Cocaine- and Amphetamine-Regulated Transcript Is Localized in Pituitary Lactotropes and Is Regulated during Lactation
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《内分泌学杂志》
Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037
Abstract
Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART-producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing a high-affinity antiserum we generated against CART-(62–102), was used to identify CART-producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (-ir) was localized in lactotropes; minor populations of CART-ir cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin-containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocriptine, a potent dopamine receptor agonist, were examined to determine whether CART mRNA expression and protein release are regulated in a similar fashion as prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased after bromocriptine treatment of dispersed rat anterior pituitary cells in culture. To explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and nonlactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked on d 10 of lactation and returned to baseline values 10 d after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.
Introduction
COCAINE- AND AMPHETAMINE-regulated transcript (CART) was originally identified by differential display PCR as an mRNA transcript that was up-regulated in the striatum of rats after acute administration of cocaine and amphetamine (1, 2). Since this initial discovery, CART has been implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors (3, 4, 5, 6, 7). CART is a highly expressed peptide that is widely distributed throughout the central nervous system (CNS), gut, pituitary, adrenal, and pancreas (5, 8, 9). In the brain, CART immunoreactivity (-ir) has been localized in cell bodies, fibers, and varicosities in the olfactory bulb, cortex, hypothalamus, mesencephalon, and brainstem (10, 11). CART-ir fibers are in close opposition to fenestrated capillaries in the external zone of the median eminence, and CART protein is found in the anterior and posterior pituitary (11). In the periphery, CART is localized in pancreatic islets (9), enteric neurons in the gut (12), and the adrenal medulla (11).
The cellular localization of CART has been extensively studied in the rat CNS. In the medulla, CART is colocalized with adrenergic and -MSH-expressing neurons (13). CART is found in oxytocin (OT)- and vasopressin (AVP)-containing neurons in the magnocellular paraventricular nucleus (PVN), TRH-containing cells are found in the parvocellular PVN, and CART-ir cells colocalize with proopiomelanocortin in the arcuate nucleus (10, 14, 15). CART-ir has also been shown to colocalize with urocortin 1 in cells of the Edinger Westphal nucleus (16).
In contrast, the cellular localization of pituitary CART has not been clearly defined. CART mRNA is expressed in the anterior pituitary; however, CART mRNA expression has not been reported in the intermediate and posterior pituitary gland (17). Immunohistochemistry (IHC) studies revealed a varied distribution of CART in the anterior pituitary gland. Koylu et al. (11) reported that the location and intensity of CART-ir in the anterior pituitary varied dependent on the antiserum used. Kuriyama et al. (18) recently demonstrated that CART-ir is localized in gonadotropes, whereas Stanely et al. (19) reported that CART was expressed in corticotropes. In the posterior pituitary, CART-ir is localized in a high density of varicose fibers, and CART-ir has not been detected in the intermediate lobe of the pituitary gland (11).
In the present study we generated antiserum against CART-(62–102) to clarify the anatomical distribution of CART in the pituitary gland. A sensitive RIA was developed, and real-time PCR was employed to identify factors that regulate CART mRNA transcription and protein release from anterior pituitary cells. In addition, we compared levels of CART in the hypothalamus, pituitary, and systemic circulation of lactating and nonlactating rats to explore the physiological roles of pituitary CART.
Materials and Methods
Animals and tissue preparation
Adult male and female Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN), were used in the experiments described in this study. Animals were maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) and provided rat chow (Harlan-Teklad, Madison, WI) and water ad libitum. Animals were anesthetized with an overdose of choral hydrate (1 g/kg body weight, ip) and perfused transcardially with 150 ml saline, followed by 350 ml 4% paraformaldehyde in borate buffer (pH 9.5). After perfusion, brains and whole pituitaries were removed and postfixed in 25% sucrose in the same fixative at 4 C overnight. Tissues were then quickly frozen on dry ice, sectioned at 25 μm using a sliding microtome, and stored in cryoprotectant at –20 C until use. The Salk Institute animal use and care committee approved all procedures described in this study.
Blood sampling
Blood samples were obtained from pregnant and lactating female Sprague Dawley rats by tail bleed. Approximately 0.3 ml blood was taken from each animal between 0800–0900 h on d 5, 12, and 18 of pregnancy and d 2, 10, 17, and 21 of lactation. Control bleeds were taken 10 d before mating and 10 d after weaning to determine baseline levels of circulating CART. Blood samples were drawn into chilled tubes containing EDTA and centrifuged, and plasma was stored at –20 C until RIA analysis.
Antiserum and controls
Antiserum was raised in rabbits immunized with a synthetic peptide encoding rat [Nle67]CART-(62–102) conjugated to human -globulins via bisdiazotized benzidine using a protocol previously described in detail for inhibin subunits (20). In all experiments, antiserum was used in an unpurified form at the indicated dilutions. The specificity of immunostaining was evaluated using primary antiserum preabsorbed overnight at 4 C with 0–300 μM synthetic rat CART-(55–102) or CART-(62–102) before incubation with brain sections. CART-(55–102) and CART-(62–102) were synthesized in this laboratory by solid phase methodologies (21).
RIA
The analog [Nle67]CART-(62–102) was radiolabeled with 125I using chloramine T and purified by HPLC with a 0.1% trifluoroacetic acid-acetonitrile solvent system for use as a tracer in the RIA. The procedure for the CART RIA was similar to that described in detail for inhibin subunits (20). Briefly, rabbit 6838 anti-CART-(62–102) serum was used at a 1:150,000 final dilution, and synthetic CART-(55–102) and CART-(62–102) were used as standards. Rat tissues were acid extracted and partially purified with octadecyl silica cartridges as previously described (20). Lyophilized tissue extracts were reconstituted in assay buffer, pH was checked and adjusted if necessary, and three to seven dose levels were tested. Cell secretion medium was tested without dilution at multiple doses. Free tracer was separated from tracer bound to antibody with the addition of sheep antirabbit -globulins and 10% (wt/vol) polyethylene glycol. The minimum detectable dose and EC50 for CART-(55–102) ranged from 2–3 and 50–60 pg/tube, respectively. Results were calculated using a logit/log RIA data processing program of Faden, Hutson, Munson, and Rodbard (National Institute of Child Health and Human Development, Reproductive Research Branch, National Institutes of Health).
Gel filtration chromatography
Pooled male and female rat plasma was partially purified using octadecyl silica cartridges as previously described (20). The purified plasma was applied to a fast protein liquid chromatography system equipped with a Superdex 75 HR 10/30 column (Amersham Biosciences Corp., Piscataway, NJ). The column was eluted with 30% acetonitrile/0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. Aliquots of fractions were lyophilized and tested in the CART RIA. In separate runs, the column was calibrated using various peptide and protein standards.
Immunoblotting
Rat pituitary glands were acid extracted and partially purified with octadecyl silica cartridges as previously described (20). Lyophilized proteins were resuspended in 1x sample buffer [50 mM Tris (pH 6.8), 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, and 10% glycerol]. Samples were boiled for 5 min, and proteins were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel (Invitrogen Life Technologies, Inc., Carlsbad, CA). Electrophoresed proteins were subsequently transferred onto nitrocellulose membranes and then probed with anti-CART-(62–102) (PBL 6838) or anti-CART-(55–102) (Phoenix Pharmaceuticals, Belmont, CA) overnight at 4 C. Membranes were washed in PBS with 0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated antirabbit IgG (Amersham Biosciences, Little Chalfont, UK). Immunoreactive proteins were visualized using Super Signal West Pico chemiluminescent substrate (Pierce Chemical Co., Rockford, IL).
IHC
Brain and whole pituitary sections were incubated in one or a combination of the following antisera: rabbit anti-CART-(62–102) (1:10,000; PBL 6838); guinea pig anti-PRL, -GH, -FSH, -TSH, and -ACTH (1:12,000; National Institutes of Health, Bethesda, MD); guinea pig anti-AVP (1:15,000; Peninsula Laboratories, San Carlos, CA) or mouse anti-OT (1:7,500; Chemicon International, Temecula, CA) in potassium PBS (KPBS) with 0.4% Triton X-100 at 4 C for 48 h. The tissues were then rinsed in KPBS and incubated in one or a combination of the following secondary antibodies: biotinylated donkey antirabbit IgG (1:700; Jackson ImmunoResearch Laboratories, West Grove, PA), fluorescein-conjugated donkey antirabbit IgG, rhodamine red X-conjugated antimouse IgG, or Cy3-conjugated antiguinea pig IgG (1:500; Jackson ImmunoResearch Laboratories) in KPBS with 0.4% Triton X-100 for 1 h at room temperature. Tissues incubated with biotinylated secondary antibodies were subjected to 1-h incubation at room temperature in avidin-biotin complex solution (Vectastain ABC Elite kit, Vector Laboratories, Inc., Burlingame, CA). The antibody-peroxidase complex was visualized with 3,3-diaminobenzidine (0.2 mg/ml) and 3% H2O2 (0.83 μl/ml) in 175 mM sodium acetate solution. When staining had reached an appropriate intensity, the tissue was rinsed in KPBS and mounted on gelatin-coated glass slides. Slides were dehydrated through graded alcohols, cleared in xylenes, and coverslipped with DPX mountant (Electronic Microscope Science, Fort Washington, PA). Adjacent series of brain sections were processed for Nissl staining for reference purposes.
Rat anterior pituitary cell cultures
Anterior pituitary cells from male Sprague Dawley rats were prepared by dispersion with collagenase as previously described (22). Cultures were plated on poly-L-lysine-coated coverslips in 24-well culture plates for immunocytochemical analysis or in 48-well plates for protein expression and release studies. Cultures were plated at a density of 1.5 x 105 cells/culture well and were allowed to recover for 72 h in complete medium [Pit Julep (PJ)] supplemented with 2% fetal bovine serum and appropriate growth factors (22).
Before immunocytochemical analysis, pituitary cells in culture were rinsed with KPBS and then fixed for 20 min with 4% paraformaldehyde at room temperature. Cultures were washed in KPBS and processed for CART, GH, TSH, FSH, ACTH, and PRL-ir following the procedures described above for brain and pituitary sections. Before the examination of CART expression and protein release, pituitary cells were washed three times with PJ and 0.1% BSA and equilibrated for 1 h. Cells were then washed with PJ and 0.1% BSA and treated in triplicate with bromocriptine (0.1–100 nM) or forskolin (1 μM) for 3, 6, and 12 h at 37 C. Media were collected after each incubation time point for measurement of secreted CART by RIA. Cells were harvested, and mRNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Inc.) to determine CART mRNA levels.
