning 7052 (Kovar) glass (1.65 mm OD, 0.8 mm ID) to give tip openings of 1- to 1.5-µm diam. The tips of the patch pipettes were fire polished and coated with Sylgard 184 elastomer (Dow-Corning). Channel currents were amplified with a List EPC-7 patch clamp (Medical Systems) and recorded on videotape with an Instrutech VR-10 digital recorder for subsequent analysis with pClamp software (Axon Instruments). For dwell-time measurements the data were filtered at 2-4 kHz with a four-pole Bessel filter. The channel detection threshold was set midway between the baseline and the open channel level.
The Ca2+ currents in Lymnaea heart ventricle
cells are very small and difficult to detect. To amplify the current
flux through the Ca2+ channels, 60.5 mM NaCl and 1 mM EGTA
was used in the patch pipettes. It was established that with the
removal of external Ca2+ Na+ substitutes for
Ca2+ as the permeant ion through the Ca2+
channels in these cells (Brezden and Gardner 1990;
Brezden et al. 1991
). Consequently, although the
channels are referred to as Ca2+ channels, it should be
emphasized that the currents through these channels were in fact
carried by Na+ ions. At least two different
non-voltage-gated Ca2+ channels were previously recorded
in dissociated heart ventricle cells (Brezden and Gardner
1990
; Brezden et al. 1991
). They were separated
according to their single channel conductance into large conductance
(LG) and small conductance (SG) channels. The data presented in this
paper are from recordings of LG. These channels have previously been
shown to have at least two subconductance states with channel openings
consisting of a mixture of flickery and longer-duration openings
(Brezden et al. 1991
). Thus the apparent mixture of
unitary currents depicted in Fig. 3 does in fact represent different
modes of a single channel. Data were analyzed from recordings with a
single LG channel in the pipette.
Synthetic FMRFamide was obtained from Sigma Chemical. SEEPLY was synthesized and purified by Alta Bioscience (Dept. of Biochemistry, University of Birmingham, UK). The peptides were applied to an area away from the patch pipette by means of separate glass superfusion pipettes with the tips positioned at distances ranging from 20 to 50 µm from the cell. The pipette openings were ~1.5 µm in diameter. The pipette contents were ejected with a gas-driven automatic syringe pump that was controlled by a timing circuit. An electronic valve system allowed ejection of the contents from either of the two pipettes or both simultaneously. Marker dye studies showed that the superfusate traveled a distance of ~100 µm in ~0.5 s with the application of 3 lb. pressure.
Early experiments were performed with 107 to 10
6
M SEEPLY and 10
7 M FMRFamide. However, the results
were more consistent when the SEEPLY concentration was increased to
10
5 M. Although the results were qualitatively similar at
all SEEPLY concentrations, the data that were analyzed quantitatively
and are presented in this paper used 10
5 M SEEPLY.
Data presentation
All data were obtained from cell-attached patches. Most of the experimental data are presented as scatter plots that were derived from single sweeps of the channel activity traces. Each scatter plot represents the activity of a single channel. In these plots the duration of each individual channel opening is plotted along a time axis, designating the point in time at which the event occurred. This format provides a clear visual presentation of changes in the levels of channel activity with time and during different treatments. Note, however, that the compression of the timescale in the scatter plots may create the appearance that some events occurred simultaneously. Events that appear to be vertically aligned were in fact separated by time intervals that were too short to be clearly resolved in these scatter plots.
Quantitative results of the population data were obtained by calculating the probability of the channel being open (P(open)) for the duration of the treatment (usually 120 s), thus forming a mean P(open) value for each treatment.
Statistical analysis of data
Cells for each experiment described in RESULTS were obtained from at least four different dissections with each dissociation being the result of enzyme treatment of at least five different hearts. Cells from each separate dissociation were divided equally among four separate 35-mm petri dishes. Although data for a particular experiment were obtained from recordings of a large number of different cells, those cells were obtained from relatively few dissociations (minimum of 4 per experiment). Although we saw no gross changes in the behavior of cells from one dissociation to the next, there existed the possibility that cells recorded from a "poor" dissociation could significantly influence the population data yielding misleading results. To nullify this possible source of error, levels of significance were determined by using the number of dissociations as the N number for determining the degrees of freedom. P(open) times from cells from each dissociation were initially averaged to obtain a mean P(open) time for each dissociation. Values from different dissociations were then averaged to yield a mean P(open) value for the experiment. Significant differences among treatments were determined by using t-tests.
