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Wisconsin Regional Primate Research Center (E.T., K.L.K., K.M., P.C.) and Department of Pediatrics (E.T.), University of Wisconsin, Madison, Wisconsin 53715
Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: terasawa{at}primate.wisc.edu
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-conotoxin GVIA (1
µM); 3) the intracellular Ca2+ stimulant,
ryanodine (1 µM), stimulated LHRH release, whereas the
intracellular Ca2+ transporting adenosine triphosphatase
antagonist, thapsigargin (1 and 10 µM), did not yield
consistent results; and 4) carbonyl cyanide
p-trifluoromethoxyphenyl-hydrazone (1 µM), a
mitochondrial Ca2+ mobilizer, stimulated LHRH release,
whereas ruthenium red, a mitochondrial Ca2+ uptake
inhibitor, did not induce consistent results. These results indicate
that: 1) the presence of extracellular Ca2+ is essential
for LHRH neurosecretion; 2) Ca2+ enters the cell via L-type
channels but not N-type channels; and 3) mobilization of intracellular
Ca2+ from inositol 1,4,5-triphosphate-sensitive stores, as
well as mitochondrial stores, seem to contribute to LHRH release in
these cells. |
Introduction |
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One of the most significant obstructions to progress in studying the cellular mechanism of the LHRH pulse-generating system is the fact that LHRH neurons are small and are scattered widely over the preoptic area and the hypothalamus, intermingled with other neurons and neuroglia. Although cell lines that express the mouse LHRH gene and human LHRH gene promoter have been established (12, 13) and made a significant contribution to our understanding on LHRH pulse-generation, these cells are not primary LHRH neurons and are of mouse origin. Accordingly, we have established a primary cell culture system for LHRH neurons derived from the olfactory placode of the monkey at embryonic age (E)35–37 (14). Because, in rhesus monkeys, LHRH neurons arise from the olfactory placode/pit, outside the brain, at an early age (E32–37) during a relatively long period (168 days) of embryonic development (15, 16), this culture system contains a large number of LHRH neurons and a relatively small number of non-LHRH neurons. As a first step in studying the underlying mechanisms of LHRH pulse generation, we examined: 1) whether the neurons in this culture system release LHRH in a pulsatile manner; and 2) whether extracellular Ca2+ and intracellular Ca2+ pools play a role in LHRH neurosecretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A few days before maximum sex-skin color change, female rhesus monkeys were placed with a fertile male until the breakdown of sex-skin color. Pregnancy was determined by ultrasound examination and uterine palpation. The day of pregnancy was designated as day 0 of gestation (E0), based on the estimated day of the LH surge, which usually occurs 2 days before the breakdown of sex-skin color. The age of fetuses was assessed by the record of sex-skin color changes and the size and the developmental characteristics of fetuses, precisely described in a previous study (16). Fetuses were delivered by cesarean section, under halothane anesthesia. A total of 15 fetuses, at E35–37, were used in this study. All experiments presented in this manuscript were performed following the standards established by the Animal Welfare Act and the document entitled: Principals for Use of Animals and Guide for the Care and Use of Laboratory Animals. The protocol for this study was reviewed and approved by the Animal Care and Use Committee, University of Wisconsin.
Tissue preparations and culture conditions
Before dissection of fetuses, the crown-rump length was
measured, the developmental characteristics were observed under
stereomicroscope, and the developmental stage was determined as
described previously (16). Three tissue areas from the fetuses were
used for culture: the nasal area, which included the olfactory pit
(placode); and two parts of the migratory pathway, divided into ventral
and dorsal (Mpv and Mpd, respectively) segments, as described
previously (14). Mpv included mostly the terminal nerve but sometimes
encroached upon the telencephalon because of individual variation of
the shape of the telencephalic vesicle, whereas Mpd consisted of the
basal part of the telencephalon. These tissues were dissected out into
chilled sterile imidazole-buffered L15 medium (Gibco, Grand Island, NY)
using a stereomicroscope, under a plexiglass enclosure, with a very
fine watchmakers’ forceps, a scalpel, and a fine iris scissors.
