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Effect of hypophysectomy on pituitary adenylate cyclase activating polypeptide gene expression in the rat hypothalamus
Peptides, Vol. 16, No. 3, pp. 407-413, 1995 Copyright 8 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0196-9781/95 $9.50 + .o
Pergamon
0196-9781(94)00198-7
Effect of Hypophysectomy on Pituitary Adenylate Cyclase Activating Polypeptide Gene Expression in the Rat Hypothalamus YUJIN
SHUTO,*t
ANIK6
SOMOGYVARI-VIGH,*t
HARUO
ONDAfl
AND
AKIRA
ARIMURA*t$$’
*U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, Departments qf fMedicine, $Physiology, and §Anatomy, Tulane University School of Medicine, New Orleans, LA 70112, and j/Discovery Research Laboratories 1, Takeda Chemical Industries Ltd., Tsukuba, Ibaraki 300-42, Japan Received
12 August
1994
SHUTO, Y., A. SOMCOYVARI-VIGH, H. ONDA AND A. ARIMURA. Effect of hypophysectomy on pituitary odenylute cyclase activating polypeptide gene expression in the rut hypothalamus. PEPTIDES 16(3) 407-413, 1995.-Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family. It stimulates adenylate cyclase in cultured rat pituitary cells, which have PACAP-specific receptors and expression of pituitary hormones. Therefore, PACAP is c:onsidered as a hypophysiotropic hormone. If so, them might be a feedback regulatory mechanism between pituitary hormones and hypothalamic PACAP. In the present study, we used nuclear run-on and RNase protection assays to examine whether transcription of the PACAP gene in the rat hypothalamus would change after hypophysectomy. PACAP levels in the hypothalamus were also determined by radioimmunoassay. The transcriptional rate of the PACAP gene and PACAP mRNA content decreased 1 and 2 weeks after hypophysectomy. Radioimmunoassayable PACAP levels in the hypothalamus also decreased after hypophysectomy. These findings suggest that the reduced rate of PACAP gene transcription after hypophysectomy causes the decreased mRNA .md peptide levels in the hypothalamus. Replacement with GH, PRL, T.,. corticosterone, and testosterone significantly restored PACAP mRNA levels in hypophysectomized rats to those in control animals. The results suggest that feedback regulation takes place between pituitary hormones or pituitary-dependent factors and hypothalamic PACAP. PACAP
Hypophysectomy
Hypothalamus
Rat
Transcription
PITUITARY adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family (18). It stimulates adenylate cyclase in cultured pituitary cells (18), and its specific receptors are present in the pituitary cells (10,26). PACAP increases the release of GH, PRL, and ACTH from perifused rat pituitary cells, but does not alter the secretion of anterior pituitary hormone in static rat pituitary monolayer cultures (18). Other investigators have reported that PACAP slightly stimulates GH release but inhibits prolactin secretion as determined by reverse hemolytic assay (11). Moreover, PACAP and LHRH exhibit a synergistic effect on LH and FSH release in static rat pituitary cell culture (8), but PACAP does not affect the action of GRF or CRF on GH or ACTH release, respectively (1). Tatsuno et al. (24) have demonstrated that PACAP stimulates the release of interleukin 6 (IL6) from cultured rat pituitary cells, possibly from folliculo-stellate cells, through a CAMP-dependent protein kinase A pathway, suggesting that these cell types in the pituitary are also targets for PACAP. An immunohistochemical study showed that, after
’ Requests for reprints should be addressed Belle Chasse, LA 70037.
to Dr. Akira Arimura,
hypophysectomy, the intensity of PACAP-like immunoreactivity increased in the nerve fibers in the median eminence and the perikarya in the paraventricular and supraoptic nuclei ( 13). These data suggest that hypophysectomy increases the synthesis of PACAP in these neurons or suppresses the release of PACAP from the nerve terminals. However, immunohistochemistry does not necessarily precisely show the quantitative pattern of PACAP biosynthesis or gene expression. Therefore, we examined PACAP content as well as the expression of the PACAP gene in hypothalami from normal and hypophysectomized rats. The combined hormone treatment (GH, PRL, T4, corticosterone, and testosterone) was also made in hypophysectomized rats, and its effect on PACAP tnRNA
levels
was examined. METHOD
Animals Both hypophysectomized 200-250 g) were purchased
U.S.-Japan
407
Biomedical
Research
and intact male rats (CD strain; from Charles River Breeding Lab-
Laboratories,
Tulane University
Hebert Center,
408
SHUT0
Rat PACAJ cDNA
oratories (Wilmington, MA). Animals were housed in a light (lights on at 0600 h, lights off at 1800 h)- and temperature-controlled room and fed standard Purina Lab Chow and water ad lib. Hypophysectomized rats were allowed to drink tap water containing 0.9% NaCl and were given sugar cubes. Completeness of the hypophysectomy was assessed by visual inspection of the sella turcica after the animals were sacrificed. Rats were killed by decapitation 1 or 2 weeks after surgery under ketamine-xylazine anesthesia. The hypothalamic area (rostra1 portion; preoptic area, caudal portion; mamillary complex), including the paraventricular and supraoptic nuclei, was immediately removed and placed in liquid nitrogen. The tissues were kept at -80°C until analysis. All animal studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Hormone Replacement
ET AL.