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RESULTS |
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SEEPLY alone does not affect Ca2+ channel activity
The application of 107 to 10
5 M SEEPLY
alone to cells that were not previously exposed to FMRFamide or SEEPLY
(naive cells) did not have any significant effect on Ca2+
channel activity (28 cells). In five of these experiments where patches
were subsequently challenged with FMRFamide, occasional spontaneous
openings could be recorded (P(open) 0.00008 ± 0.0008; means ± SE) but these were not affected by the
application of SEEPLY (P(open) SEEPLY
0.0002 ± 0.0003; P > 0.05; n = 4; see Fig. 1B). However,
FMRFamide did promote a significant increase in LG Ca2+
channel activity in those patches where SEEPLY had previously been
shown to have no affect (P(open) 0.0985 ± 0.011; n = 4) (Fig. 1, A and B).
Previous experiments demonstrated that the application of 10
7
M FMRFamide to naive heart ventricle cells is capable of
activating the same type of Ca2+ channel (LG)
(Brezden et al. 1991
). Taken together these data indicate that naive cells were capable of responding to FMRFamide but
not to SEEPLY with increased Ca2+ channel activity (see
also Brezden et al. 1991
).
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Ca2+ channel activity is lost after successive applications of FMRFamide
Although LG Ca2+ channels could be activated by
FMRFamide, the response to this peptide was invariably lost after
several successive applications (Brezden et al. 1991).
Figure 2 shows the response of an
isolated heart cell to five successive 15-s applications of 10
7
M FMRFamide (30 cells). The first and second applications were associated with a relatively large increase in Ca2+ channel
activity. However, the FMRFamide-induced activity was greatly reduced
by the third application, and the cell became essentially nonresponsive
to the fifth application. Washing the cells between applications did
not prevent this loss of response nor could the response be restored by
washing the cells for
1 h with normal saline.
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We have therefore shown that FMRFamide can regulate LG Ca2+ channel activity over two distinct timescales. Over a relatively fast timescale FMRFamide increases the P(open) time of the these channels. However, on repeated applications FMRFamide loses this ability, suggesting that over a relatively slow timescale its main effect is to decrease the P(open) time of these channels.
In the following sections we will show that the effects of SEEPLY on FMRFamide-activated Ca2+ channel activity also occurs over two distinct timescales.
SEEPLY prevents the of loss of response to repeated applications of FMRFamide
Whereas SEEPLY alone did not activate LG Ca2+ channel
activity, it did prevent the loss of response of these Ca2+
channels to FMRFamide (12 cells; n = 5). The
experimental data traces presented in Fig.
3 show that the response to FMRFamide was
maintained if the cell was also exposed to SEEPLY. This cell was
exposed to five 240-s application cycles consisting of a 120-s application of 107 M FMRFamide followed by a 120-s
application of 10
5 M SEEPLY + 10
7 M
FMRFamide for a total exposure time to FMRFamide of 20 min. Only the
fifth (last) exposure cycle is shown. The channel activity at ~41 s
into the fifth application of FMRFamide is shown in Fig. 3A.
After ~90 s of exposure the activity subsided considerably (Fig.
3B) before the application of FMRFamide + SEEPLY at 120 s. The trace in Fig. 3C shows the increased level of
activity ~20 s after the exposure to SEEPLY + FMRFamide. The
response to FMRFamide persisted even up to 20 min of continuous
exposure, in contrast to the rapid loss of response observed in the
absence of SEEPLY (cf. Fig. 2). In Fig. 4
the population data from the experiments described in Figs. 2 and 3 are
plotted as a histogram allowing a comparison of the mean
P(open) times for the fifth application of both
FMRFamide (P(open) 4.9 × 10
6 ±
5.72 × 10
7; n = 7) and FMRFamide + SEEPLY (P(open) 0.024 ± 0.003;
n = 5). Statistical analysis of these data showed that
channel activity after the fifth application of FMRFamide + SEEPLY was
significantly greater than that seen after the fifth application of
FMRFamide alone (P < 0.05). Although a statistical
comparison of the data was only performed after the fifth application
of the peptides, SEEPLY-treated cells were seen to respond to FMRFamide
for as long as the patch was viable, which was
1 h.
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It is important to note that, although the coapplication of SEEPLY and FMRFamide can prevent the loss of Ca2+ channel activity seen when FMRFamide is repeatedly applied alone, once FMRFamide induced a loss of response it cannot be recovered by the subsequent application of SEEPLY (data not shown).
