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Department of Psychiatry, Mental Retardation Research Center, University of California School of Medicine, Los Angeles, California 90024-1759
Address all correspondence and requests for reprints to: Dr. James A. Waschek, 68–225 NPI, Department of Psychiatry, University of California, 760 Westwood Plaza, Los Angeles, California 90024. E-mail: jwaschek{at}mednet.ucla.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies by Olcese et al. (3) demonstrated that
PACAP induced GnRH secretion from immortalized mouse hypothalamic GnRH
cells via action on the PACAP-preferring PAC1 receptor.
Intraventricular and iv injection of PACAP enhanced GnRH gene
expression in the rat hypothalamus, an action that was blocked by a
PAC1 antagonist (4). In addition to its hypothalamic
effects, PACAP injected systemically into rats elevated serum levels of
LH, apparently due in part to enhanced release of LH from the anterior
pituitary (5). In the cultured pituitary cells, PACAP treatment
stimulated LH and FSH release (6, 7), elevated and reduced the
expression of gonadotropin 
-unit and FSH ß-unit messenger RNA
(mRNA), respectively (8), and increased the size of LH ß-unit mRNA
(8). PACAP was also reported to interact with GnRH to regulate the
activity of pituitary gonadotropes (9, 10). In addition, PACAP is
expressed in the rat testis, where it appears to stimulate the
development of germ cells (11) and to enhance fetal testicular
steroidogenesis (12). These results in mammals suggest that PACAP might
be a part of the hypothalamic-pituitary-gonadal feedback circuit.
In amphibians, several lines of evidence also suggest a physiological role for PACAP in the reproductive system. With the use of a rabbit antibody to ovine PACAP-27, Yon et al. (13, 14) determined the distribution of PACAP-27-containing cells in the preoptic area of the frog Rana ridibunda hypothalamus and reported the presence of dense PACAP-27-containing fibers in the external zone of the median eminence. Recently, in situ hybridization in Xenopus (15) and radioreceptor binding (16) in Rana revealed abundant PAC1 receptor mRNA and PACAP-27-binding sites, respectively, in the both the hypothalamus and distal lobe of pituitary. Pharmacological studies in Rana indicated that PACAP stimulated a dose-dependent cAMP accumulation in both hypothalamic slices and isolated distal lobe pituitary cells (14). Furthermore, cultured frog pituitary cells responded to PACAP treatment with a marked elevation of intracellular Ca2+, suggesting the involvement of calcium mobilization in regulating the function of pituitary cells (17). In the periphery, PACAP has been demonstrated to stimulate the secretion of corticosteroids from cultured cells of the frog adrenal glands (18). It is not yet clear, however, whether PACAP functions in the frog testis. Nonetheless, the results of studies on PACAP action on frog hypothalamic and pituitary cells imply that PACAP may be involved in reproductive feedback.
To begin to determine the physiological role of PACAP in the gonadal feedback axis, we analyzed changes in PACAP mRNA expression in the frog anterior preoptic area of the hypothalamus after acute castration. Because frog PACAP mRNA sequences have not been identified, we first cloned PACAP complementary DNA (cDNA) from a Xenopus brain cDNA library and determined the mRNA distribution in the brain. We found that PACAP mRNA was present in several areas of the brain, and that the expression of PACAP and PAC1 receptor mRNA in the anterior preoptic area is up-regulated in male frogs after acute castration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The temperatures and
times for 30 PCR cycles were 94 C denaturing for 30 sec, 50 C annealing
for 45 sec, and 72 C extension for 45 sec, with a final 72 C elongation
for 5 min. PCR reaction solution (10 µl) was run on 1.5% agarose gel
to visualize the products. The resulting 250-bp cDNA fragments were
ligated into PCR 2.1 TA-vector (Invitrogen) and
sequenced.
