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Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037
Address all correspondence and requests for reprints to: Wylie W. Vale, Ph.D., Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu.
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Introduction |
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CART has been reported to regulate behavioral and physiological responses to stress. CART is expressed in both the magnocellular and parvocellular subnuclei of the PVN. CART immunoreactivity is present in oxytocinergic neurons in the magnocellular PVN, and CART immunoreactive cells have been observed in the anterior and central regions of the parvocellular PVN (15). CART and corticotropin-releasing factor (CRF), a 41-amino-acid peptide that is the principal regulator of the hypothalamic-pituitary-adrenal (HPA) axis (16), do not colocalize in the PVN of the hypothalamus (15). However, CART immunoreactivity is found in areas known to regulate CRF, including the bed nucleus of the stria terminalis, the amygdala, the hippocampus, and the arcuate and periventricular nuclei of the hypothalamus (3, 15, 17). Central administration of CART results in a robust induction of cFOS expression in CRF-containing neurons of the PVN, along with an accompanied increase in circulating levels of corticosterone (15, 18). Infusion of CART significantly increases CRF release from static hypothalamic explants, and direct injection of CART into the PVN increases plasma ACTH and corticosterone levels (18). CART has also been shown to induce anxiogenic-like behaviors and decrease social interaction in mice (19).
Findings from anatomical studies suggest that CART may directly regulate hormone secretion from the anterior pituitary. CART immunoreactive fibers are found in close opposition to fenestrated capillaries in the external zone of the median eminence (14), and CART immunoreactive cells are found in both the anterior and posterior pituitary (20). CART neurons in the periventricular nucleus, supraoptic nucleus, and PVN internalize systemically administered neuronal tracers and have been shown to colocalize with known hypothalamic releasing factors (21, 22). Importantly, CART has been shown to be secreted into the portal circulation after sodium nitroprusside-induced hypotension (21).
To further elucidate the role of CART in the regulation of the HPA axis, we compared the effects of central and peripheral injection of CART(55–102) on ACTH release from the anterior pituitary. The effects of the CRF receptor antagonist Astressin B were examined to determine the contributions of CRF receptors in CART regulation of ACTH release. CRF and arginine vasopressin (AVP) mRNA expression were also examined to determine whether CART regulates these hypothalamic releasing factors. Last, dispersed rat pituitary cells were used to investigate the effects of CART(55–102) on CRF- and AVP-mediated ACTH release from pituitary corticotropes.
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Materials and Methods |
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Peptides
CART(55–102), rat/human CRF, and the nonselective CRF receptor antagonist Astressin B were synthesized in this laboratory by solid-phase methodologies (24, 25). Peptides were first dissolved in water and diluted to desired concentrations using sterile 0.9% NaCl containing 0.1% crystalline BSA (ICN Biochemicals, Aurora, OH). The peptide CART(55–102) corresponds to amino acids(55–102) of the long splice variant of CART and is identical with CART(42–89) found in the short splice variant (3). CART(55–102) was used in this study because it has been shown to be biologically active in both the central nervous system (7, 26, 27, 28) and the anterior pituitary (20).
Peptide injections and sample collection
On the morning of each bioassay, indwelling catheters and intracerebroventricular (icv) connectors were attached to sampling syringes, and animals were left undisturbed for 3 h before collection of basal blood samples (1100–1200 h). Food was not available to the animals during the acclimation and experimental periods. After collection of basal blood samples, rats were injected with CART(55–102) (iv, 0.09–9.0 nmol/kg; icv, 1 µg), CRF (iv, 9.0 nmol/kg; icv, 1 µg), or 0.9% saline containing 0.1% BSA (vehicle). In select experiments, the CRF receptor antagonist Astressin B (50 µg/kg) (24) was administered by iv injection, 20 min before icv injection of CART(55–102), CRF, or vehicle control. Blood samples (0.3 ml) were collected through iv cannulae in undisturbed rats and immediately replaced with an equivalent volume of apyrogenic isotonic saline. Blood samples were drawn into chilled tubes containing EDTA and were centrifuged, and plasma was stored at –20 C until assays.