Therapy
The combined treatment with GH, PRL, T.,, corticosterone, and testosterone was given to the hypophysectomized rats for the hormone replacement. Three-week release pellets containing T4 (15 mg), corticosterone (100 mg), and testosterone (5 mg) were obtained from Innovative Research (Toledo, OH) and implanted subcutaneously under ketamine-xylazine anesthesia. Rat GH (25 pg) and rat PRL (50 ,ug) were kindly provided by the NIDDK and the National Hormone and Pituitary Program. Rats were injected subcutaneously with GH (25 pg/injection, twice daily) and PRL (50 &injection, twice daily). Four days after hypophysectomy, a group of rats was given replacement therapy. Control animals were injected with vehicle and subjected to skin incision without pellet placement. Animals were sacrificed by decapitation under ket&nine-xylazine anesthesia after 10 days of hormone replacement (2 weeks after hypophysectomy). RNA Isolation and Preparation
of Probes
Poly(A)+ RNA was extracted from rat hypothalami using Poly A Tract System 1000 (Promega Corporation, Madison, WI). pRB3Z3, containing the 670-base pair Pst I/EcoR I fragment of rat PACAP cDNA (21), was used to make 32P-labeled cRNA transcripts (Fig. 1). The plasmid was linearized with Hind III and transcribed with [~Y-~*P]UTP(Du Pont NEN Research Products, Boston, MA) using MAXIscriptTM T7 Kit (Ambion Inc., Austin, TX). To make the probe for p-actin, pTRI-@Actin (Ambion Inc., Austin, TX) containing the 250-base pair fragment of the mouse p-actin gene was transcribed as described above. After the RNA transcription reaction, the probes were treated with DNase I and electrophoresed through 5% polyacrylamide/8 M urea gels. The gel was then exposed to X-ray film, and the area of the gel that contained the full-length labeled transcripts was excised. The elution of the probes was performed overnight in the buffer [0.5 M NH40Ac, 1 mM EDTA, and 0.2% sodiumdodecyl sulfate (SDS)] at 37°C. RNase Protection Assay RNase protection assays were performed using RPA II@@Kit (Ambion Inc., Austin, TX). Poly(A)+ RNA (2 pg) from each hypothalamus was coprecipitated with 6 X 104 cpm of 32P-labeled PACAP probe prepared as described above. The same cpm of p-actin probe was also coprecipitated with 0.1 pg of poly(A)+ RNA. Hybridization was then performed at 45°C overnight. RNase digestions were carried out at 37°C for 30 min. The protected fragments were precipitated and size separated on 5% polyacrylamide/8 M urea gels. The gels were exposed to X-ray film at -70°C overnight. The levels of RNA in each lane were quantified using a real-time video analysis system (Hamamatsu C2400
FIG. 1. Organization of the pRB3Z3. The region corresponding to mature PACAP is indicated with a box. The 670-base pair Pst I/&OR I fragment of PACAP cDNA was subcloned into PGEM-3Zf(-) @omega Corporation, Madison, WI). The plasmid was linearized and transcribed with T7 RNA polymerase
to prepare the ‘ZP-labeled cRNA probe.
video camera, DataTranslation Frame Grabber video card, Macintosh Quadra 950, and “NIH IMAGE 1.44” software). For each sample, PACAP signal strength was normalized to the corresponding @actin signal. Nuclear Run-On Transcription
Assay
The nuclear run-on assay was performed as described by White et al. (27) with minor modifications. Hypothalami were obtained from hypophysectomized and control rats as described above. Nuclei were isolated from individual hypothalami. The transcription reaction was performed in the presence of 1 X lo7 isolated nuclei, 20 mM Tris-HCl (pH 7.9), 20% glycerol, 140 mhf KCl, 10 mM MgCl*, 1 n-&f ditbiothreitol, 0.1 mg/ml creatine phosphokinase, 8.5 m&f phosphocreatine, 2 mM each ATP, CTP, and GTP, 1 U/ml RNasin, and 160 ,uCi [cY-~*P]UTP (800 Ci/ mmol). The reactions were allowed to proceed for 30 min at 30°C. The reaction mixture was then digested with DNase I and proteinase K, followed by RNA extraction with phenol-chloroform. RNA was precipitated two times with ammonium acetate and ethanol. Aliquots of synthesized RNA were counted with a liquid scintillation counter. The immobilized template for assaying the radiolabeled transcripts was pRB38Pl containing the entire coding region of the rat PACAP gene (21). pUC118, the vector of pRB38P1, was also used as a control. These plasmids were denatured and immobilized on nitrocellulose. Prehybridization was carried out for 20 h at 42°C in the hybridization buffer [0.2% SDS, 50 mM TES (pH 7.4), 0.3 M NaCl, 10 mh4 EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.1% sodium phosphate, and 0.1 mg/ml yeast tRNA]. After prehybridization, the filters were incubated for 48 h at 42°C in the hybridization buffer containing the total transcripts (1 X lo6 cpm). The filters were then washed two times in 2 X SSC (1 X SSC; 0.15 M NaCl and 0.015 h4 Na citrate, pH 7.0) and 0.1% SDS for 30 min at room temperature, and two times in 0.2 x SSC and 0.1% SDS for 10 min at 50°C. The filters were exposed to X-ray film at -70°C for 10 days. The autoradiographic dots were quantified as described above. The transcriptional rate of the PACAP gene was calculated after subtracting the nonspecific background hybridized to pUCl18.
PACAP
EXPRESSION
AWER
409
HYPOPHYSECTOMY
control
hypox 1 w hypox 2w B
FIG. 2. Effect of hypophysectomy on hypothalamic PACAP mRNA levels. (A) Autoradiographs of the RNase protection assay. Lanes l-3: control; lanes 4-6: 1 week after hypophysectomy; lanes 7-9: 2 weeks after hypophysectomy. The positions of the RNA size markers are shown to the left of gel. (B) Quantification of PACAP mRNA levels. Data are the mean t SE of RNA from three individual rat hypothalami. mRNA levels in arbitrary units are shown as a percent of the control values. hypox, hypophysectomized rats. Statistical significance was examined1 by one-way ANOVA. *p < 0.05 vs. the control group.
Radioimmunoassay The tissues were homogenized in 1 ml of 20% trifluoroacetic acid using a glass homogenizer and aliquots were taken for the protein assay. The homogenates were then centrifuged at 15,000 x g for 30 min. The supernatants were collected and evaporated by a Speed-Vat Concentrator (Sa-
vant were
Instruments, reconstituted
Inc.,
Hicksville,
NY).