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-Ziplox brain cDNA library (15) was
prepared with the use of both oligo(deoxythymidine) and random primers
(Stratagene, La Jolla, CA). Phage plaques were transferred
to nylon membranes (Micron Separations, Westboro, MA), exposed to
denaturing solution (2 M NaOH/1.5 M NaCl),
washed in 1.5 M NaCl/1 M Tris-HCl, and then
soaked in 2 x SSC (0.3 M NaCl/0.3 M
sodium citrate) for 5 min each time. DNA was fixed on membranes by
baking the membranes for 2 h at 80 C. A
[32P]deoxy-CTP-labeled probe was made from the
original PCR-amplified PACAP cDNA using a random primers DNA labeling
kit (Life Technologies, Inc.). Overnight hybridization was
performed at 42 C. Clones that remained hybridized after low (2 x
SSC/0.2% SDS) and high stringency (0.2 x SSC/0.2% SDS) washes
were selected. Plasmid DNA was excised from positive plaques and
sequenced initially with vector-specific primers and then with primers
corresponding to sequenced portions of the cDNA.
Tissue distribution of PACAP revealed by Northern blot and
RT-PCR
Total RNA was isolated from the brain, lung, muscle, intestine,
liver, and testis using TRIzol reagent. Total RNA (25 µg) of each
tissue was fractionated on a 1.5% agarose-formaldehyde gel, blotted to
nylon membrane, and fixed on the membrane by baking at 80 C for 2
h. The membrane was hybridized with the original 250-bp PCR product
32P-labeled using a random primers DNA labeling
kit. Hybridization was performed at 42 C as previously described (20).
The blot was washed sequentially in 2 x SSC/0.5% SDS and
0.2 x SCC/0.5% SDS at 55 C (twice each, for 15 min each time)
and exposed for 16 h in a PhosphorImager screen
(Molecular Dynamics, Inc., Sunnyvale, CA). Signals were
analyzed with ImageQuant software.
Total RNA (1 µg) from the above different tissues was subjected to
RT-PCR to amplify a 300-bp PACAP cDNA fragment (corresponding to
sequence 529–837 in Fig. 1
) using sense and antisense primers
(5'-CACTCAGATGGAATCTTC-3' and 5'-GTGCATTCTCTAGTGC-3', respectively).
After an initial 2-min denaturation at 94 C, 30 thermal cycles of 40
sec at 94 C, 30 sec at 52 C, and 40 sec at 72 C were performed. PCR
products were checked by 1.5% agarose gel electrophoresis and
transferred to a nylon membrane. To verify the PCR products, an
oligonucleotide, 5'-GGACGGCGTGTAGCATATTTG-3' (sequence from 643–652 in
Fig. 1), was end labeled with [
-32P]ATP and
hybridized overnight with the membrane at 37 C using the same buffer.
The blot was washed in 2 x SSC/0.5% SDS at 38 C three times for
10 min each time and exposed in a PhosphorImager screen for 16 h.
Signals were examined with ImageQuant software.
Distribution of GHRH/PACAP mRNA in the frog (Xenopus)
brain
Five adult male frogs were anesthetized with ketamine (0.5 g/kg)
and perfused through the heart with 4% paraformaldehyde in 0.1
M PBS. Brains were postfixed overnight with the same
fixative. Alternate coronal sections of 15 µm thickness throughout
brain were cut for in situ hybridization. Sections were
mounted on slides and treated with 0.25% acetic anhydride in 0.1
M triethanolamine. After washing in PBS, the
sections were incubated for 2 h at 60 C with a prehybridization
solution (20) and hybridized overnight at the same temperature with
33P-labeled PACAP antisense or sense riboprobes.
The original 250-bp PCR product cloned into PCR-2.1
(Invitrogen, San Diego, CA) was used as the template to
synthesize the antisense and sense riboprobes (20). After
hybridization, sections were washed with 2 x SSC for 1 h at
37 C and then incubated for 30 min with 0.8 mg/ml ribonuclease A in a
solution containing 500 mM NaCl, 10
mM Tris, and 1 mM EDTA.
Slides were washed with 1 x SSC for 15 min, dehydrated in
ascending alcohol, air-dried, and exposed to Kodak film
(Eastman Kodak Co., Rochester, NY). Two days later, the
films were developed, and the slides were dipped in Kodak
NTB-2 emulsion. After a 6-day exposure, slides were developed and
counterstained with 0.1% cresyl violet.
