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Department of Physiology, David Geffen School of Medicine at University of California-Los Angeles, Los Angeles, California 90095-1751
Address all correspondence and requests for reprints to: Nancy L. Wayne, Department of Physiology, Room 53-231, Center for Health Sciences, David Geffen School of Medicine at University of California-Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1751. E-mail: nwayne{at}mednet.ucla.edu.
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Introduction |
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The BCNs are an excellent model for investigating the functional relationship between changes in membrane potential (Vm), prolonged neurohormone secretion, and neurohormone biosynthesis because they are large in size and easily identifiable in intact CNS, making electrophysiological recordings simple, and their secretion of large amounts of hormone and synthesis of that hormone can be assayed quantifiably (1, 2). Synaptic stimulation of BCNs stimulates a pattern of repetitive and synchronous action-potential firing, called an electrical afterdischarge, which triggers exocytotic secretion of the neuropeptide egg-laying hormone (ELH) (3, 4, 5, 6). Our previous work showed that activation of an afterdischarge, which typically lasts 10–30 min in duration, leads to prolonged elevations in ELH secretion and persistent membrane depolarization, both of which continue for at least 40 min after the end of action-potential firing (6, 7). Blocking Ca2+ influx inhibits action-potential firing but has no effect on either ELH secretion or the timing of the post-afterdischarge membrane repolarization (7, 8, 9), suggesting that afterdischarge-induced Ca2+ influx is not playing an important role in maintaining prolonged ELH release. Furthermore, activation of an afterdischarge leads to a robust increase in the rate of ELH synthesis through stimulation of translation of an abundant and stabile supply of ELH mRNA (10). This earlier work suggests the possibility that persistent membrane depolarization both maintains prolonged ELH secretion in a Ca2+-independent manner and stimulates ELH synthesis to replenish supplies lost due to its secretion. To test this hypothesis in the present study, BCN preparations were treated with a low Na+ artificial seawater (ASW) to rapidly repolarize Vm, and the effects on Ca2+ signaling, ELH secretion, and ELH biosynthesis were determined. Further experiments using low Ca2+ ASW were conducted to determine the role of Ca2+ influx on ELH biosynthesis.
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Materials and Methods |
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Bag cell preparations (n = 6–7 per group) were maintained and treated with the following solutions. Unless otherwise noted, chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). The pH of all external solutions ranged from 7.65–7.80; the pH of solutions used in the intracellular recording/microinjection electrode was 7.4. Filtered, normal ASW contained 395 mM NaCl, 10 mM KCl, 10 mM CaCl2, 50 mM MgCl2, 28 mM Na2SO4, 30 mM HEPES, and 5000 U/liter penicillin-streptomycin. Na+ concentration was reduced to 15% in the low Na+ ASW, which contained 12 mM NaCl, 10 mM KCl, 10 mM CaCl2, 50 mM MgCl2, 30 mM HEPES, and 438 mM N-methyl-D-glucamine. Free Ca2+ concentration was reduced to 39 nM in the low Ca2+/EGTA ASW (8), which contained 419 mM NaCl, 10 mM KCl, 5 mM CaCl2, 50 mM MgCl2, 28 mM Na2SO4, 30 mM HEPES, and 10 mM EGTA. Two-percent Protenate (Baxter Healthcare Corp., Glendale, CA) and a cocktail of peptidase inhibitors (25 mg/100 ml each: bacitracin, type II-0 ovomucoid/ovoinhibitor trypsin inhibitor from chicken egg white, and type III-0 ovomucoid trypsin inhibitor from chicken egg white) were added to ASW solutions before the secretion experiments. Osmolarities of the ASW solutions (1025 mOsm) were checked with a Vapro osmometer (Wescor, Inc., Logan, UT) to make sure that they were equivalent. The microelectrode solution contained 0.5 M KCl and 10 mM HEPES. The calcium-indicator dye Fura-PE3 (TEF Labs, Inc., Austin, TX) was dissolved in the 0.5-M KCl solution for a concentration of 10 mM in the microelectrode, which minimized the volume needed for microinjection.