In situ hybridization
Animals were rapidly decapitated, and whole brains were removed and frozen on dry ice. Coronal sections (20 μm) were cut on a cryostat, thaw-mounted onto glass slides, and stored at –80 C until use. Antisense cRNA probes were transcribed from linearized cDNA templates corresponding to bp 495–679 of the rat CART gene. [35S]UTP (PerkinElmer Life Sciences, Emeryville, CA) was incorporated into cRNA probes during transcription, and the specific activity of the probes was determined to be approximately 2 x 108 dpm/μg cRNA. The saturating concentration for the probes used for assays was 0.3 μg/ml/kb.
The procedure used for in situ hybridization was described previously (23). Briefly, brain sections were fixed in 4% paraformaldehyde and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2% standard saline sulfate (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air dried. The slides were then exposed to cRNA probes overnight in humidified chambers at 55 C. After incubation, the slides were washed in SSC of increasing stringency, in ribonuclease, and then in 0.1% SSC at 63 C; dehydrated through a graded series of alcohols; and dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed from 7–14 d at 4 C, and developed. After development, the slides were counterstained with cresyl violet.
Tissue analysis
Tissue sections and pituitary cultures were initially examined using an E600 light microscope (Nikon, Tokyo, Japan). The cellular localization of CART with pituitary hormones and hypothalamic neuropeptides was determined using a Fluoview confocal system on an IX70 microscope (Olympus, Melville, NY) with the following laser excitation lines: 488 nm for fluorescein and 568 nm for rhodamine red X and Cy3. Dichroic/emission filters for detection were 500 nm LP/515–565nm for fluorescein, and 575 nm LP/590 nm LP for rhodamine red X and Cy3. These filter combinations resulted in very low levels of cross-talk between fluorochrome signals.
The extent of colocalization between CART and pituitary hormones was ascertained from six separate pituitary cultures stained for CART and individual pituitary hormones. The total numbers of cells containing 1) CART-ir, 2) the pituitary hormone of interest, and 3) colocalization of CART-ir and a specific hormone were determined for each culture. The results are presented as the percentage of CART-ir cells that contain individual pituitary hormones.
Autoradiograms for CART in situ hybridization were visualized under dark-field illumination using a E600 light microscope (Nikon, Tokyo, Japan) and analyzed with Image-Pro Plus imaging software (Media Cybernetics, San Diego, CA). Integrated density values of hybridized neurons of each side of the supraoptic nucleus (SON) and PVN were measured in at least five consecutive sections for each animal. Nonlinearity of radioactivity in the emulsion was evaluated by comparing density values with a calibration curve created from autoradiograms of known dilutions of the radiolabeled probes immobilized on glass slides in 2% gelatin fixed with 4% paraformaldehyde and exposed and developed simultaneously with the in situ hybridization autoradiograms.
All images were captured using a digital camera (Photometrics, Huntington Beach, CA) and Image-Pro Plus imaging software (Media Cybernetics, San Diego, CA). The images were cropped and adjusted to balance brightness and contrast in Adobe Photoshop (version 5.5; Adobe Systems, San Jose, CA) before import into Canvas (version 6.0) for assembly into plates.
RT and real-time PCR analysis
CART mRNA levels in anterior pituitary cells were quantified using real-time PCR analysis. After deoxyribonuclease treatment, a constant amount of RNA (1 μg) was added to a reverse transcriptase mixture (SuperScript II RNase H-Reverse Transcriptase, Invitrogen Life Technologies, Inc.). To test for possible pseudo gene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. Levels of CART expression were determined with primers for CART (sense, 5'-GTAAACGCATTCCGATCTATGAGA-3'; antisense, 5'-CCGATCCTGGCCCCTTT) and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense, 5'-GGAAGGGCTCATGACCACAGT-3'; antisense, 5'-CACAGTCTTCTGAGTGGCAGTGAT-3'). Reaction mixtures contained TaqMan Universal Master Mix with SYBR Green (Sigma-Aldrich Corp., St. Louis, MO), 80 nM primers, and 1 ng cDNA in a final reaction volume of 50 μl. PCRs were performed in 96-well plates on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) with sequence detection system software. For each biological sample, real-time PCRs were performed in duplicate, and CART expression was normalized to GAPDH.
Statistical analysis
Statistical analyses were performed using unpaired Student’s t tests and one-way ANOVAs as indicated for each study. Tukey analysis tests were used after ANOVA to make comparisons between groups at a particular time point and between time points within a particular group. Differences were considered statistically significant at P < 0.05.
Results
CART-ir in rat brain and peripheral tissues
We generated antiserum against a synthetic peptide encoding rat CART-(62–102), which allowed us to determine the cellular localization of CART in the pituitary gland. The affinity and specificity of the antiserum were examined by IHC, immunoblotting, and size-exclusion chromatography. Immunohistochemical analysis revealed that anti-CART-(62–102) stained cell bodies and fibers in multiple regions of the rat brain, including the olfactory bulb, nucleus accumbens, amygdala, thalamus, hypothalamus, and several nuclei in the mesencephalon and brainstem. Preabsorption of anti-CART-(62–102) with 20 μg/ml CART-(55–102) or CART-(62–102) completely eliminated CART staining in the SON and Edinger Westphal nucleus, demonstrating the specificity of anti-CART-(62–102) (Fig. 1).
A RIA was developed using CART-(62–102) antiserum to determine the levels of CART protein in tissues and biological fluids. The CART RIA is highly sensitive, with a detection limit of 2–3 pg/tube. As shown in Fig. 2A, both synthetic CART-(62–102) (EC50, 30 pg/tube) and CART-(55–102) (EC50, 50 pg/tube) displaced tracer bound to antibody in a similar manner. CART-like ir was measured in acid-extracted rat tissues partially purified using octadecyl silica cartridges. The highest concentrations of CART were detected in the posterior pituitary and hypothalamus, moderate levels were found in the cerebellum and pancreas, and low levels were observed in the heart and spleen. The approximate concentrations of CART per milligram of partially purified tissue extract were as follows: 2.8 μg/mg posterior pituitary, 950 ng/mg hypothalamus, 35 ng/mg cerebellum, 7 ng/mg pancreas, 0.7 ng/mg heart, and 0.08 ng/mg spleen (Fig. 2A).
To examine the specificity of anti-CART-(62–102), we employed gel exclusion chromatography to determine the molecular mass of CART-ir products in rat plasma. As shown in Fig. 2B, anti-CART-(62–102) recognized a single immunoreactive species of approximately 5.8 kDa. This is in agreement with the predicted molecular mass of that reported for biologically active CART-(55–102).
Cellular localization of CART-ir in rat anterior pituitary gland
Immunoblot analysis was used to verify that CART peptides were present in the rat pituitary gland. Partially purified protein extracts from whole rat pituitaries were probed with antiserum we generated against CART-(62–102) and commercially available antiserum against CART-(55–102) used by other researchers (18). Both antisera detected bands of approximately 5 and 8 kDa in pituitary extracts and had strong immunoreactivity with synthetic CART-(55–102) (Fig. 3A).
IHC was employed to examine CART protein distribution in whole pituitary sections from adult male rats. Moderate levels of CART-ir were detected throughout the anterior pituitary. The majority of CART-ir was distributed around the lateral edge of the anterior lobe (Fig. 3B). Preabsorption of anti-CART-(62–102) with 20 μg/ml CART-(55–102) completely eliminated CART staining in the pituitary, confirming the specificity of the antiserum (Fig. 3D).
The cellular localization of CART in the anterior pituitary was determined by double-label IHC using rabbit anti-CART-(62–102) together with antisera raised in guinea pigs against pituitary hormones (ACTH, FSH, GH, PRL, and TSH) in dispersed rat anterior pituitary cells in culture. The majority of CART-ir was localized in lactotropes, with minor populations of CART-ir cells identified as somatotropes and corticotropes (Fig. 4A). As summarized in Fig. 4B, we determined that 82% of CART-immunopositive cells also contained PRL, 10% of cells contained GH, and 5% of CART-ir cells expressed ACTH. Less than 1% of CART-ir cells contained TSH or FSH (Fig. 4B). The percentage of specific anterior pituitary cell types that contained CART-ir was also determined; this revealed that 25% of lactotropes, 5% of somatotropes, and 3% of corticotropes contained CART-ir (Fig. 4C). To ensure that the observed cellular localization of CART-ir in cultured anterior pituitary cells was representative of the intact pituitary, we determined the cellular localization of CART-ir in whole pituitary sections and dispersed anterior pituitary cells from female rats. In all preparations, similar distribution and localization patterns were observed.
Distribution of CART-ir in posterior pituitary and hypothalamus
Strong immunostaining for CART was detected in tightly packed varicose fibers throughout the posterior lobe of the pituitary, and CART-ir was not detected in the intermediate lobe of the pituitary (Fig. 3C). Double-label IHC analysis of whole pituitary sections revealed that a large population of OT fibers colocalized CART-ir in the posterior pituitary (Fig. 5A). We next examined the distribution of CART-ir in magnocellular neurons of the SON and PVN that contribute axons to the posterior pituitary. A large number of CART-ir neurons colocalized with OT in the SON. Curiously, very few CART-ir cells colocalized with OT in the PVN (Fig. 5A). In contrast to OT, a small number of AVP fibers contained CART-ir in the posterior pituitary (Fig. 5B). Only a small percentage of magnocellular neurons in the SON stained for both AVP and CART. CART-ir was not detected in AVP neurons in the magnocellular division of the PVN (Fig. 5B). Similar distribution and localization patterns were observed in both the posterior pituitary and hypothalamus of male and female rats.
Effects of bromocriptine on CART mRNA expression and protein release from anterior pituitary
The effects of bromocriptine, a potent dopamine (DA) receptor agonist, on CART mRNA expression and protein release were examined to determine whether CART and PRL are regulated by similar mechanisms in the anterior pituitary. Anterior pituitary cultures were treated with varying concentrations of bromocriptine (0.1–100 nM) or forskolin (1 μM) for 3, 6, and 12 h at 37 C. CART mRNA expression levels were determined by quantitative real-time PCR and normalized to the housekeeping gene GAPDH. A significant dose-dependent reduction in CART mRNA expression was observed after 12-h incubation with bromocriptine (Fig. 6A). No significant changes in CART mRNA expression were observed in cultures incubated with bromocriptine for 3 and 6 h (data not shown). As expected, forskolin, an activator of adenyl cyclase, significantly increased CART mRNA levels in anterior pituitary cells (Fig. 6A).