The ability of SEEPLY to prevent the loss of response of the LG Ca2+ channel to repeated applications of FMRFamide is suggestive that its actions occur over a realtively slow timescale. However, SEEPLY like FMRFamide was also capable of either up- or down-regulating FMRFamide-activated Ca2+ channel activity over a relatively fast timescale.
SEEPLY can up-regulate the FMRFamide response
The presence of an LG Ca2+ channel in a patch could be
recognized by unitary inward currents that appeared on the formation of
a gigaseal. These inward currents usually disappeared shortly after
seal formation but could be reactivated by superfusing the cell with
FMRFamide (Brezden et al. 1991). FMRFamide-induced
activity was often characterized by bursts of channel activity (e.g.,
see Figs. 1 and 5). However, the first
application of FMRFamide to different naive cells produced markedly
different degrees of Ca2+ channel activity. Sometimes the
FMRFamide-evoked activity was relatively low. When the
P(open) value of FMRFamide-induced activity was
0.004 ± 0.002 (13 cells; n = 4) the application
of SEEPLY during FMRFamide treatment promoted an increase in
Ca2+ channel activity. Figure 5 shows the data from an
experiment where the response to FMRFamide was initially weak. The
subsequent application of 10
5 M SEEPLY during FMRFamide
exposure significantly increased the Ca2+ channel activity
(P < 0.05).
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As is evident from the scatter plots, in the absence of FMRFamide
Ca2+ channel open dwell times were generally very brief
(e.g., see Fig. 1). The application of FMRFamide or FMRFamide + SEEPLY
not only increased the frequency of the channel openings but also appeared to increase the open dwell time. However, the low frequency of
the channel openings in naive cells in these experiments made it
impossible to examine this observation statistically. In earlier investigations, where this question was addressed directly, it was
shown that the open channel dwell-time kinetics were identical for
spontaneous and FMRFamide-activated channels with Na+ as
the charge carrier (Brezden and Gardner 1990;
Brezden et al. 1991
).
The Ca2+ channel density in isolated Lymnaea heart cells appeared to be relatively low as usually only a single channel was present in a patch, and no inward channel currents could be detected in ~20% of the cells. In the absence of any background spontaneous activity it was assumed that no Ca2+ channels were present. However, in two cells where a single 300-s application of FMRFamide alone initially failed to activate Ca2+ channels, the subsequent application of SEEPLY conferred a response to FMRFamide (Fig. 6), suggesting that Ca2+ channels were present in these patches but the channels were not initially responsive to FMRFamide. Therefore although SEEPLY alone had no effect on Ca2+ channel activity it was able to induce FMRFamide sensitivity to previously nonresponsive channels. The small number of cells in which this phenomenon was observed prevented any detailed statistical analysis.
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SEEPLY can down-regulate the FMRFamide response
Our evidence suggests that SEEPLY can also down-regulate, or
reduce, the level of FMRFamide-activated LG Ca2+ channel
activity. When the P(open) value of
FMRFamide-induced activity was "high" (mean = 0.058 ± 0.017; 8 cells; n = 4) the application of SEEPLY during
FMRFamide treatment resulted in a reduction of the frequency of channel
openings. An example of this type of result is shown in Fig.
7 where the application of 105
M SEEPLY during FMRFamide exposure significantly reduced
FMRFamide-induced Ca2+ channel activity (P < 0.05). There also appeared to be a reduction in the open dwell time
of the channel, but as in the previous example there were not enough
data to perform a statistical analysis.
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SEEPLY can up- and down-regulate the FMRFamide response in the same cell
Up- and down-regulation of the FMRFamide response by SEEPLY
was often seen in the same cell. In the experiment shown in Fig. 8, 107 M FMRFamide was
applied continuously, and 10
5 M SEEPLY was applied at
regular intervals for 240 s with intervening periods when
FMRFamide was applied alone. The repeated applications of SEEPLY
maintained a basic responsiveness to FMRFamide (see Figs. 3 and 4) on
top of which the shorter term effects of SEEPLY application could be
seen. The first SEEPLY application produced a significant increase in
Ca2+ channel activity (P < 0.05, t-test); the second application was associated with a
reduction in activity (P < 0.05, t-test).
It should be noted that, although SEEPLY can both up- and down-regulate FMRFamide-induced Ca2+ channel activity, spontaneous bursts
or spontaneous cycling of activity was not observed during long (2 min)
applications of SEEPLY (Figs. 3 and 4) within the lifetime of the
patches (
1 h).