Sample collection for ELH secretion and electrophysiology recording
Details of this procedure are described in Michel and Wayne (7). Briefly, the abdominal ganglion containing the bilateral BCN clusters and attached pleurovisceral connective nerves were dissected from the animal and placed in a flow-through recording chamber filled with 1 ml ASW solution. Temperature in the recording chamber was maintained at 21–22 C throughout the experiments. The artery leading to the abdominal ganglion and BCN clusters was cannulated, and solutions were then perfused throughout the experiment at a rate of 10 µl/min. At the end of each experiment, Fast Green (Sigma Chemical Co., St. Louis, MO) was added to the perfusate, and its passage through abdominal ganglion and BCN clusters was monitored to determine whether the perfusion was successful. Perfusion through the artery assured that ELH was maximally flushed out of the vascular space and into the surrounding medium and that solution was delivered rapidly to the intact bag-cell clusters (6, 8). Solution surrounding the preparation was completely exchanged via a low-flow perfusion pump every 5 min, starting during the pretreatment period and ending 90 min after onset of the afterdischarge. Therefore, each sample contained 5 min worth of secretory material. Previous work showed that this perfusion set-up provided 95% recovery of a known amount of protein by the second 5-min fraction (7). Samples were stored at -20 C until RIA.
Afterdischarges were electrically stimulated with a suction electrode placed on the pleurovisceral connective nerve, and standard procedures for extracellular recording of BCN compound action potentials and intracellular recording of Vm were used. Only BCNs showing resting Vm of at least -40 mV were used. Electrical recording began at least 10 min before electrical stimulation. Treatment with the low Na+ ASW began 60 sec after onset of the afterdischarge and continued until the end of the experiment. We used ionic manipulation of Vm because injection of hyperpolarizing current into a single BCN does not spread sufficiently to the other electrotonically connected neurons in the cluster. The particular low Na+ treatment used in these experiments was chosen because it caused an approximate 15-mV hyperpolarization from resting levels in pilot studies (data not shown). Microinjection of the calcium indicator dye Fura-PE3 was achieved by valve-controlled pressure application through the same microelectrode used for monitoring Vm.
Optical imaging of cytosolic Ca2+ ([Ca2+]i)
Details of this procedure are described in Michel and Wayne (7). Briefly, BCNs were imaged using a cooled charge-coupled-device camera (Sensicam; PCO Computer Optics, Kelheim, Germany) controlled by a personal computer-based imaging and analysis software (Slide-Book; Intelligent Imaging Innovations, Denver, CO). Cells were filled with Fura-PE3 calcium indicator dye by microinjection until the fluorescence intensity measured at 340 nm over 20 msec reached 2500 arbitrary units. This provided a sufficient signal without overloading the cells (7). The same microelectrode used for monitoring Vm was also used for microinjection, so Vm and [Ca2+]i were measured simultaneously from the same BCN. The fluorescence of Fura-PE3 was excited alternatively at wavelengths of 340 nm (F340) and 380 nm (F380) using a rotating filter wheel (Lambda 10–2; Sutter Instrument Co., Novato, CA). Emitted light was collected through a dichroic filter, and optical images (12 bit) were acquired every 15 sec. The Ca2+ data are shown as the concentration of free intracellular Ca2+ ([Ca2+]i) and were calculated using the following equation: [Ca2+]free = Kd x ß x (R - Rmin)/(Rmax - R) (12), with R being the ratio of F340 to F380, Rmin = 0.182, Rmax = 2.727, and ß = 2.073, as previously calculated (7).
RIA of ELH secretion
Concentrations of ELH in ASW were measured using the RIA procedure described by Wayne and Wong (6). The limit of detection of the assays was 1.1 ± 0.2 ng/ml (251 pM; 2 SD from buffer control values of 100 µl aliquots). The intraassay coefficient of variation of quadruplicate samples containing 18 ± 1.7 and 48 ± 4.9 ng/ml averaged 16%, and the interassay coefficient of variation of these samples averaged 32%. Samples from experimental and control preparations were placed in the same assay to eliminate bias due to interassay variability.