Tissues were then cut into very small pieces (<0.5 mm3),
and four to five pieces of tissue were plated onto 22 x 22
mm2 Thermanox plastic coverslips (Nalge Nunc International, Rochester, NY) that had been previously coated
with a layer of dried rat-tail collagen and sterilized under a UV light
(17). Cultures on coverslips were maintained in 35-mm tissue culture
dishes (Corning, Inc., Corning, NY) containing Medium 199
(Gibco) supplemented with 10% FBS (Hyclone Laboratories, Inc., Logan, UT), 0.6% glucose, and 75 µg/ml gentamicin and
were incubated at 37 C with 1.5% CO2-98.5% air (18).
Serum was stripped with dextran-coated charcoal (18) to remove steroids
and other small molecules (19). Medium was replaced every 3–4 days at
the beginning of cultures and every 1–2 days after the establishment
of cultures. Cultures were maintained up to 5 weeks, and experiments
were conducted after at least 2 weeks in culture, mostly between 2–4
weeks. From one fetus, usually four pairs of cultures from the
olfactory placode, four pairs from the Mpv, and six pairs from the Mpd
were obtained.
Perifusion experiments
Two to 4 weeks after the initiation of culture, coverslips with
cells were mounted in Sykes-Moore chambers, described by Martinez de la
Escalera et al. (20); i.e. two coverslips were
placed, face-to-face, separated by a rubber O-ring, forming a chamber
with a vol of 200 µl. Cultured cells were perifused with a modified
Krebs-Ringer phosphate buffer (21), with 0.05% BSA and 0.1% glucose
(pH = 7.4), under 95% O2-5% CO2 at 37 C.
Before the initiation of sample collection, cells were perifused with
medium for at least 20 min. Perifusates were collected at either 32 or
25 µl/min (see below) in 10-min fractions for 3–6 h using the
ACUSYST (Endotronics, Inc., Minneapolis, MN) perifusion
system. All samples were stored at -70 C until assay for LHRH. After
the perifusion experiment, cells were fixed with 2% paraformaldehyde
(pH = 7.6) and immunostained for LHRH and neuron-specific enolase
(see below).
Experimental designs
LHRH pulsatility and the effects of K+.
Perifusates were collected continuously in 10-min fractions for 5
h. At the end of the perifusion, the effects of high K+ on
LHRH release were examined by infusing 56 mM K+
for 20 min. In addition, the dose response of the effects of
K+ were tested at concentrations of 30, 56, and 75
mM. Samples in this experiment were obtained at a rate of
320 µl/10 min, aliquoted in duplicates at 150 µl/each, and stored
at -70 C for later RIA.
Effects of veratridine and tetrodotoxin (TTX). Effects of the Na+ channel opener, veratridine at 5 µM, and the Na+ channel blocker, TTX at 1 µM and 5 µM, on LHRH release were examined by infusing each individually for 20 min. The ability of TTX to block veratridine’s effects was further tested by infusing veratridine for 20 min together with TTX, or 20 min after the initiation of TTX perifusion. In addition, the effects of TTX on K+-induced LHRH release were examined. Samples were collected at 250 µl/10 min and aliquoted at 200 µl for RIA in single.
Role of extracellular Ca2+ in LHRH neurosecretion. Effects of low Ca2+ were examined by exposing cells to either normal perifusion solution (media) with Ca2+ concentration at 1.25 mM or reduced Ca2+ concentration at 20 nM. In the latter case, salt balance was maintained by adjusting Mg2+ concentration to 6 mM. Samples were collected at 250 µl/10 min and aliquoted at 200 µl for RIA in single.
