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Endocrinology, doi:10.1210/en.2003-0845
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Endocrinology Vol. 145, No. 5 2542-2550
Copyright © 2004 by The Endocrine Society

Cocaine- and Amphetamine-Regulated Transcript Peptide in the Rat Anterior Pituitary Gland Is Localized in Gonadotrophs and Suppresses Prolactin Secretion

Genshin Kuriyama, Susumu Takekoshi, Katsuyoshi Tojo, Yoshikatsu Nakai, Michael J. Kuhar and R. Yoshiyuki Osamura

Division of Diabetes and Endocrinology (G.K., K.T.), Department of Internal Medicine, The Jikei University School of Medicine, Tokyo 105-8461, Japan; Department of Pathology (S.T., R.Y.O.), Tokai University School of Medicine, Kanagawa 259-1193, Japan; Division of the Science of Nursing (Y.N.), College of Medical Technology, Kyoto University, Kyoto 606-8507, Japan; and Yerkes Regional Primate Research Center of Emory University (M.J.K.), Atlanta, Georgia 30329

Address all correspondence and requests for reprints to: Robert Yoshiyuki Osamura, M.D., Ph.D., Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan. E-mail: osamura{at}is.icc.u-tokai.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cocaine- and amphetamine-regulated transcript (CART) mRNA and CART peptide are abundant in the hypothalamic nuclei that control anterior pituitary function. CART peptide has also been localized in the anterior pituitary gland itself, although its role in pituitary function has not as yet been elucidated.

In the present study, we investigated the localization and function of CART peptide in the anterior pituitary gland. Immunohistochemical observations revealed that CART peptide colocalized with FSH and LH in gonadotroph cells but that it was absent from the other hormone-producing cells. Immunoelectronmicroscopy suggested that CART peptide and gonadotropin were colocalized in the same secretory granules. CART peptide suppressed prolactin release from dispersed anterior pituitary cells 15 min after its addition into the media [basal production, 234.9 ± 14.6 ng/ml vs. CART 55–102 peptide 100 nM, 125.0 ± 18.2 ng/ml (P < 0.01, n = 5)]. Prolactin release was suppressed by CART in a dose-related manner; on the other hand, CART peptide did not affect the secretion of other anterior pituitary hormones. CART peptide synthesis by these cells was elevated 15 min after the addition of leptin to the media (100 nM), as determined by immunoblotting, but LHRH (10 nM) did not significantly affect CART peptide expression.

These findings suggest that CART synthesis in the anterior pituitary may be stimulated by leptin and that CART peptide may play a role in the regulation of anterior pituitary hormone secretion in the rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COCAINE- AND AMPHETAMINE-regulated transcript (CART) peptide was originally isolated as the product of a transcript that was elevated in the rat striatum after acute injection of psycostimulant drugs (1). CART mRNA is highly abundant in the hypothalamus and is widely expressed in the brain, adrenal gland, D cells of the pancreatic islets, and anterior pituitary gland (1, 2, 3, 4, 5, 6, 7), suggesting that it plays an important role in neuroendocrine and endocrine regulation.

Studies have shown that CART peptide acts as a neurotransmitter in the brain, and data suggest that it plays a role in feeding behavior, sensory processing, learning, memory, development, drug addiction, the stress response, and reproduction (1, 4, 8, 9, 10). CART mRNA and CART immunoreactivity have been reported to colocalize in hypothalamic nuclei that are known to modulate pituitary function. CART neurons have been shown to coexist with proopiomelanocortin neurons in the arcuate nucleus hypothalamus (ARH), melanocyte-concentrating hormone neurons in the lateral hypothalamic area, and TRH in the paraventricular nucleus (PVN) (11, 12, 13). In addition to influencing the release of a number of hypothalamic-releasing factors and neuropeptides, studies also support a role for CART peptide as a downstream regulator of leptin (8, 10, 12, 14).