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The results of experiments with 32 applications of SEEPLY and SEEPLY + FMRFamide to 13 different cells (from 4 dissociations; a minimum of 20 different hearts) are summarized in Fig. 9. The first important observation is that the application of FMRFamide allowed us to distinguish two populations of cells whose mean P(open) times are significantly different from one another (P < 0.05). In the case of cells that showed up-regulation (20 episodes) the mean Ca2+ channel P(open) during FMRFamide treatment was 0.004 ± 0.002. The coapplication of FMRFamide and SEEPLY significantly increased the P(open) to 0.027 ± 0.011 (P < 0.05). Where down-regulation was observed (12 episodes) the mean P(open) was 0.058 ± 0.017 during FMRFamide treatment, which was significantly decreased to 0.013 ± 0.004 by superfusion with a combination of FMRFamide and SEEPLY (P < 0.05). It should be noted that there is no significant difference between the P(open) values of the two FMRFamide + SEEPLY data sets, indicating that SEEPLY functions to maintain FMRFamide-activated Ca2+ channel activity at some defined level.
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SEEPLY's ability to cause both up- and down-regulation of
FMRFamide-gated channel activity is extremely unusual, and so it was
important to have unequivocal validation of the data. Because there
were fewer examples of down-regulation there existed the possibility,
although remote, that these episodes were the result of either a single
"poor" dissociation or a difference in the properties of cells in
different petri dishes. To eliminate batch variability as a factor we
performed a further statistical analysis on the data. This involved
randomly assigning the data from each cell to one of four groups rather
than assigning them to groups based on the dissociation they came from.
As described previously, both FMRFamide-treated groups were
significantly different from one another (P < 0.05),
indicating that we were dealing with two distinct populations of cells.
In cells that showed up-regulation P(open) times
were increased from 4.39 × 103 ± 7.98 × 10
4 to 2.67 × 10
2 ± 6.45 × 10
3 (P < 0.05), whereas cells in which
down-regulation was observed showed a decrease in the mean
P(open) time from 5.78 × 10
2 ± 1.4 × 10
2 to 1.25 × 10
2 ± 2.74 × 10
3 (P < 0.05). By using this second type of analysis, there was still no
significant difference between the two FMRFamide + SEEPLY data
sets, indicating that up- and down-regulation of channel activity was
to a common range of values. This more rigorous analysis clearly
demonstrates that SEEPLY's effects on FMRFamide-gated channel activity
were not due to problems with particular batches of cells.
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DISCUSSION |
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SEEPLY modulates FMRFamide-action in Lymnaea heart muscle cells
Our observation that FMRFamide can over a fast timescale
increase the P(open) time of the LG
Ca2+ channel provides confirmation of previous work by
Brezden et al. (1991). The application of SEEPLY alone to isolated
heart muscle cells had no effect on LG Ca2+ channel
activity. However, when SEEPLY was applied together with FMRFamide to
isolated heart muscle cells, FMRFamide-induced Ca2+ channel
activity was either increased or decreased, depending on the level of
FMRFamide-induced activity present immediately before the coapplication
of the two peptides. In only 1 of 32 applications was the channel
activity not significantly changed. To quantify these results with the
probability of the channel being open as an indicator of activity, it
was found that when P(open) was <0.004 ± 0.002, SEEPLY + FMRFamide increased the Ca2+ channel
activity, whereas if P(open) was >0.058 ± 0.017 the application of SEEPLY + FMRFamide decreased the
FMRFamide-induced activity. It is unlikely that down-regulation was the
result of competition between SEEPLY and FMRFamide for the same
receptor or of dilution of the FMRFamide by SEEPLY because both up- and
down-regulation were observed in the same cell. Both up- and
down-regulation are examples of the actions of SEEPLY that occur over a
relatively fast timescale.
