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Department of Physiology, University of California School of Medicine, Los Angeles, California 90095-1751
Address all correspondence and requests for reprints to: Dr. Nancy L. Wayne, Department of Physiology, Room 53–231 CHS, University of California School of Medicine, 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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A 10- to 30-min afterdischarge stimulates ELH secretion over a 1- to 2-h period, during which time hundreds to thousands of nanograms of ELH are released (13, 14). Previous work showed that newly synthesized ELH is preferentially released within 24 h of being made, and that this new hormone contributes to half of the total amount of ELH released in response to afterdischarge (14). This suggests that the synthetic capacity of the bag cell neurons is quite high, especially during the breeding season when animals are laying eggs and secreting large amounts of ELH on a near-daily basis (15, 16). For bag cell neurons to secrete a sufficient amount of ELH to stimulate egg laying, the releasable pool of hormone would need to be rapidly replenished. The primary purpose of this study was to test the hypothesis that the afterdischarge, which triggers loss of ELH through secretion, ultimately replenishes releasable pools by up-regulating the ELH biosynthetic pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental design
Effect of afterdischarge on ELH synthesis. To determine
whether the afterdischarge stimulates ELH synthesis, bilateral bag cell
clusters (n = 6–9/group) were dissected from animals and
separated so that one cluster served as a control for the other.
Experimental clusters were placed in filtered artificial sea water
(ASW; 395 mM NaCl, 10 mM KCl, 10 mM
CaCl2, 50 mM MgCl2, 28
mM Na2SO4, 30 mM HEPES,
and 5000 U/liter penicillin-streptomycin, pH 7.7) and electrically
stimulated to afterdischarge at time zero. Four-hour incubations in 100
µCi/ml [3H]leucine (Amersham, Aylesbury, UK) were
initiated 0, 4, and 8 h after the onset of afterdischarge to
radiolabel newly synthesized peptides during those time periods.
Control preparations were incubated with [3H]leucine
simultaneously, but were not stimulated to afterdischarge. After the
4-h incubation, clusters were processed for immunoprecipitation and
measurement of newly synthesized ELH (see below).
Effect of afterdischarge on the level of ELH messenger RNA (mRNA). Afterdischarges were stimulated either in vivo or in vitro, and the effect on levels of ELH mRNA was monitored over a 0- to 8-h period. In in vivo experiments, animals (n = 4–5/group) were injected with atrial gland peptides A and B (1–2 µg/g BW; see below). These peptides activate cells in the cerebral and pleural ganglia that, in turn, transmit neural signals to the bag cells in the abdominal ganglia to stimulate an afterdischarge (19). Egg-laying and its related behaviors induced by peptides A and B were observed. Importantly, peptides A and B do not activate egg laying by acting directly on the ovotestis. Therefore, any egg laying in response to injection was due to simulation of a bag cell afterdischarge and downstream events leading to egg laying. Animals were killed 0, 15, 30, 60, 120, 240, and 480 min after the injection, and bag cell clusters were dissected and homogenized. Total RNA was isolated and processed for Northern blot analysis of ELH mRNA.
To better monitor the onset of afterdischarge, we also performed the above experiment in vitro. Bilateral bag cell clusters (n = 3/group) were separated; one of the clusters was stimulated to afterdischarge by electrical stimulation or by treatment with 11–22 µg/ml peptides A and B. The other cluster served as an unstimulated control. In the experiments using peptides A and B to activate an afterdischarge, the paired cerebral and pleural ganglia were also dissected and separated along with the bag cell clusters so that the connection between the ganglia and ipsilateral bag cell cluster remained intact. Bag cell clusters were homogenized 15, 30, 60, 120, and 240 min after the onset of afterdischarge, and total mRNA was isolated and processed for Northern blot analysis of ELH mRNA.
Effect of transcription inhibitor on ELH synthesis. To determine whether the effect of afterdischarge on ELH synthesis requires transcriptional processes, three treatment groups were tested: afterdischarge alone (n = 6), afterdischarge plus actinomycin D (transcription inhibitor; Sigma Chemical Co., Inc., St. Louis, MO; n = 7), and actinomycin D alone (n = 7). Again, one of the bilateral clusters served as an unstimulated and drug-untreated control. All clusters were incubated for 4 h in ASW containing 100 µCi/ml [3H]leucine with or without 50 µg/ml actinomycin D to radiolabel newly synthesized peptides. After incubation, clusters were processed for immunoprecipitation and measurement of newly synthesized ELH.