The
samples
in 1 ml of distilled water and lyophilized twice. The residues were dissolved in RIA assay buffer and PACAP was assayed as previously described (2). The aliquots of the homogenate were lyophilized and the protein was assayed using the Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA).
410
SHUT0
ET AL
FIG. 3. Autoradiographs of the nuclear run-on assay. The hybridization probe was the rat PACAP cDNA (pRB38Pl) containing the entire coding region of the PACAP gene. pUCll8 is the vector of pRB38Pl and served as a hybridization control. (A )One week after hypophysectomy. Lanes l-3: control; lanes 4-6: 1 week after hypophysectomy. (B) Two weeks after hypophysectomy. Lanes 1-3: control; lanes 4-6: 2 weeks after hypophysectomy.
Statistical Analysis
Effect of Hypophysectomy PACAP Gene
Results were presented as the mean + SE. Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan New Multiple Range or Student’s t-test. A value ofp < 0.05 was considered significant.
RESULTS
Effect of Hypophysectomy
on the Content of PACAP tnRNA
Because of low levels of PACAP mRNA in total hypothalamic RNA, PACAP mRNA levels were measured using solution hybridization followed by nuclease protection, which is more sensitive than Northern blot analysis (15). Hypophysectomy appeared to decrease the content of PACAP mRNA [Fig. 2(A)]. The major protected bands were found at 670 nucleotides, and a few minor bands were also present [Fig. 2(A)]. Changing the concentrations of RNase did not change this pattern. Because the PACAP probe contains the 3’ noncoding region (Fig. l), these minor fragments might result from the use of alternate polyadenylation sites. Quantitative data were obtained by image analysis. PACAP mRNA levels decreased following hypophysectomy to 70.3 ? 10.5% of control values after 1 week and to 60.0 5 6.2% of control values after 2 weeks [Fig. 2(B)]. A significant decrease was seen 2 weeks after hypophysectomy @ < 0.05).
on the Transcriptional
Rate of the
To determine whether the decreased PACAP mRNA levels after hypophysectomy were due to an altered transcriptional rate of the gene, the nuclear run-on assay was performed. Clear activity was observed in the control rats, but the dots from hypophysectomized rats were very faint [Fig. 3(A,B)]. No hybridization was observed with the plasmid devoid of PACAP (pUC118). Figure 4(A,B) shows the quantification of the autoradiographs. The rate of PACAP gene transcription decreased to 69.4 + 15.1% 1 week after and to 45.1 -C 4.6% 2 weeks after hypophysectomy [Fig. 4(A,B)J. A significant decrease was seen 2 weeks after hypophysectomy (p < 0.05). Effect of Hypophysectomy
on PACAP-Like Immunoreactivity
Radioimmunoassayable PACAP in the hypothalamus also decreased to 80.3 ? 10.6% 1 week after and to 73.5 2 7.3% 2 weeks after hypophysectomy (Fig. 5). The decrease at 2 weeks was significant (p < 0.05), being a similar tendency as that for mRNA levels and transcriptional rates. Effect of Replacement Therapy on the Content of PACAP mRNA in Hypophysectomized Rat The combined replacement therapy with GH, prolactin, T4, corticosterone, and testosterone significantly increased the con-
PACAP
EXPRESSION
control
AFTER
411
HYPOPHYSECTOMY
* II
hypox 1w
hypox
hypox
hypox2~
control
6
A
control
PIG. 4. Effect of hypophysectomy on the rate of hypothalamic PACAP gene transcription. Data are the mean + SE. The transcriptional rate in arbitrary units is shown as a percent of the control values. Statistical significance was examined by Student’s r-test. *p < 0.05 vs. the control group. (A) One week after hypophysectomy. (B) Two weeks after hypophysectomy.
tent of PACAP mRNA in the hypothalamus of 2-week postoperative hypophysectomized rats compared to the untreated hypophysectomized animals @ < 0.05) (Fig. 6). DISCUSSION The present study demonstrated that hypophysectomy decreased the transcriptional rate of the PACAP gene and the levels of both its mRNA and peptide in the hypothalamus.
Positive or negative feedback regulation by pituitary hormones was proposed for several hypothalamic hypophysiotropic peptides after hypophysectomy, as shown in Table 1 (3,5,6,9,12,14,16,17,19,22,23,25,28). The levels of these hypophysiotropic peptides and their mRNA in the hypothalamus changed in different ways after hypophysectomy. Hypophysectomy reduced LHRH levels and its mRNA as demonstrated for PACAP (Table 1). Testosterone treatment of hypophysectomized rats raised LHRH content to that of intact rats, suggesting that the reduction of hypothalamic LHRH after hypophysectomy was due mainly to loss of testosterone secretion (6). To test whether pituitary hormones and/or pituitary-dependent factors may exert their feedback effect on hypothalamic PACAP by influencing the expression of the PACAP
G&PRLJ.,C,T
PIG. 6. Effect of replacement therapy (GH; PRL; T4; C, corticosterone; and T, testosterone) on the content of PACAP mRNA in hypophysectomized rats. Data are the mean ? SE of RNA from three individual rat hypothalami. mRNA levels in arbitrary units are shown as a percent of the control values. hypox, hypophysectomized rats. Statistical significance was examined by one-way ANOVA. *p < 0.05 vs. the control group. +p < 0.05 vs. the hypox group.