CART peptide was shown to significantly stimulate the release of CRH, TRH, and neuropeptide Y from hypothalamic explants in vitro but significantly reduce {alpha}MSH release (15). Others have reported that CART peptide stimulated pulsatile GnRH secretion from prepubertal female rat hypothalamic tissue in vitro (10, 14, 16). Data from in vivo studies suggested that centrally administered CART peptide activated CRH neurons in the rat PVN and resulted in a rise in plasma ACTH and corticosterone (15, 17). These findings suggest that CART peptide directly regulates hormone release along the hypothalamic-pituitary-adrenal, thyroid, and gonadal axes.

CART mRNA was reported to have been detected in the anterior pituitary by Northern blot analysis, although no expression was detected in the posterior pituitary (18). Immunoblot analysis demonstrated localization of CART peptide in both the anterior and posterior pituitary gland (19). Immunohistochemical analysis revealed moderately stained clusters of CART peptide-positive cells in the anterior pituitary, no staining in the intermediate lobe, and a very high density of stained varicose fibers in the posterior pituitary (7).

To further define the relationship between CART peptide and the regulation of anterior pituitary hormone secretion, we performed RT-PCR and immunoblot analysis on rat pituitary gland extracts. We also used double-labeling techniques to immunohistochemically localize CART peptide, anterior pituitary hormones (GH, FSH, LH, TSH, ACTH, and prolactin), and folliculostellate (FS) cells. CART localization in the anterior pituitary gland was further assessed by immunoelectronmicroscopy. Finally, we examined the effects of leptin on CART peptide expression as well as the effects of CART peptide on the release of anterior pituitary hormones in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissues
The following experiments were conducted in strict accordance with National Institute of Health guidelines (7th ed., 1996) and the published standards of animal care and experimentation at our university. Adult male Wistar rats weighing 200–250 g were housed individually and maintained on a 12-h light, 12-h dark schedule and given free access to food and water. For collection of the brains and pituitary glands, the rats were anesthetized under diethylether inhalation and then decapitated.

CART mRNA expression analysis by RT-PCR
Eight animals were decapitated and their brains and pituitary glands harvested, washed, and dissected in ice-cold 0.01 M PBS. Total RNA from the hypothalamus and either the whole pituitary or isolated anterior and posterior lobes was extracted using a TRIzol reagent kit (Life Technologies, Inc., Rockville, MD) and analyzed for the presence of CART mRNA by RT-PCR.

cDNA was prepared using a Ready-To-Go T-primed first-strand kit (Amersham Biosciences Corp., Piscataway, NJ). The sequence of the rat CART oligo primer was 5'-oligo 5'-CTC AAG AGT AAA CGC ATT CC-3', 3'-oligo 5'-ACA AGC ACT TCA AGA GGA AA-3'. The housekeeping gene, ß-actin (Promega Co., Madison, WI) consisted of the following forward and reverse primers, respectively: 5'-oligo 5'-TCA TGA AGT GTG ACG TTG ACA TCC GT-3', 3'-oligo 5'-CCT AGA AGC ATT TGC GGT GCA CGA TG-3'. These were used as the internal standard. The PCR was performed as follows: denaturation at 95 C for 90 sec, followed by 30 cycles at 94 C for 30 sec, 54 C for 60 sec, and 72 C for 60 sec. After 30 cycles, an additional cycle at 72 C for 120 sec was run. The PCR products were analyzed by 2% agarose gel electrophoresis with ethidium bromide staining.

