The Growth Hormone (GH) - Semantic Scholar

0013-7227/98/$03.00/0 Endocrinology Copyright © 1998 by The Endocrine Society

Vol. 139, No. 5 Printed in U.S.A.

The Growth Hormone (GH) Gene Is Expressed in the Lateral Hypothalamus: Enhancement by GH-Releasing Hormone and Repression by Restraint Stress H. YOSHIZATO, T. FUJIKAWA, H. SOYA, M. TANAKA,

AND

K. NAKASHIMA

Department of Biochemistry (H.Y., T.F., M.T., K.N.), Faculty of Medicine, and the Department of Health and Physical Education (H.S.), Faculty of Education, Mie University, 2–174 Edobashi, Tsu, Mie 514-8507, Japan ABSTRACT Recent studies suggest that GH may modulate emotion, behavior, or stress response by its direct actions on the brain, and possible expression of the GH gene in the brain has been predicted. In this study we have investigated whether and where the GH gene is expressed in the brain and how it is regulated. Ribonuclease protection assay and 59-rapid amplification of complementary DNA ends-PCR analyses indicated that the GH gene was expressed in rat brain, initiating at the identical transcription start point as that for pituitary GH gene expression. The brain GH messenger RNA was pre-

G

H HAS attracted increasing interest for its neuronal or neuroendocrine actions on the central nervous system (1, 2). Recent investigations have suggested the involvement of GH in the regulation of emotion, behavior, stress response, neurotransmitters, neuropeptides, or other hormones (3–5). The presence of GH receptors was also demonstrated in the central nervous system, including the choroid plexus, hippocampus, and hypothalamus (6 – 8). Our previous work (9) has shown that expression of GH receptor messenger RNA (mRNA) in the brain is regulated by restraint stress in the water (RSW). In the cerebrospinal fluid (CSF), GH-like peptides are detected by an immunological method (10, 11). These GH molecules in CSF are considered to be transported from the circulatory system across the choroid plexus by a GH receptor-mediated mechanism (12). In the brain, immunoreactive GH molecules can be detected in regions such as hypothalamus, amygdaloid nucleus, hippocampus, and cortex (10, 11). However, it is not clear whether these immunoreactive GH-like peptides are synthesized in situ or transported from other regions of brain or CSF. On the other hand, in situ complementary DNA (cDNA) mRNA hybridization analysis has shown the presence of positive signals for GH mRNAlike sequences in wide areas of the brain, except the amygdaloid nucleus (13). Similarly, GH cDNA-like fragments have been successfully amplified by the reverse transcription-PCR (RT-PCR) technique, with total RNA preparations extracted from both hypothalamic and extrahypothalamic Received October 27, 1997. Address all correspondence and requests for reprints to: Dr. Kunio Nakashima, Department of Biochemistry, Mie University Faculty of Medicine, 2–174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail: [email protected].

dominantly detected in the lateral hypothalamus (lh) by in situ reverse transcription-PCR analysis. GH gene expression in the brain was significantly enhanced by GH-releasing hormone administration and was rapidly repressed by exposure to restraint stress in the water, whereas the changes in pituitary GH messenger RNA contents in these circumstances were relatively smaller. The results of the present study suggest that the brain GH is predominantly expressed in lh under the control of physiological conditions to play a role in the modulation of brain functions. (Endocrinology 139: 2545–2551, 1998)

brain tissues (14). These results strongly suggest that the GH gene is also expressed in the brain. However, the exact loci of the GH expression, the transcription start point (tsp) of the gene, and the regulatory mechanisms of its transcription in the brain are still unclear. We show here, by ribonuclease (RNase) protection assay, 59-rapid amplification of cDNA ends (RACE) and sequencing, and in situ RT-PCR analysis, that the brain GH gene is predominantly expressed in the lateral hypothalamus (lh), initiating at the same tsp as in the pituitary. We also present evidence for regulation of brain GH expression by neuroendocrine and psychophysical circumstances. Materials and Methods Animals Adult male Sprague-Dawley rats (8 weeks old) were purchased from SLC (Shizuoka, Japan) and housed at a constant temperature of 22 C with a 12-h light/12-h dark cycle (lights on at 0700 h) for at least 1 week before experiments. Food and water were available ad libitum. The experiments reported here were conducted according to the principles set forth in the Guides to the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council [DHEW publication (NIH) 85–23], and Mie University Faculty of Medicine.