Castration-induced changes in PACAP and PAC1 receptor
mRNA expression in the hypothalamic anterior preoptic area
Seven male frogs were castrated, and three other male frogs were
sham operated under anesthesia with ketamine (0.5 g/kg). After
castration, three male frogs were injected sc with 0.1 mg testosterone
in sesame oil, and this injection was repeated at 12-h intervals until
perfusion. This dose was used because previous studies (21, 22) showed
that serum levels of testosterone in the castrated frogs receiving this
dose were about 2 times higher than those in normal frogs. Three days
after operation, all frogs were fixed by perfusion with 4%
paraformaldehyde in 0.1 M PBS. Brains were postfixed with
the same fixative. Alternate coronal sections (15 µm) throughout the
anterior preoptic area of the hypothalamus were cut for in
situ hybridization. The anterior preoptic area was chosen for
study because it appears to be critical for regulating the pituitary
gonadotropes. For example, neurons immunoreactive to mammalian GnRH
were restricted to this area in frog (23), and castration induced a
rapid decrease in the immunoreactivity of GnRH neurons in this region
(22). Antisense and sense RNA probes were made from the original
PCR-amplified 250-bp PACAP cDNA and a 300-bp Xenopus
PAC1 cDNA (15). All sections for control, castrated, and
testosterone-treated animals were analyzed in the same assay. The
procedure for in situ hybridization has been described
above. After hybridization, slides were immersed in Kodak
NTB-2 emulsion (diluted 1:1 with distilled water at 37 C) and exposed
for 7 days. After developing, slides were counterstained with 0.1%
cresyl violet and coverslipped.
Semiquantitative analyses of in situ hybridization signals
Semiquantification of 1) the number of hybridization-positive
neurons and 2) the relative mRNA intensities (the number of silver
grains per hybridizing cell) was determined in the anterior preoptic
area of the hypothalamus in control frogs and in castrated frogs with
or without testosterone replacement. All sections containing this area
from each animal were included in the analysis. The intensities of mRNA
signals were also calculated in neurons in the dorsal pallium and
medial amygdala, the structures immediately adjacent to the
anterior preoptic area on the same sections. In those areas, the
numbers of total and hybridization-positive neurons were counted
manually in each section. A cell that contained silver grains at least
3 times higher than background was considered positive. The abundance
of silver grains over a neuron was calculated by counting manually the
number of silver grains over a cell and subtracting the average number
of silver grains over an equivalent size area occupied by background
grains on the same section. Data from each hybridizing neuron on each
section were pooled and divided by the number of hybridizing cells.
This gave the average number of silver grains per cell for each
section. For each treatment group, a mean was obtained by calculating
the average of all sections. The mean values of positive cells per
section and silver grains per cell from the above three groups were
used for statistical analysis. Significance of difference was assessed
by one-way ANOVA followed by Dunnett’s test, and difference was
established at the level of P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To verify these results, we
sequenced all 14 other clones, the sizes of which ranged from 450-1200
bp. None had nucleotide sequence that was different from that shown in
Fig. 1.
The full-length PACAP cDNA consisted of about 2-kb nucleotides. The
first in-frame ATG of the cDNA (24) appears to represent the start
codon of a 513-bp single open reading frame of 171 amino acids. Within
the encoded peptide is a 46-amino acid peptide that has high similarity
to GHRH and another peptide with near identity to PACAP-38. An
alignment of the deduced peptide sequence of the GHRH/PACAP cDNA with
known sequences for these peptides showed high identity to PACAP and
GHRH in other species (Fig. 2). The
putative signal peptide (the first C-terminal 26 amino acids) of the
frog cDNA exhibited 61% identity with that of human and rat. The
Xenopus GHRH-like peptide was 72% identical to a chicken
and salmon GHRH, 52% identical to rat GHRH, and 48% identical to a
human or mouse PACAP-related peptide (PRP). The PACAP-38 showed the
greatest degree of identity (97%) to that of human and rat, with only
1 amino acid substitution of isoleucine for valine at position 35 (Fig. 2).
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), or testis (data not shown). The
size of the main hybridizing band was almost the same as that of 18S
ribosomal RNA (
2 kb). To confirm the Northern analyses, RT-PCR was
used to amplify PACAP mRNA sequence from the same tissues including
testis. A band of predicted size (300 bp) was amplified from brain
only. This hybridized with a 32P-labeled internal
primer after Southern transfer (Fig. 3).