Static incubations of hypothalamic explants
A static hypothalamic explant system was used as previously described by Stanley et al. (18). Male Sprague Dawley rats (200–250 g) were killed by rapid decapitation, and hypothalami were removed and placed into 24-well cell culture plates containing 1 ml artificial cerebrospinal fluid (aCSF) (20 mM NaHCO3, 126 mM NaCl, 0.09 mM NaH2PO4, 6 mM KCl, 1.4 mM CaCl2, 0.09 mM MgSO4, 5 mM glucose, and 0.18 mg/ml ascorbic acid) equilibrated with 95% O2-5% CO2. The hypothalami were bordered anteriorly by the optic chiasm, posteriorly by the rostral edge of the mammillary bodies, and laterally by the hypothalamic sulci. Hypothalamic explants were equilibrated in aCSF for 30 min at 37 C in a humidified atmosphere of air with 5% CO2. Explants were then transferred to 24-well culture plates containing aCSF with CART(55–102) (10 nM and 1 µM), forskolin (100 nM), or aCSF alone and incubated for 2 or 6 h at 37 C. After peptide incubations, total RNA was extracted from hypothalamic tissue using Trizol RNA Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH) following methods specified by the manufacturer.
Rat anterior pituitary cell bioassays
Anterior pituitary cells from male Sprague Dawley rats were prepared by dispersion with collagenase and plated in 48-well plates (1.5 x 105 cells/well) as previously described (29). Before initiating experiments, the cells were allowed to recover for 72 h in complete medium [ß Pit Julep (ßPJ)] supplemented with 2% fetal bovine serum and appropriate growth factors (29). The cells were washed three times with ßPJ + 0.1% BSA and equilibrated for 1 h. Cells were then washed with ßPJ + 0.1% BSA and treated in triplicate with test peptides. Media were collected, after 3 h, for secreted ACTH measurement; and intracellular cAMP concentration was determined from cells that were extracted after 30 min of incubation with test peptides.
Immunoassays
Plasma and pituitary culture medium ACTH concentrations were measured using a commercially available two-site immunoradiometric assay (Nichols Institute Diagnostics, San Clemente, CA), which has been validated for the measurement of rat ACTH (30). Corticosterone levels in rat plasma were determined using a commercially available RIA kit (MP Biomedicals, Inc., Orangeburg, NY) following methods specified by the manufacturer. Intracellular levels of cAMP were measured by RIA (Biomedical Technologies, Stoughton, MA) following methods specified by the manufacturer. Values for pmoles per well cAMP were determined using Prism 3.0 software (GraphPad Inc., San Diego, CA) from three independent experiments.
RT-PCR analysis
CRF and AVP mRNA levels in hypothalamic explant tissue were quantified using RT-PCR. After deoxyribonuclease treatment, a constant amount of RNA (5 µg) was added to a reverse transcriptase (RT) mixture (SuperScript II RNase H-Reverse Transcriptase, Life Technologies, Inc. Invitrogen Corp., San Diego, CA). To test for possible pseudo gene or genomic DNA contamination, either the RT enzyme or RNA was omitted from the reaction tube. Upon completion of the RT, 500 ng of the RT mixture was added to separate PCR mixtures containing 100 pmol CRF (sense: 5'-GAAGAGAAAGGGGAAAGGCAAAGA-3'; antisense: 5'-GCGGTGAGGGGCGTGGAGTT-3') or AVP (sense: 5'-CCTCACCTCTGCCTGCTACTT-3'; antisense 5'-GGGGGCGATGGCTCAGTAGAC-3') oligonucleotide primers, 100 µM deoxynucleotide triphosphate, 3 mM MgCl2, 1x Taq buffer, and 2 U Taq DNA Polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd., London, UK) in a total vol of 50 µl. The PCRs were amplified for 35 cycles using a programmable thermal controller (PTC-200, MJ Research Inc., Watertown, MA). Each cycle included denaturation at 94 C for 30 sec, annealing at 62 C for 30 sec (CRF), annealing at 58 C for 30 sec (AVP), and primer extension at 72 C for 1 min. PCR products (403 bp for CRF and 440 bp for AVP) were separated on a 2% agarose gel, visualized with ethidium bromide, photographed, and quantified using the ImageQuant software program (version 1.2, Molecular Dynamics Inc., Sunnyvale, CA). The expression of the ribosomal protein S-16 served as an internal control (31).