gene, hypophysectomized animals were treated with a combination of hormone replacement (GH, prolactin, Tq, corticosterone, and testosterone). The replacement therapy significantly restored PACAP mRNA levels in hypophysectomized rats to those in intact rats. Because there exists a feedback regulation between hypothalamic peptides and pituitary hormones or pituitary-dependent factors, it is possible that one or more of the replaced hormones may regulate PACAP gene expression through these feedback mechanisms. However, we cannot rule out the possibility that a decrease in general metabolism induced by hypophysectomy contributed, at least partly, to the decrease in the transcriptional rate of the PACAP gene, and the levels of PACAP mRNA and PACAP immunoreactivity that we have observed. PACAP appears to be different from classical releasing factors in terms of its action on pituitary cells. Although PACAP stimulates adenylate cyclase in the rat pituitary cells, it did not alter the secretion of any known anterior pituitary hormone in static rat pituitary monolayer cultures (18). PACAP and LHRH had a synergistic effect on LH and FSH release in static rat pituitary cell culture (8), but PACAP did not affect the action of GRF or CRF on GH or ACTH release, respectively (1). PACAP increased the release of GH, PRL, and ACTH from perifused rat pituitary cells. However, PACAP injected IV into rats did not stimulate secretion of any of the pituitary hormones except that
TABLE
1
OF HYFOPHYSECTOMY ON HYPOTHALAMIC NEUROPEPTIDES
EFFECT
control
hypox
lw hypox 2w
5. Effect of hypophysectomy on the PACAP immunoreactivity. Data are the mean -C SE of immunoreactivity from six individual rat hypotbalami. The immunoreactivity is shown as ng/mg protein. Statistical significance was examined by one-way ANOVA. *p < 0.05 vs. the control group. FIG.
Neuropeptides
Immunoreactivity
mRNA Content
PACAP GRP Somatostatin CRP LHRH TRH VIP
1 -1 1 +t 1 1-t t
1 t +l +t 1 t t
t, increase;
1, decrease;
+ no significant
Transcriptional Rate
References
1 17,28 22, 25, 28 9, 19 3, 6, 16 12, 14, 23 5, 13 change.
412
SHUT0
it released both PRL and GH following injection into lactating rats (20) and that it stimulated PRL and GH release in hypothalamus-lesioned rats (11). Others have reported that PACAP stimulated the expression of the PRL gene in the rat GH3 pituitary cell line, which was directed by the PRL promoter (7). POMC mRNA expression was also demonstrated to be induced by PACAP in the AtT-20/D16v corticotrope cell line (4). These data suggest that PACAP may be a regulator of the gene transcription of pituitary hormones rather than a releasing factor. On the other hand, our previous data demonstrated that more than 90% of folliculo-stellate cells bound biotinylated PACAP, suggesting that folliculo-stellate cells are a major target for PACAP (26). PACAP stimulated the release of IL-6 from cultured rat pituitary cells, possibly from folliculo-stellate cells (24). Other known hypothalamic factors, including CRF, GRF, TRH, and LHRH, failed to alter the production of IL-6. Because IL-6 was reported to stimulate ACTH when injected IV and to alter the release of PRL, GH, FSH, and LH in cultures of pituitary cells (24), PACAP might have the potency to release pituitary hormones through IL-6. These findings support the view that PACAP may be an auxiliary releasing factor of pituitary hormones and that, in certain conditions (e.g., lactating rats), it may regulate the release of pituitary hormones through some factor released from folliculo-stellate cells. As shown in Table 1, the changes in mRNA content after hypophysectomy do not necessarily reflect the levels of peptides. For LHRH and somatostatin, the levels of both peptide and mRNA decreased after hypophysectomy. In contrast, GRF mRNA levels were increased after hypophysectomy, whereas hypophysectomy resulted in a decrease in GRF content. These data illustrate the difficulties in using the peptide content data as an index of the synthetic capacity for a peptide after hypophysectomy (22). The changes in peptide content may be attributable to a change in transcription, translation, processing, degradation, or release. The changes in mRNA levels are also a reflection of changes in transcription and/or mRNA stability. Because the results of the nuclear run-on assay agree with those of the RNase protection assay and radioimmunoassay, it may be reasonable to conclude that the reduced rate of PACAP gene transcription results in a decrease in both mRNA and peptide levels in the hypothalamus after hypophysectomy. Thus, modulation of PACAP gene transcription could be one mechanism of feedback regula-
ET AL.
tion of hypothalamic PACAP by some pituitary hormones or pituitary-dependent factors. Our previous immunohistochemical study showed that the intensity of PACAP-immunoreactive staining in the internal zone of the median eminence and in the perikarya of the paraventricular and supraoptic nuclei enhanced after hypophysectomy compared to the control rats (13). At the same time, abundant PACAP-immunoreactive fibers appeared in the external zone of the median eminence after hypophysectomy. In contrast, the present RIA data showed that PACAP-like immunoreactivity decreased after hypophysectomy. The discrepancy between the previous immunohistochemical results and the results in the present study is difficult to explain, but it could be explained in two ways. First, because the antibody used in RIA was different from that used in immunohistochemistry, radioimmunoassayable PACAP may not be necessarily the same as PACAP-like immunoreactivity demonstrated by immunohistochemistry. We often experienced that an antibody useful for RIA was not always good for immunohistochemistry. Second, hypophysectomy could result in alteration in the plasticity of PACAP neurons in the hypothalamus, altering the distribution of PACAP immunostaining. Considering that our previous immunohistochemistry was not a quantitative study, the altered distribution of PACAP immunostaining might mask an overall decrease in immunoreactivity in the whole hypothalamus. In summary, we have used nuclear run-on and RNase protection assays to assess PACAP gene transcription in response to hypophysectomy. We have demonstrated that hypophysectomy decreased the transcriptional rate of the PACAP gene, its transcripts, and the levels of PACAP in the hypothalamus, suggesting that the reduced rate of PACAP gene transcription results in decreased mRNA and peptide levels in the hypothalamus. Replacement therapy with GH, prolactin, T.,, corticosterone, and testosterone significantly restored the content of PACAP mRNA in hypophysectomized rats. Our observations imply that feedback regulation of hypothalamic PACAP is mediated by some pituitary hormones and/or pituitary-dependent factors. ACKNOWLEDGEMENTS
We wish to thank Drs. Sandor Vigh, Richard Harlan, and Carol Phelps for their valuable advice. This study was supported in part by NIH grant DK 09094 and a grant from Takeda Chemical Industries, Ltd.