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). The hybridization signals
of antisense probes over specific cells were much higher than that of
the sense probe (Fig. 4B). In the olfactory bulb, signals were highly
concentrated in the mitral cell layer but sparse in the granular or
glomerular cell layer (Fig. 4, C and D, and Fig. 5, A and B). The accessory olfactory bulb
also contained obvious mRNA signals (Fig. 5, C and D). Throughout the
anterior/posterior axis of the pallium, dense PACAP mRNA signals were
found primarily in the dorsal and lateral parts, but rarely in the
ventral portion (Fig. 5, C and D). Other telencephalon structures on
the same section, such as medial septum and dorsal striatum, did not
contain obvious PACAP mRNA signals. In the anterior diencephalon, a
moderate level of PACAP mRNA was found in the hypothalamic anterior
preoptic area, although strong signals were situated in the lateral
pallium (Fig. 5, E and F) and lateral amygdala (not shown). In the
middle/posterior diencephalon, abundant mRNA signals were observed in
the ventral hypothalamus, ventral thalamus, and dorsolateral thalamic
nucleus (Fig. 5, G and H). We also detected strong signals in the
infundibular nucleus.
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, A and B) and nuclei of oculomotor
nerve (data not shown). Moderate to weak PACAP mRNA was also found in
the optic tectum. Starting from the posterior mesencephalon, signals
were detected throughout the stratum griseum superficiale tecti.
Although no PACAP gene transcripts were localized over the pituitary
(Fig. 6, C and D), strong hybridization signals were found in nuclei of
isthmi and posteroventral tegmentum on the same sections (Fig. 6, C and
D). No expression of PACAP mRNA was evident in the cerebellar Purkinje,
molecular, and granule cell layers. Beginning from the medulla, PACAP
mRNA was markedly distributed in the griseum central rhombencephali,
nuclei of the trigeminal nerve (Fig. 6, E and F) and nuclei of
cerebellum. In posterior medulla, gene transcripts were abundant in the
trigeminal nerve nucleus and cuneate nucleus (Fig. 6, G and H). PACAP
mRNA in the spinal cord was weakly present mainly in the deep portion
of the ventral horn and the medial ventral horn (data not shown).
Castration-induced changes in the expression of PACAP and
PAC1 receptor mRNAs
The hypothalamic anterior preoptic area in the frog begins just
rostral to the disappearance of anterior commissure on coronal
sections. In intact animals, about 39.2 ± 2.6 (mean ±
SEM) cells in the anterior preoptic area on each section
contained clustered PACAP mRNA signals, most of which were situated in
the dorsal and middle portions (Fig. 7, A, D, and G). The average density of signals in these cells over
background is shown in Table 1. After
castration, the number of PACAP-containing cells in the anterior
preoptic area of the castrated frogs showed no significant change
(41.3 ± 1.8). However, the density of hybridized signals over
cells was 29% higher than that in control animals (Table 1). The
elevation of the PACAP gene expression after castration appeared more
evident in the dorsal and middle portions of the anterior preoptic area
than in the ventral part of this nucleus (Fig. 7, B, E, and H).
Testosterone treatment after castration prevented castration-induced
increased PACAP mRNA expression (Fig. 7, C, F, and I). The silver
grain density in the anterior preoptic area of hormone-treated
castrated frogs was not markedly different from that in control frogs
(Table 1).
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and Fig. 8). Testosterone
treatment reversed the increased expression of PAC1
receptor mRNA induced by castration (Table 1). The mean density of
silver grains in testosterone-treated castrated frogs was not
significantly different from that in control frogs. Castration of male
frogs or testosterone treatment of castrated frogs did not induce
significant changes in the intensity of PACAP or PAC1 mRNA
in the cells situated in the lateral amygdala or dorsal pallium
compared with that of control animals (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The peptide sequences of Xenopus PACAP-27 and PACAP-38 are conserved 100% and 97%, respectively, with the human counterparts (1). The difference in PACAP-38 is in only one amino acid at position 35, consistent with the report by Chartrel et al. (29), who sequenced PACAP-38 peptide in the frog Rana ridibunda by chemical methodology. The brain distribution and biological functions of PACAP have been examined in the hypothalamus, pituitary, and adrenal gland of Rana (13, 14, 18, 29). Thus, the PACAP-38 encoded by the GHRH/PACAP mRNA reported here probably represents the major functional PACAP-38 in the Xenopus brain. PACAP mRNA in all species examined to date encodes two neuropeptides on the precursor, PACAP in all species and another peptide, either a GHRH-like peptide in nonmammal vertebrates (30, 31) or PRP in mammals (32). Because the upstream peptide in Xenopus PACAP mRNA shows high identity to nonmammalian GHRH but low identity to the rat or human PRP, we conclude that this peptide is likely to function in Xenopus as a GH-releasing factor.