Intracerebroventricular (icv) injection of hormones Rats were anesthetized with sodium pentobarbital (50 mg/kg, ip), and a stainless steel cannula was implanted stereotaxically into the left lateral ventricle. The icv injection of GH-releasing hormone (GRH; Peptide Institute, Osaka, Japan) in 5 ml PBS was performed by the sudden drop method. At 2 h after the injection, rats under anesthesia were killed by decapitation. The cerebrum was promptly removed from the cerebellum and pituitary gland, quickly frozen in liquid nitrogen, and stored at 280 C until used. Blood was obtained by heart puncture or decapitation and used for measurements of plasma GH and ACTH.

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FIG. 1. Schematic organization of the rat GH gene and cDNA, and localizations of cDNA primers and cRNA probe. The TATAAA sequence starting at position 230 and cDNA are indicated by boxes. cRNA used for RNase protection assay is shaded. Nucleotide 11, representing the tsp in the pituitary GH gene expression (22), is shown. The arrows are the locations of primers used for the PCR or RACE. The positions of Pit-1/GHF-1-binding elements are marked by P.

RSW Rats were exposed to RSW as described by Takagi and Okabe (15). At 10 weeks of age, rats were put into individual small restraining wire net cages and immersed in water to the chest. The temperature of the water was kept constant at 23 6 1 C. After 2 or 4 h of RSW, rats were removed from the cages and decapitated. The cerebrum, pituitary gland, and blood were removed as described above.

Determination of plasma hormones Plasma GH and ACTH concentrations were determined in duplicate by a double antibody RIA. Standard hormones and antibodies were supplied by the NIDDK Hormone Distribution Program. The minimal values detected for ACTH and GH were 1.5 pg/ml and 0.76 ng/ml, respectively.

RNA preparation Total RNA was extracted from rat brain by the guanidium isothiocyanate-phenol-chloroform method, as described by Chomczynski and Sacchi (16). RNA was dissolved in diethylpyrocarbonate (DEPC)-treated water, and the concentrations were determined spectrophotometrically at 260 nm. Polyadenylated [poly(A)1] RNA was prepared with oligo(deoxythymidine) [oligo(dT)] latex (Takara, Siga, Japan) according to the manufacturer’s instruction.

Northern blot hybridization Northern blot hybridization was carried out as previously described (17). Five micrograms of total RNA derived from the pituitary were separated by electrophoresis on 1% agarose gel containing 2.2 m formaldehyde, transferred to a nylon membrane by the capillary transfer method, and immobilized on the membrane by baking. The membranes were prehybridized with denatured salmon sperm DNA (0.2 mg/ml) in 6 3 SET [1 m NaCl, 6 mm EDTA, 0.1 m Tris-HCl (pH 7.5), 0.2% SDS, and 2 3 Denhart’s solution] at 42 C for 4 h and hybridized with the 32P end-labeled oligonucleotide probes for GH and b-actin mRNAs in the same solution at 37 C overnight. The membranes were washed twice with 6 3 SSC (standard saline citrate) at 50 C for 20 min and twice with 6 3 SSC at 55 C for 20 min, then exposed to x-ray film at 280 C for 16 –24 h with an intensifying screen. The radioactivity of the Northern blot hybridization signals on the membrane was determined by a bioimage analyzer (BAS2000, Fuji Film, Japan).

RNase protection assay for 59-noncoding regions of rat GH mRNAs A rat GH-specific cDNA fragment of 388 bp that contained the region from 2147 upstream from the cap site to the 1241 downstream site of rat GH gene lacking introns I and II was constructed with a genomic PCR fragment and a RT-PCR fragment subcloned in a T/A-type PCR cloning vector (pCR II plasmid vector, Invitrogen, San Diego, CA) and used as the template for complementary RNA (cRNA) synthesis (Fig. 1). The cRNA probe (504 nucleotides) was generated with Sp6 RNA polymerase in the presence of [a-32P]CTP, transcribing the 388-bp fragment of rat GH

FIG. 2. RNase protection assay of 59-end regions of GH mRNAs expressed in rat brain and pituitary. Lane 1, Pituitary total RNA (1 mg); lane 2, liver total RNA (50 mg); lane 3, brain total RNA (25 mg); lane 4, brain total RNA (50 mg); lane 5, brain poly(A)1 RNA (5 mg). P, cRNA probe; M, DNA size marker (pUC19/MspI). cDNA and 116 bp of EcoRV-linearized pCR II plasmid vector. GH mRNA species in the rat brain and pituitary were determined by RNase protection assay on total or poly(A)1 RNA from the cerebrum and pituitary hybridized with the RNA probe. After digestion with RNases A and T1 at 30 C for 30 min, the reaction mixtures were treated with proteinase K at 37 C for 15 min. The reaction products were then separated by electrophoresis in a 6% acrylamide-8 m urea DNA sequence gel. The gel was dried and subjected to autoradiography at 280 C with an intensifying screen. Molecular size marker fragments deriving from MspI digestion of pUC19 were radiolabeled by a T4 kinase 59-end labeling kit (Nippon Gene, Toyama, Japan) with [g-32P]ATP.