Bromocriptine had significant effects on CART protein release from anterior pituitary cultures. The accumulation of released CART was readily detectable in culture medium by RIA analysis. The release of CART into the culture medium was significantly decreased after 3 h of incubation with bromocriptine. Specifically, the concentration of CART was 200 ± 18 pg/ml for controls, 77 ± 10 pg/ml for 0.1 nM bromocriptine, 67 ± 6 pg/ml for 10 nM bromocriptine, and 61 ± 6 pg/ml for 100 nM bromocriptine (Fig. 6B). Bromocriptine had similar effects on CART release after 6- and 12-h incubations of anterior pituitary cells in culture (data not shown). Similar to mRNA expression, CART protein release was significantly increased after incubation with 1 μM forskolin (Fig. 6B).
Regulation of CART during lactation
Our findings revealed that CART is colocalized with PRL and OT, important regulators of lactation (24), and suggested that CART may potentially function to regulate lactation. To investigate this hypothesis, we examined the effects of lactation on CART mRNA expression in the hypothalamus and anterior pituitary. The levels of CART mRNA expression in the SON and PVN of lactating (d 1) and nonlactating female rats (diestrous controls) were determined by in situ hybridization. CART mRNA was localized in magnocellular neurons throughout the SON and in both the parvocellular and magnocellular divisions of the PVN (Fig. 7A). Density values were determined by image analysis, and levels of CART mRNA were increased 2-fold in the SON of lactating rats (Fig. 7B). Interestingly, no significant changes in CART mRNA expression were observed in either division of the PVN in lactating rats compared with nonlactating controls (Fig. 7B). The effects of lactation on CART mRNA expression in the anterior pituitary were determined by quantitative real-time PCR. Levels of CART mRNA were increased by approximately 5-fold in the anterior pituitary of lactating rats compared with nonlactating controls (Fig. 7C).
Serum CART was measured by RIA during pregnancy and lactation in rats to determine whether circulating CART levels correlated with changes in mRNA expression in the SON and anterior pituitary. Blood was serially collected by tail bleed between 0800–0900 h to avoid confounds with the diurnal pattern of CART in the circulation of Sprague Dawley rats (25). Serum CART remained constant throughout pregnancy, with a mean value of 116 ± 4 pg/ml (Fig. 7D). Immunoreactive CART levels were significantly elevated by d 2 of lactation (162 ± 14 pg/ml), and peak levels were detected on d 10 of lactation (255 ± 14 pg/ml). Levels of circulating CART returned to baseline values (150 ± 11 pg/ml) 10 d after pups were weaned (Fig. 7D).
Discussion
The anatomical distribution and specific cell types that express CART have been extensively studied in the rat CNS (10, 11, 13, 14, 15, 16). Findings from anatomical studies have aided in elucidation of the physiological roles of CART in the CNS (3, 6, 7, 26, 27, 28). In contrast, the anatomical localization of CART in the pituitary gland remains ill defined. The cellular localization of CART in the pituitary has varied in published reports, and physiological roles for pituitary CART have yet to be determined (11, 18, 19). In the present study, antiserum we generated against CART-(62–102) was used to examine the cellular localization of CART. The affinity and specificity of this antiserum were determined using IHC, immunoblotting, and size-exclusion chromatography. Analysis of the rat brain revealed that anti-CART-(62–102) stained multiple regions previously reported to contain CART mRNA and protein (10, 14, 29, 30, 31). Anti-CART-(62–102) and a commercially available antiserum against CART-(55–102) recognized similar proteins of approximately 5 and 8 kDa in pituitary extracts. These findings are in agreement with previous studies that reported that CART-ir peptides of a similar size are found in pituitary lysates (8, 18). Furthermore, anti-CART-(62–102) had strong immunoreactivity with a single species in rat serum with a molecular weight that corresponds to the biologically active form of CART (1). These findings demonstrate that the anti-CART-(62–102) we produced has high affinity and selectivity for endogenous rat CART.
An RIA was developed to determine the levels of CART protein in tissues and biological fluids. The CART RIA is highly sensitive, and both synthetic CART-(62–102) and CART-(55–102) displaced tracer bound to antibody in a similar manner. The relative concentration and distribution of CART protein observed in the CNS and peripheral tissues correlated well with findings from previous reports (1, 8, 19, 32).
In the anterior pituitary, the vast majority CART-ir cells contained PRL, and a small population expressed GH and ACTH. These findings support previous studies that demonstrated that high levels of CART mRNA are found in GH3 cells, a PRL- and GH-secreting cell line derived from a rat anterior pituitary tumor (33, 34, 35). Our results expand upon findings by Stanley et al. (19), who reported that 28% of CART-expressing cells contained ACTH-ir, but did not identify other anterior pituitary cells that expressed CART mRNA. It should be noted that we observed a smaller percentage of CART-ir cells expressing ACTH. Differences in the percentage of anterior pituitary cells that colocalized CART and ACTH can be attributed to the sensitivity of the detection methods used in these studies. IHC was used to detect CART in our studies, whereas Stanley et al. (19) used in situ hybridization to examine CART distribution in the anterior pituitary gland. In contrast, Kuriyama et al. (18) reported a divergent pattern of cellular localization for CART in the anterior pituitary. They found that CART colocalized with FSH- and LH-containing cells, but not GH-, TSH-, ACTH-, or PRL-containing cells. The discrepancy in the pattern of colocalization observed in our study and the report from Kuriyama et al. (18) may stem from different affinities and epitope recognition sites of the particular antibodies used in each study. Koylu et al. (11) also reported multiple antiserum-dependent staining patterns for CART in the pituitary gland.
Axons of magnocellular neurons located in the SON and PVN form the supraopticohypophyseal tract, which terminates in the posterior lobe of the pituitary gland (36). Immunohistochemical analysis revealed that anti-CART-(62–102) stained a large number of supraopticohypophyseal axon fibers in the posterior pituitary that contained OT. In the SON, CART-ir neurons colocalized with OT. In agreement with Larsen et al. (37), we observed little CART-ir in OT neurons in the PVN, and CART was not detected in AVP neurons. Similar patterns of colocalization for CART in magnocellular neurons have previously been reported (14, 37); however, we are the first to compare the localization of CART with OT and AVP in fibers of the posterior pituitary.
Anatomical studies revealed that the majority of anterior pituitary CART was localized to lactotropes. PRL is secreted from pituitary lactotropes in an episodic manner (38) under the tonic inhibition of DA released from tuberoinfundibular neurons in the hypothalamus (39, 40). DA binds D2-type receptors on lactotropes, resulting in a decrease in adenylate cyclase activity and a subsequent decrease in PRL mRNA transcription and protein release (41). We investigated the effects of DA receptor activation on CART mRNA transcription and protein release to determine the physiological significance of the observed cellular localization in the anterior pituitary. A significant inhibition of CART mRNA expression and protein release was detected in anterior pituitary cells after treatment with bromocriptine. These findings demonstrate that CART and PRL may be regulated by similar mechanisms in the anterior pituitary.
Prolactin regulates a wide array of physiological processes associated with lactation (42). Oxytocin is a powerful galactokinetic hormone that is essential for the process of lactation (43). Our findings reveal that CART is colocalized with these potent lactation regulatory hormones in the pituitary gland and hypothalamus. In addition, in vitro studies demonstrate that CART mRNA expression and protein release are regulated similarly to PRL. To expand upon these findings, we examined the effects of lactation on CART mRNA expression in the anterior pituitary, SON, and PVN. Levels of CART mRNA were significantly increased in the anterior pituitary and SON of lactating rats compared with nonlactating controls. Consistent with the lack of colocalization between CART and OT, no significant changes in CART mRNA expression were observed in the PVN of lactating rats. Together, these results suggest that CART may play a role in the regulation of lactation.
Prolactin secretion is largely dependent upon levels of DA; however, PRL transcription and protein release are also regulated by feedback signals from target tissues, hypothalamic peptides, and local factors in the pituitary gland (44). TRH, OT, and vasoactive intestinal peptide have been shown to stimulate PRL release from anterior pituitary cells in vitro (45, 46, 47). Endothelin additionally exerts biphasic effects on PRL, both inhibiting and stimulating secretion in vitro (48). CART has been shown to modulate PRL release from the anterior pituitary. Kuriyama et al. (18) reported that CART suppresses PRL release from anterior pituitary cultures, and Raptis et al. (49) recently reported that CART inhibits TRH-induced PRL secretion. These studies together with findings detailed in this report suggest that pituitary CART may function as an autocrine/paracrine factor that regulates the release of PRL when levels of DA are suppressed, such as during lactation.
The endocrine control of lactation is a complex physiological process requiring the proper regulation of mammogenesis, lactogenesis, galactopoiesis, and galactokinesis (24). A wide array of hormones, growth factors, and proteins are involved in the regulation of milk production and release. Multiple factors act as circulating hormones and paracrine autocrine factors to stimulate and inhibit various phases of mammary gland development and lactation (24, 50). In addition to the hypothalamus and pituitary, CART is present in the systemic circulation at physiological levels (19, 25). We measured CART in serum from pregnant and lactating rats to determine whether levels of circulating CART correlated with changes in mRNA expression in the anterior pituitary and hypothalamus. Serum levels of CART remained stable throughout pregnancy; however, significant increases in circulating CART were detected during lactation. The findings presented in this study implicate CART as a novel candidate factor involved in the regulation of lactation. CART may potentially act as an autocrine/paracrine factor regulating PRL release from the anterior pituitary gland, or it could function as a circulating hormone involved in the process of lactation. Additional studies, including the identification of specific target tissues and a receptor for CART, are required to more clearly elucidate the physiological roles of this protein.
Acknowledgments
We thank Dr. Jozsef Gulyas for the synthesis and purification of the peptides used in these studies, Dr. Louise Bilezikjian for critical reading of the manuscript, and Sandra Guerra for assistance with preparation of this manuscript.
