Intermedin/Adrenomedullin-2 Inhibits Growth Hormone Release from Cultured, Primary Anterior Pituitary Cells
http://www.100md.com
《内分泌学杂志》
Saint Louis University, Pharmacological and Physiological Science, St. Louis, Missouri 63104
Abstract
Intermedin (IMD), a novel member of the adrenomedullin (AM), calcitonin gene-related peptide (CGRP), amylin (AMY) peptide family, has been reported to act promiscuously at all the known receptors for these peptides. Like AM and CGRP, IMD acts in the circulation to decrease blood pressure and in the brain to inhibit food intake, effects that could be explained by activation of the known CGRP, AM, or AMY receptors. Because AM, CGRP, and AMY have been reported to affect hormone secretion from the anterior pituitary gland, we examined the effects of IMD on GH, ACTH, and prolactin secretion from dispersed anterior pituitary cells harvested from adult male rats. IMD, in log molar concentrations ranging from 1.0 pM to 100 nM, failed to significantly alter basal release of the three hormones. Similarly, IMD failed to significantly alter CRH-stimulated ACTH or TRH-stimulated prolactin secretion in vitro. However, IMD concentration-dependently inhibited GHRH-stimulated GH release from these cell cultures. The effects of IMD, although requiring higher concentrations, were as efficacious as those of somatostatin and, like somatostatin, may be mediated, at least in part, by decreasing cAMP accumulation. These actions of IMD were not shared by other members of the AM-CGRP-AMY family of peptides, suggesting the presence of a novel, unique IMD receptor in the anterior pituitary gland and a potential neuroendocrine action of IMD to interact with the hypothalamic mechanisms controlling growth and metabolism.
Introduction
INTERMEDIN/ADRENOMEDULLIN-2 is a novel member of the adrenomedullin (AM) peptide family that has been reported to act promiscuously on all the receptors described to date for AM family peptides (1, 2). The peptide was independently cloned by two groups (1, 3). Roh and colleagues (1) discovered intermedin (IMD) through phylogenetic profiling and demonstrated that it was abundantly expressed in the intermediate lobe of the pituitary. Takei et al. (3) cloned mammalian AM-2 based on its homology to pufferfish AM-2. IMD (as it will be referred to here) is abundantly expressed in stomach, kidney, hypothalamus, and pituitary and can be found in the general circulation, suggesting that IMD could act as a local autocrine or paracrine factor or as a circulating hormone (1, 3, 4).
Initial reports concluded that IMD was able to activate multiple receptors, including the receptors for AM, calcitonin gene-related peptide (CGRP), and amylin (AMY) (1, 2). All these receptors are comprised of a seven-transmembrane-spanning, G protein-coupled receptor in association with a single transmembrane spanning receptor activity-modifying protein (RAMP) (reviewed in Ref.5). There are three members of the RAMP family, and each confers unique specificity to the G protein-coupled receptor by directly or indirectly influencing ligand binding. The calcitonin receptor (CTR)-like receptor (CRLR) in association with RAMP1 is a CGRP-preferring receptor, whereas the CRLR in association with RAMP2 or -3 is an AM receptor. The CRLR alone is a null receptor. The CTR alone binds calcitonin; however, the CTR in association with RAMP1, -2, or -3 comprises the AMY receptor family. Because IMD was reported to induce cAMP accumulation in vitro in cells engineered to express the CRLR or CTR in association with any RAMP (1, 2), it would be reasonable to assume that IMD would have physiological actions similar to those of AM, CGRP, and AMY.
IMD has indeed been reported to have many actions similar to those of AM and CGRP. When given iv, IMD lowers blood pressure (1, 3, 4). This action can be partially blocked with CGRP or AM antagonists. Like AM and CGRP, intracerebroventricular injection of IMD elevates blood pressure and heart rate via activation of the sympathetic nervous system (4). The central actions of IMD on blood pressure were most similar to those of CGRP and could be partially attenuated with the CGRP receptor antagonist, CGRP8–37 (4). Like CGRP (6), both ip and intracerebroventricular administration of IMD inhibited food intake (1, 4). Central administration of IMD also inhibited water intake (4), an action similar to that of AM (7). Because of the similarities in the actions of IMD, AM, and CGRP and the reports that these peptides share common receptor components, we examined the effects of IMD in cultured anterior pituitary cells, where AM, CGRP, and AMY have all been reported to have trophic actions.