RT-PCR The RT reaction was carried out at 42 C for 1 h in 25 ml of the reaction mixture containing 5 mg total RNA, 25 pmol oligo(dT) primer (Pharmacia Biotech, Uppsala, Sweden), 25 pmol random primer (BRL, Bethesda, MD), 75 mm KCl, 50 mm Tris-HCl (pH 7.5), 3 mm MgCl2, 10 mm dithiothreitol, 2 mm deoxy (d)-NTPs, 10 U RNase inhibitor (Wako, Osaka, Japan), and 100 U Moloney murine leukemia virus reverse transcriptase (BRL). One microliter of the reaction mixture was amplified by

GH EXPRESSION IN BRAIN AND REPRESSION BY STRESS 35 cycles of PCR on a thermal cycler (PC-800, Astec, Fukuoka, Japan) in the reaction buffer containing 50 mm KCl, 10 mm Tris-HCl (pH 7.5), 1.5 mm MgCl2, 200 mm dNTPs, 0.01% gelatin, 50 pm of sense and antisense primer pairs for GH or b-actin, and 1 U Taq DNA polymerase (PerkinElmer, Foster City, CA) in the final volume of 25 ml. The hot start method was employed for this PCR, and each cycle consisted of denaturing at 94 C for 45 sec, annealing at 60 C for 2 min, and extension at 72 C for 2 min. The PCR reaction was completed by addition of the extension reaction at 72 C for 8 min before termination. The PCR for b-actin mRNA was performed for 25 cycles. The primers used for RT-PCR of GH mRNA

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were 59-CTGTTTGCCAATGCTGTGCT-39 (primer 1 for the 59-end) and 59-GTCTTCCAGCTCCTGCATCA-39 (primer 2 for the 39-end), as shown in Fig. 1. The predicted length of amplified fragments is 360 bp. For b-actin mRNA, the primers 59-TTGTAACCAACTGGGACGATATGG-39 (for the 59-end) and 59-GATCTTGATCTTCATGGTGCTAGG-39 (for the 39-end) were used. The predicted length of the amplified fragments is 764 bp.

Southern blot hybridization After amplification by PCR, 5 ml of the reaction products were separated by electrophoresis in 1.6% agarose gels, transferred onto nylon membranes (New England Nuclear Research Products, Boston, MA) overnight at room temperature in 10 3 SSC (1.5 m NaCl and 0.15 m Na citrate, pH 7.4), and cross-linked to the membranes. The membranes were prehybridized with denatured salmon sperm DNA (0.2 mg/ml) at 37 C for 2 h in 6 3 SET and hybridized with 32P end-labeled oligonucleotide probes for GH and b-actin cDNAs in the same solution at 37 C for 8 h. The membranes were washed twice with 6 3 SSC at 50 C for 20 min and exposed to x-ray film at room temperature for 16 –24 h with an intensifying screen. Radioactivities on the membrane were also determined using a BAS2000 bioimage analyzer.

Analysis of rat brain GH mRNA 59-end by RACE

FIG. 3. Detection of rat brain GH mRNA by the RT-PCR method. A, Ethidium bromide staining of the amplified rat GH cDNA fragments. B, Autoradiogram of Southern hybridization of the amplified GH cDNA fragments. Lane M, DNA size marker (100-bp ladder); lane 1, RT-PCR products with pituitary total RNA (0.05 mg); lane 2, brain RT-PCR products with total RNA (5 mg); lane 3, liver RT-PCR products with total RNA (5 mg).