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Pergamon
0196-9781(94)00198-7
Effect of Hypophysectomy on Pituitary Adenylate Cyclase Activating Polypeptide Gene Expression in the Rat Hypothalamus YUJIN
SHUTO,*t
ANIK6
SOMOGYVARI-VIGH,*t
HARUO
ONDAfl
AND
AKIRA
ARIMURA*t$$’
*U.S.-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, Departments qf fMedicine, $Physiology, and §Anatomy, Tulane University School of Medicine, New Orleans, LA 70112, and j/Discovery Research Laboratories 1, Takeda Chemical Industries Ltd., Tsukuba, Ibaraki 300-42, Japan Received
12 August
1994
SHUTO, Y., A. SOMCOYVARI-VIGH, H. ONDA AND A. ARIMURA. Effect of hypophysectomy on pituitary odenylute cyclase activating polypeptide gene expression in the rut hypothalamus. PEPTIDES 16(3) 407-413, 1995.-Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family. It stimulates adenylate cyclase in cultured rat pituitary cells, which have PACAP-specific receptors and expression of pituitary hormones. Therefore, PACAP is c:onsidered as a hypophysiotropic hormone. If so, them might be a feedback regulatory mechanism between pituitary hormones and hypothalamic PACAP. In the present study, we used nuclear run-on and RNase protection assays to examine whether transcription of the PACAP gene in the rat hypothalamus would change after hypophysectomy. PACAP levels in the hypothalamus were also determined by radioimmunoassay. The transcriptional rate of the PACAP gene and PACAP mRNA content decreased 1 and 2 weeks after hypophysectomy. Radioimmunoassayable PACAP levels in the hypothalamus also decreased after hypophysectomy. These findings suggest that the reduced rate of PACAP gene transcription after hypophysectomy causes the decreased mRNA .md peptide levels in the hypothalamus. Replacement with GH, PRL, T.,. corticosterone, and testosterone significantly restored PACAP mRNA levels in hypophysectomized rats to those in control animals. The results suggest that feedback regulation takes place between pituitary hormones or pituitary-dependent factors and hypothalamic PACAP. PACAP
Hypophysectomy
Hypothalamus
Rat
Transcription
PITUITARY adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family (18). It stimulates adenylate cyclase in cultured pituitary cells (18), and its specific receptors are present in the pituitary cells (10,26). PACAP increases the release of GH, PRL, and ACTH from perifused rat pituitary cells, but does not alter the secretion of anterior pituitary hormone in static rat pituitary monolayer cultures (18). Other investigators have reported that PACAP slightly stimulates GH release but inhibits prolactin secretion as determined by reverse hemolytic assay (11). Moreover, PACAP and LHRH exhibit a synergistic effect on LH and FSH release in static rat pituitary cell culture (8), but PACAP does not affect the action of GRF or CRF on GH or ACTH release, respectively (1). Tatsuno et al. (24) have demonstrated that PACAP stimulates the release of interleukin 6 (IL6) from cultured rat pituitary cells, possibly from folliculo-stellate cells, through a CAMP-dependent protein kinase A pathway, suggesting that these cell types in the pituitary are also targets for PACAP. An immunohistochemical study showed that, after
’ Requests for reprints should be addressed Belle Chasse, LA 70037.
to Dr. Akira Arimura,
hypophysectomy, the intensity of PACAP-like immunoreactivity increased in the nerve fibers in the median eminence and the perikarya in the paraventricular and supraoptic nuclei ( 13). These data suggest that hypophysectomy increases the synthesis of PACAP in these neurons or suppresses the release of PACAP from the nerve terminals. However, immunohistochemistry does not necessarily precisely show the quantitative pattern of PACAP biosynthesis or gene expression. Therefore, we examined PACAP content as well as the expression of the PACAP gene in hypothalami from normal and hypophysectomized rats. The combined hormone treatment (GH, PRL, T4, corticosterone, and testosterone) was also made in hypophysectomized rats, and its effect on PACAP tnRNA
levels
was examined. METHOD
Animals Both hypophysectomized 200-250 g) were purchased
U.S.-Japan
407
Biomedical
Research
and intact male rats (CD strain; from Charles River Breeding Lab-
Laboratories,
Tulane University
Hebert Center,
408
SHUT0
Rat PACAJ cDNA
oratories (Wilmington, MA). Animals were housed in a light (lights on at 0600 h, lights off at 1800 h)- and temperature-controlled room and fed standard Purina Lab Chow and water ad lib. Hypophysectomized rats were allowed to drink tap water containing 0.9% NaCl and were given sugar cubes. Completeness of the hypophysectomy was assessed by visual inspection of the sella turcica after the animals were sacrificed. Rats were killed by decapitation 1 or 2 weeks after surgery under ketamine-xylazine anesthesia. The hypothalamic area (rostra1 portion; preoptic area, caudal portion; mamillary complex), including the paraventricular and supraoptic nuclei, was immediately removed and placed in liquid nitrogen. The tissues were kept at -80°C until analysis. All animal studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Hormone Replacement
ET AL.