It is known that the genomic organization of Xenopus laevis is tetraploid, so two distinct PACAP mRNAs were expected to be present (33). We recently identified an alternate form of PACAP mRNA from the Xenopus brain cDNA library. In this mRNA, the region encoding the first 32 amino acids of GHRH is deleted, whereas the PACAP-encoding sequence is intact (with 1 amino acid substitution compared with that of Xenopus GHRH/PACAP mRNA). The nucleotide sequence of the open reading frame (not shown) and the deduced polypeptide precursor sequence in this mRNA were 95% and 94% identical, respectively, to those of the Xenopus GHRH/PACAP mRNA. Although we do not know whether these two mRNAs originate from 2 different genes, the overall nucleotide sequences of the two Xenopus PACAP precursor mRNAs reported here are too dissimilar outside the truncated region to have arisen by alternative splicing. One possible hypothesis is that two different PACAP genes are present in Xenopus, one of which transcribes a primary gene transcript that is spliced to a form truncated in the GHRH-encoding region (34, 35). As the nucleotide and deduced peptide sequence of this alternate PACAP mRNA differs significantly from that encoding the full GHRH/PACAP precursor, its origin, distribution, and biological and functional characteristics in Xenopus need to be clarified.
Distribution of GHRH/PACAP mRNA in Xenopus brain
Our Northern and RT-PCR analyses revealed the presence of PACAP
mRNA in the brain, but not in several other tissues. Although Yon
et al. (13) used rabbit antiovine PACAP-27 antibody to study
PACAP ligand distribution in the frog Rana ridibunda brain,
the detailed location of PACAP mRNA in a frog has not been reported.
Our results in Xenopus laevis revealed a wide distribution
of highly clustered mRNA signals over specific cells in the brain. The
overall distribution of PACAP gene expression in the Xenopus
brain is somewhat similar to that in mammals (36, 37). For example,
PACAP mRNA is present in the nucleus of ventral hypothalamus, the
dorsal thalamus, and the nucleus of cerebellum in both
Xenopus and rat. However, PACAP mRNA was not observed in the
bed nucleus of the stria terminalis and the medial septum of
Xenopus brain, unlike in the rat (38). These differences
suggest that PACAP performs additional functions in mammals that are
associated with these latter structures.
Compared with an immunohistochemical study in Rana ridibunda that used a PACAP-27 antibody (13), we noted similarities as well as several differences in the localization of PACAP-containing cells. Both methodologies showed PACAP-expressing cells in the ventral hypothalamus, thalamus, and torus semicircularis. Yon et al. (13), however, did not note PACAP-containing cells in the pallium, stratum griseum superficial tecti, nucleus of oculomotor nerve, and nucleus of cerebellum, whereas we observed strong mRNA signals in these areas. On the other hand, we did not identify marked hybridization signals in the pallial commissure, the bed nuclei of the pallial commissure, and the nuclei of accumbens. The discrepancies might be due to the differences in the frog species examined or in the methodologies.
The distribution of PACAP mRNA in many brain areas overlapped with that of PAC1 receptor mRNA (15) in Xenopus and with that of PACAP-27-binding sites (16) in Rana. These structures include the dorsal and lateral pallium, ventral hypothalamic nucleus, dorsal and ventral thalamus, nucleus of cerebellum, nucleus isthmi, and torus semicircularis. Although PAC1 receptor mRNA was moderately localized in the Purkinje cell layer of the cerebellum, PACAP mRNA was not found in this layer. In the olfactory bulb, PACAP mRNA was mainly distributed in the mitral cell layer, whereas PAC1 receptor mRNA was mainly in the granule cell and glomerular layer. The distribution patterns of PACAP and PAC1 mRNA in Xenopus brain might suggest important PACAP signaling and target fields. In fact, PAC1 receptor mRNA showed a wider distribution than PACAP in the Xenopus brain, suggesting multiple target sites for PACAP cells.