Footnotes
This work was supported by National Institute of Drug Abuse Grant DA-017550-02, National Institute of Diabetes and Digestive and Kidney Diseases Grant P01-DK-26741, the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation, and the Foundation for Research. W.W.V. is a senior investigator with the Foundation for Research.
S.M.S, J.M.V., C.J.D., R.E.F., C.L., and A.C. have nothing to declare. W.W.V. is a cofounder and a board member of Neurocrine Biosciences, Inc., and Acceleron Pharmaceuticals, Inc.
First Published Online December 8, 2005
Abbreviations: AVP, Vasopressin; CART, cocaine- and amphetamine-regulated transcript; CNS, central nervous system; DA, dopamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; -ir, immunoreactivity, immunoreactive; KPBS, potassium PBS; OT, oxytocin; PJ, Pit Julep; PVN, paraventricular nucleus; SSC, standard saline sulfate; SON, supraoptic nucleus.
Accepted for publication November 23, 2005.
References
Douglass J, McKinzie AA, Couceyro P 1995 PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J Neurosci 15:2471–2481
Adams LD, Gong W, Vechia SD, Hunter RG, Kuhar MJ 1999 CART: from gene to function. Brain Res 848:137–140
Kristensen P, Judge ME, Thim L, Ribel U, Christjansen KN, Wulff BS, Clausen JT, Jensen PB, Madsen OD, Vrang N, Larsen PJ, Hastrup S 1998 Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393:72–76
Lambert PD, Couceyro PR, McGirr KM, Dall Vechia SE, Smith Y, Kuhar MJ 1998 CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29:293–298
Kuhar MJ, Dall Vechia SE 1999 CART peptides: novel addiction- and feeding-related neuropeptides. Trends Neurosci 22:316–320
Kuhar MJ, Adams S, Dominguez G, Jaworski J, Balkan B 2002 CART peptides. Neuropeptides 36:1–8
Smith SM, Vaughan JM, Donaldson CJ, Rivier J, Li C, Chen A, Vale WW 2004 Cocaine- and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism. Endocrinology 145:5202–5209
Thim L, Kristensen P, Nielsen PF, Wulff BS, Clausen JT 1999 Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat. Proc Natl Acad Sci USA 96:2722–2727
Wierup N, Kuhar M, Nilsson BO, Mulder H, Ekblad E, Sundler F 2004 Cocaine- and amphetamine-regulated transcript (CART) is expressed in several islet cell types during rat development. J Histochem Cytochem 52:169–177
Elias CF, Lee CE, Kelly JF, Ahima RS, Kuhar M, Saper CB, Elmquist JK 2001 Characterization of CART neurons in the rat and human hypothalamus. J Comp Neurol 432:1–19
Koylu EO, Couceyro PR, Lambert PD, Ling NC, DeSouza EB, Kuhar MJ 1997 Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. J Neuroendocrinol 9:823–833
Ekblad E, Kuhar M, Wierup N, Sundler F 2003 Cocaine- and amphetamine-regulated transcript: distribution and function in rat gastrointestinal tract. Neurogastroenterol Motil 15:545–557
Wittmann G LZ, Lechan RM, Fekete C 2005 Origin of cocaine- and amphetamine-regulated transcript (CART)-containing axons innervating hypophysiotropic corticotropin-releasing hormone (CRH)-synthesizing neurons in the rat. Endocrinology 146:2985–2991
Vrang N, Larsen PJ, Clausen JT, Kristensen P 1999 Neurochemical characterization of hypothalamic cocaine-amphetamine-regulated transcript neurons. J Neurosci 19:RC5
Fekete C, Mihaly E, Luo LG, Kelly J, Clausen JT, Mao Q, Rand WM, Moss LG, Kuhar M, Emerson CH, Jackson IM, Lechan RM 2000 Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting. J Neurosci 20:9224–9234
Kozicz T 2003 Neurons colocalizing urocortin and cocaine and amphetamine-regulated transcript immunoreactivities are induced by acute lipopolysaccharide stress in the Edinger-Westphal nucleus in the rat. Neuroscience 116:315–320
Murphy KG, Abbott CR, Mahmoudi M, Hunter R, Gardiner JV, Rossi M, Stanley SA, Ghatei MA, Kuhar MJ, Bloom SR 2000 Quantification and synthesis of cocaine- and amphetamine-regulated transcript peptide (79–102)-like immunoreactivity and mRNA in rat tissues. J Endocrinol 166:659–668
Kuriyama G, Takekoshi S, Tojo K, Nakai Y, Kuhar MJ, Osamura RY 2004 CART Peptide in the rat anterior pituitary gland is localized in gonadotrophs and suppresses prolactin secretion. Endocrinology 145:2542–2550
Stanley SA, Murphy KG, Bewick GA, Kong WM, Opacka-Juffry J, Gardiner JV, Ghatei M, Small CJ, Bloom SR 2004 Regulation of rat pituitary cocaine- and amphetamine-regulated transcript (CART) by CRH and glucocorticoids. Am J Physiol 287:E583–E590
Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Voglmayr JK, Bardin CW, Rivier C, Vale W 1989 Detection and purification of inhibin using antisera generated against synthetic peptide fragments. Methods Enzymol 168:588–617
Gulyas J, Rivier C, Perrin M, Koerber SC, Sutton S, Corrigan A, Lahrichi SL, Craig AG, Vale W, Rivier J 1995 Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor. Proc Natl Acad Sci USA 92:10575–105759
Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton D, Rivier C, Rivier J 1983 Assay of corticotropin releasing factor. Methods Enzymol 103:565–577
Kageyama K, Li C, Vale WW 2003 Corticotropin-releasing factor receptor type 2 messenger ribonucleic acid in rat pituitary: localization and regulation by immune challenge, restraint stress, and glucocorticoids. Endocrinology 144:1524–1532
Buhimschi CS 2004 Endocrinology of lactation. Obstet Gynecol Clin North Am 31:963–979
Vicentic A, Dominguez G, Hunter RG, Philpot K, Wilson M, Kuhar MJ 2004 Cocaine- and amphetamine-regulated transcript peptide levels in blood exhibit a diurnal rhythm: regulation by glucocorticoids. Endocrinology 145:4119–4124
Vrang N, Larsen PJ, Kristensen P, Tang-Christensen M 2000 Central administration of cocaine-amphetamine-regulated transcript activates hypothalamic neuroendocrine neurons in the rat. Endocrinology 141:794–801
Stanley SA, Small CJ, Murphy KG, Rayes E, Abbott CR, Seal LJ, Morgan DG, Sunter D, Dakin CL, Kim MS, Hunter R, Kuhar M, Ghatei MA, Bloom SR 2001 Actions of cocaine- and amphetamine-regulated transcript (CART) peptide on regulation of appetite and hypothalamo-pituitary axes in vitro and in vivo in male rats. Brain Res 893:186–194
Vrang N, Larsen PJ, Tang-Christensen M, Larsen LK, Kristensen P 2003 Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids. Brain Res 965:45–50
Dallvechia-Adams S, Smith Y, Kuhar MJ 2001 CART peptide-immunoreactive projection from the nucleus accumbens targets substantia nigra pars reticulata neurons in the rat. J Comp Neurol 434:29–39
Koylu EO, Couceyro PR, Lambert PD, Kuhar MJ 1998 Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J Comp Neurol 391:115–132
Fekete C, Wittmann G, Liposits Z, Lechan RM 2004 Origin of cocaine- and amphetamine-regulated transcript (CART)-immunoreactive innervation of the hypothalamic paraventricular nucleus. J Comp Neurol 469:340–350
Kuhar MJ, Yoho LL 1999 CART peptide analysis by Western blotting. Synapse 33:163–171
Barrett P, Morris MA, Moar KM, Mercer JG, Davidson JA, Findlay PA, Adam CL, Morgan PJ 2001 The differential regulation of CART gene expression in a pituitary cell line and primary cell cultures of ovine pars tuberalis cells. J Neuroendocrinol 13:347–352
Dominguez G, Lakatos A, Kuhar MJ 2002 Characterization of the cocaine- and amphetamine-regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP-dependent signaling pathway in GH3 cells. J Neurochem 80:885–893
Tashjian Jr AH, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352
Vandesande F, Dierickx K 1975 Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat. Cell Tissue Res 164:153–162
Larsen PJ, Seier V, Fink-Jensen A, Holst JJ, Warberg J, Vrang N 2003 Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit. J Neuroendocrinol 15:219–226
Meites J 1977 Neuroendocrine control of prolactin in experimental animals. Clin Endocrinol (Oxf) 6(Suppl):9S–18S
MacLeod RM, Lehmeyer JE 1974 Studies on the mechanism of the dopamine-mediated inhibition of prolactin secretion. Endocrinology 94:1077–1085
Ruberg M, Enjalbert A, Arancibia S, Kordon C 1981 Regulation of prolactin secretion at the pituitary level. Exp Brain Res(Suppl 3):182–199
Albert PR, Neve KA, Bunzow JR, Civelli O 1990 Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion. J Biol Chem 265:2098–2104
Jaffe RB, Yuen BH, Keye Jr WR, Midgley Jr AR 1973 Physiologic and pathologic profiles of circulating human prolactin. Am J Obstet Gynecol 117:757–773
Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM 1996 Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci USA 93:11699–11704
Samson WK, Taylor MM, Baker JR 2003 Prolactin-releasing peptides. Regul Pept 114:1–5
Samson WK, Said SI, Snyder G, McCann SM 1980 In vitro stimulation of prolactin release by vasoactive intestinal peptide. Peptides 1:325–332
Samson WK, Lumpkin MD, McCann SM 1986 Evidence for a physiological role for oxytocin in the control of prolactin secretion. Endocrinology 119:554–560
Lamberts SW, Macleod RM 1990 Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 70:279–318
Kanyicska B, Sellix MT, Freeman ME 2001 Autocrine regulation of prolactin secretion by endothelins: a permissive role for estradiol. Endocrine 16:133–137
Raptis S, Fekete C, Sarkar S, Rand WM, Emerson CH, Nagy GM, Lechan RM 2004 Cocaine- and amphetamine-regulated transcript co-contained in thyrotropin-releasing hormone (TRH) neurons of the hypothalamic paraventricular nucleus modulates TRH-induced prolactin secretion. Endocrinology 145:1695–1699
Lamote I, Meyer E, Massart-Leen AM, Burvenich C 2004 Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution. Steroids 69:145–159(Sean M. Smith, Joan M. Vaughan, Cynthia )
Abstract
Cocaine- and amphetamine-regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART-producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing a high-affinity antiserum we generated against CART-(62–102), was used to identify CART-producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (-ir) was localized in lactotropes; minor populations of CART-ir cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin-containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocriptine, a potent dopamine receptor agonist, were examined to determine whether CART mRNA expression and protein release are regulated in a similar fashion as prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased after bromocriptine treatment of dispersed rat anterior pituitary cells in culture. To explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and nonlactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked on d 10 of lactation and returned to baseline values 10 d after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.