Effects of transcription inhibitor and translation inhibitor on the level of ELH mRNA. To test whether the effect of actinomycin D on ELH synthesis in Exp 3 was due to a changed level of ELH mRNA and whether translation played a role in maintaining ELH mRNA levels, bilateral bag cell clusters were divided in two: one cluster was part of an experimental treatment group, and the other served as a control. Three groups of experimental clusters (n = 4–5/group) were treated as described in Exp 3, except no [3H]leucine was added (i.e. afterdischarge alone, afterdischarge plus actinomycin D, and actinomycin D alone). The fourth group (n = 4) was treated with 53 µg/ml anisomycin (translation inhibitor; Sigma Chemical Co.) without electrical stimulation. All clusters were homogenized after 4-h incubations; total RNA was isolated and processed for Northern blot analysis of ELH mRNA.
Stability of ELH mRNA. One of the bilateral clusters was treated with 50 µg/ml actinomycin D for 2, 4, 8, 16, and 32 h (n = 4–5/group), and the other served as an untreated control. We have demonstrated in a previous study that bag cell clusters maintained in culture for up to 5 days showed normal electrical and secretory properties (14). Therefore, bag cell functions should not decline over the course of 32 h. After incubation, the clusters were homogenized; total RNA was isolated and processed for Northern blot analysis of ELH mRNA.
Stimulation and recording
The procedures were described previously (13). Briefly, bag cell
afterdischarges were stimulated with a suction electrode placed on the
pleurovisceral connective nerve (10–40 V, 6 Hz, 40 msec/pulse, 10-sec
duration) and were monitored with another suction electrode placed on
the bag cell cluster. A Gould Instruments Systems Ltd.
(Valley View, OH) bioelectric amplifier with chart recorder and a
Hitachi Scientific Instruments (Woodbury, NY) digital
storage oscilloscope were used to amplify and record the compound
action potentials.
Extraction of peptides A and B
The extraction of peptides A and B was performed according to
the method described by Heller et al. (20). Briefly, 40
atrial glands were dissected from animals and homogenized in 150 ml
0.01 M Tris-HCl (pH 8.5). The homogenate was centrifuged,
and the supernatant was transferred to another tube. Then, ammonium
sulfate was added to the homogenate to reach 45% saturation. The
mixture was stirred for 5 h at 4 C, followed by centrifugation.
The collected precipitate was dissolved in 0.01 M sodium
phosphate, pH 6.5, containing 6 M urea, applied to a
1.5 x 100-cm Bio-Gel A-0.5 m column (Bio-Rad Laboratories, Hercules, CA), and eluted with the same buffer.
One-milliliter fractions were collected, and their protein contents
were measured spectrophotometrically. Three peaks were obtained from
the protein content profile, and peptides A and B were in the second
peak. Fractions from this peak were combined and desalted with an
Econo-PacI0 DG desalting column (Bio-Rad Laboratories).
The protein content of the extract was determined using the
bicinchoninic acid protein assay reagent (Pierce,
Rockford, IL), and the resulting concentration was 1111 µg/ml.
Concentrations of 1–2 µg/g BW consistently triggered bag cell
afterdischarges in vivo, and a concentration of 11 µg/ml
triggered bag cell afterdischarges in vitro.
Quantitation of ELH synthesis
After incubation with [3H]leucine, bag cell
clusters were washed and then homogenized in a glass microtissue
grinder with 600 µl homogenizing buffer [150 mM NaCl, 50
mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% SDS, 5
mM EDTA, 1 mM phenymethylsulfonylfluoride, 50
µM leupeptin, and 0.24 trypsin inhibitor unit
aprotinin]. After incubation on ice for 30 min, samples were
centrifuged, and supernatant was removed. An aliquot of the supernatant
was removed to measure total protein content using the bicinchoninic
acid protein assay reagent. Another aliquot of the supernatant was
immunocleared by adding 10 µl rabbit serum (Sigma Chemical Co.) and 50 µl protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ) and was rocked at 4 C for 4 h.
Afterward, samples were centrifuged, and supernatant was removed. Four
50-µl aliquots were removed from the supernatant, and 950 µl buffer
were added. Then, 5 µl ELH antibody (13) were added to two of the
aliquots, and an equal volume of rabbit serum was added to two others
to serve as controls. Samples were rocked for 2 h, then incubated
overnight after the addition of protein A-Sepharose. Afterward, samples
were centrifuged, and the pellets were washed five times with the
buffer. Then, 200 µl buffer were added, and samples were heated at
100 C for 5 min. The 3H radioactivity incorporated into ELH
was measured by liquid scintillation counting.