Immunoblot analysis
The hypothalamus and whole pituitary glands or separated anterior and posterior pituitary lobes (n = 5 in each group) were homogenized immediately after being harvested from 10 animals using a Potter-Elvehjem homogenizer in 10 volumes (wt/vol) of lysate buffer containing 1% sodium dodecyl sulfate. The lysate was then solubilized in sample buffer [25 mM Tris-HCl (pH 6.5), 5% glycerol, 1% sodium dodecyl sulfate, 0.05% bromophenol blue, and 1% ß-mercaptoethanol]. Lysates were subjected to SDS-PAGE; a prestained protein marker (Broad Range, Bio-Rad Laboratories, Hercules, CA) was run on the outside lane to verify the molecular weight of the CART peptide. The samples were then transferred to nitrocellulose membranes. Membranes were blocked with 3% BSA in 0.01 M PBS for 1 h at 37 C. After three washes with 0.01 M PBS, the membranes were incubated overnight at 4 C with either rabbit antirat pro-CART peptide antiserum (specific for the CART 26–38, which is unique to the long isoform of CART; 2025A, Novo Nordisk A/S, Copenhagen, Denmark) (5) or rabbit antirat CART 55–102 peptide antiserum (specific for the physiologically active form of CART; Phoenix Pharmaceuticals Inc., Belmont, CA) and diluted in PBS containing 5% BSA and 0.1% Tween 20; antiserum specificity was evaluated immunohistochemically. After five washes with 0.05% Tween 20 in 0.01 M PBS, the membranes were incubated with horseradish peroxidase-labeled antirabbit goat polyclonal IgG (Amersham Biosciences) and diluted 1:5000 in 0.05% Tween 20 in 0.01 M PBS for 1 h at room temperature. The immunoreactive bands were developed using an enhanced chemiluminescence system (Amersham Biosciences).

Immunohistochemistry
Five animals were perfused with 100 mM phosphate buffer (pH 7.4), containing 4% paraformaldehyde under chloral hydrate anesthesia. After perfusion, the brains and pituitary glands were harvested and stored in perfusion buffer overnight at 4 C. The tissues were then paraffinized and 4 µm-sections were prepared.

Immunohistochemistry was performed on these sections after they were deparaffinization, heated to 100 C in a microwave for 13 min in citric monohydrate buffer (pH 6.0), and treated with 100% methanol containing 0.3% H2O2 for 30 min at room temperature. The sections were washed twice in 0.01 M PBS for 10 min and incubated with 4% normal goat serum and 1% BSA. The sections were then incubated with rabbit antirat CART 55–102 peptide antiserum (Phoenix Pharmaceuticals) diluted 1:4000 in 1% BSA-PBS overnight at 4 C. As a specificity control for the antirat CART 55–102 antiserum, negative controls were run in which the primary antibody was replaced with normal rabbit serum. Furthermore, some slides were also preabsorbed with CART 55–102 peptide. The sections were then washed 10 times by incubation with 0.01 M PBS for 10 min. The sections were then incubated with goat antirabbit IgG conjugated to horseradish peroxidase-labeled-dextran polymer (Envision kits, Dako Co., Carpinteria, CA) for 60 min at room temperature. After being washed 20 times in 0.01 M PBS, the sections were developed in 3,3'-diaminobenzidine (DAB) solution containing 0.006% hydrogen peroxide (H2O2) for 3–5 min at room temperature. The above staining was also carried out on frozen sections using a previously described methodology (7).

After DAB color development, we proceeded with the second phase of the double-labeling procedure in the pituitary tissues. To thoroughly remove the antibodies used in the CART immunolabeling process, tissues were immersed in glycine-HCl buffer (pH 2.2) for 2 h, followed by a 30-min wash in 0.01 M PBS. The sections were then processed for second labeling with antibodies to pituitary hormones and the S-100 protein, which is a marker of FS cells. The primary antibodies that were directed against the pituitary hormones were: rabbit antirat GH (1:1000 dilution; NIH); antirat FSHß (1:2000 dilution; NIH), antirat LHß (1:2000 dilution; NIH), TSHß (1:5000 dilution; NIH), and prolactin (1:1000 dilution; NIH); and mouse antirat ACTH (1:5000 dilution; Dako). Rabbit anti-cow S-100 protein antibody (Dako), directed against FS cells, was used at a 1:200 dilution. All sections were incubated with their primary antibodies overnight at 4 C. The sections were washed 10 times and then incubated with secondary antibody. The secondary antibodies that were used were as follows: alkaline phosphatase-conjugated swine antirabbit IgG (Dako) at a 1:20 dilution for GH, FSHß, LHß, TSHß, prolactin, and S-100; alkaline phosphatase-conjugated rabbit antimouse IgG (Dako) at a 1:20 dilution for ACTH. All sections were incubated with these secondary antibodies for 60 min at room temperature and were then developed in 4-benzoylamino-2, 5-dimethoxybenzene-diazonium chloride hemisalt solution for 4–10 min at room temperature. After air drying, the slides were washed in distilled water, dehydrated, and coverslipped. Negative controls consisted of slides that were incubated only with the alkaline phosphatase-conjugated secondary antibodies after being washed with 0.1 M glycine-HCl buffer (pH 2.2) for 2 h to remove anti-CART antiserum/secondary antibodies complexes, after which they were treated with the 4-benzoylamino-2, 5-dimethoxybenzene-diazonium chloride hemisalt solution. A similar double-labeling procedure as the one just outlined was also carried out using antiserum against pro-CART (2025A, Novo Nordisk).