Introduction
COCAINE- AND AMPHETAMINE-regulated transcript (CART) was originally identified by differential display PCR as an mRNA transcript that was up-regulated in the striatum of rats after acute administration of cocaine and amphetamine (1, 2). Since this initial discovery, CART has been implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors (3, 4, 5, 6, 7). CART is a highly expressed peptide that is widely distributed throughout the central nervous system (CNS), gut, pituitary, adrenal, and pancreas (5, 8, 9). In the brain, CART immunoreactivity (-ir) has been localized in cell bodies, fibers, and varicosities in the olfactory bulb, cortex, hypothalamus, mesencephalon, and brainstem (10, 11). CART-ir fibers are in close opposition to fenestrated capillaries in the external zone of the median eminence, and CART protein is found in the anterior and posterior pituitary (11). In the periphery, CART is localized in pancreatic islets (9), enteric neurons in the gut (12), and the adrenal medulla (11).
The cellular localization of CART has been extensively studied in the rat CNS. In the medulla, CART is colocalized with adrenergic and -MSH-expressing neurons (13). CART is found in oxytocin (OT)- and vasopressin (AVP)-containing neurons in the magnocellular paraventricular nucleus (PVN), TRH-containing cells are found in the parvocellular PVN, and CART-ir cells colocalize with proopiomelanocortin in the arcuate nucleus (10, 14, 15). CART-ir has also been shown to colocalize with urocortin 1 in cells of the Edinger Westphal nucleus (16).
In contrast, the cellular localization of pituitary CART has not been clearly defined. CART mRNA is expressed in the anterior pituitary; however, CART mRNA expression has not been reported in the intermediate and posterior pituitary gland (17). Immunohistochemistry (IHC) studies revealed a varied distribution of CART in the anterior pituitary gland. Koylu et al. (11) reported that the location and intensity of CART-ir in the anterior pituitary varied dependent on the antiserum used. Kuriyama et al. (18) recently demonstrated that CART-ir is localized in gonadotropes, whereas Stanely et al. (19) reported that CART was expressed in corticotropes. In the posterior pituitary, CART-ir is localized in a high density of varicose fibers, and CART-ir has not been detected in the intermediate lobe of the pituitary gland (11).
In the present study we generated antiserum against CART-(62–102) to clarify the anatomical distribution of CART in the pituitary gland. A sensitive RIA was developed, and real-time PCR was employed to identify factors that regulate CART mRNA transcription and protein release from anterior pituitary cells. In addition, we compared levels of CART in the hypothalamus, pituitary, and systemic circulation of lactating and nonlactating rats to explore the physiological roles of pituitary CART.
Materials and Methods
Animals and tissue preparation
Adult male and female Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN), were used in the experiments described in this study. Animals were maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) and provided rat chow (Harlan-Teklad, Madison, WI) and water ad libitum. Animals were anesthetized with an overdose of choral hydrate (1 g/kg body weight, ip) and perfused transcardially with 150 ml saline, followed by 350 ml 4% paraformaldehyde in borate buffer (pH 9.5). After perfusion, brains and whole pituitaries were removed and postfixed in 25% sucrose in the same fixative at 4 C overnight. Tissues were then quickly frozen on dry ice, sectioned at 25 μm using a sliding microtome, and stored in cryoprotectant at –20 C until use. The Salk Institute animal use and care committee approved all procedures described in this study.
Blood sampling
Blood samples were obtained from pregnant and lactating female Sprague Dawley rats by tail bleed. Approximately 0.3 ml blood was taken from each animal between 0800–0900 h on d 5, 12, and 18 of pregnancy and d 2, 10, 17, and 21 of lactation. Control bleeds were taken 10 d before mating and 10 d after weaning to determine baseline levels of circulating CART. Blood samples were drawn into chilled tubes containing EDTA and centrifuged, and plasma was stored at –20 C until RIA analysis.
Antiserum and controls
Antiserum was raised in rabbits immunized with a synthetic peptide encoding rat [Nle67]CART-(62–102) conjugated to human -globulins via bisdiazotized benzidine using a protocol previously described in detail for inhibin subunits (20). In all experiments, antiserum was used in an unpurified form at the indicated dilutions. The specificity of immunostaining was evaluated using primary antiserum preabsorbed overnight at 4 C with 0–300 μM synthetic rat CART-(55–102) or CART-(62–102) before incubation with brain sections. CART-(55–102) and CART-(62–102) were synthesized in this laboratory by solid phase methodologies (21).
RIA
The analog [Nle67]CART-(62–102) was radiolabeled with 125I using chloramine T and purified by HPLC with a 0.1% trifluoroacetic acid-acetonitrile solvent system for use as a tracer in the RIA. The procedure for the CART RIA was similar to that described in detail for inhibin subunits (20). Briefly, rabbit 6838 anti-CART-(62–102) serum was used at a 1:150,000 final dilution, and synthetic CART-(55–102) and CART-(62–102) were used as standards. Rat tissues were acid extracted and partially purified with octadecyl silica cartridges as previously described (20). Lyophilized tissue extracts were reconstituted in assay buffer, pH was checked and adjusted if necessary, and three to seven dose levels were tested. Cell secretion medium was tested without dilution at multiple doses. Free tracer was separated from tracer bound to antibody with the addition of sheep antirabbit -globulins and 10% (wt/vol) polyethylene glycol. The minimum detectable dose and EC50 for CART-(55–102) ranged from 2–3 and 50–60 pg/tube, respectively. Results were calculated using a logit/log RIA data processing program of Faden, Hutson, Munson, and Rodbard (National Institute of Child Health and Human Development, Reproductive Research Branch, National Institutes of Health).
Gel filtration chromatography
Pooled male and female rat plasma was partially purified using octadecyl silica cartridges as previously described (20). The purified plasma was applied to a fast protein liquid chromatography system equipped with a Superdex 75 HR 10/30 column (Amersham Biosciences Corp., Piscataway, NJ). The column was eluted with 30% acetonitrile/0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. Aliquots of fractions were lyophilized and tested in the CART RIA. In separate runs, the column was calibrated using various peptide and protein standards.
Immunoblotting
Rat pituitary glands were acid extracted and partially purified with octadecyl silica cartridges as previously described (20). Lyophilized proteins were resuspended in 1x sample buffer [50 mM Tris (pH 6.8), 100 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, and 10% glycerol]. Samples were boiled for 5 min, and proteins were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel (Invitrogen Life Technologies, Inc., Carlsbad, CA). Electrophoresed proteins were subsequently transferred onto nitrocellulose membranes and then probed with anti-CART-(62–102) (PBL 6838) or anti-CART-(55–102) (Phoenix Pharmaceuticals, Belmont, CA) overnight at 4 C. Membranes were washed in PBS with 0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated antirabbit IgG (Amersham Biosciences, Little Chalfont, UK). Immunoreactive proteins were visualized using Super Signal West Pico chemiluminescent substrate (Pierce Chemical Co., Rockford, IL).
IHC
Brain and whole pituitary sections were incubated in one or a combination of the following antisera: rabbit anti-CART-(62–102) (1:10,000; PBL 6838); guinea pig anti-PRL, -GH, -FSH, -TSH, and -ACTH (1:12,000; National Institutes of Health, Bethesda, MD); guinea pig anti-AVP (1:15,000; Peninsula Laboratories, San Carlos, CA) or mouse anti-OT (1:7,500; Chemicon International, Temecula, CA) in potassium PBS (KPBS) with 0.4% Triton X-100 at 4 C for 48 h. The tissues were then rinsed in KPBS and incubated in one or a combination of the following secondary antibodies: biotinylated donkey antirabbit IgG (1:700; Jackson ImmunoResearch Laboratories, West Grove, PA), fluorescein-conjugated donkey antirabbit IgG, rhodamine red X-conjugated antimouse IgG, or Cy3-conjugated antiguinea pig IgG (1:500; Jackson ImmunoResearch Laboratories) in KPBS with 0.4% Triton X-100 for 1 h at room temperature. Tissues incubated with biotinylated secondary antibodies were subjected to 1-h incubation at room temperature in avidin-biotin complex solution (Vectastain ABC Elite kit, Vector Laboratories, Inc., Burlingame, CA). The antibody-peroxidase complex was visualized with 3,3-diaminobenzidine (0.2 mg/ml) and 3% H2O2 (0.83 μl/ml) in 175 mM sodium acetate solution. When staining had reached an appropriate intensity, the tissue was rinsed in KPBS and mounted on gelatin-coated glass slides. Slides were dehydrated through graded alcohols, cleared in xylenes, and coverslipped with DPX mountant (Electronic Microscope Science, Fort Washington, PA). Adjacent series of brain sections were processed for Nissl staining for reference purposes.
Rat anterior pituitary cell cultures
Anterior pituitary cells from male Sprague Dawley rats were prepared by dispersion with collagenase as previously described (22). Cultures were plated on poly-L-lysine-coated coverslips in 24-well culture plates for immunocytochemical analysis or in 48-well plates for protein expression and release studies. Cultures were plated at a density of 1.5 x 105 cells/culture well and were allowed to recover for 72 h in complete medium [Pit Julep (PJ)] supplemented with 2% fetal bovine serum and appropriate growth factors (22).
Before immunocytochemical analysis, pituitary cells in culture were rinsed with KPBS and then fixed for 20 min with 4% paraformaldehyde at room temperature. Cultures were washed in KPBS and processed for CART, GH, TSH, FSH, ACTH, and PRL-ir following the procedures described above for brain and pituitary sections. Before the examination of CART expression and protein release, pituitary cells were washed three times with PJ and 0.1% BSA and equilibrated for 1 h. Cells were then washed with PJ and 0.1% BSA and treated in triplicate with bromocriptine (0.1–100 nM) or forskolin (1 μM) for 3, 6, and 12 h at 37 C. Media were collected after each incubation time point for measurement of secreted CART by RIA. Cells were harvested, and mRNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Inc.) to determine CART mRNA levels.