Ca2+ channels. To examine the role of
Ca2+ channels in LHRH release, the voltage-sensitive L-type
channel agonist Bay K 8644 at 0.1, 1, and 10 µM, and the
voltage sensitive L-type channel antagonist nifedipine at 1 and 5
µM, and the voltage sensitive N-type Ca2+
channel antagonist -conotoxin GVIA at 1 µM were
infused for 20 min. For control, vehicle (perifusion medium) was
similarly infused. Samples were collected at a rate of 250 µl/10 min
and aliquoted at 200 µl for RIA in single.
Role of intracellular Ca2+. To examine the role of intracellular Ca2+ stores on LHRH release, thapsigargin (1 and 10 µM) [a mobilizer of inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+ stores], ryanodine (1 µM) (a mobilizer of ryanodine sensitive Ca2+ stores), carbonyl cyaninide p-trifluoromethoxyphenyl-hydrazone (FCCP, 1 µM) (a mitochondrial Ca2+ releaser), and ruthenium red (10 µM) (a mitochondrial Ca2+ uptake inhibitor) were infused for 20 min. Samples were collected at a rate of 250 µl/10 min and aliquoted at 200 µl for RIA in single.
All chemicals, except for Bay K 8644 (Research Biochemicals International, Natick, MA), were obtained from Sigma Chemical Co. (St. Louis, MO).
LHRH assay
LHRH concentrations in media samples were measured in 150-µl
(duplicates) or 200-µl (single), with RIA, using antiserum R-1245 (a
gift from Dr. T. Nett, Colorado State University, Fort Collins, CO), as
described previously (8, 22). Synthetic LHRH (Richelieu Laboratory,
Inc., Montréal, Canada) was used for both the trace and the
reference standard. The standard curve was constructed using the
culture medium as a part of the assay buffer. The sensitivity of the
assay, at 95% binding, was 0.05 pg/tube. Intra- and interassay
coefficients of variation were 8.3% and 11.4%, respectively.
Immunocytochemistry
All cultures were immunostained after perifusion experiments, as
described previously (14). Cultured cells were gently rinsed with PBS
and fixed with 2% paraformaldehyde in PBS for 30 min at room
temperature. They were rinsed thoroughly with PBS and then were placed
in 10% normal goat serum in PBS for 2 h at room temperature.
Cells were exposed to primary antibody for 48 h at 4 C. For LHRH
staining, the polyclonal antiserum LR-1, which primarily recognizes
mammalian LHRH (a gift of Dr. Benoit, University of Montréal,
Montréal, Canada) or GF6, which recognizes several forms of LHRH
(a gift of Dr. Sherwood, University of British Victoria, Victoria,
Canada; see Ref. 16) was used at a dilution of 1:10000 and 1:6000,
respectively. Cultured cells were again rinsed thoroughly with PBS and
were incubated with biotinylated anti- rabbit IgG for 1.5 h at
room temperature. After rinsing in PBS, avidin-biotinylated peroxidase
complex (Vector Laboratory, Burlingame, CA) in PBS was applied
for 1.5 h at room temperature, followed by rinses in Tris-buffered
saline. For visualization, 3,3'-diaminobenzidine, and H2O2, in
Tris-buffer, were applied. As a positive control, LHRH neurons in the
hypothalamus of rhesus monkeys, at various ages, were also stained.
To stain neuron-specific protein, cells were further exposed to antibody against neuron-specific enolase (NSE, INCSTAR Corp., Stillwater, MN) at a dilution of 1:1000. Double staining was accomplished using a similar procedure, except for a different chromagen (Vector SG complex, Vector Laboratory).