Over a fast timescale FMRFamide was capable of increasing the P(open) time of the LG Ca2+ channel; however, repetitive applications of FMRFamide demonstrated that over a slower timescale its main effect was to decrease channel activity and eventually cause a complete loss of response. This led to the possibility that the down-regulation of channel activity that we saw in response to application of SEEPLY (Figs. 7 and 8, 2nd SEEPLY application) might in fact be due to these slower effects of FMRFamide. However, this is unlikely to be the case as the coapplication of SEEPLY and FMRFamide prevented any loss of FMRFamide-induced channel activity. The ability of SEEPLY to prevent a loss of response occurred over a relatively slow timescale and was distinct from its faster effects (up- and down-regulation). It was interesting to note that, although SEEPLY was unable to reverse the loss of response seen after repeated applications of FMRFamide, it was capable of inducing FMRFamide sensitivity to two cells that were initially unresponsive after a single application of FMRFamide. The prevention of the loss of the FMRFamide response by SEEPLY and the ability of SEEPLY to confer FMRFamide sensitivity to previously nonresponsive cells can be considered as further examples of up-regulation. The mechanism underlying the initial lack or subsequent loss of response to FMRFamide is not known but is unlikely to involve a common mechanism as SEEPLY is unable to reverse the loss of response seen after repeated applications of FMRFamide.
The ability of SEEPLY to both up- and down regulate FMRFamide-activated LG channel activity suggests that there may be a physiological "set-point" value for P(open) of the Ca2+ channel that the release of transmitter(s) is aiming to achieve. It appears that FMRFamide alone cannot produce a predictable Ca2+ channel P(open) response on a target muscle cell but that FMRFamide plus SEEPLY can achieve this goal. If the response to FMRFamide was low then SEEPLY increased P(open); if the response to FMRFamide was high then SEEPLY reduced P(open) (Figs. 7-9). This implies that a mechanism with the properties of a negative feedback system is operating to achieve a specific value of P(open) for the Ca2+ channel. A more accurate way of describing these results would be to consider that a range of P(open) values is achieved by the feedback mechanism rather than a specific set point or single value. We assume that normally FMRFamide and SEEPLY would be released together to allow the proposed control system to operate, and we recently obtained evidence in our laboratory to show the Ca2+-dependant corelease of both peptides from the heart (A. C. Dobbins, unpublished data).
The nature of this feedback mechanism is not known, and it is
postulated here solely as a working hypothesis. Up- and down-regulation of the activity of a nonspecific cationic channel was previously described by Wilson and Kaczmarek (1993). By using excised inside-out patches from the bag cell neurons of Aplysia the authors
demonstrated that application of the catalytic subunit of protein
kinase A (PKA) was capable of either up- or down-regulating channel
activity, depending on the initial activity of the channel. The authors referred to the change in activity as "mode switching" as
application of PKA either changed channel activity from a bursting (low
P(open)) mode to a high activity (high
P(open)) mode or converted a channel in
high-activity mode to one with bursting kinetics and a correspondingly lower P(open). The initial activity state of the
channel was shown to be a consequence of tyrosine phosphorylation, with
phosphorylated channels exhibiting bursting kinetics and
unphosphorylated channels being tonically active. Mode switching was
regulated by the actions of a PKA-regulated tyrosine phosphatase. In
the absence of sufficient data it is impossible to determine whether
SEEPLY is inducing mode switching of the sort described Wilson and
Kaczmarek (1993)
, although previous work by Brezden et al. (1991)
suggests this is unlikely. However, our observation that the LG
Ca2+ channel appears to be regulated in a similar way is
suggestive that this may be a common means of regulating channel activity.
Previous work on molluscan muscle has shown that modulation either
involves different neuropeptides affecting different channel types or
the same channel to different degrees (Brezina et al. 1994a,b
) or acting at the presynaptic site to regulate
transmitter release (Cropper et al. 1988
, 1990
). As is the case
for FMRFamide, the action of SEEPLY is also likely to be mediated by a
second messenger as modulation of the FMRFamide response is evident
when SEEPLY is applied outside the patch pipette. In neither case are the messenger systems associated with either FMRFamide or SEEPLY known.
However, the complex interactions of the two peptides and their
different roles, SEEPLY modulating the ability of FMRFamide to open
Ca2+ channels, may mean that different second-messenger
systems are involved.
Comparison with other systems
FMRFamide activates sarcolemmal Ca2+ channels in
isolated Lymnaea heart cells, and application of FMRFamide
often causes contraction in the same cells (Brezden et al.
1991). Application of FMRFamide to the whole heart increases
the rate of myogenic heart beat as well as the underlying tonus
(Buckett et al. 1990a
). Although the effects of
FMRFamide are likely to be complex, it is reasonable, on the basis of
comparison with other muscle systems, that the increased influx of
Ca2+ would be associated with an increase in the strength
of contraction either by direct association of externally derived
Ca2+ with the contractile apparatus or by
Ca2+-activated release of Ca2+ from
intracellular stores. Invertebrate smooth and cardiac muscle contraction is blocked by dihydropyridines and other Ca2+
channel blockers, and entry of extracellular Ca2+ is
equally important in a variety of molluscan muscles that were examined
(e.g., Hill et al. 1970
; Ram et al.