In situ hybridization
Animals were rapidly decapitated, and whole brains were removed and frozen on dry ice. Coronal sections (20 μm) were cut on a cryostat, thaw-mounted onto glass slides, and stored at –80 C until use. Antisense cRNA probes were transcribed from linearized cDNA templates corresponding to bp 495–679 of the rat CART gene. [35S]UTP (PerkinElmer Life Sciences, Emeryville, CA) was incorporated into cRNA probes during transcription, and the specific activity of the probes was determined to be approximately 2 x 108 dpm/μg cRNA. The saturating concentration for the probes used for assays was 0.3 μg/ml/kb.
The procedure used for in situ hybridization was described previously (23). Briefly, brain sections were fixed in 4% paraformaldehyde and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2% standard saline sulfate (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air dried. The slides were then exposed to cRNA probes overnight in humidified chambers at 55 C. After incubation, the slides were washed in SSC of increasing stringency, in ribonuclease, and then in 0.1% SSC at 63 C; dehydrated through a graded series of alcohols; and dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed from 7–14 d at 4 C, and developed. After development, the slides were counterstained with cresyl violet.
Tissue analysis
Tissue sections and pituitary cultures were initially examined using an E600 light microscope (Nikon, Tokyo, Japan). The cellular localization of CART with pituitary hormones and hypothalamic neuropeptides was determined using a Fluoview confocal system on an IX70 microscope (Olympus, Melville, NY) with the following laser excitation lines: 488 nm for fluorescein and 568 nm for rhodamine red X and Cy3. Dichroic/emission filters for detection were 500 nm LP/515–565nm for fluorescein, and 575 nm LP/590 nm LP for rhodamine red X and Cy3. These filter combinations resulted in very low levels of cross-talk between fluorochrome signals.
The extent of colocalization between CART and pituitary hormones was ascertained from six separate pituitary cultures stained for CART and individual pituitary hormones. The total numbers of cells containing 1) CART-ir, 2) the pituitary hormone of interest, and 3) colocalization of CART-ir and a specific hormone were determined for each culture. The results are presented as the percentage of CART-ir cells that contain individual pituitary hormones.
Autoradiograms for CART in situ hybridization were visualized under dark-field illumination using a E600 light microscope (Nikon, Tokyo, Japan) and analyzed with Image-Pro Plus imaging software (Media Cybernetics, San Diego, CA). Integrated density values of hybridized neurons of each side of the supraoptic nucleus (SON) and PVN were measured in at least five consecutive sections for each animal. Nonlinearity of radioactivity in the emulsion was evaluated by comparing density values with a calibration curve created from autoradiograms of known dilutions of the radiolabeled probes immobilized on glass slides in 2% gelatin fixed with 4% paraformaldehyde and exposed and developed simultaneously with the in situ hybridization autoradiograms.
All images were captured using a digital camera (Photometrics, Huntington Beach, CA) and Image-Pro Plus imaging software (Media Cybernetics, San Diego, CA). The images were cropped and adjusted to balance brightness and contrast in Adobe Photoshop (version 5.5; Adobe Systems, San Jose, CA) before import into Canvas (version 6.0) for assembly into plates.
RT and real-time PCR analysis
CART mRNA levels in anterior pituitary cells were quantified using real-time PCR analysis. After deoxyribonuclease treatment, a constant amount of RNA (1 μg) was added to a reverse transcriptase mixture (SuperScript II RNase H-Reverse Transcriptase, Invitrogen Life Technologies, Inc.). To test for possible pseudo gene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. Levels of CART expression were determined with primers for CART (sense, 5'-GTAAACGCATTCCGATCTATGAGA-3'; antisense, 5'-CCGATCCTGGCCCCTTT) and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense, 5'-GGAAGGGCTCATGACCACAGT-3'; antisense, 5'-CACAGTCTTCTGAGTGGCAGTGAT-3'). Reaction mixtures contained TaqMan Universal Master Mix with SYBR Green (Sigma-Aldrich Corp., St. Louis, MO), 80 nM primers, and 1 ng cDNA in a final reaction volume of 50 μl. PCRs were performed in 96-well plates on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) with sequence detection system software. For each biological sample, real-time PCRs were performed in duplicate, and CART expression was normalized to GAPDH.
Statistical analysis
Statistical analyses were performed using unpaired Student’s t tests and one-way ANOVAs as indicated for each study. Tukey analysis tests were used after ANOVA to make comparisons between groups at a particular time point and between time points within a particular group. Differences were considered statistically significant at P < 0.05.
Results
CART-ir in rat brain and peripheral tissues
We generated antiserum against a synthetic peptide encoding rat CART-(62–102), which allowed us to determine the cellular localization of CART in the pituitary gland. The affinity and specificity of the antiserum were examined by IHC, immunoblotting, and size-exclusion chromatography. Immunohistochemical analysis revealed that anti-CART-(62–102) stained cell bodies and fibers in multiple regions of the rat brain, including the olfactory bulb, nucleus accumbens, amygdala, thalamus, hypothalamus, and several nuclei in the mesencephalon and brainstem. Preabsorption of anti-CART-(62–102) with 20 μg/ml CART-(55–102) or CART-(62–102) completely eliminated CART staining in the SON and Edinger Westphal nucleus, demonstrating the specificity of anti-CART-(62–102) (Fig. 1).
A RIA was developed using CART-(62–102) antiserum to determine the levels of CART protein in tissues and biological fluids. The CART RIA is highly sensitive, with a detection limit of 2–3 pg/tube. As shown in Fig. 2A, both synthetic CART-(62–102) (EC50, 30 pg/tube) and CART-(55–102) (EC50, 50 pg/tube) displaced tracer bound to antibody in a similar manner. CART-like ir was measured in acid-extracted rat tissues partially purified using octadecyl silica cartridges. The highest concentrations of CART were detected in the posterior pituitary and hypothalamus, moderate levels were found in the cerebellum and pancreas, and low levels were observed in the heart and spleen. The approximate concentrations of CART per milligram of partially purified tissue extract were as follows: 2.8 μg/mg posterior pituitary, 950 ng/mg hypothalamus, 35 ng/mg cerebellum, 7 ng/mg pancreas, 0.7 ng/mg heart, and 0.08 ng/mg spleen (Fig. 2A).
To examine the specificity of anti-CART-(62–102), we employed gel exclusion chromatography to determine the molecular mass of CART-ir products in rat plasma. As shown in Fig. 2B, anti-CART-(62–102) recognized a single immunoreactive species of approximately 5.8 kDa. This is in agreement with the predicted molecular mass of that reported for biologically active CART-(55–102).
Cellular localization of CART-ir in rat anterior pituitary gland
Immunoblot analysis was used to verify that CART peptides were present in the rat pituitary gland. Partially purified protein extracts from whole rat pituitaries were probed with antiserum we generated against CART-(62–102) and commercially available antiserum against CART-(55–102) used by other researchers (18). Both antisera detected bands of approximately 5 and 8 kDa in pituitary extracts and had strong immunoreactivity with synthetic CART-(55–102) (Fig. 3A).
IHC was employed to examine CART protein distribution in whole pituitary sections from adult male rats. Moderate levels of CART-ir were detected throughout the anterior pituitary. The majority of CART-ir was distributed around the lateral edge of the anterior lobe (Fig. 3B). Preabsorption of anti-CART-(62–102) with 20 μg/ml CART-(55–102) completely eliminated CART staining in the pituitary, confirming the specificity of the antiserum (Fig. 3D).
The cellular localization of CART in the anterior pituitary was determined by double-label IHC using rabbit anti-CART-(62–102) together with antisera raised in guinea pigs against pituitary hormones (ACTH, FSH, GH, PRL, and TSH) in dispersed rat anterior pituitary cells in culture. The majority of CART-ir was localized in lactotropes, with minor populations of CART-ir cells identified as somatotropes and corticotropes (Fig. 4A). As summarized in Fig. 4B, we determined that 82% of CART-immunopositive cells also contained PRL, 10% of cells contained GH, and 5% of CART-ir cells expressed ACTH. Less than 1% of CART-ir cells contained TSH or FSH (Fig. 4B). The percentage of specific anterior pituitary cell types that contained CART-ir was also determined; this revealed that 25% of lactotropes, 5% of somatotropes, and 3% of corticotropes contained CART-ir (Fig. 4C). To ensure that the observed cellular localization of CART-ir in cultured anterior pituitary cells was representative of the intact pituitary, we determined the cellular localization of CART-ir in whole pituitary sections and dispersed anterior pituitary cells from female rats. In all preparations, similar distribution and localization patterns were observed.
Distribution of CART-ir in posterior pituitary and hypothalamus
Strong immunostaining for CART was detected in tightly packed varicose fibers throughout the posterior lobe of the pituitary, and CART-ir was not detected in the intermediate lobe of the pituitary (Fig. 3C). Double-label IHC analysis of whole pituitary sections revealed that a large population of OT fibers colocalized CART-ir in the posterior pituitary (Fig. 5A). We next examined the distribution of CART-ir in magnocellular neurons of the SON and PVN that contribute axons to the posterior pituitary. A large number of CART-ir neurons colocalized with OT in the SON. Curiously, very few CART-ir cells colocalized with OT in the PVN (Fig. 5A). In contrast to OT, a small number of AVP fibers contained CART-ir in the posterior pituitary (Fig. 5B). Only a small percentage of magnocellular neurons in the SON stained for both AVP and CART. CART-ir was not detected in AVP neurons in the magnocellular division of the PVN (Fig. 5B). Similar distribution and localization patterns were observed in both the posterior pituitary and hypothalamus of male and female rats.
Effects of bromocriptine on CART mRNA expression and protein release from anterior pituitary
The effects of bromocriptine, a potent dopamine (DA) receptor agonist, on CART mRNA expression and protein release were examined to determine whether CART and PRL are regulated by similar mechanisms in the anterior pituitary. Anterior pituitary cultures were treated with varying concentrations of bromocriptine (0.1–100 nM) or forskolin (1 μM) for 3, 6, and 12 h at 37 C. CART mRNA expression levels were determined by quantitative real-time PCR and normalized to the housekeeping gene GAPDH. A significant dose-dependent reduction in CART mRNA expression was observed after 12-h incubation with bromocriptine (Fig. 6A). No significant changes in CART mRNA expression were observed in cultures incubated with bromocriptine for 3 and 6 h (data not shown). As expected, forskolin, an activator of adenyl cyclase, significantly increased CART mRNA levels in anterior pituitary cells (Fig. 6A).