Analysis of CART localization in anterior pituitary cells by immunoelectronmicroscopy
Immunohistochemistry was performed on frozen sections as described above. Antibody incubations were carried out overnight at 4 C. After incubation with secondary antibody, the sections were washed as above and were then immersed in 0.1% glutaraldehyde for 5 min. The sections were washed again in 0.01 M PBS for 15 min and immersed in DAB solution without H2O2 for 30 min. The sections were then immersed in DAB solution containing 0.006% H2O2 for 5 min and placed in a 2% osmium tetroxide solution in 0.1 M phosphate buffer (pH 7.4) for 90 min. They were then washed in buffer for 10 min and dehydrated in a graded series of alcohol (~50–100%), after which they were embedded in Epon-filled Beem capsules. Ultrathin sections were collected from the surface of the plastic-embedded tissues and examined with an electron microscope (JEM-1200 EX, JEOL Ltd., Tokyo, Japan).

Effects of CART 55–102 peptide on hormone release from dispersed anterior pituitary cells
Analysis of the effects of CART peptide on pituitary hormone release was performed as previously described (20, 21, 22, 23, 24). Briefly, pituitary glands were rapidly removed from 81 animals and the anterior pituitary glands dissected free of their attached neurointermediate lobes. These anterior pituitary glands were placed in cold DMEM that contained 0.11 g/liter sodium pyruvate, 4.5 g/liter glucose, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.3% BSA, and 5 g/liter HEPES (pH 7.4). The glands were then cut into approximately 0.5- to 1.0-mm blocks using a sterile razor blade, which were then transferred with some media into conical tubes and centrifuged for 5 min at 120 x g. The supernatants were then removed and the tissues incubated in 10 ml of dissociation medium that consisted of DMEM with 0.3% trypsin and 0.16% deoxyribonuclease (DNase) for 20 min at 37 C, after which the tissues were again centrifuged for 5 min at 120 x g. After discarding the supernatants, the tissue blocks were resuspended in 5 ml DMEM that contained 10% fetal bovine serum and 0.16% DNase. The tissues completely dispersed after they were pipetted through a flame-polished disposable pipette. The cells were then seeded in poly L-lysine-coated 96-well tissue culture plates (5.25 x 104/well, determined by hemocytometer counts) and maintained in 5% CO2/95% air at 37 C for a 48-h recovery period, after which they were washed twice in DMEM containing 0.1% BSA. After a 2-h preincubation period, the media were replaced with 220 µl DMEM containing one of the following appropriate test substances, i.e. 100 nM CART peptide, 10 nM GH-releasing factor, 10 nM LHRH, 10 nM TRH, 5 nM corticotropin-releasing factor, or 10 nM prolactin-releasing peptide (all of which were purchased from Peptide Institute Inc., Osaka, Japan). The dose of CART was determined from previous experiments, which showed that 100 nM CART 55–102 peptide significantly stimulated the release of CRH, TRH, and neuropeptide Y from hypothalamic explants (15).

This being said, we also examined the effects of 100 nM or less CART on these cells. The cells were incubated at 37 C for either 15 or 30 min or 4 h. The concentrations of anterior pituitary hormones in the culture media were measured by RIA (GH, FSH, LH, TSH, and prolactin were measured using the Biotrak RIA system (Amersham Bioscience), whereas ACTH was measured by using the ALLEGRO ACTH kit (Nichols Institute Diagnostics, San Clemente, CA).