1984
). This suggests that the entry of extracellular
Ca2+ is required for contraction of molluscan muscles.
In Lymnaea, FMRFamide is thought to be acting as the
major transmitter for a pair of excitatory heart motor neurons called the Ehe cells (Buckett et al.
1990c). Immunocytochemical, radioimmunological (Buckett
et al. 1990a
,c
), and molecular analysis (Santama et al. 1995
) showed that FMRFamide is present in the cell body of the Ehe cells and nerve terminals in the heart.
Large quantities of FMRFamide are present in the heart (mean
concentration of 31.8 pmol/mg wet weight of tissue) (Buckett et
al. 1990a
). Application of FMRFamide to the heart closely
mimics the effect of neuronal stimulation so that both can initiate
beating in a quiescent heart or modulate beat rate and tonus in a
similar manner. In this system FMRFamide is considered to act as a
primary transmitter and is therefore behaving in a similar fashion to
vasoactive intestinal peptide (Wakade et al. 1991
) in
vertebrates. This is unlike the invertebrate muscle systems that were
studied such as the buccal musculature of Aplysia where ACh
is the primary transmitter in several types of motor neurons
(Cohen et al. 1978
). In the Aplysia accessory
radula closer (ARC) system, ACh causes contraction by depolarizing the
muscle and causing Ca2+ influx (Brezina and Weiss
1993
; Ram and Parti 1985
), but in
Lymnaea FMRFamide a peptidergic rather than a classical, low
molecular weight transmitter causes Ca2+ influx by
activating a membrane Ca2+ channel. In the
Lymnaea heart ACh inhibits the heartbeat and is the
transmitter of a completely different motor neuron type (Buckett
et al. 1990b
).
As well as primary transmitters such as ACh (Cohen et al.
1978) and glutamate (Bishop et al. 1991
),
invertebrate motor neurons also contain neuropeptides that act as ion
channel modulators. In crayfish proctolin is colocalized with glutamate
(Bishop et al. 1984
, 1987
) in three excitatory motor
neurons innervating the muscle of the animal's abdomen. Proctolin does
not itself induce tension in the muscle but acts directly on the muscle
to amplify the effect of glutamate. Only when glutamate first
depolarizes the muscle membrane does proctolin modulate muscle
contraction. Bishop et al. (1991)
showed that proctolin is able to act
on Ca2+ channels, which are first activated by
depolarization, to prolong their period of activity. This probably
plays an important role in the potentiation of tension in the crayfish
muscle. In some ways this action of proctolin resembles that of SEEPLY
in Lymnaea heart muscle cells. SEEPLY alone cannot open
Ca2+ channels but when combined with FMRFamide
influences the activity of the Ca2+ channels. However,
unlike proctolin, SEEPLY appears to be able to up- or down-regulate
Ca2+ channel activity, depending on the initial response to
FMRFamide. This is seen as part of a negative feedback system to
maintain a particular level of Ca2+ channel activity,
whereas in the case of proctolin the muscle response is always
potentiated and prolonged by the action of the modulator. Similar
enhancement of a Ca2+ current was recently reported in the
ARC muscle of Aplysia by Brezina et al. (1994a)
. The
modulator myomodulin increases the size of a voltage-gated
Ca2+ current in the muscle and modulates the contraction of
the muscle evoked by the primary transmitter ACh. The complexity of the
system is increased by the fact that myomodulin can also activate a
K+ current in the same muscle cells (Brezina et al.
1994b
). The possibility that FMRFamide may also influence more
than one channel type in Lymnaea heart muscle was not yet
examined but could account for the complexity of the FMRFamide effect
on the whole heart (increase in tonus, beat rate, and ability to
initiate beating).
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ACKNOWLEDGMENTS |
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B. Brezden and D. R. Gardner were supported by Natural Sciences and Engineering Research Council of Canada Grant A-4290. P. R. Benjamin and M. S. Yeoman were supported by Biotechnology and Biological Sciences Research Council Grant GR/J33234.
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FOOTNOTES |
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Address for reprint requests: P. R. Benjamin, Sussex Center for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton, E. Sussex BN1 9QG, U. K.
Received 6 October 1997; accepted in final form 15 December 1998.
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REFERENCES |
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