Data analysis
The number of LHRH cells in each culture was directly counted,
under a light microscope, with computer-aided image analysis. For
analysis, the data were excluded if a culture did not contain LHRH
immunopositive cells. This happened with Mpd cultures, especially in
younger fetuses (E35). LHRH pulses were characterized using the PULSAR
algorithm (23). The parameters used to characterize the LHRH pulses
were similar to those described previously (8). Based on the LHRH assay
sensitivity (0.33 pg/ml in 150 µl sample and 0.25 pg/ml in 200 µl
sample), LHRH pulses at less than 0.4 pg/ml, revealed by PULSAR
algorithm, were discarded. We did not include the data from Mpd
cultures for the characterization of LHRH release (Table 1
), because a sufficient number of
experiments were not conducted for statistical evaluation. For the
statistical analysis of the effects of high K+, TTX,
veratridine, low Ca2+, and Ca2+ channel
agonists and antagonists on LHRH release, the data from the three
groups (olfactory placode and both migratory pathways) were combined,
because there were no differences among the groups. The effects of high
K+, TTX, veratridine, low Ca2+,
Ca2+ channel agonists and antagonists, and intracellular
Ca2+ modulators, on LHRH release were tested using ANOVA
with repeated-measures, followed by post hoc Tukey’s tests.
Mean LHRH levels, during the 30 min immediately before the challenge of
chemicals, were calculated as the baseline level. Statistical
significance was attained at P < 0.05.
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Results |
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). The pattern
of LHRH release (Fig. 1) and the interpulse interval (Table 1
) of both
cultures were very similar. Overall mean (±SEM) of
the interpulse interval (n = 13) was 46.6 ± 4.0 min (Table 1). Mean LHRH release and the pulse amplitude (Table 1) of both
cultures were also not different (Table 1). Cultures from the dorsal
migratory pathway also exhibited similar LHRH pulses (data not shown).
In general, LHRH levels were higher when a larger number of LHRH
immunopositive cells were present in cultures.
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, left); but in two of seven olfactory placode cultures and
one of six migratory ventral pathway cultures, exceptionally large
responses were observed (Fig. 1, right). Again there were no
differences in the magnitude of LHRH release, in response to high
K+, between olfactory placode and migratory pathway
cultures (Table 1). Cultures exposed to 56 mM
K+ exhibited a significant (P < 0.01)
increase in LHRH release over the baseline levels (Fig. 2). LHRH response to K+
occurred in a dose-dependent manner (Fig. 3). The Na+ channel opener,
veratridine (5 µM), stimulated LHRH release (Fig. 4B), and this increase (over 80%
increase from preinfusion levels) was significantly higher than the
baseline level (n = 6, P < 0.02, data not shown);
but the veratridine-induced LHRH release was prevented by the
Na+ channel blocker, TTX, at doses of 1–5 µM
(Fig. 4B). TTX occasionally suppressed baseline LHRH release, but the
effects were not consistent. In contrast, the high
K+-induced LHRH release was not suppressed by TTX (1–5
µM, Fig. 4A).
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, top). LHRH pulses, revealed
by PULSAR, were consistently observed before, but not after, exposure
to low Ca2+ (Fig. 5, top left). Means of hourly
LHRH release, during the 3 h of low Ca2+ exposure,
were significantly (P < 0.05) lower than those before
low Ca2+ (Fig. 5, top right), as well as those
during the corresponding period in cultures exposed to normal
Ca2+ (Fig. 5, bottom right). In contrast, in
cultures exposed to normal Ca2+, LHRH pulses continued
throughout the experiment (Fig. 5, bottom left). Means of
hourly LHRH release, before and after exposure to normal
Ca2+ solution (1.25 mM), were not significantly
different (Fig. 5, bottom right).
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and 7A;
P < 0.01), whereas the L-type channel blocker,
nifedipine, suppressed LHRH release (Figs. 6B and 7B; P
< 0.05). In contrast, neither the N-type channel blocker,
-conotoxin (Figs. 6C and 7C), nor vehicle infusion (Figs. 6 and 7D),
induced any significant effect on LHRH release.
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and 9A; P < 0.05). FCCP, a
chemical which releases Ca2+ from mitochondrial stores,
stimulated LHRH release (Figs. 8B and 9B; P < 0.05);
but ruthenium red, a mitochondrial Ca2+ uptake inhibitor,
failed to induce any consistent change in LHRH release (data not
shown).