Statistical analysis
Statistical analyses were performed by repeated-measures ANOVA for data in Figs. 1
, 2, and 3 and one-way ANOVA for the factors depicted in Figs. 4 and 5. Tukey analysis tests were used 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.
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Results |
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). Circulating levels of ACTH peaked 30 min after icv injection (481 ± 122 vs. 93 ± 14 pg/ml; CART vs. vehicle), remained elevated for 60 min, and subsequently declined to baseline values (Fig. 1A). Corticosterone levels peaked 60 min after icv injection (460 ± 29 vs. 179 ± 62 ng/ml; CART vs. vehicle) of CART(55–102) and remained elevated throughout the 120-min observation period (Fig. 1B). The effect of CART was also compared with the potent ACTH secretagogue CRF. As expected, central injection of CRF (1 µg) produced significant increases in circulating levels of ACTH (484 ± 82 vs. 101 ± 21 pg/ml; CRF vs. vehicle) and corticosterone (505 ± 73 vs. 234 ± 54 pg/ml; CRF vs. vehicle) (Fig. 1). Interestingly, central administration of CART(55–102) produced elevations in plasma ACTH and corticosterone that were similar in time course and potency to CRF (Fig. 1).
CART(55–102) was administered by iv injection to investigate the possible direct effects of this protein on ACTH release from pituitary corticotropes. Peripheral administration of CART(55–102) (0.09–9.0 nmol/kg) failed to produce significant increases in circulating levels of ACTH or corticosterone at any concentration examined throughout the 120-min sampling period (Fig. 2). In contrast, iv injection of CRF (9.0 nmol/kg) produced significant elevations in plasma ACTH and corticosterone (Fig. 2). Increases in plasma ACTH were observed at 5 min (210 ± 40 vs. 22 ± 10 pg/ml; CRF vs. vehicle) and remained elevated through 120 min, when levels returned to baseline values (Fig. 2A). Corticosterone levels peaked 90 min after iv injection (480 ± 82 vs. 76 ± 23 ng/ml; CRF vs. vehicle) and remained elevated through 120 min of observation (Fig. 2B). Last, plasma ACTH and corticosterone levels were low and did not differ significantly between treatment groups before injections (Figs. 1 and 2).
CART(55–102) elicits ACTH secretion by CRF receptor-dependent mechanisms
The potent CRF receptor antagonist Astressin B (32) was administered by iv injection to determine whether CART(55–102) elicits ACTH secretion via a CRF receptor-dependent mechanism. Administration of Astressin B (50 µg/kg) 20 min before icv injection of CART(55–102) significantly reduced plasma ACTH levels at all time points examined (Fig. 3). However, slight elevations in ACTH levels were observed in Astressin B-treated animals at 15 and 30 min after icv injection of CART(55–102) when compared with baseline values (Fig. 3A). Marked decreases in circulating levels of corticosterone were detected in Astressin B pretreated animals from 60–120 min after central injection of CART(55–102) (Fig. 3B). In vehicle (saline, iv) controls, central administration of CART(55–102) produced increases in plasma ACTH and corticosterone (Fig. 3) similar to those described in the previous section (Fig. 1).
Astressin B was equally effective in inhibiting ACTH and corticosterone secretion induced by either CART(55–102) or CRF. Peripheral administration of Astressin B significantly reduced circulating levels of ACTH induced by icv injection of CRF (Fig. 3A). Astressin B had similar inhibitory effects on corticosterone levels after central injection of CRF (Fig. 3B). Peripheral administration of Astressin B did not significantly affect baseline levels of ACTH or corticosterone (Fig. 3).