Therapy
The combined treatment with GH, PRL, T.,, corticosterone, and testosterone was given to the hypophysectomized rats for the hormone replacement. Three-week release pellets containing T4 (15 mg), corticosterone (100 mg), and testosterone (5 mg) were obtained from Innovative Research (Toledo, OH) and implanted subcutaneously under ketamine-xylazine anesthesia. Rat GH (25 pg) and rat PRL (50 ,ug) were kindly provided by the NIDDK and the National Hormone and Pituitary Program. Rats were injected subcutaneously with GH (25 pg/injection, twice daily) and PRL (50 &injection, twice daily). Four days after hypophysectomy, a group of rats was given replacement therapy. Control animals were injected with vehicle and subjected to skin incision without pellet placement. Animals were sacrificed by decapitation under ket&nine-xylazine anesthesia after 10 days of hormone replacement (2 weeks after hypophysectomy). RNA Isolation and Preparation
of Probes
Poly(A)+ RNA was extracted from rat hypothalami using Poly A Tract System 1000 (Promega Corporation, Madison, WI). pRB3Z3, containing the 670-base pair Pst I/EcoR I fragment of rat PACAP cDNA (21), was used to make 32P-labeled cRNA transcripts (Fig. 1). The plasmid was linearized with Hind III and transcribed with [~Y-~*P]UTP(Du Pont NEN Research Products, Boston, MA) using MAXIscriptTM T7 Kit (Ambion Inc., Austin, TX). To make the probe for p-actin, pTRI-@Actin (Ambion Inc., Austin, TX) containing the 250-base pair fragment of the mouse p-actin gene was transcribed as described above. After the RNA transcription reaction, the probes were treated with DNase I and electrophoresed through 5% polyacrylamide/8 M urea gels. The gel was then exposed to X-ray film, and the area of the gel that contained the full-length labeled transcripts was excised. The elution of the probes was performed overnight in the buffer [0.5 M NH40Ac, 1 mM EDTA, and 0.2% sodiumdodecyl sulfate (SDS)] at 37°C. RNase Protection Assay RNase protection assays were performed using RPA II@@Kit (Ambion Inc., Austin, TX). Poly(A)+ RNA (2 pg) from each hypothalamus was coprecipitated with 6 X 104 cpm of 32P-labeled PACAP probe prepared as described above. The same cpm of p-actin probe was also coprecipitated with 0.1 pg of poly(A)+ RNA. Hybridization was then performed at 45°C overnight. RNase digestions were carried out at 37°C for 30 min. The protected fragments were precipitated and size separated on 5% polyacrylamide/8 M urea gels. The gels were exposed to X-ray film at -70°C overnight. The levels of RNA in each lane were quantified using a real-time video analysis system (Hamamatsu C2400
FIG. 1. Organization of the pRB3Z3. The region corresponding to mature PACAP is indicated with a box. The 670-base pair Pst I/&OR I fragment of PACAP cDNA was subcloned into PGEM-3Zf(-) @omega Corporation, Madison, WI). The plasmid was linearized and transcribed with T7 RNA polymerase
to prepare the ‘ZP-labeled cRNA probe.
video camera, DataTranslation Frame Grabber video card, Macintosh Quadra 950, and “NIH IMAGE 1.44” software). For each sample, PACAP signal strength was normalized to the corresponding @actin signal. Nuclear Run-On Transcription
Assay
The nuclear run-on assay was performed as described by White et al. (27) with minor modifications. Hypothalami were obtained from hypophysectomized and control rats as described above. Nuclei were isolated from individual hypothalami. The transcription reaction was performed in the presence of 1 X lo7 isolated nuclei, 20 mM Tris-HCl (pH 7.9), 20% glycerol, 140 mhf KCl, 10 mM MgCl*, 1 n-&f ditbiothreitol, 0.1 mg/ml creatine phosphokinase, 8.5 m&f phosphocreatine, 2 mM each ATP, CTP, and GTP, 1 U/ml RNasin, and 160 ,uCi [cY-~*P]UTP (800 Ci/ mmol). The reactions were allowed to proceed for 30 min at 30°C. The reaction mixture was then digested with DNase I and proteinase K, followed by RNA extraction with phenol-chloroform. RNA was precipitated two times with ammonium acetate and ethanol. Aliquots of synthesized RNA were counted with a liquid scintillation counter. The immobilized template for assaying the radiolabeled transcripts was pRB38Pl containing the entire coding region of the rat PACAP gene (21). pUC118, the vector of pRB38P1, was also used as a control. These plasmids were denatured and immobilized on nitrocellulose. Prehybridization was carried out for 20 h at 42°C in the hybridization buffer [0.2% SDS, 50 mM TES (pH 7.4), 0.3 M NaCl, 10 mh4 EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.1% sodium phosphate, and 0.1 mg/ml yeast tRNA]. After prehybridization, the filters were incubated for 48 h at 42°C in the hybridization buffer containing the total transcripts (1 X lo6 cpm). The filters were then washed two times in 2 X SSC (1 X SSC; 0.15 M NaCl and 0.015 h4 Na citrate, pH 7.0) and 0.1% SDS for 30 min at room temperature, and two times in 0.2 x SSC and 0.1% SDS for 10 min at 50°C. The filters were exposed to X-ray film at -70°C for 10 days. The autoradiographic dots were quantified as described above. The transcriptional rate of the PACAP gene was calculated after subtracting the nonspecific background hybridized to pUCl18.
PACAP
EXPRESSION
AWER
409
HYPOPHYSECTOMY
control
hypox 1 w hypox 2w B
FIG. 2. Effect of hypophysectomy on hypothalamic PACAP mRNA levels. (A) Autoradiographs of the RNase protection assay. Lanes l-3: control; lanes 4-6: 1 week after hypophysectomy; lanes 7-9: 2 weeks after hypophysectomy. The positions of the RNA size markers are shown to the left of gel. (B) Quantification of PACAP mRNA levels. Data are the mean t SE of RNA from three individual rat hypothalami. mRNA levels in arbitrary units are shown as a percent of the control values. hypox, hypophysectomized rats. Statistical significance was examined1 by one-way ANOVA. *p < 0.05 vs. the control group.
Radioimmunoassay The tissues were homogenized in 1 ml of 20% trifluoroacetic acid using a glass homogenizer and aliquots were taken for the protein assay. The homogenates were then centrifuged at 15,000 x g for 30 min. The supernatants were collected and evaporated by a Speed-Vat Concentrator (Sa-
vant were
Instruments, reconstituted
Inc.,
Hicksville,
NY).