Effects of hypothalamic factors and leptin on CART 55–102 peptide production by dispersed anterior pituitary cells
Dispersed anterior pituitary cells from 25 animals were incubated in the presence of hypothalamic factors and leptin (100 nM) (Peptide Institute) for 15 min, 30 min, and 4 h, after which the cells and supernatants were harvested and examined for the presence of CART peptide using the immunoblot method described above. This immunoblot analysis was repeated three times and evaluated hypothalamic factors and leptin effect for CART synthesis.

Statistics
Values are expressed as the mean ± SEM. Data from the pituitary hormone assays were compared by using a paired t test. In all cases, P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CART mRNA expression analysis by RT-PCR
After amplification by RT-PCR, CART mRNA, with the expected size of 160 bp, was detected in the anterior pituitary gland. CART mRNA was expressed strongly in the anterior pituitary but was undetectable in the posterior pituitary. The housekeeping gene ß-actin served as the internal control, whereas the hypothalamus was included as a positive control (Fig. 1Go).



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  		FIG. 1. Expression of CART mRNA in the rat pituitary by RT-PCR. The product marked with an arrow (160-bp band) was detected in whole pituitary and the anterior, but not posterior, lobe. The housekeeping gene, ß-actin, served as the internal standard.

 
Immunoblot analysis
To determine whether the CART peptide was also present in the rat brain and pituitary tissues, immunoblot analysis was carried out using lysates of whole pituitary as well as the individual anterior and posterior pituitary glands (Fig. 2Go, A and B). Rabbit antirat pro-CART antiserum detected two bands at approximately 8 and 10 kDa (Fig. 2AGo), whereas rabbit antirat CART 55–102 antiserum detected two bands at approximately 5 and 8 kDa in the anterior pituitary (Fig. 2BGo); staining was less intense in the hypothalamus neither. Similar bands were detected in the posterior pituitary, but their staining was also weak.



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  		FIG. 2. Immunoblot analysis of CART peptide in the rat hypothalamus, whole pituitary, anterior pituitary, and posterior pituitary. A prestained protein marker was used to verify the molecular weight of the peptide bands. A, Rabbit antirat pro-CART peptide antiserum (raised against CART 26–38, the 13-amino acid insert unique to the long isoform of CART) identified two peptide bands. The upper arrow indicates the 10-kDa band of the CART peptide and the lower arrow indicates the 8-kDa band. Lane 1, hypothalamus; lane 2, whole pituitary; lane 3, anterior pituitary; lane 4, posterior pituitary. B, Rabbit antirat active CART (CART 55–102 peptide) antiserum also detected two peptide bands. The upper arrow indicates the 8-kDa band and the lower arrow indicates the 5-kDa band. The lower band is considered to be the active CART 55–102 peptide. Lane 1, positive control (recombinant CART 55–102 peptide); lane 2, hypothalamus; lane 3, whole pituitary; lane 4, anterior pituitary; lane 5, posterior pituitary.

 
Immunohistochemistry
Absorption controls for the antiserum against CART 55–102 gave the expected results (Fig. 3Go). Immunohistochemistry revealed CART 55–102 peptide immunoreactivity in the hypothalamus and in both the anterior and posterior pituitary glands (Fig. 4Go, B–F, I, and J). Negative controls in which normal rabbit serum was substituted for primary antibody failed to show any specific staining (Fig. 4Go, A, G, and H). In the hypothalamus, CART peptide immunoreactive neurons were particularly abundant in the PVN, the supraoptic nucleus, the zona incerta, the anterior periventricular nucleus, and the ARH (Fig. 4BGo) [corresponding to level 26 in the atlas by Swanson (25)]. On the other hand, a moderate density of stained cells was found in the anterior hypothalamic nucleus, and some scattered cells were found in the ventromedial nucleus hypothalamus (Fig. 4BGo). CART peptide immunoreactivity in these areas appeared as granular-type staining in neuronal perikarya (Fig. 4Go, C and E). The highest density of stained fibers was found in the external layer of the median eminence (Fig. 4Go, D and F). Staining of neuronal fibers was less intense in paraffin, compared with frozen sections, although the staining of neuronal cell bodies and fibers was comparable using these two methodologies.