Effects of CART(55–102) on CRF and AVP mRNA expression in hypothalamic tissue
Central administration of CART has been reported to induce phosphorylation of transcription factors and activate cFOS expression in CRF neurons (27, 33). These findings led us to examine the effects of CART(55–102) on CRF and AVP expression in static hypothalamic explants. Hypothalamic explants were treated with CART(55–102) (10 nM and 1µM) or forskolin (100 nM) for 2 and 6 h at 37 C. Expression levels of CRF and AVP were determined by semiquantitative RT-PCR. A significant 2-fold up-regulation of CRF mRNA was detected in hypothalamic explants after 6 h of treatment with CART(55–102) (Fig. 4). CART(55–102) had a similar effect on AVP expression in hypothalamic explants (Fig 4). AVP expression was up-regulated approximately 2-fold after a 6-h incubation with CART(55–102) (Fig. 4B). Both the 10-nM and 1-µM doses of CART(55–102) were equally effective in inducing expression of CRF and AVP in hypothalamic tissue (Fig. 4). Forskolin, an activator of adenyl cyclase, was used as a positive control and significantly increased expression of both CRF and AVP in hypothalamic explants (Fig 4). Significant differences were not detected in CRF or AVP expression after 2 h of incubation with CART(55–102) or forskolin in hypothalamic tissue (data not shown).
Effect of CART(55–102) on ACTH release and second-messenger activation in dispersed rat pituitary cells
To determine whether CART has direct effects on ACTH release, CART(55–102) was added to dispersed rat anterior pituitary cells at doses ranging from 1–100 nM. CART(55–102) did not affect the release of ACTH at any concentration examined. A high dose (100 nM) of CART(55–102) was then incubated with CRF and AVP to establish whether CART has any synergistic or inhibitory effects on these well-known ACTH secretagogues. Relatively high concentrations of CRF and AVP, 50 nM and 500 nM, respectively, were used in these experiments to ensure a robust release of ACTH from dispersed pituitary cells. As expected, administration of CRF and AVP significantly increased the release of ACTH from dispersed pituitary cells when compared with basal levels (Fig 5A). CART(55–102) did not significantly affect CRF- or AVP-mediated ACTH secretion (Fig. 5A). Similarly, CART(55–102) did not significantly affect cAMP accumulation in dispersed pituitary cells in the presence or absence of CRF and AVP (Fig. 5B).
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Discussion |
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Central administration of CART(55–102) markedly increased plasma concentrations of ACTH and corticosterone compared with vehicle-injected controls. Our findings were consistent with previous results reported by Vrang et al. (27), who observed similar robust increases in circulating corticosterone after central administration of CART. Stanely et al. (18) also reported increases in circulating levels of ACTH and corticosterone after icv injection of CART; however, the duration and peak levels of hormone elevation were significantly lower than those we observed. Differences in CART(55–102)-induced ACTH and corticosterone release observed by other groups may be attributed to differences in experimental protocols and activity of synthetic peptides used for injections.
The effects of peripheral injection of CART(55–102) were examined to exclude the possibility that icv injection of CART(55–102) increased ACTH through direct interaction with pituitary corticotropes. Peripheral injection of CART(55–102) did not significantly affect circulating levels of ACTH or corticosterone. This suggests that CART regulates activity of the HPA axis indirectly through central mechanisms and not through direct interaction with the pituitary corticotropes.
Central injection of CART(55–102) and CRF at equimolar doses elicits similar patterns of plasma ACTH and corticosterone elevation. Also, central injection of CART has been shown to induce cFOS expression in CRF neurons (27), and administration of CART into the PVN produced significant increases in plasma ACTH (18). These results suggest that CART-induced release of ACTH may be dependent on CRF. Astressin B has been reported to suppress CRF receptor-mediated increases in ACTH concentration for as long as 120 min after administration in vivo (34). We used this long-lasting CRF receptor antagonist to investigate the role of CRF receptors in CART(55–102)-induced changes in ACTH and corticosterone levels. Peripheral administration of Astressin B attenuated CART(55–102), and CRF induced increases in ACTH and corticosterone levels. These new findings reveal that CART(55–102)-induced ACTH secretion is dependent on CRF receptor activation in the anterior pituitary gland.