FIG. 4. Stimulation of GH mRNA expressions in rat brain and pituitary by GRH administration. GRH or PBS was icv injected into rats 2 h before analysis. A, GH and b-actin mRNA expressions in rat brain detected by RT-PCR-Southern blot hybridization. Lanes 1–3, Treated with PBS alone; lanes 4 – 6, treated with 0.5 mg GRH in PBS; lanes 7–9, treated with 5.0 mg GRH in PBS. B, GH and b-actin mRNA expressions in the pituitary detected by Northern blot hybridization. Lanes 1 and 2, Treated with PBS alone; lanes 3 and 4, treated with 0.5 mg GRH in PBS; lanes 5 and 6, treated with 5.0 mg GRH in PBS. Autoradiograms were shown in the upper panels in A and B, and the radioactivities determined by a BAS2000 bioimage analyzer (Fuji film) were expressed as photostimulated luminescence value (PSL) minus background (PSL-BG) in the lower panels. Values are the mean of four determinations 6 SE. *, P , 0.05; **, P , 0.01 (vs. the control values).

The RACE procedure was carried out as described previously (18), using a cDNA amplification kit (Marathon, Clontech, Palo Alto, CA). Five micrograms of poly(A)1 RNA were reverse transcribed as described above except for the use of 20 pmol GH-specific antisense primer (primer 2 in Fig. 1) instead of oligo(dT) and the random primer. Second strand synthesis was performed with a cocktail of Escherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase. After creation of blunt ends with T4 DNA polymerase, the double-stranded (ds) cDNA was ligated to the Marathon cDNA adapter that was partially double stranded and was phosphorylated at the 59-end to facilitate blunt end ligation of the adapter to both ends of the ds cDNA by T4 DNA ligase. Adapter-ligated ds cDNA was amplified by PCR using an adapter-

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FIG. 5. Repression of GH mRNA levels in rat brain and pituitary during exposure to RSW. A, GH and b-actin mRNA levels in the brain detected by RT-PCR. Lanes 1– 4, Control rats; lanes 5– 8, rats exposed to RSW for 2 h; lanes 9 –11, rats exposed to RSW for 4 h. B, GH and b-actin mRNA levels in the pituitary detected by Northern blot analysis. Lanes 1 and 2, Control; lanes 3 and 4, rats exposed to 2 h of RSW; lanes 5 and 6, rats exposed to RSW for 4 h. Autoradiograms and radioactivities determined were shown as described in Fig. 4.

specific primer and a GH specific primer in the reaction buffer described above. The reaction products of 5 ml were separated in 1.6% agarose gels and Southern blotted. One microliter of the products was subcloned directly into a T/A-type PCR cloning vector, screened by colony hybridization using a 32P-labeled GH-specific oligonucleotide probe, and sequenced with a sequencing kit (ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction, Perkin-Elmer).

In situ RT-PCR In situ RT-PCR was performed as described previously (19, 20). The sections (20 mm thick) were fixed in 4% paraformaldehyde-0.1 m phosphate buffer for 2 h at room temperature; washed in PBS, dehydrated in an ascending ethanol concentration series (70%, 90%, 95%, and 100%), 100% chloroform, and 100% ethanol; and air-dried. The sections were subjected to protease digestion with proteinase K [2 mg/ml in 0.1 m Tris-HCl (pH 7.5) and 50 mm EDTA] at 37 C for 15 min. Proteinase K was inactivated by washing the slide with DEPC-treated water for 1 min and with 100% ethanol for 1 min and was air-dried. Sections were then treated with 10 U RNase-free deoxyribonuclease (DNase) in 0.1 m sodium acetate and 5 mm MgSO4 (pH 7.4), while covered with plastic coverslips in a humidity chamber at 37 C overnight. After DNase digestion, the sections were washed with DEPC-treated water for 1 min, then with 100% ethanol, and air-dried. Firstly, the in situ RT reaction was performed in a reaction buffer containing 1 mm 39-specific primer (primer 2 in Fig. 1), 75 mm KCl, 50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 10 mm dithiothreitol, 1 mm dNTPs, 10 U RNase inhibitor, and 50 U Moloney murine leukemia virus reverse transcriptase while covered with coverslips for 30 min in a humidity chamber at 42 C. The sections were washed with 100% ethanol for 5 min and air-dried. The negative control section was digested with DNase and was not treated with RT. The positive control section was not digested by DNase, and the genomic DNA was amplified by PCR. In situ PCR was then carried out using the GeneAmp kit and a thermal cycler (GeneAmp In Situ PCR System 1000, Perkin-Elmer). The reaction solution contained 50 mm KCl, 10 mm TrisHCl (pH 7.5), 4.5 mm MgCl2, 200 mm dNTPs, 0.01% gelatin, 20 mm digoxigenin-dUTP, 50 pm each of sense and antisense primers for GH mRNA (primers 3 and 4, respectively, in Fig. 1), and 2 U Taq DNA