Measurement of ELH biosynthesis
Bilateral BCN clusters were dissected from animals and separated so that one cluster served as a genetically matched control for the other. Clusters were either treated with normal ASW, low Na+ ASW, or low Ca2+ ASW. Depending on the experiment, some preparations were stimulated to afterdischarge. Because the two clusters were separated, it was not possible to perfuse solutions through the abdominal-ganglion artery because it was severed. However, the resulting breach in the surrounding connective sheath allowed solutions to diffuse into the cluster, and Vm repolarized in response to low Na+ ASW in the bath just 1–2 min later than in response to the perfusion seen in the secretion experiment. Newly synthesized proteins were radiolabeled by placing individual BCN clusters in 50 µl of either normal ASW, low Na+ ASW, or low Ca2+ ASW containing 1% glucose and 1.5 mCi/ml [35S]methionine (specific activity > 1000 Ci/mmol; Amersham, Arlington Heights, IL) at room temperature. For unknown reasons, there is high variability in radiotracer uptake between animals but little variability in its uptake between pairs of BCN clusters from the same animal. Therefore, each animal must act as its own control. After a 4-h incubation period, individual BCN clusters were homogenized with 500 µl homogenizing buffer as described previously (10). An aliquot of the supernatant was removed to measure total protein content by using BCA Protein Assay Reagents (Pierce, Rockford, IL) and equal amounts of total protein for pairs of control and experimental clusters (20–50 µg) were loaded onto the gel. Samples were immunoprecipitated with an antibody directed against ELH (6, 13) for detecting newly synthesized ELH-related proteins (ELH prohormone, intermediate processing products, and ELH peptide). This antibody is the same as that used in the ELH RIA and has been described extensively in previous work; it specifically recognizes the N-terminal portion of the ELH peptide (6). Newly synthesized proteins were separated by SDS-PAGE and visualized by autoradiography as described previously (10). The intensity of the bands that appeared on the film was quantified by densitometric measurement with National Institutes of Health Image program (Bethesda, MD).
Data analysis
Values in figures and text are shown as the mean ± SEM, unless otherwise indicated. Baseline values for Vm and ELH secretion were defined as the mean + 2 SD of those values before electrical stimulation. Baseline values for [Ca2+]i were defined as the mean + 2 SD of those values before electrical stimulation and 10 min before the end of the experiment. Because there was typically a slope in resting Ca2+ levels (7), data for [Ca2+]i were detrended using IGOR data analysis program (WaveMetrics, Inc., Lake Oswego, OR) before calculating baseline. The decay time constant (
) of [Ca2+]i is the time point at which a single-exponential curve has fallen to 1/e of its maximal value, where e = 2.718281828 ( analysis done using the IGOR program). The Mann-Whitney U test was used to compare values between normal ASW- and low Na+-treated preparations in the secretion study. Values were considered significantly different if P 
0.05. In the ELH biosynthesis experiments, significant differences between treated preparations and their matched controls were analyzed using a 95% confidence interval test. Between-group differences of percent control data in the low Na+ experiment were analyzed by Kruskal-Wallis ANOVA followed by Mann-Whitney U test pairwise comparison. Between-group difference of percent control data in the low Ca2+ experiment was analyzed by Mann-Whitney U test. Values were considered significantly different if P 0.05.
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Results |
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; 21.5 ± 5.3 mV from rest within the first minute after the end of the afterdischarge). The low Na+ treatment caused a significant decrease in the time for Vm to repolarize, after the start of the afterdischarge, to resting levels compared with controls (8.95 ± 1.33 min vs. 56.67 ± 13.73 min; P < 0.003). It also led to a significant decrease in the duration of afterdischarge compared with controls (4.14 ± 0.37 min vs. 8.00 ± 1.37 min; P < 0.02). Vm eventually became hyperpolarized relative to pretreatment resting levels in all preparations treated with the low Na+ solution (60 min after onset of afterdischarge, BCNs showed Vm of -65 ± 5 mV compared with -50 ± 3 mV resting pretreatment Vm).