The
samples
in 1 ml of distilled water and lyophilized twice. The residues were dissolved in RIA assay buffer and PACAP was assayed as previously described (2). The aliquots of the homogenate were lyophilized and the protein was assayed using the Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA).
410
SHUT0
ET AL
FIG. 3. Autoradiographs of the nuclear run-on assay. The hybridization probe was the rat PACAP cDNA (pRB38Pl) containing the entire coding region of the PACAP gene. pUCll8 is the vector of pRB38Pl and served as a hybridization control. (A )One week after hypophysectomy. Lanes l-3: control; lanes 4-6: 1 week after hypophysectomy. (B) Two weeks after hypophysectomy. Lanes 1-3: control; lanes 4-6: 2 weeks after hypophysectomy.
Statistical Analysis
Effect of Hypophysectomy PACAP Gene
Results were presented as the mean + SE. Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan New Multiple Range or Student’s t-test. A value ofp < 0.05 was considered significant.
RESULTS
Effect of Hypophysectomy
on the Content of PACAP tnRNA
Because of low levels of PACAP mRNA in total hypothalamic RNA, PACAP mRNA levels were measured using solution hybridization followed by nuclease protection, which is more sensitive than Northern blot analysis (15). Hypophysectomy appeared to decrease the content of PACAP mRNA [Fig. 2(A)]. The major protected bands were found at 670 nucleotides, and a few minor bands were also present [Fig. 2(A)]. Changing the concentrations of RNase did not change this pattern. Because the PACAP probe contains the 3’ noncoding region (Fig. l), these minor fragments might result from the use of alternate polyadenylation sites. Quantitative data were obtained by image analysis. PACAP mRNA levels decreased following hypophysectomy to 70.3 ? 10.5% of control values after 1 week and to 60.0 5 6.2% of control values after 2 weeks [Fig. 2(B)]. A significant decrease was seen 2 weeks after hypophysectomy @ < 0.05).
on the Transcriptional
Rate of the
To determine whether the decreased PACAP mRNA levels after hypophysectomy were due to an altered transcriptional rate of the gene, the nuclear run-on assay was performed. Clear activity was observed in the control rats, but the dots from hypophysectomized rats were very faint [Fig. 3(A,B)]. No hybridization was observed with the plasmid devoid of PACAP (pUC118). Figure 4(A,B) shows the quantification of the autoradiographs. The rate of PACAP gene transcription decreased to 69.4 + 15.1% 1 week after and to 45.1 -C 4.6% 2 weeks after hypophysectomy [Fig. 4(A,B)J. A significant decrease was seen 2 weeks after hypophysectomy (p < 0.05). Effect of Hypophysectomy
on PACAP-Like Immunoreactivity
Radioimmunoassayable PACAP in the hypothalamus also decreased to 80.3 ? 10.6% 1 week after and to 73.5 2 7.3% 2 weeks after hypophysectomy (Fig. 5). The decrease at 2 weeks was significant (p < 0.05), being a similar tendency as that for mRNA levels and transcriptional rates. Effect of Replacement Therapy on the Content of PACAP mRNA in Hypophysectomized Rat The combined replacement therapy with GH, prolactin, T4, corticosterone, and testosterone significantly increased the con-
PACAP
EXPRESSION
control
AFTER
411
HYPOPHYSECTOMY
* II
hypox 1w
hypox
hypox
hypox2~
control
6
A
control
PIG. 4. Effect of hypophysectomy on the rate of hypothalamic PACAP gene transcription. Data are the mean + SE. The transcriptional rate in arbitrary units is shown as a percent of the control values. Statistical significance was examined by Student’s r-test. *p < 0.05 vs. the control group. (A) One week after hypophysectomy. (B) Two weeks after hypophysectomy.
tent of PACAP mRNA in the hypothalamus of 2-week postoperative hypophysectomized rats compared to the untreated hypophysectomized animals @ < 0.05) (Fig. 6). DISCUSSION The present study demonstrated that hypophysectomy decreased the transcriptional rate of the PACAP gene and the levels of both its mRNA and peptide in the hypothalamus.
Positive or negative feedback regulation by pituitary hormones was proposed for several hypothalamic hypophysiotropic peptides after hypophysectomy, as shown in Table 1 (3,5,6,9,12,14,16,17,19,22,23,25,28). The levels of these hypophysiotropic peptides and their mRNA in the hypothalamus changed in different ways after hypophysectomy. Hypophysectomy reduced LHRH levels and its mRNA as demonstrated for PACAP (Table 1). Testosterone treatment of hypophysectomized rats raised LHRH content to that of intact rats, suggesting that the reduction of hypothalamic LHRH after hypophysectomy was due mainly to loss of testosterone secretion (6). To test whether pituitary hormones and/or pituitary-dependent factors may exert their feedback effect on hypothalamic PACAP by influencing the expression of the PACAP
G&PRLJ.,C,T
PIG. 6. Effect of replacement therapy (GH; PRL; T4; C, corticosterone; and T, testosterone) on the content of PACAP mRNA in hypophysectomized rats. Data are the mean ? SE of RNA from three individual rat hypothalami. mRNA levels in arbitrary units are shown as a percent of the control values. hypox, hypophysectomized rats. Statistical significance was examined by one-way ANOVA. *p < 0.05 vs. the control group. +p < 0.05 vs. the hypox group.