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  		FIG. 3. Absorption controls for the anti-CART 55–102 antiserum. A, CART 55–102 peptide, 0.3 µg/ml; anti-CART 55–102 antiserum, 5 µg/ml. B, CART 55–102 peptide, 0.61 µg/ml; anti-CART 55–102 antiserum, 5 µg/ml. C, CART 55–102 peptide, 1.21 µg/ml; anti-CART 55–102 antiserum, 5 µg/ml. D, CART 55–102 peptide, 2.43 µg/ml; anti-CART 55–102 antiserum, 5 µg/ml. E, CART 55–102 peptide, 4.87 µg/ml; anti-CART 55–102 antiserum, 5 µg/ml.

 


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  		FIG. 4. Immunohistochemical localization of CART peptide in the hypothalamus (B–F) and pituitary gland (I and J). AHN, Anterior hypothalamic nucleus; ME, median eminence; V3, third ventricle. Negative controls are shown in A, G, and H. Neuronal cells and fibers in the PVN of a paraffin section are shown at higher magnification in C, whereas comparable regions in a frozen section are shown in E. Similarly, neurons in the ARH are shown in both paraffin and frozen sections (D and F, respectively). Whereas cellular staining was comparable between paraffin and frozen sections, the staining of fibers was less intense in paraffin section. The median eminence, shown in D and F, showed dense fibrous staining in its external layer. In the anterior pituitary, CART-positive cells were found mainly in peripheral regions, with some of these cells located around blood vessels (I). In the posterior pituitary, stained varicose fibers were observed throughout the lobe, whereas CART-positive cell bodies were not found (J).

 
In the anterior pituitary, CART-positive cells were found mainly in peripheral areas and surrounding blood vessels (Fig. 4IGo). On the other hand, in the posterior pituitary, dense, fibrous staining was observed that was restricted to varicose fibers that were present throughout the lobe, whereas no staining of cell bodies was observed (Fig. 4JGo).

Double labeling using antiserum for pro-CART and antianterior pituitary hormone or anti-FS cell antibodies showed that 80.2 ± 1.2% of FSH/LH-immunoreactive cells (averaged by counting 200 cells per one slide; n = 5) (Fig. 5Go, B and C) but not GH, TSH, ACTH, or prolactin immunoreactive cells, colocalized with CART staining (Fig. 5Go, D–G); negative controls gave the expected results (Fig. 5AGo). Furthermore, CART staining did not colocalize with FS cell staining (Fig. 5HGo). Double labeling using antiserum against CART 55–102 peptide and antianterior pituitary hormone or anti-FS cell antibodies showed similar results; 20.6 ± 1.4% of gonadotrophs (averaged by counting as described above) colocalized with CART (Fig. 6Go, B and C). Immunoelectron microscopic analysis revealed that CART-positive cells were frequently adjacent to prolactin cells. CART 55–102 peptide and LH were localized in many granules of the similar cells, respectively, which suggested that CART 55–102 peptide and gonadotropin were present in the same granule (Fig. 7Go, A and B).



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  		FIG. 5. Double immunolabeling for pro-CART peptide and anterior pituitary hormones (B–H). A, Negative control. B and C, Colocalization of pro-CART peptide with FSH or LH was observed in approximately 80% of gonadotropin-immunopositive cells as a mixture of brown (pro-CART) and blue (FSH, LH) color. D–H, The double immunostaining of pro-CART (brown) and GH, TSH, ACTH, prolactin (PRL), or S-100 (all of which stained blue) did not reveal any colocalization.

 


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  		FIG. 6. Double immunolabeling for CART 55–102 peptide and anterior pituitary hormones (B–H). A, Negative control. B and C, Colocalization of CART peptide with FSH or LH was observed in approximately 20% of gonadotropin-immunopositive cells as a mixture of brown (CART) and blue (FSH, LH) color. D–H, Double immunostaining of CART (brown) and GH, TSH, ACTH, prolactin (PRL), or S-100 (all of which stained blue) did not reveal any colocalization.

 


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  		FIG. 7. Analysis of colocalization of CART peptide (A) and LH (B) by immunoelectron microscopy. The data suggest that CART peptide and gonadotropin may be localized in the same granules. Scale bars, 1 µm.