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). There was no significant effect on peak [Ca2+]i (476.8 ± 46 nM vs. 475.1 ± 57.8 nM), total [Ca2+]i (7.51 ± 1.42 µM vs. 10.68 ± 1.56 µM), and duration of [Ca2+]i above baseline (32.2 ± 9.6 min vs. 37.5 ± 7.2 min) compared with controls. However, there was a significant decrease in the decay time constant () compared with controls (2.63 ± 0.34 min vs. 4.81 ± 1.18 min; P < 0.04). This more rapid decay time in [Ca2+]i is a reflection of the shortened afterdischarge. Previous work suggested that the post-afterdischarge elevation in [Ca2+]i is due to release of Ca2+ from intracellular stores (7).
Low Na+ treatment did not inhibit ELH secretion (Fig. 1C). There was no significant effect on total ELH secreted above baseline (3.87 ± 1.30 µg/ml vs. 2.12 ± 0.34 µg/ml) or duration of ELH secretion above baseline (69 ± 10 min vs. 79 ± 7 min) compared with controls. Unexpectedly, there was a significant increase in peak ELH levels compared with controls (1.06 ± 0.31 µg/ml vs. 0.33 ± 0.08 µg/ml; P = 0.05). Previous work showed that there was no correlation between the duration of membrane depolarization and the total amount of ELH secreted (7). Similarly, our present findings show that premature membrane repolarization does not lead to a premature decline in ELH secretion or a decrease in the amount of ELH released. This finding indicates that persistent membrane depolarization is not driving the prolonged phase of ELH secretion.
Effect of low Na+ treatment on ELH biosynthesis
Experiments were designed to assess the effects of rapid repolarization and inhibition of action-potential firing on afterdischarge-induced ELH synthesis. Bilateral BCN clusters were separated; one cluster served as the experimental treatment preparation, and the other cluster served as the untreated control (unstimulated in normal ASW). There were four treatment groups. The first group was stimulated to afterdischarge and maintained in normal ASW containing [35S]methionine for 4 h as a positive control. The second group was unstimulated but maintained in low Na+ ASW containing [35S]methionine for 4 h to hyperpolarize Vm. The third group was stimulated to afterdischarge in normal ASW and then switched to low Na+ ASW 60 sec after onset of action-potential firing and maintained in low Na+ ASW containing [35S]methionine for 4 h to rapidly repolarize/hyperpolarize Vm and shorten the duration of the afterdischarge. The fourth group was stimulated to afterdischarge in normal ASW, allowed to exhibit the average duration of an afterdischarge (10 min), then switched to low Na+ ASW for 15 min to allow full repolarization to resting Vm, and then switched back to normal ASW containing [35S]methionine for the remainder of the 4-h incubation time (Fig. 2). Fifteen minutes of low Na+ ASW at the end of the afterdischarge led to a premature repolarization of Vm without altering the duration of the afterdischarge (Fig. 2).
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shows the effect of low Na+ treatment on ELH biosynthesis. Stimulating an afterdischarge in the presence of normal ASW leads to a significant increase in ELH synthesis as observed previously (10). Treatment with low Na+ ASW, starting 60 sec after the onset of the afterdischarge and continuing for 4 h, blocked the stimulating effects of the afterdischarge on ELH synthesis. Furthermore, just treatment with the low Na+ ASW for 4 h, which leads to a 15-mV hyperpolarization compared with resting Vm (data not shown), significantly suppressed basal levels of ELH synthesis. Pilot work showed that 4-h treatment with low Na+ ASW did not impair the health of the BCNs because they were able to show a normal afterdischarge after ASW washout (data not shown). The design of these experiments did not distinguish between an effect of premature termination of the afterdischarge and an effect of inhibiting post-afterdischarge depolarization on ELH synthesis. Furthermore, the relatively long duration of treatment (4 h) could lead to nonspecific effects on ELH synthesis. Therefore, a more refined design was implemented in which the low Na+ treatment was brief and allowed for a normal-duration afterdischarge without the prolonged depolarization. Five of six preparations in this treatment group ended their afterdischarges before low Na+ treatment was initiated. This treatment blocked the stimulatory effect of afterdischarge on ELH synthesis compared with untreated controls. Between-group analysis of the data revealed that the group stimulated to afterdischarge had significantly greater levels of ELH synthesis than all groups treated with low Na+, with or without afterdischarge (Fig. 3). These results indicate that it is the prolonged post-afterdischarge depolarization, and not the afterdischarge per se, that stimulates ELH biosynthesis. Furthermore, normal resting levels of Vm play an important role in maintaining basal levels of ELH synthesis.