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), but only a small number of
LHRH-negative neuronal cells were present. Cultures of the migratory
pathway, however, contained LHRH neurons, as well as a large number of
non-LHRH neurons (Fig. 10B) and some glia (data not shown). In all
cultures, a large number of epithelial cells and fibroblasts were also
present.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence indicate that LHRH neurons seem to have an
endogenous pulse-generating mechanism. First, GT-1 cells release LHRH
in a pulsatile manner, with interpulse intervals of approximately
22–30 min (24, 25, 26). LHRH neurons, isolated from the adult male rat
brain by immunocytochemical treatments, release LHRH at approximately
19-min intervals (27). These interpulse intervals are similar to those
reported for LH pulses in mice and rats (28, 29), but they are
different from those in primates (30). Second, in the present study, it
is shown that LHRH neurons, in cultures derived from the embryonic
olfactory placode of the rhesus monkey, release the decapeptide in a
pulsatile manner. In fact, the interpulse interval of cultured LHRH
neurons from monkey embryos at E35–37 is very similar to that reported
in adult monkeys in vivo (8, 30, 31). Third,
electrophysiological studies in primary LHRH neurons from the terminal
nerve in adult fish (32), from the mouse embryonic olfactory placode
(33), and in GT-1 cells (34, 35) indicate that LHRH cells exhibit
spontaneous oscillatory action potentials. Whether these oscillatory
action potentials are related to LHRH neurosecretion is unknown.
Fourth, individual LHRH cells, from the monkey olfactory placode,
exhibited spontaneous intracellular Ca2+ oscillations with
a unique interpulse interval in each cell (36). Moreover, individual
LHRH cells, exhibiting intracellular Ca2+ oscillations with
their own rhythm, synchronize at an interval (37) similar to that of
LHRH release in vitro in this study and in vivo
(8). Finally, a preliminary study (38) from our laboratory shows that
the oscillatory pattern of intracellular Ca2+ was altered
by high K+, veratridine, low extracellular
Ca2+, nifedipine, ryanodine, and FCCP (but not normal
extracellular Ca2+ and -conotoxin) in a manner similar
to what we have observed for LHRH release in this study. Therefore, it
is hypothesized that the intracellular Ca2+ oscillations in
LHRH neurons are a manifestation of neurosecretion. This hypothesis is
supported by the well-documented observation in which intracellular
Ca2+ oscillations are associated with insulin secretion in
pancreatic ß-cells (39, 40).
The understanding of communication between individual LHRH neurons is limited to speculation. It has been proposed that GT-1 cells can release LHRH synchronously by communication through synapses, gap-junctions, electrical couplings (26, 41), or diffusible substances (25). The presence of synapses and dye-coupling between GT-1 cells has been reported (26), and connexin 26 (a protein associated with gap-junctions) was found in GT-1 cells (41). Moreover, the observation that GT-1 cells (25) or LHRH cells (this study), grown on two separate coverslips enclosed within one chamber, release LHRH with distinct, presumably synchronized, pulses indicates that cells may communicate with diffusible substances such as LHRH and nitric oxide. It has been reported that changes in the extracellular concentration of LHRH in vitro determine the pulse frequency of LHRH release in GT-1 cells (42) and that nitric oxide synthase messenger RNA (mRNA) is present in GT-1 cells (43). In a preliminary study, we also found that our culture from the olfactory placode contained cells immunopositive to connexin 32, another protein associated with gap-junctions (Terasawa, unpublished observation). Nonetheless, the mechanism of LHRH neuronal network communication requires further study.
Veratridine, a fast Na+ channel opener, stimulated LHRH release; whereas TTX, a fast Na+ channel blocker, suppressed the veratridine-induced LHRH release. High K+ also consistently stimulated LHRH release, although the size of response was variable. The reason for this variable LHRH response is unclear. It is possible that LHRH release was indirectly influenced by the concomitant depolarization of inhibitory neurons in cultures, by high K+. However, this speculation is clouded by the fact that LHRH responses to high K+ were similar in placode and migratory pathway cultures, even though the number of non-LHRH neurons differs. Nonetheless, LHRH responses were dose-dependent on extracellular K+ concentrations. These data suggest that membrane depolarization with K+ or through Na+ channel opening induces neurosecretion and that a greater membrane depolarization results in a higher amount of LHRH release from these neurons. When veratridine allows Na+ influx, the Ca2+/Na+ pump is activated, and Ca2+ influx occurs. This Ca2+ influx would trigger the cascade of intracellular events leading to neurosecretion.
