FMRFamide-Activated Ca2+ Channels in Lymnaea Heart Cells Are Modulated by "SEEPLY," a Neuropeptide Encoded on the Same Gene

B. L. Brezden,1 M. S. Yeoman,2 D. R. Gardner,1 and P. R. Benjamin2

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-7 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SEEPLY alone does not affect Ca2+ channel activity

The application of 10-7 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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Fig. 1. The neuropeptide SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY") alone does not activate Ca2+ channels. A: application of 10-5 M SEEPLY for 120 s did not affect the Ca2+ channel activity. However, the subsequent application of 10-7 M FMRFamide alone for 120 s did promote an increase in the channel activity. The data are shown as a scatter plot where each point represents a single Ca2+ channel opening along the time axis. B: bar chart showing the mean P(open) times of Ca2+ channel activity under control conditions (spontaneous openings) or after the application of 10-5 M SEEPLY or 10-7 M FMRFamide. Bars represent the means ± SE; asterisk indicates P < 0.05.

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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Fig. 2. Ability of FMRFamide to activate Ca2+ channels in isolated heart cells is reduced and eventually lost with successive applications of this peptide. The heavy horizontal bars mark the time and duration of application of 10-7 M FMRFamide (5 separate15-s applications). The first application of FMRFamide promoted a large increase in Ca2+ channel activity. However, the response to FMRFamide was reduced during subsequent applications and was virtually abolished by the last application. There was no intervening wash between FMRFamide applications in this experiment.

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 10-7 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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Fig. 3. SEEPLY prevents the loss of response to FMRFamide. Successive applications of 10-7 M FMRFamide and 10-5 M SEEPLY + 10-7 M FMRFamide were not associated with a loss of response, as was the case when FMRFamide was applied alone. The experimental records are extracts from the last 4 min of a 20-min experiment. FMRFamide was applied throughout. SEEPLY + FMRFamide was applied at regular intervals of 120 s. A and B: declining activity in the presence of FMRFamide alone. C: increased activity after the application of FMRFamide in the presence of SEEPLY. Inward currents are represented by downward deflections. Scale bar: 200 ms, 2 pA.



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Fig. 4. Comparison of channel activity after repeated applications of FMRFamide or FMRFamide + SEEPLY. The mean P(open) times of the Ca2+ channel are plotted after the fifth application of either 10-7 M FMRFamide or 10-7 M FMRFamide + 10-5 M SEEPLY. Bars represent the means ± SE; asterisk indicates P < 0.05.

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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Fig. 5. SEEPLY can up-regulate the FMRFamide response; 10-7 M FMRFamide was applied throughout (top bar). Initially the FMRFamide-induced Ca2+ channel activity was relatively low. The subsequent application of 10-5 M SEEPLY + 10-7 M FMRFamide at 80 s (bottom bar) was associated with an increase in the Ca2+ channel activity.

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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Fig. 6. SEEPLY confers FMRFamide sensitivity to cells that are initially not responsive to FMRFamide. In this experiment the cell was exposed to 10-7 M FMRFamide for 420 s (top bar). There was no response to FMRFamide alone during the first 300 s of application. However, the coapplication of 10-5 M SEEPLY + 10-7 M FMRFamide at 300 s (bottom bar) was associated with an increase in Ca2+ channel activity.

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 10-5 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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Fig. 7. SEEPLY can down-regulate the FMRFamide response; 10-7 M FMRFamide was applied throughout (top bar). Initially the FMRFamide-induced Ca2+ channel activity was relatively high. The subsequent application of 10-5 M SEEPLY + 10-7 M FMRFamide at 120 s (bottom bar) was associated with a decrease in the Ca2+ channel activity.

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, 10-7 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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Fig. 8. SEEPLY can up- or down-regulate FMRFamide-induced Ca2+ channel activity in the same cell; 10-7 M FMRFamide was applied throughout (top bar); 10-5 M SEEPLY + 10-7 M FMRFamide was applied for 240 s at 240-s intervals (bottom bars). The first application of SEEPLY + FMRFamide was associated with increased Ca2+ channel activity, whereas the second SEEPLY + FMRFamide application decreased this activity.

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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Fig. 9. Summary of the results showing up- and down-regulation of the FMRFamide-induced Ca2+ channel activity by SEEPLY. This figure summarizes the results with alternating applications of 10-7 M FMRFamide and 10-5 M SEEPLY + 10-7 M FMRFamide to isolated heart cells. The data are shown as the probability of the channel being open during the exposure to FMRFamide alone and to exposure to SEEPLY + FMRFamide as calculated over the interval of either FMRFamide or SEEPLY + FMRFamide exposure (60-240 s). Values plotted are the means ± SE. In both instances the FMRFamide and FMRFamide + SEEPLY data bars are significantly different from each other at the P < 0.05 level. Mean P(open) values for the 2 FMRFamide + SEEPLY bars are not significantly different. The 1 instance where SEEPLY + FMRFamide did not significantly change the channel activity is not included in this figure.

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 × 10-3 ± 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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ABSTRACT
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