polymerase. Twenty-five microliters of the reaction mixture were dropped on the section, and the section was covered with a siliconerubber diaphragm that could make a space on the section. The specimen was clamped over by a thin stainless steel clip and set to the thermal cycler. The PCR cycle, consisting of denaturing at 94 C for 40 sec, annealing at 60 C for 1 min, and extension at 72 C for 2 min, was hot started and carried out for 20 cycles. After the in situ PCR reaction, the coverslips were removed, and the sections were washed twice with 100% ethanol for 5 min, air dried, and washed with 1 3 SSC containing 0.2% BSA at 52 C for 3 min. The sections were then treated with antidigoxigenin antibody conjugated with alkaline phosphatase at a dilution of 1:200 in 0.1 m Tris-HCl (pH 7.5) and 0.1 m NaCl at 37 C for 30 min. The specimen was washed with the detection solution [0.1 m Tris-HCl (pH 9.5) and 0.1 m NaCl] and incubated in the detection solution added with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt (NTB/BCIP) chromagen at 37 C for 10 min.

Statistical analysis The data were analyzed for statistical significance using the Macintosh SuperANOVA program and were expressed as the mean 6 se. The significance of differences between the values was analyzed using Scheffe’s post-hoc test, and P , 0.05 was considered significant.

Results Detection of GH gene expression in the brain and analysis of 59-end and sequence of the brain GH mRNA

Expression of the GH gene in the brain was detected by RNase protection assay. The protected bands specific for GH mRNA derived from both pituitary and brain were detected as signal bands of same size at 241 bp, whereas RNA from the liver gave no positive band (Fig. 2). This result indicated that GH mRNA was expressed in the brain in the same size as in the pituitary. The GH mRNA expression in the brain

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was detected more clearly by RT-PCR, which also yielded no positive band with the liver RNA preparation (Fig. 3). Nucleotide sequencing of both the sense and antisense strands of GH cDNA cloned from the brain tissue showed sequences identical to those reported for pituitary GH cDNA (21, 22). The nucleotide sequence of the 59-end of brain GH mRNA analyzed by 59-RACE was also identical to that in the pituitary (data not shown).

TABLE 1. Plasma ACTH and GH levels during RSW ACTH (pg/ml)

Pre (4) 2-h RSW (4) 4-h RSW (3)

112.86 6 3.36 229.78 6 10.25a 202.97 6 18.83a

GH (ng/ml)

10.88 6 1.98 1.09 6 0.28a 0.77 6 0.28a

The number of animals in each group is shown in parentheses. Results are the mean 6 SE. a P , 0.01 compared with Pre value.

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Effect of GRH administration on GH mRNA expression in the brain

As shown in Fig. 4, icv injections of GRH significantly elevated the expression levels of GH mRNA in the brain. Two hours after the GRH injections, the brain GH mRNA detected by RT-PCR (Fig. 4A) and the pituitary GH mRNA detected by Northern blotting (Fig. 4B) were both increased significantly. However, the levels of b-actin mRNA in the brain and pituitary were not changed during these treatments. Effect of RSW on GH mRNA expression in the brain

The GH mRNA level in the brain was significantly decreased by stress. When rats were exposed to RSW, the GH mRNA level in the brain was remarkably suppressed in 4 h (Fig. 5A). On the contrary, the pituitary GH mRNA was not significantly decreased during the treatment (Fig. 5B). The expression levels of b-actin mRNA in the brain and pituitary were not changed by RSW. During RSW, the plasma GH

FIG. 6. Detection of GH mRNA in the brain by in situ RT-PCR. Each panel depicts in situ PCR, where digoxigenin-dUTP (dig-dUTP) has been incorporated into amplified cDNA. A and D, Sections treated with DNase and RT. GH mRNA was detected in the region of the lh from a nonstressed rat by in situ RT-PCR (dark stain). B, Same area and same treatment as in A after 4 h of RSW. C, Negative control section; same area as in A treated with DNase but not with RT. Magnification: A–C, 320; D, 3100. Arrows indicate GH mRNA-positive cells, and bars indicate 100 mm in each panel. fx, Fornix; opt, optic tract; ic, internal capsula.