Autoradiography
To verify the above ELH immunoprecipitation procedure, 2 vol
tricine sample buffer (Bio-Rad Laboratories) were added,
and the proteins contained in samples after the immunoprecipitation
procedure were separated electrophoretically on a 16.5% T-3.3%
C tricine-SDS-polyacrylamide gel with 5% stacking and 10%
spacer gels (21). In the previous sentence, T refers to total solids
content and C refers to the ratio of cross-linker to acrylamide
monomer. Gels were washed with 5% glacial acetic acid and 5%
isopropyl alcohol three times (15 min each time) and with running water
for another 15 min. The gels were then placed in Autofluor (National
Diagnostics, Atlanta, GA) for 2 h, vacuum-dried, placed on
x-ray films (Eastman Kodak Co., Rochester, NY), and
exposed at -70 C.
Quantitation of total protein and total RNA synthesis
To test the effect of anisomycin on total protein synthesis, bag
cell clusters were separated and incubated in ASW containing 1%
glucose and 100 µCi/ml [3H]leucine with or without 53
µg/ml anisomycin for 4 h and then washed. Clusters were
homogenized with homogenizing buffer as described above. After
incubation on ice for 30 min, samples were centrifuged for 15 min, and
supernatant was removed. Aliquots of radiolabeled samples in duplicate
were added with an equal volume of 1 mg/ml BSA. Proteins were
precipitated with 25% trichloroacetic acid (TCA) and incubated on ice
for 30 min. They were centrifuged, and the pellets were washed three
times with 5% TCA. Finally, 0.5 N NaOH was added, and
total radiolabeled proteins were measured by liquid scintillation
counting.
To test the effect of actinomycin D on total RNA synthesis, bag cell clusters were separated and incubated in ASW containing 1% glucose and 100 µCi/ml [3H]uridine with or without 50 µg/ml actinomycin D for 4 h and then washed. Clusters were homogenized with buffer containing 10 mM Tris (pH 7.50), 150 mM NaCl, 1% SDS, and 1 mM EDTA. After incubation on ice for 5 min, samples were centrifuged, and the supernatant was transferred to another tube. Duplicate 50-µl aliquots were removed, 2 µg transfer RNA (Sigma Chemical Co.) and 200 µl 10% TCA were added, and the samples were incubated on ice for 30 min. They were centrifuged, and the pellets were washed three times with 10% TCA. Then, 10 µl perchloric acid (68%; Sigma Chemical Co.) and 200 µl water were added. The samples were heated at 90 C for 15 min, and radiolabeled total RNA was measured by liquid scintillation counting.
Quantitation of ELH mRNA
Total RNA was prepared using a modified method described by
Chirgwin et al. (22). Briefly, tissues were homogenized with
a lysis buffer containing 4 M guanidinium isothiocyanate,
25 mM sodium acetate, 0.5% lauroylsarcosine, and 0.1
M ß-mercaptoethanol. Total RNA was then extracted with
phenol-chloroform and precipitated with ethanol. A mRNA isolation kit
(Boehringer Mannheim, Indianapolis, IN) was used to purify bag cell
mRNA according to the manufacturer’s protocol. Equal amounts of total
RNA or mRNA were denatured in gel buffer containing 50% formamide and
2.2 M formaldehyde by heating for 10 min at 65 C. Tracking
dye was added, and the samples were subjected to electrophoresis in a
denaturing 1% agarose gel in a buffer containing 20 mM
sodium morpholinopropanesulfonic acid (MOPS), 5 mM sodium
acetate, 5 mM sodium EDTA (pH 7.0), and 2.2 M
formaldehyde. After electrophoresis, total RNA or mRNA was transferred
to Hybond-N+ nylon membranes (Amersham) using
the Turboblotter transfer system (Schleicher & Schuell, Inc., Keene, NH). The nylon membranes were then baked for 2
h at 80 C and processed for hybridization to a 32P-labeled
ELH complementary DNA (cDNA) or Aplysia actin cDNA for
4 h at 65 C, using the Rapid-hyb buffer (Amersham).
The 32P-labeled probes were prepared using the Rediprime
DNA labeling system (Amersham). The ELH cDNA and
Aplysia actin cDNA [provided by Gregg Nagle, University of
Texas Medical Branch (Galveston, TX), and Luc DesGroseillers,
University of Montréal (Montréal, Canada), respectively]
were both cloned into the Bluescript vector. After the initial
hybridization (with either ELH cDNA or actin cDNA), the nylon membranes
were washed for autoradiography. For a second hybridization (with
either actin cDNA or ELH cDNA), the first probe was stripped from the
nylon membranes with boiling hot 0.5% SDS, followed by the same
hybridization procedures described above. The intensities of ELH mRNA
and actin mRNA bands were measured with a densitometric system
(AlphaImager 2000, Alpha Innotech Co., San Leandro, CA). The data were
expressed as the densitometric value of ELH or actin divided by that of
18S ribosomal RNA (rRNA) loaded onto the gel. The 18S rRNA was also
measured densitometrically from photographs of the gels stained by
ethidium bromide before transfer.