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Treatment with low Ca2+/EGTA ASW starting 60 sec after onset of the afterdischarge prevented activity-dependent stimulation of ELH synthesis (Fig. 4). Treatment with low Ca2+/EGTA ASW in the absence of electrical stimulation inhibited basal levels of ELH synthesis. There was no significant difference in the levels of ELH synthesis between BCNs treated with low Ca2+/EGTA ASW in the presence or absence of afterdischarge (Fig. 4). These results indicate that Ca2+ influx is required for afterdischarge-induced ELH synthesis. The data also show the importance of normal calcium homeostasis in regulating basal ELH synthesis.
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Discussion |
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The kinetics of Vm and kinetics of transmitter or protein/peptide release often correlate well, especially under circumstances in which changes in these cellular events are relatively rapid and take place over the course of milliseconds to seconds (15, 16, 17). However, the results from our previous work (7), as well as the present findings, indicate that the slow kinetics of BCN Vm do not correspond well with the kinetics of ELH release. The present study showed that premature repolarization of Vm shortly after onset of the afterdischarge did not inhibit ELH secretion, indicating that post-afterdischarge membrane depolarization plays no role in maintaining post-afterdischarge secretion of ELH. These findings were unexpected because, in many other cell types, treatments that either hyperpolarize or repolarize Vm lead to inhibition of exocytosis. For example, hyperpolarizing treatment inhibits prolactin secretion from pituitary lactotrophs (18), neurotransmitter release from crayfish neuromuscular junction (19), and insulin secretion from rat pancreatic ß cells (20); and repolarization of Vm inhibits exocytosis in frog saccular hair cells (21). In BCNs, premature repolarization/hyperpolarization of Vm in BCNs did not inhibit ELH secretion; rather, the hyperpolarizing treatment led to an increase in peak ELH release. This is similar to what has been reported in rat neurosecretosomes in which removal of extracellular Na+ stimulated both basal and calcium-induced neurohormone release (22). Although calcium imaging did not reveal an increase in [Ca2+]i that could account for this enhanced ELH secretion, it is possible that the smaller volume of the neurites is more susceptible to changes in Ca2+ flux than the much larger volume of the soma where we were imaging. Because [Ca2+]i was not measured at BCN neurites or nerve terminals in this study, the effect of low Na+ treatment at or near release sites remains unknown. It is important to note that, although the low Na+ treatment was initiated 60 sec after onset of the afterdischarge and was continued throughout the remainder of the experiment, only peak levels of ELH release were potentiated by this treatment, which coincided with the timing of membrane repolarization. The bulk of ELH secretion was unaffected by continued low Na+ treatment and the resulting membrane hyperpolarization. The effect of continued low Na+ treatment on Ca2+ can be temporary because the intracellular Na+ necessary for the Na+/Ca2+ exchanger will be depleted at some point, resulting in a suppression of [Ca2+]i at release sites. Our previous work showed that [Ca2+]i in BCN neurites declines to baseline before the end of ELH secretion (7), suggesting that the calcium signal is not playing an important role in maintaining ELH release. A caveat to this interpretation is that we have been measuring global changes in [Ca2+]i in the neurite or soma, but there might be highly localized changes in [Ca2+]i that are critical for secretion, for instance at the inner plasma membrane, that we cannot measure with our current technique.