Bromocriptine had significant effects on CART protein release from anterior pituitary cultures. The accumulation of released CART was readily detectable in culture medium by RIA analysis. The release of CART into the culture medium was significantly decreased after 3 h of incubation with bromocriptine. Specifically, the concentration of CART was 200 ± 18 pg/ml for controls, 77 ± 10 pg/ml for 0.1 nM bromocriptine, 67 ± 6 pg/ml for 10 nM bromocriptine, and 61 ± 6 pg/ml for 100 nM bromocriptine (Fig. 6B). Bromocriptine had similar effects on CART release after 6- and 12-h incubations of anterior pituitary cells in culture (data not shown). Similar to mRNA expression, CART protein release was significantly increased after incubation with 1 μM forskolin (Fig. 6B).
Regulation of CART during lactation
Our findings revealed that CART is colocalized with PRL and OT, important regulators of lactation (24), and suggested that CART may potentially function to regulate lactation. To investigate this hypothesis, we examined the effects of lactation on CART mRNA expression in the hypothalamus and anterior pituitary. The levels of CART mRNA expression in the SON and PVN of lactating (d 1) and nonlactating female rats (diestrous controls) were determined by in situ hybridization. CART mRNA was localized in magnocellular neurons throughout the SON and in both the parvocellular and magnocellular divisions of the PVN (Fig. 7A). Density values were determined by image analysis, and levels of CART mRNA were increased 2-fold in the SON of lactating rats (Fig. 7B). Interestingly, no significant changes in CART mRNA expression were observed in either division of the PVN in lactating rats compared with nonlactating controls (Fig. 7B). The effects of lactation on CART mRNA expression in the anterior pituitary were determined by quantitative real-time PCR. Levels of CART mRNA were increased by approximately 5-fold in the anterior pituitary of lactating rats compared with nonlactating controls (Fig. 7C).
Serum CART was measured by RIA during pregnancy and lactation in rats to determine whether circulating CART levels correlated with changes in mRNA expression in the SON and anterior pituitary. Blood was serially collected by tail bleed between 0800–0900 h to avoid confounds with the diurnal pattern of CART in the circulation of Sprague Dawley rats (25). Serum CART remained constant throughout pregnancy, with a mean value of 116 ± 4 pg/ml (Fig. 7D). Immunoreactive CART levels were significantly elevated by d 2 of lactation (162 ± 14 pg/ml), and peak levels were detected on d 10 of lactation (255 ± 14 pg/ml). Levels of circulating CART returned to baseline values (150 ± 11 pg/ml) 10 d after pups were weaned (Fig. 7D).
Discussion
The anatomical distribution and specific cell types that express CART have been extensively studied in the rat CNS (10, 11, 13, 14, 15, 16). Findings from anatomical studies have aided in elucidation of the physiological roles of CART in the CNS (3, 6, 7, 26, 27, 28). In contrast, the anatomical localization of CART in the pituitary gland remains ill defined. The cellular localization of CART in the pituitary has varied in published reports, and physiological roles for pituitary CART have yet to be determined (11, 18, 19). In the present study, antiserum we generated against CART-(62–102) was used to examine the cellular localization of CART. The affinity and specificity of this antiserum were determined using IHC, immunoblotting, and size-exclusion chromatography. Analysis of the rat brain revealed that anti-CART-(62–102) stained multiple regions previously reported to contain CART mRNA and protein (10, 14, 29, 30, 31). Anti-CART-(62–102) and a commercially available antiserum against CART-(55–102) recognized similar proteins of approximately 5 and 8 kDa in pituitary extracts. These findings are in agreement with previous studies that reported that CART-ir peptides of a similar size are found in pituitary lysates (8, 18). Furthermore, anti-CART-(62–102) had strong immunoreactivity with a single species in rat serum with a molecular weight that corresponds to the biologically active form of CART (1). These findings demonstrate that the anti-CART-(62–102) we produced has high affinity and selectivity for endogenous rat CART.
An RIA was developed to determine the levels of CART protein in tissues and biological fluids. The CART RIA is highly sensitive, and both synthetic CART-(62–102) and CART-(55–102) displaced tracer bound to antibody in a similar manner. The relative concentration and distribution of CART protein observed in the CNS and peripheral tissues correlated well with findings from previous reports (1, 8, 19, 32).
In the anterior pituitary, the vast majority CART-ir cells contained PRL, and a small population expressed GH and ACTH. These findings support previous studies that demonstrated that high levels of CART mRNA are found in GH3 cells, a PRL- and GH-secreting cell line derived from a rat anterior pituitary tumor (33, 34, 35). Our results expand upon findings by Stanley et al. (19), who reported that 28% of CART-expressing cells contained ACTH-ir, but did not identify other anterior pituitary cells that expressed CART mRNA. It should be noted that we observed a smaller percentage of CART-ir cells expressing ACTH. Differences in the percentage of anterior pituitary cells that colocalized CART and ACTH can be attributed to the sensitivity of the detection methods used in these studies. IHC was used to detect CART in our studies, whereas Stanley et al. (19) used in situ hybridization to examine CART distribution in the anterior pituitary gland. In contrast, Kuriyama et al. (18) reported a divergent pattern of cellular localization for CART in the anterior pituitary. They found that CART colocalized with FSH- and LH-containing cells, but not GH-, TSH-, ACTH-, or PRL-containing cells. The discrepancy in the pattern of colocalization observed in our study and the report from Kuriyama et al. (18) may stem from different affinities and epitope recognition sites of the particular antibodies used in each study. Koylu et al. (11) also reported multiple antiserum-dependent staining patterns for CART in the pituitary gland.
Axons of magnocellular neurons located in the SON and PVN form the supraopticohypophyseal tract, which terminates in the posterior lobe of the pituitary gland (36). Immunohistochemical analysis revealed that anti-CART-(62–102) stained a large number of supraopticohypophyseal axon fibers in the posterior pituitary that contained OT. In the SON, CART-ir neurons colocalized with OT. In agreement with Larsen et al. (37), we observed little CART-ir in OT neurons in the PVN, and CART was not detected in AVP neurons. Similar patterns of colocalization for CART in magnocellular neurons have previously been reported (14, 37); however, we are the first to compare the localization of CART with OT and AVP in fibers of the posterior pituitary.
Anatomical studies revealed that the majority of anterior pituitary CART was localized to lactotropes. PRL is secreted from pituitary lactotropes in an episodic manner (38) under the tonic inhibition of DA released from tuberoinfundibular neurons in the hypothalamus (39, 40). DA binds D2-type receptors on lactotropes, resulting in a decrease in adenylate cyclase activity and a subsequent decrease in PRL mRNA transcription and protein release (41). We investigated the effects of DA receptor activation on CART mRNA transcription and protein release to determine the physiological significance of the observed cellular localization in the anterior pituitary. A significant inhibition of CART mRNA expression and protein release was detected in anterior pituitary cells after treatment with bromocriptine. These findings demonstrate that CART and PRL may be regulated by similar mechanisms in the anterior pituitary.
Prolactin regulates a wide array of physiological processes associated with lactation (42). Oxytocin is a powerful galactokinetic hormone that is essential for the process of lactation (43). Our findings reveal that CART is colocalized with these potent lactation regulatory hormones in the pituitary gland and hypothalamus. In addition, in vitro studies demonstrate that CART mRNA expression and protein release are regulated similarly to PRL. To expand upon these findings, we examined the effects of lactation on CART mRNA expression in the anterior pituitary, SON, and PVN. Levels of CART mRNA were significantly increased in the anterior pituitary and SON of lactating rats compared with nonlactating controls. Consistent with the lack of colocalization between CART and OT, no significant changes in CART mRNA expression were observed in the PVN of lactating rats. Together, these results suggest that CART may play a role in the regulation of lactation.
Prolactin secretion is largely dependent upon levels of DA; however, PRL transcription and protein release are also regulated by feedback signals from target tissues, hypothalamic peptides, and local factors in the pituitary gland (44). TRH, OT, and vasoactive intestinal peptide have been shown to stimulate PRL release from anterior pituitary cells in vitro (45, 46, 47). Endothelin additionally exerts biphasic effects on PRL, both inhibiting and stimulating secretion in vitro (48). CART has been shown to modulate PRL release from the anterior pituitary. Kuriyama et al. (18) reported that CART suppresses PRL release from anterior pituitary cultures, and Raptis et al. (49) recently reported that CART inhibits TRH-induced PRL secretion. These studies together with findings detailed in this report suggest that pituitary CART may function as an autocrine/paracrine factor that regulates the release of PRL when levels of DA are suppressed, such as during lactation.
The endocrine control of lactation is a complex physiological process requiring the proper regulation of mammogenesis, lactogenesis, galactopoiesis, and galactokinesis (24). A wide array of hormones, growth factors, and proteins are involved in the regulation of milk production and release. Multiple factors act as circulating hormones and paracrine autocrine factors to stimulate and inhibit various phases of mammary gland development and lactation (24, 50). In addition to the hypothalamus and pituitary, CART is present in the systemic circulation at physiological levels (19, 25). We measured CART in serum from pregnant and lactating rats to determine whether levels of circulating CART correlated with changes in mRNA expression in the anterior pituitary and hypothalamus. Serum levels of CART remained stable throughout pregnancy; however, significant increases in circulating CART were detected during lactation. The findings presented in this study implicate CART as a novel candidate factor involved in the regulation of lactation. CART may potentially act as an autocrine/paracrine factor regulating PRL release from the anterior pituitary gland, or it could function as a circulating hormone involved in the process of lactation. Additional studies, including the identification of specific target tissues and a receptor for CART, are required to more clearly elucidate the physiological roles of this protein.
Acknowledgments
We thank Dr. Jozsef Gulyas for the synthesis and purification of the peptides used in these studies, Dr. Louise Bilezikjian for critical reading of the manuscript, and Sandra Guerra for assistance with preparation of this manuscript.
Footnotes
This work was supported by National Institute of Drug Abuse Grant DA-017550-02, National Institute of Diabetes and Digestive and Kidney Diseases Grant P01-DK-26741, the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation, and the Foundation for Research. W.W.V. is a senior investigator with the Foundation for Research.