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concentration was also remarkably decreased, whereas the plasma ACTH concentration was significantly increased (Table 1). Localization of GH mRNA in the brain analyzed by in situ RT-PCR

We carried out in situ RT-PCR analysis on localization of GH mRNA in the brain. GH mRNA-positive cells were predominantly detected in the sections from the region of the lh treated with DNase and RT (Fig. 6A). These signals for GH mRNA in lh were clearly decreased after the rats were subjected to RSW for 4 h (Fig. 6B). In the negative control section that was treated with DNase but not with RT, there was no signal-positive cell (Fig. 6C). The GH mRNA-positive cells were small or medium-sized round and oval cells, and the signals were seen in their cytoplasm (Fig. 6D). Discussion

As well as in the pituitary gland, low levels of GH gene expression and immunoreactive GH production have been reported in hematopoietic/lymphoid cells (23–25) and brain tissues (13, 14). A recent report has shown that the mammary gland also expresses the GH gene (26). The present study clearly demonstrates that the rat brain expresses the GH gene predominantly in the lh, as assessed by RNase protection assay, RT-PCR, and in situ RT-PCR and also shows that GH gene transcription in the brain is initiated at the same tsp as that in the pituitary, as assessed by 59-RACE and sequencing. We further demonstrate here that GH gene expression in the brain is significantly stimulated by GRH and remarkably repressed by stress. It is well known that gene expression and secretion of GH in the pituitary is stimulated by GRH and reduced by somatostatin (27). Many reports also show that stress induces the hypothalamic somatostatin expression to inhibit the production and secretion of GH in the pituitary of the rat (28, 29). In this study, RSW indeed depressed the plasma GH level remarkably, but it did not lower the pituitary GH mRNA content significantly. This might be due to much slower turnover of GH mRNA in the pituitary than in the brain, as suggested previously (14). The results of this study suggest that the brain GH mRNA expression is promptly regulated by GRH and somatostatin. In the pituitary gland, the transcription factor Pit-1/GHF-1 (Pit-1) is responsible for expressions of GH, PRL, and TSH (30, 31). In some hematopoietic and lymphoid cells, Pit-1 is shown to be expressed and has been suggested to be involved in the gene expressions of GH and PRL (32). Rat lymphocyte GH cDNA analysis has suggested that the GH gene expression in the immune tissue may be regulated by transcription factors similar to those described in the pituitary (23). A recent study has also shown that human neutrophils can express the GH-N gene and Pit-1b proteins (33). Other studies, however, suggest that GH gene expression in the bone marrow of Snell mice is independent of Pit-1 (34), and that PRL gene expression in human lymphocytes and endometrial stroma is independent of Pit-1 (35). In the brain, the dependence on Pit-1 of GH or PRL gene expression has not yet been elucidated clearly, and the difficulty of detecting Pit-1 mRNA in the brain by in situ hybridization (32) or

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RT-PCR (36) has yielded speculations that the gene expression for GH (37) and PRL (36) in the brain may be independent of Pit-1. However, our preliminary experiments using the RT-PCR technique indicate that the Pit-1 gene is clearly expressed in the brain of the chicken (Tanaka, M., I. Yamamoto, and K. Nakashima, unpublished results) or the rat (Yoshizato, H., and K. Nakashima, unpublished results). This finding and the results of the present study suggest that GH gene expression in the brain may also be dependent on the transcription factor Pit-1. GH gene transcripts were localized predominantly in the lh of the rat brain in this study. A previous study reported that signals of in situ hybridization using 3H-labeled GH cDNA probes are recognized in wide areas of the brain, including outside the hippocampus, caudate putamen, or basal cortex (13). However, we could not detect significant signals in these regions in our in situ RT-PCR experiments, probably because of an insufficient concentration of GH mRNA in these areas under the conditions employed. The GH mRNA expression in lh was significantly decreased by exposing the animals to RSW, whereas the plasma GH level was also decreased remarkably. The decrease in the plasma GH level is considered to be due to the inhibition of GH secretion in the pituitary by stress-induced somatostatin (27– 29). These and the present results suggest that the GH expression in lh is also inhibited by stress-induced somatostatin. Neurons from the hippocampus and amygdala nucleus directly project to cells in the lh area, and lh neurons project to these regions indirectly (38). The amygdaloid area is known to mediate emotion and behavior, and the hippocampus is involved in memory and cognitive function manifestation. The GH molecules expressed in the lh may have some roles in these neuronal or neuroendocrine brain functions. Acknowledgments The authors thank the U.S. National Hormone and Pituitary Program and Dr. A. F. Palow of the NIDDK for providing rat GH and ACTH RIA kits.

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