Potential role of PACAP in gonadal axis feedback regulation in
Xenopus
Earlier studies in mammals demonstrated that PACAP regulates the
function of endocrine cells in the hypothalamus (3), pituitary (6, 7, 8),
and testis (11, 39). In the frog brain, several studies have suggested
potential actions of PACAP on the hypothalamus (15, 16) and pituitary
(17, 29). However, no studies appear to have addressed whether PACAP
can regulate the function of the testis in the frog. Our Northern
hybridization and RT-PCR analyses did not reveal the presence of
detectable PACAP or PAC1 receptor mRNA in the testis (not
shown), suggesting that the PACAP and PAC1 mRNAs reported
here may not execute obvious local effects on the adult testis in
Xenopus. Although we cannot exclude the possible presence of
other forms of PACAP or PAC1 receptor variants in the male
frog testis, our results suggest that PACAP might affect the frog
gonadal axis feedback mainly in the hypothalamus and possibly the
pituitary. The latter expresses a PACAP-specific (PAC1)
(15) as well as a vasoactive intestinal polypeptide/PACAP receptor
(40).
Previous studies in the male rat indicated that acute castration
resulted in up-regulated expression of GnRH mRNA in the hypothalamic
preoptic area (41, 42, 43) and promoted the release of FSH and LH from the
pituitary (44, 45, 46). In other studies, testosterone treatment of
castrated animals prevented castration-induced release of GnRH from the
hypothalamus (47) and release of LH (48) and FSH (49) from the
pituitary. It is generally agreed that hypothalamic GnRH neurons and
pituitary gonadotropes receive information from multiple hormonal and
synaptic inputs and integrate these to appropriately regulate
reproductive function. Previous studies indicated that PACAP treatment
enhanced hypothalamic GnRH mRNA levels (4), stimulated pituitary LH and
FSH release (5-7), and elevated the expression of
gonadotropin -unit (8). PACAP was also reported to interact with
GnRH to regulate the activation of pituitary gonadotropes (9, 10).
Thus, PACAP may be part of the reproductive regulator network by way of
its direct or indirect effects on hypothalamic GnRH cells and on
pituitary gonadotropes.
In the frog it has been demonstrated that the mammalian GnRH neurons are relatively confined to the anterior preoptic area (50). GnRH immunoreactivity in cells in this area is down-regulated after removal of endogenous testosterone, apparently due to the continuous secretion of GnRH (51). These studies may imply a functional role for the neurons in the hypothalamic anterior preoptic area in the control of GnRH secretion or synthesis in the frog brain. Our results in Xenopus showed that short-term castration induced an increase in the expression of PACAP and PAC1 receptor mRNA in the Xenopus anterior preoptic area. These effects were blocked by testosterone replacement, suggesting that deficiency of endogenous androgen, rather than other testicular factors, was responsible for the changes in PACAP and PAC1 receptor mRNA expression. The induction of PACAP and PAC1 receptor gene expression in the frog preoptic area after androgen loss may result in increased activity of PACAP on hypothalamic GnRH cells and pituitary gonadotropes. This would be expected to lead to enhanced synthesis and release of GnRH and LH/FSH. This hypothesis was supported by the fact that frog PACAP-38 stimulated LH release from cultured Rana pituitary cells (46). The castration induction of the PACAP ligand/receptor system may thus be a part of a normal feedback response associated with the feedback control of the gonadal axis.
On the other hand, it is of interest that GHRH in Xenopus is encoded on the same mRNA as PACAP. Previous studies indicated that GHRH might have a role in regulating reproduction. For example, human GHRH stimulated the release of LH and FSH from cultured rat pituitary cells (52), and GHRH secreted from rat testis interstitial and germ cells appears to directly modulate the function of testis Sertoli cells (53, 54). The distribution of GHRH immunoreactivity in the frog has been described (55), and a teleost receptor relatively specific for a GHRH-like peptide has recently been cloned (56). Furthermore, human GHRH has been shown to stimulate cAMP accumulation and to enhance GH release in the frog pituitary (57). Moreover, it seems possible that GHRH has wider functions in the frog brain. In any case, it will be of interest to determine whether GHRH executes effects separate from those of PACAP on the frog gonadal feedback axis.
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Acknowledgments |
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Footnotes |
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2 The GenBank access numbers for GHRH/PACAP and
PAC1 mRNA are AF187878 and AF187877, respectively.
3 The first two authors made equal contributions to this
work.
Received December 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1
cells. Ann NY Acad Sci 805:112–120[Abstract]
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