S.M.S, J.M.V., C.J.D., R.E.F., C.L., and A.C. have nothing to declare. W.W.V. is a cofounder and a board member of Neurocrine Biosciences, Inc., and Acceleron Pharmaceuticals, Inc.
First Published Online December 8, 2005
Abbreviations: AVP, Vasopressin; CART, cocaine- and amphetamine-regulated transcript; CNS, central nervous system; DA, dopamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; -ir, immunoreactivity, immunoreactive; KPBS, potassium PBS; OT, oxytocin; PJ, Pit Julep; PVN, paraventricular nucleus; SSC, standard saline sulfate; SON, supraoptic nucleus.
Accepted for publication November 23, 2005.
References
Douglass J, McKinzie AA, Couceyro P 1995 PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J Neurosci 15:2471–2481
Adams LD, Gong W, Vechia SD, Hunter RG, Kuhar MJ 1999 CART: from gene to function. Brain Res 848:137–140
Kristensen P, Judge ME, Thim L, Ribel U, Christjansen KN, Wulff BS, Clausen JT, Jensen PB, Madsen OD, Vrang N, Larsen PJ, Hastrup S 1998 Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature 393:72–76
Lambert PD, Couceyro PR, McGirr KM, Dall Vechia SE, Smith Y, Kuhar MJ 1998 CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29:293–298
Kuhar MJ, Dall Vechia SE 1999 CART peptides: novel addiction- and feeding-related neuropeptides. Trends Neurosci 22:316–320
Kuhar MJ, Adams S, Dominguez G, Jaworski J, Balkan B 2002 CART peptides. Neuropeptides 36:1–8
Smith SM, Vaughan JM, Donaldson CJ, Rivier J, Li C, Chen A, Vale WW 2004 Cocaine- and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism. Endocrinology 145:5202–5209
Thim L, Kristensen P, Nielsen PF, Wulff BS, Clausen JT 1999 Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat. Proc Natl Acad Sci USA 96:2722–2727
Wierup N, Kuhar M, Nilsson BO, Mulder H, Ekblad E, Sundler F 2004 Cocaine- and amphetamine-regulated transcript (CART) is expressed in several islet cell types during rat development. J Histochem Cytochem 52:169–177
Elias CF, Lee CE, Kelly JF, Ahima RS, Kuhar M, Saper CB, Elmquist JK 2001 Characterization of CART neurons in the rat and human hypothalamus. J Comp Neurol 432:1–19
Koylu EO, Couceyro PR, Lambert PD, Ling NC, DeSouza EB, Kuhar MJ 1997 Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. J Neuroendocrinol 9:823–833
Ekblad E, Kuhar M, Wierup N, Sundler F 2003 Cocaine- and amphetamine-regulated transcript: distribution and function in rat gastrointestinal tract. Neurogastroenterol Motil 15:545–557
Wittmann G LZ, Lechan RM, Fekete C 2005 Origin of cocaine- and amphetamine-regulated transcript (CART)-containing axons innervating hypophysiotropic corticotropin-releasing hormone (CRH)-synthesizing neurons in the rat. Endocrinology 146:2985–2991
Vrang N, Larsen PJ, Clausen JT, Kristensen P 1999 Neurochemical characterization of hypothalamic cocaine-amphetamine-regulated transcript neurons. J Neurosci 19:RC5
Fekete C, Mihaly E, Luo LG, Kelly J, Clausen JT, Mao Q, Rand WM, Moss LG, Kuhar M, Emerson CH, Jackson IM, Lechan RM 2000 Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting. J Neurosci 20:9224–9234
Kozicz T 2003 Neurons colocalizing urocortin and cocaine and amphetamine-regulated transcript immunoreactivities are induced by acute lipopolysaccharide stress in the Edinger-Westphal nucleus in the rat. Neuroscience 116:315–320
Murphy KG, Abbott CR, Mahmoudi M, Hunter R, Gardiner JV, Rossi M, Stanley SA, Ghatei MA, Kuhar MJ, Bloom SR 2000 Quantification and synthesis of cocaine- and amphetamine-regulated transcript peptide (79–102)-like immunoreactivity and mRNA in rat tissues. J Endocrinol 166:659–668
Kuriyama G, Takekoshi S, Tojo K, Nakai Y, Kuhar MJ, Osamura RY 2004 CART Peptide in the rat anterior pituitary gland is localized in gonadotrophs and suppresses prolactin secretion. Endocrinology 145:2542–2550
Stanley SA, Murphy KG, Bewick GA, Kong WM, Opacka-Juffry J, Gardiner JV, Ghatei M, Small CJ, Bloom SR 2004 Regulation of rat pituitary cocaine- and amphetamine-regulated transcript (CART) by CRH and glucocorticoids. Am J Physiol 287:E583–E590
Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Voglmayr JK, Bardin CW, Rivier C, Vale W 1989 Detection and purification of inhibin using antisera generated against synthetic peptide fragments. Methods Enzymol 168:588–617
Gulyas J, Rivier C, Perrin M, Koerber SC, Sutton S, Corrigan A, Lahrichi SL, Craig AG, Vale W, Rivier J 1995 Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor. Proc Natl Acad Sci USA 92:10575–105759
Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton D, Rivier C, Rivier J 1983 Assay of corticotropin releasing factor. Methods Enzymol 103:565–577
Kageyama K, Li C, Vale WW 2003 Corticotropin-releasing factor receptor type 2 messenger ribonucleic acid in rat pituitary: localization and regulation by immune challenge, restraint stress, and glucocorticoids. Endocrinology 144:1524–1532
Buhimschi CS 2004 Endocrinology of lactation. Obstet Gynecol Clin North Am 31:963–979
Vicentic A, Dominguez G, Hunter RG, Philpot K, Wilson M, Kuhar MJ 2004 Cocaine- and amphetamine-regulated transcript peptide levels in blood exhibit a diurnal rhythm: regulation by glucocorticoids. Endocrinology 145:4119–4124
Vrang N, Larsen PJ, Kristensen P, Tang-Christensen M 2000 Central administration of cocaine-amphetamine-regulated transcript activates hypothalamic neuroendocrine neurons in the rat. Endocrinology 141:794–801
Stanley SA, Small CJ, Murphy KG, Rayes E, Abbott CR, Seal LJ, Morgan DG, Sunter D, Dakin CL, Kim MS, Hunter R, Kuhar M, Ghatei MA, Bloom SR 2001 Actions of cocaine- and amphetamine-regulated transcript (CART) peptide on regulation of appetite and hypothalamo-pituitary axes in vitro and in vivo in male rats. Brain Res 893:186–194
Vrang N, Larsen PJ, Tang-Christensen M, Larsen LK, Kristensen P 2003 Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids. Brain Res 965:45–50
Dallvechia-Adams S, Smith Y, Kuhar MJ 2001 CART peptide-immunoreactive projection from the nucleus accumbens targets substantia nigra pars reticulata neurons in the rat. J Comp Neurol 434:29–39
Koylu EO, Couceyro PR, Lambert PD, Kuhar MJ 1998 Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J Comp Neurol 391:115–132
Fekete C, Wittmann G, Liposits Z, Lechan RM 2004 Origin of cocaine- and amphetamine-regulated transcript (CART)-immunoreactive innervation of the hypothalamic paraventricular nucleus. J Comp Neurol 469:340–350
Kuhar MJ, Yoho LL 1999 CART peptide analysis by Western blotting. Synapse 33:163–171
Barrett P, Morris MA, Moar KM, Mercer JG, Davidson JA, Findlay PA, Adam CL, Morgan PJ 2001 The differential regulation of CART gene expression in a pituitary cell line and primary cell cultures of ovine pars tuberalis cells. J Neuroendocrinol 13:347–352
Dominguez G, Lakatos A, Kuhar MJ 2002 Characterization of the cocaine- and amphetamine-regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP-dependent signaling pathway in GH3 cells. J Neurochem 80:885–893
Tashjian Jr AH, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352
Vandesande F, Dierickx K 1975 Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat. Cell Tissue Res 164:153–162
Larsen PJ, Seier V, Fink-Jensen A, Holst JJ, Warberg J, Vrang N 2003 Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit. J Neuroendocrinol 15:219–226
Meites J 1977 Neuroendocrine control of prolactin in experimental animals. Clin Endocrinol (Oxf) 6(Suppl):9S–18S
MacLeod RM, Lehmeyer JE 1974 Studies on the mechanism of the dopamine-mediated inhibition of prolactin secretion. Endocrinology 94:1077–1085
Ruberg M, Enjalbert A, Arancibia S, Kordon C 1981 Regulation of prolactin secretion at the pituitary level. Exp Brain Res(Suppl 3):182–199
Albert PR, Neve KA, Bunzow JR, Civelli O 1990 Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion. J Biol Chem 265:2098–2104
Jaffe RB, Yuen BH, Keye Jr WR, Midgley Jr AR 1973 Physiologic and pathologic profiles of circulating human prolactin. Am J Obstet Gynecol 117:757–773
Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM 1996 Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci USA 93:11699–11704
Samson WK, Taylor MM, Baker JR 2003 Prolactin-releasing peptides. Regul Pept 114:1–5
Samson WK, Said SI, Snyder G, McCann SM 1980 In vitro stimulation of prolactin release by vasoactive intestinal peptide. Peptides 1:325–332
Samson WK, Lumpkin MD, McCann SM 1986 Evidence for a physiological role for oxytocin in the control of prolactin secretion. Endocrinology 119:554–560
Lamberts SW, Macleod RM 1990 Regulation of prolactin secretion at the level of the lactotroph. Physiol Rev 70:279–318
Kanyicska B, Sellix MT, Freeman ME 2001 Autocrine regulation of prolactin secretion by endothelins: a permissive role for estradiol. Endocrine 16:133–137
Raptis S, Fekete C, Sarkar S, Rand WM, Emerson CH, Nagy GM, Lechan RM 2004 Cocaine- and amphetamine-regulated transcript co-contained in thyrotropin-releasing hormone (TRH) neurons of the hypothalamic paraventricular nucleus modulates TRH-induced prolactin secretion. Endocrinology 145:1695–1699
Lamote I, Meyer E, Massart-Leen AM, Burvenich C 2004 Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution. Steroids 69:145–159(Sean M. Smith, Joan M. Vaughan, Cynthia )