gene, hypophysectomized animals were treated with a combination of hormone replacement (GH, prolactin, Tq, corticosterone, and testosterone). The replacement therapy significantly restored PACAP mRNA levels in hypophysectomized rats to those in intact rats. Because there exists a feedback regulation between hypothalamic peptides and pituitary hormones or pituitary-dependent factors, it is possible that one or more of the replaced hormones may regulate PACAP gene expression through these feedback mechanisms. However, we cannot rule out the possibility that a decrease in general metabolism induced by hypophysectomy contributed, at least partly, to the decrease in the transcriptional rate of the PACAP gene, and the levels of PACAP mRNA and PACAP immunoreactivity that we have observed. PACAP appears to be different from classical releasing factors in terms of its action on pituitary cells. Although PACAP stimulates adenylate cyclase in the rat pituitary cells, it did not alter the secretion of any known anterior pituitary hormone in static rat pituitary monolayer cultures (18). PACAP and LHRH had a synergistic effect on LH and FSH release in static rat pituitary cell culture (8), but PACAP did not affect the action of GRF or CRF on GH or ACTH release, respectively (1). PACAP increased the release of GH, PRL, and ACTH from perifused rat pituitary cells. However, PACAP injected IV into rats did not stimulate secretion of any of the pituitary hormones except that
TABLE
1
OF HYFOPHYSECTOMY ON HYPOTHALAMIC NEUROPEPTIDES
EFFECT
control
hypox
lw hypox 2w
5. Effect of hypophysectomy on the PACAP immunoreactivity. Data are the mean -C SE of immunoreactivity from six individual rat hypotbalami. The immunoreactivity is shown as ng/mg protein. Statistical significance was examined by one-way ANOVA. *p < 0.05 vs. the control group. FIG.
Neuropeptides
Immunoreactivity
mRNA Content
PACAP GRP Somatostatin CRP LHRH TRH VIP
1 -1 1 +t 1 1-t t
1 t +l +t 1 t t
t, increase;
1, decrease;
+ no significant
Transcriptional Rate
References
1 17,28 22, 25, 28 9, 19 3, 6, 16 12, 14, 23 5, 13 change.
412
SHUT0
it released both PRL and GH following injection into lactating rats (20) and that it stimulated PRL and GH release in hypothalamus-lesioned rats (11). Others have reported that PACAP stimulated the expression of the PRL gene in the rat GH3 pituitary cell line, which was directed by the PRL promoter (7). POMC mRNA expression was also demonstrated to be induced by PACAP in the AtT-20/D16v corticotrope cell line (4). These data suggest that PACAP may be a regulator of the gene transcription of pituitary hormones rather than a releasing factor. On the other hand, our previous data demonstrated that more than 90% of folliculo-stellate cells bound biotinylated PACAP, suggesting that folliculo-stellate cells are a major target for PACAP (26). PACAP stimulated the release of IL-6 from cultured rat pituitary cells, possibly from folliculo-stellate cells (24). Other known hypothalamic factors, including CRF, GRF, TRH, and LHRH, failed to alter the production of IL-6. Because IL-6 was reported to stimulate ACTH when injected IV and to alter the release of PRL, GH, FSH, and LH in cultures of pituitary cells (24), PACAP might have the potency to release pituitary hormones through IL-6. These findings support the view that PACAP may be an auxiliary releasing factor of pituitary hormones and that, in certain conditions (e.g., lactating rats), it may regulate the release of pituitary hormones through some factor released from folliculo-stellate cells. As shown in Table 1, the changes in mRNA content after hypophysectomy do not necessarily reflect the levels of peptides. For LHRH and somatostatin, the levels of both peptide and mRNA decreased after hypophysectomy. In contrast, GRF mRNA levels were increased after hypophysectomy, whereas hypophysectomy resulted in a decrease in GRF content. These data illustrate the difficulties in using the peptide content data as an index of the synthetic capacity for a peptide after hypophysectomy (22). The changes in peptide content may be attributable to a change in transcription, translation, processing, degradation, or release. The changes in mRNA levels are also a reflection of changes in transcription and/or mRNA stability. Because the results of the nuclear run-on assay agree with those of the RNase protection assay and radioimmunoassay, it may be reasonable to conclude that the reduced rate of PACAP gene transcription results in a decrease in both mRNA and peptide levels in the hypothalamus after hypophysectomy. Thus, modulation of PACAP gene transcription could be one mechanism of feedback regula-
ET AL.
tion of hypothalamic PACAP by some pituitary hormones or pituitary-dependent factors. Our previous immunohistochemical study showed that the intensity of PACAP-immunoreactive staining in the internal zone of the median eminence and in the perikarya of the paraventricular and supraoptic nuclei enhanced after hypophysectomy compared to the control rats (13). At the same time, abundant PACAP-immunoreactive fibers appeared in the external zone of the median eminence after hypophysectomy. In contrast, the present RIA data showed that PACAP-like immunoreactivity decreased after hypophysectomy. The discrepancy between the previous immunohistochemical results and the results in the present study is difficult to explain, but it could be explained in two ways. First, because the antibody used in RIA was different from that used in immunohistochemistry, radioimmunoassayable PACAP may not be necessarily the same as PACAP-like immunoreactivity demonstrated by immunohistochemistry. We often experienced that an antibody useful for RIA was not always good for immunohistochemistry. Second, hypophysectomy could result in alteration in the plasticity of PACAP neurons in the hypothalamus, altering the distribution of PACAP immunostaining. Considering that our previous immunohistochemistry was not a quantitative study, the altered distribution of PACAP immunostaining might mask an overall decrease in immunoreactivity in the whole hypothalamus. In summary, we have used nuclear run-on and RNase protection assays to assess PACAP gene transcription in response to hypophysectomy. We have demonstrated that hypophysectomy decreased the transcriptional rate of the PACAP gene, its transcripts, and the levels of PACAP in the hypothalamus, suggesting that the reduced rate of PACAP gene transcription results in decreased mRNA and peptide levels in the hypothalamus. Replacement therapy with GH, prolactin, T.,, corticosterone, and testosterone significantly restored the content of PACAP mRNA in hypophysectomized rats. Our observations imply that feedback regulation of hypothalamic PACAP is mediated by some pituitary hormones and/or pituitary-dependent factors. ACKNOWLEDGEMENTS
We wish to thank Drs. Sandor Vigh, Richard Harlan, and Carol Phelps for their valuable advice. This study was supported in part by NIH grant DK 09094 and a grant from Takeda Chemical Industries, Ltd.
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