Statistical analysis
All values were expressed as the mean ± SEM.
Significant differences between experimental and untreated controls
were analyzed using a 95% confidence interval test. Significant
differences between treatment groups (data presented as a percentage of
the tissue-matched control values) were analyzed by Kruskal-Wallis
ANOVA, followed by Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This
effect was no longer significant during the 8- to 12-h period following
initiation of afterdischarge. The total protein content in bag cell
clusters was not significantly different between control and
experimental groups at all three time periods.
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). In the second part of the
experiment, bag cell clusters were either electrically stimulated or
treated with peptide A and B extract in vitro. The data were
very similar between these two treatments. The results again showed
that ELH mRNA levels were not significantly different from those in
controls (data not shown). This suggests that the effect of
afterdischarge on ELH synthesis did not occur at the transcriptional
level.
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and Fig. 3). However, this increase was blocked by
treatment with actinomycin D. In addition, treatment with actinomycin D
lowered basal levels of ELH synthesis compared with those in untreated
controls. ELH synthesis (indicated as a percentage of the control
value) in the afterdischarge plus actinomycin D group was not
significantly different from that in the group treated only with
actinomycin D; however, both groups showed significantly lower ELH
synthesis than the group stimulated with afterdischarge (see Table 1).
Overall, these findings suggest that transcription of a non-ELH gene is
required for normal levels of basal ELH synthesis. In addition, the
data suggest that the afterdischarge-induced increase in ELH synthesis
is mediated by transcription of a non-ELH gene.
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and Fig. 3) was similar to that on ELH synthesis, suggesting
that the effects of actinomycin D were not specific. Afterdischarge
also suppressed total protein synthesis, but had the opposite effect on
ELH synthesis. To test the possibility that bag cell neurons were damaged by actinomycin D, one of the clusters was first treated with actinomycin D for 4 h, then electrically stimulated to afterdischarge. For all the clusters tested (n = 3), the length and the pattern of afterdischarge were not significantly different from those in the untreated controls (28 ± 10 vs. 17 ± 10 min; by t test). This suggests that the inhibitory effect of actinomycin D on ELH synthesis was not due to cellular damage.
Effects of transcription and translation inhibitors on the level of
ELH mRNA
As actinomycin D suppressed basal ELH synthesis and blocked the
stimulatory effect of afterdischarge on ELH synthesis, we next
investigated whether this was caused by a decrease in ELH mRNA levels.
Bag cell clusters were electrically stimulated to afterdischarge,
electrically stimulated and treated with actinomycin D, treated with
actinomycin D only, or treated with the translation inhibitor
anisomycin only. The results (Fig. 4)
showed that 4 h after treatment, ELH mRNA levels remained
unchanged in all four experimental groups. Incomplete inhibition of
pharmacological effects was unlikely, because 50 µg/ml actinomycin D
blocked 93% of RNA synthesis, and 53 µg/ml anisomycin blocked 97%
of protein synthesis in bag cells. In addition, increasing the
actinomycin D dose to 500 µg/ml had no significant effect on the
level of ELH mRNA (data not shown). Thus, the data indicate that ELH
mRNA levels are not influenced by inhibition of transcription or
translation during the 4-h experimental period.
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). Isolated bag cell clusters have been
shown to exhibit normal electrical and secretory properties for days in
culture medium (14). This result further demonstrates the durable
nature of these neuroendocrine cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, the effect of afterdischarge on ELH synthesis was not due to enhanced transcription of the ELH gene, because the level of ELH mRNA remained unchanged for up to 8 h after the onset of afterdischarge. ELH mRNA levels were also not affected by treatment with transcription and translation inhibitors for up to 4 h. This suggests that transcriptional and translational processes are not required to maintain normal levels of ELH mRNA over several hours. Thus, the stimulatory effect of afterdischarge on ELH synthesis for up to 8 h after the onset of afterdischarge is likely to occur at the level of ELH translation. In pancreatic ß-cells, glucose induces insulin secretion and increases proinsulin biosynthesis (31). This effect of glucose on proinsulin biosynthesis also occurs at the translational level (32). However, when the stimulation of glucose continues for a longer period (>6 h), the preproinsulin mRNA level (33) and its stability (34) increase. In our previous study (14), 4 consecutive days of afterdischarge caused a decrease in the bag cell content of ELH. Thus, increased ELH biosynthesis appears to be unable to fully compensate for secretion-induced loss of ELH. It is possible that regulatory control may occur at the level of ELH gene transcription or act to enhance ELH mRNA stability when there is greater depletion of ELH stores over several days.