The effects of CART(55–102) on CRF and AVP expression were examined to identify potential interactions of CART with these known ACTH secretagogues. Incubation of CART(55–102) significantly increased CRF and AVP mRNA expression in hypothalamic tissue. The demonstration that CART up-regulates expression of CRF and AVP expands upon earlier observations that central injection of CART increases phosphorylation of the transcription factor cAMP response element binding protein (33) and activation of the immediate early gene cFOS in hypothalamic CRF neurons (27, 35). The extended time period required to increase CRF and AVP expression in hypothalamic tissue (6 h) suggests that changes in message expression may contribute to, but are likely not the primary mechanism responsible for, mediating the rapid effects of CART on ACTH release. Instead, CART induced up-regulation of CRF and AVP expression may function to replace and maintain the stores of these potent ACTH secretagogues after chronic activation of the HPA axis. The hypothalamic explants used in this study did not distinguish between the effects of CART on AVP expression in parvocellular and magnocellular neuronal populations. Further studies are required to determine whether CART has differential effects on the various populations of AVP-expressing neurons in the hypothalamus. In addition, the characterization of the mechanism of action for CART is severely limited because a receptor for CART has not been characterized.
Our studies provide strong evidence that CART regulates activity of the HPA axis through interaction with CRF and not by direct stimulation of ACTH release from pituitary corticotropes. It is well known that AVP interacts with CRF synergistically in the anterior pituitary to stimulate ACTH secretion from corticotropes (36, 37). Thus, it is possible that, in the presence of other ACTH secretagogues, CART may regulate ACTH secretion at the level of the anterior pituitary. In the present study, primary dispersed rat anterior pituitary cells were employed to investigate the effects of CART(55–102) on CRF- and AVP-mediated ACTH release. Incubation of CART(55–102) did not significantly affect ACTH release from dispersed anterior pituitary cells in the presence or absence of CRF and AVP. These results are supported by the recent findings of Kuriyama et al. (20) that CART does not significantly affect ACTH release from dispersed anterior pituitary cells.
In keeping with the wealth of sensory information capable of initiating behavioral responses to stress, CRF neurons located in the PVN of the hypothalamus are extensively innervated (38, 39). Several neurotransmitters and neuropeptides, in addition to CRF and AVP, have been shown to regulate activation of the HPA axis. Norepinephrine has been shown to increase both CRF and ACTH release (40, 41, 42). Serotonergic neurons located in the median raphe nuclei innervate PVN neurons (39, 43), and administration of serotonin receptor agonists increases release of ACTH and corticosterone (44, 45). Several neuropeptides, including vasoactive intestinal peptide (46, 47), neuropeptide Y (48, 49), and cholecystokinin (50, 51), also regulate components of the HPA axis. It is conceivable that CART may function as a modulator of CRF expression and release and plays a role in the regulation of the physiological responses to a vast number of environmental and internal stressors.
In summary, the present study revealed that central injection of CART(55–102) markedly increased plasma concentrations of ACTH and corticosterone, whereas peripheral injection had no significant effect on these hormones. Peripheral administration of Astressin B, a CRF receptor antagonist, attenuated CART(55–102)-mediated increases in ACTH and corticosterone levels. Furthermore, expression of CRF and AVP mRNA were significantly up-regulated in hypothalamic tissue after incubation with CART(55–102). These findings clearly demonstrate that CART regulates activity of the HPA axis through a CRF-dependent central mechanism and not by means of direct interaction with pituitary corticotropes.
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Acknowledgments |
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Footnotes |
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Abbreviations: aCSF, Artificial cerebrospinal fluid; AVP, arginine vasopressin; CART, cocaine- and amphetamine-regulated transcript; CRF, corticotropin-releasing factor; HPA, hypothalamic-pituitary-adrenal; icv, intracerebroventricular; ßPJ, ß Pit Julep; PVN, paraventricular nucleus; RT, reverse transcriptase.
Received June 2, 2004.
Accepted for publication July 16, 2004.
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