The present findings show that prolonged post-afterdischarge membrane depolarization plays a critical role in up-regulating ELH synthesis. Rapid repolarization of Vm, at either the beginning or the end of the afterdischarge, prevented the stimulus-induced increase in ELH synthesis. Allowing for a full afterdischarge but blocking the prolonged post-afterdischarge depolarization with just 15 min of low Na+ treatment was insufficient to stimulate ELH synthesis. Furthermore, because low Na+ treatment inhibited ELH synthesis but not ELH secretion, ELH secretion or ELH feedback is not playing a role in stimulating ELH synthesis. Together, these findings suggest that repetitive action-potential firing acts as a trigger for post-afterdischarge depolarization, and it is this later event that ultimately controls up-regulation of ELH synthesis. The role of membrane excitability in regulating synthesis of peptides that are targeted for secretion has been well documented in a variety of systems (23). For example, hypothalamic magnocellular oxytocin and vasopressin neurons show increased frequency of action-potential firing in response to various stimuli, such as suckling, dehydration, parturition, and hemorrhage. These same stimuli or stimuli that lead to membrane excitation also activate secretion and biosynthesis of these peptide hormones (24, 25, 26). As far as we know, the unusual nature of our present findings, a role for post-discharge Vm in controlling neuropeptide synthesis, is unlike anything described in the literature to date.
As observed in both our previous work (7) and in the present study, there is a temporal dissociation between the pattern of the Ca2+ signal and that of ELH secretion. That is, peak [Ca2+]i occurred during the early afterdischarge, whereas the bulk of ELH secretion occurred after the end of the afterdischarge. Furthermore, [Ca2+]i declined to resting levels before ELH returned to baseline. This delay between the Ca2+ signal and ELH release is not due to the delay in clearance of solution through the recording chamber because there is 95% clearance within 2 samples or 10 min (7). Although the afterdischarge-induced Ca2+ signal might be activating the early phase of ELH release, it is not driving the prolonged phase of secretion. On the other hand, our present data indicate that Ca2+ influx is required for both basal and afterdischarge-induced ELH biosynthesis. It is unclear whether there is a causal relationship between post-afterdischarge membrane depolarization and Ca2+ signaling that could account for the stimulus-induced increase in ELH synthesis. Although premature repolarization led to a decrease in the decay time of the Ca2+ signal, other characteristics of the Ca2+ signal, such as peak [Ca2+]i, total [Ca2+]i, and duration of elevated [Ca2+]i, were unaffected. It is possible that the overall Ca2+ signal in the soma does not accurately represent possible localized Ca2+ signaling that might be important for regulating ELH secretion and ELH biosynthesis. Nevertheless, the present data clearly show that Ca2+ homeostasis is playing a critical role in ELH synthesis under both basal and stimulated conditions. These findings directly contradict earlier work suggesting that Ca2+ influx inhibits ELH synthesis (27, 28). Technical differences in both the measurement of ELH biosynthesis and manipulation of [Ca2+]i are likely to account for the differences in the outcome of these studies. Work in other cell types support an important role of Ca2+ signaling in gene expression (29). If the afterdischarge-induced Ca2+ signal is playing a role in regulating activity-dependent ELH synthesis, it is not doing so through stimulating transcription of the ELH gene (10). However, it could be acting through either transcription of a non-ELH gene(s) (which is required for afterdischarge-induced stimulation of ELH synthesis) or translation of ELH mRNA (10, 30). Another possibility is that Vm and calcium signaling/homeostasis are acting through independent pathways to regulate both activity-dependent and basal ELH biosynthesis. Regardless, both post-afterdischarge depolarization and Ca2+ influx are required for activity-dependent ELH synthesis.
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Acknowledgments |
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Footnotes |
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N.L.W., W.L., and S.M. contributed equally to this work.
Abbreviations: ASW, Artificial seawater; BCN, bag cell neuron; [Ca2+]i, cytosolic Ca2+; ELH, egg-laying hormone; Vm, membrane potential.
Received October 27, 2003.
Accepted for publication December 18, 2003.
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Vm) from resting levels. B, Ca2+ signal is shown as the estimated [Ca2+]i from ASW control (mean + SEM) and low Na+-treated (mean - SEM) preparations. C, Concentrations of ELH (mean ± SEM) from perfusate collected in 5-min fractions.