Further investigation revealed that ELH mRNA levels remained stable for at least 32 h in the presence of a transcriptional inhibitor. With such a stable message, it would be more effective to regulate rapid peptide synthesis through translation of already existing mRNA rather than through transcription of the ELH gene. Transcription inhibitors, such as actinomycin D and cycloheximide, have been shown to stabilize mRNA (35), making these kinds of half-life studies not as reliable as commonly thought. Nevertheless, the data indicate that ELH mRNA is relatively stable compared with actin mRNA. The stability of mRNA is an important factor in regulating protein/peptide synthesis. This stability can be determined by the length and sequences of the 3'-untranslated regions (UTRs) of mRNA as well as by trans-acting factors that bind to specific elements of mRNA (36). In addition, the 3'-UTRs can affect translational efficiency (37, 38, 39, 40). The elements and roles of 3'-UTRs of ELH mRNA have not been investigated. It would be interesting to know whether they are responsible for its high stability and/or play a role in the enhanced ELH synthesis induced by afterdischarge.
Previous studies have shown that the bag cells release only about 10% of their ELH store in response to afterdischarge and that the remaining 90% of stored hormone contains more than 1000 ng ELH in the bilateral clusters (6, 14). The seemingly small loss of ELH due to secretion still needs to be rapidly replenished, as suggested from our earlier findings that 50% of released ELH is newly synthesized (14), and from the current study showing that afterdischarge induces a 100% increase in ELH synthesis within 4 h from the onset of stimulation. Together, these results suggest that most of the large, older store of ELH is not in a releasable form and that newly synthesized ELH is preferentially placed in the relatively small, releasable pool of hormone. In addition, the amount of ELH released is somehow coordinated with the total volume of the store. That is, when bag cell content of ELH decreases with daily stimulation of secretion, ELH release is likewise suppressed, so that the ratio of cell content to release is maintained at 10:1 (14).
Transcription of a non-ELH gene(s) appears to play an important role in regulating basal ELH synthesis, because treatment with actinomycin D alone suppressed levels of ELH synthesis compared with those in untreated controls. Furthermore, transcription of a non-ELH gene(s) seems to be involved in the afterdischarge-induced up-regulation of ELH synthesis, because treatment with actinomycin D blocked the stimulatory effect of afterdischarge on ELH synthesis such that there was no significant difference between preparations treated with actinomycin D and those stimulated to afterdischarge and also treated with the transcription inhibitor; notably, both of these groups showed significantly lower levels of ELH synthesis than the group stimulated to afterdischarge. This non-ELH gene(s) is likely, either directly or indirectly, to increase the rate of translation of already existing ELH mRNA. Activity-dependent alteration of translation initiation and elongation is not an uncommon mechanism for regulating peptide synthesis. For example, electrical stimulation of cardiocytes enhances myosin heavy chain synthesis through an increase in the rate of translation initiation (41). The effect of glucose on proinsulin translational processes has been suggested to be at the levels of translation initiation and elongation (42, 43). It has been shown that calcium impedes translation initiation in the cortical synaptoneurosomes (44). Calcium also enhances phosphorylation of elongation factor-2, which inhibits protein synthesis in cortical neurons (45). Protein kinase C increases phosphorylation of initiation factor-4E, which enhances protein synthesis in cardiocytes (46). Notably, one type of serotonin-induced facilitation in an Aplysia sensorimotor synapse is dependent on synthesis of protein, but not synthesis of mRNA (47). The serotonin-induced increase in translation that might stimulate facilitation requires activation of protein kinase C, protein kinase A, and tyrosine kinase and is blocked by rapamycin, which inhibits specific elements along the translational pathway (48). Likewise, activation of the bag cell afterdischarge triggers a cascade of cellular events, including elevation of intracellular calcium and stimulation of the cAMP and diacylglycerol second messenger pathways (1, 2). cAMP has been shown to enhance ELH synthesis (49) and processing of the ELH prohormone (30), whereas calcium and protein kinase C suppress ELH synthesis (49, 50). These second messengers might mediate the effects of afterdischarge on ELH synthesis by acting directly on translation of ELH mRNA or by activating a certain gene(s) that is important for ELH translational processes.
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Acknowledgments |
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Footnotes |
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Received June 17, 1998.
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