 
Effects of CART peptide on anterior pituitary hormones release from dispersed anterior pituitary cells
CART 55–102 peptide (100 nM) suppressed prolactin release 15 min after its addition into the media (Fig. 8AGo); specifically, basal production (234.9 ± 14.6 ng/ml, 100 ± 6.2% basal) was reduced to 125.0 ± 18.2 ng/ml (53.2 ± 14.6% basal) (P < 0.01, n = 5). On the other hand, CART 55–102 peptide did not significantly affect the release of other hormones. Although prolactin levels were similarly suppressed at 30 min, they were elevated 4 h after administration of CART peptide, although this elevation failed to reach statistical significance (P = 0.6 vs. basal at 30 min, P = 0.14 vs. basal 4 h after administration). Similarly, TSH levels were elevated 30 min after administration but this increase also failed to reach statistical significance (P = 0.15 vs. basal).



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  		FIG. 8. Time- (A) and dose-dependent (B) effects of CART peptide on the release of hormones from dispersed anterior pituitary cells. Hormone levels in the culture media were measured after incubation with CART peptide for 15 min, 30 min, or 4 h in A. A dose-response analysis of the effects of a 15 min incubation with prolactin appears in B. CART suppressed prolactin release in both a time- and dose-dependent fashion. The results are the mean ± SEM in triplicate. **, P < 0.01 vs. basal, n = 5; *, P < 0.05 vs. basal, n = 5.

 
A dose-dependent analysis of the effects of a 15-min incubation with CART 55–102 revealed that only prolactin release was significantly suppressed by a concentration of 100 nM CART (Fig. 8BGo, P = 0.04 vs. basal).

Effects of LHRH and leptin on CART peptide release from dispersed anterior pituitary cells
CART peptide synthesis in dispersed anterior pituitary cells, as determined by immunoblotting, was stimulated by leptin (100 nM) at 15 min after administration (Fig. 9Go, lane 5). On the other hand, LHRH (10 nM) did not significantly affect CART peptide synthesis.



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  		FIG. 9. Effects of leptin on CART synthesis in anterior pituitary cells using the immunoblot method. Leptin stimulated CART synthesis after a 15-min incubation, whereas LHRH had no effect on CART synthesis. Lane 1, Positive control (recombinant CART 55–102 peptide); lane 2, control (DMEM with 0.1% BSA-PBS) at 0 min; lane 3, control at 15 min; lane 4, LHRH (10 nM) at 15 min; lane 5, leptin (100 nM) at 15 min; lane 6, control at 30 min; lane 7, LHRH (10 nM) at 30 min; lane 8, leptin (100 nM) at 30 min; lane 9, control at 4 h; lane 10, LHRH (10 nM) at 4 h; lane 11, leptin (100 nM) at 4 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We herein confirmed, using RT-PCR, immunoblotting, and immunohistochemical analysis, the localization of CART mRNA and peptide in the rat hypothalamus and pituitary gland (1, 4, 7). CART mRNA was found to be expressed in the hypothalamus and anterior pituitary gland. In the hypothalamus and both the anterior and posterior parts of the pituitary, CART peptide was detected as three molecular-weight bands. The 5-kDa band could correspond to CART 55–102, which is a physiologically active fragment, whereas the 8- and 10-kDa bands may have represented the long form of the pro-CART peptide (19). The bands in the hypothalamus were less intense than those in the anterior pituitary. Whereas we expected to be able to detect pro-CART (10 kDa) using our antibody that was directed against the CART 55–102 peptide, we were unable to do so, probably because of low antibody affinity or very low peptide concentration. Immunohistochemistry showed that CART peptide localized primarily in the PVN and arcuate nucleus in the hypothalamus and in both the anterior and posterior pituitary glands. CART peptide in the posterior pituitary was assumed to have originated in CART-containing magnocellular neurons in the PVN (26).