In the present study, the effects of K+ on LHRH release were not blocked by TTX. Similar findings were reported by Mellon et al. (12) in GT-1 cells. A recent study, however, shows that exposure of LHRH neurons from cultured mouse olfactory placode, to 50 mM KCl for several days, resulted in repression of LHRH mRNA expression, whereas TTX (1 µM) not only blocked the effects of high K+ but also stimulated LHRH mRNA expression (44). Although these authors did not examine the effects of prolonged exposure to KCl and TTX on LHRH release, it is conceivable that the effects of prolonged exposure to KCl and TTX differ from the effects of short-term exposure, resulting in altered channel properties and secretory activities.
Neurosecretion in LHRH cells requires the presence of extracellular
Ca2+, because lowering the extracellular Ca2+
concentration to 20 nM abolished LHRH release. Moreover,
the present study indicates that Ca2+ enters through
voltage-sensitive L-type Ca2+ channels, but not N-type
Ca2+ channels, because the L-type Ca2+ channel
blocker nifedipine, but not the N-type Ca2+ channel blocker
-conotoxin, suppressed LHRH release. The voltage-sensitive
Ca2+ channel agonist, Bay K 8644, also stimulated LHRH
release. Perhaps, depolarization of the LHRH cell membrane with high
K+ activates the voltage-sensitive Ca2+
channels, allowing Ca2+ influx, which subsequently leads to
intracellular events for LHRH neurosecretion. The view that the
activation of the voltage-sensitive Ca2+ channels and
subsequent Ca2+ entry triggers neurosecretion from LHRH
neurons, is consistent with observations in GT-1 cells (24, 35), and
fetal hypothalami (24).
It has been well established that Ca2+ influx mobilizes intracellular Ca2+ stores, among which are an intracellular IP3-sensitive pool, and ryanodine- and caffeine-sensitive Ca2+ pools in the endoplasmic reticulum. The phenomenon is known as Ca2+-induced Ca2+ release. Thapsigargin, a Ca2+-adenosine triphosphatase reuptake inhibitor of the endoplasmic reticulum, which increases intracellular Ca2+, did not induce consistent results; whereas ryanodine stimulated LHRH release. We did not examine the effects of caffeine. These results suggest that LHRH neurons possess a ryanodine-sensitive Ca2+ pool and that mobilization of this pool from the endoplasmic reticulum can result in neurosecretion. Although the failure of LHRH release with thapsigargin may be caused by inadequate doses used in this study or to its side effects, it is possible that LHRH neurons are insensitive to thapsigargin. This view is consistent with the observation that thapsigargin was not an effective stimulator for intracellular Ca2+ oscillations in GT-1 cells (35). In contrast, FCCP, a mitochondrial Ca2+ mobilizer, clearly stimulated LHRH release, indicating that mitochondrial Ca2+ could contribute to neurosecretion.
In summary, in the present study, we have shown that LHRH neurons, derived from the embryonic nasal region, release the decapeptide in a pulsatile manner, and LHRH neurosecretion requires Ca2+ influx and subsequent intracellular events. This is only the beginning of cellular studies on LHRH neurosecretion in primates. Are the hourly rhythms of LHRH neurosecretion a consequence of the synchronization of a population of LHRH neurons? How does a network of LHRH neurons synchronize their activity, i.e. whether by the result of synaptic mechanisms or electrical coupling through gap junctions or through a diffusible substance(s)? It is our hope that answers to these questions will be forthcoming in the near future.
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Acknowledgments |
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Footnotes |
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Received August 13, 1998.
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