Our new observation was that CART peptide colocalized with gonadotropic hormone (FSH/LH) in the rat anterior pituitary. Pro-CART peptide was found in approximately 80% of gonadotrophs, whereas physiologically active CART localized in approximately 20% of gonadotrophs. The difference in distribution between pro-CART and active CART seemed to reflect differences in processing, secretion, or degradation (7). A recent study indicated that the subtilisin/kexin-like prohormone convertases, PC2 and PC1/3, might participate in CART processing in vivo (27). The distribution of these enzymes in gonadotrophs may have contributed to the differences that we detected in CART distribution. The physiological significance of the localization of CART in gonadotrophs is as yet unknown. Although we did not find an effect of LHRH on the expression of CART, it is possible that reproductive gonadal steroids such as estrogen, progesterone, and testosterone may influence CART expression. Thus, our data suggest the presence of a unique pathway by which CART may influence and/or mediate reproductive behavior. The cellular colocalization of CART and gonadotropic hormone suggested that it represented a global colocalization within the same granules. It has been reported that estrogens target most anterior pituitary hormone-secreting cells that express receptors (28); because gonadotrophs express estrogen receptors, it is conceivable that gonadal hormones in general and estrogen in particular may regulate CART release.

We also observed that leptin stimulated CART synthesis from anterior pituitary cells in vitro, supporting similar reports of leptin-induced CART release in the central nervous system (8, 10, 12, 14). Recently the CART promotor was sequenced and found to contain signal transducer and activator of transcription (9, 29), supporting the findings of others (30), who showed an effect of leptin signaling on CART gene expression. This latter finding suggests that leptin can stimulate not only CART release but also its synthesis by regulating its gene expression. Although leptin is known to be secreted by adipocytes, other studies have shown that it is also localized in primarily the TSH cells of the anterior pituitary (31, 32), in which it may be acting as a paracrine regulator of CART peptide release. Leptin was reported to have produced a dose-dependent increase in FSH and LH release from incubated dispersed anterior pituitary cells that was nearly identical with that seen with LHRH (33). Whereas those investigators did not measure CART peptide levels, their data suggest that leptin may directly affect gonadotropic hormone as well as CART release. Leptin receptors were shown to localize on a specific subtype of anterior pituitary cells (31, 32, 33, 34, 35, 36). In the ovine anterior pituitary, 90% of the gonadotrophs in the pars tuberalis were immunopositive for the leptin receptor (36). On the other hand, another group (32) found that leptin receptors were expressed primarily on GH cells, with fewer than 1% of gonadotrophs expressing the receptor. In light of these conflicting results, it is still unclear as to whether leptin activates CART directly or indirectly.

Using our in vitro system, we also found that CART peptide inhibited prolactin secretion, suggesting putative roles for CART as both an autocrine and paracrine regulator of hormone secretion in the anterior pituitary. The receptors that mediate the actions of CART peptide remain to be identified; such information would undoubtedly help to clarify the role of this peptide in anterior pituitary function.

In conclusion, we found that CART peptide colocalized with gonadotrophs in the granules of gonadotroph cells of the rat anterior pituitary and as such, might play a role in the regulation of gonadal hormone release or in the mediation of the effects of reproductive hormones such as estrogen. We also showed that leptin stimulated CART synthesis and that CART peptide suppressed prolactin secretion.


   Acknowledgments
 
The authors thank Professor Naoko Tajima (Division of Diabetes and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine) for her encouragement and support. We also thank Dr. Jes Thorn Clausen (Novo Nordisk A/S, Bagsvaerd, Denmark) for his generous gift of anti-CART antiserum (2025A, 2055A). Finally, we are grateful to Dr. Johbu Itoh for his excellent photographic assistance and Dr. Hideaki Hasegawa, Dr. Yoshiko Itoh, Mr. Masayuki Sone, Mr. Shunsuke Miyai, Mr. Noboru Egashira, Mr. Kentaro Matsuzaki, and Mr. Chiaki Wakabayashi for their technical assistance.


   Footnotes
 
This work was supported in part by a grant from the Japan Smoking Research Fundation.

Abbreviations: ARH, Arcuate nucleus hypothalamus; CART, cocaine- and amphetamine-regulated transcript; DAB, 3,3'-diaminobenzidine; FS, folliculostellate; PVN, paraventricular nucleus.

Received July 9, 2003.

Accepted for publication January 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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