Materials and Methods
Peptides and cell signaling blockers
All peptides [rat IMD1–47, GHRH, somatostatin (SRIF), CRH, TRH, AM, CGRP, and AMY] were purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). A MAPK inhibitor (PD98059) and the inactive analog (SB20274), a potassium channel blocker (glyburide), the protein kinase C (PKC) inhibitor (calphostin C), the protein kinase A inhibitor (H-89), and pertussis toxin were purchased from Calbiochem (La Jolla, CA). The phospholipase C (PLC) inhibitor and its inactive analog (U73122 and U73343, respectively) were purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Cell culture
Procedures were approved by the Saint Louis University animal care committee. Male Sprague Dawley rats (Harlan, Indianapolis, IN) were killed by rapid decapitation. Anterior pituitary glands were collected as previously described (8) into MEM containing 20 mM HEPES, 1% penicillin-streptomycin (all obtained from Invitrogen Life Technologies, Inc., Carlsbad, CA), 0.1% BSA (Sigma-Aldrich Corp.), and 0.1% trypsin (1:250; Difco, Detroit, MI), and mechanically dispersed until a single-cell suspension was obtained (37 C). Single-cell suspensions were aliquoted into 12 x 75-mm test tubes (200,000 cells/tube) and incubated for 72 h at 37 C (room air) in medium 199 (Sigma-Aldrich Corp.) containing 20 mM HEPES, 10% horse serum, and 1% penicillin-streptomycin (both obtained from Invitrogen Life Technologies, Inc.). On the day of experimentation, cells were pelleted by centrifugation (600 x g, 10 min, room temperature), and medium was removed and replaced with test medium [medium 199, 0.1% BSA, 20 mM HEPES, 1% penicillin-streptomycin, and 2.5 mM bacitracin (Sigma-Aldrich Corp.)] alone or medium containing test substances in a final volume of 0.5 ml. Cells were treated with control medium or medium containing IMD and/or SRIF 10 min before the addition of GHRH. Incubations lasted a total of 60 min and were terminated by centrifugation and collection of medium for subsequent determination of prolactin (PRL), GH, and ACTH contents by RIA. For experiments involving pertussis toxin, cells were pretreated with 10–300 ng/ml pertussis toxin for 24 h before experimentation. On the day of the experiment, cells were pelleted and placed in treatment medium without pertussis toxin (control, IMD, or SRIF), followed by the addition of GHRH 10 min later. Medium was collected as before for determination of GH levels by RIA. For cAMP determinations, after 30 min of incubation, an equal volume of ice-cold 95% ethanol was added to each tube, and the entire mixture was sonicated, then dried in a rotary evaporator before reconstitution in assay buffer. The total protein content was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). All experiments were repeated in three separate cell harvests. In each harvest, group size for each treatment was minimally six. Similar results were obtained in all three harvests.
RIAs
GH levels in incubation medium were measured using the material provided by the National Hormone and Pituitary Program (rGH-RP-2 standard). The minimum detectable hormone level was 0.5 ng/ml (defined as <90% bound/free ratio), and the inter- and intraassay coefficients of variation were 4.3% and 4.8% respectively. PRL levels were similarly determined using the National Institutes of Health kit materials (rPRL-RP-3 standard; minimum detectable hormone level, 0.5 ng/ml; all samples were run in one RIA; 7.6% intraassay coefficient of variation). The ACTH content of the incubation medium was determined using a single commercial RIA kit (rat ACTH, Phoenix Pharmaceuticals, Inc.). The ACTH assay has a minimum detectable hormone level of 1 pg/tube (intraassay coefficient of variation, 2.3%). Total cAMP levels in cells and medium were determined using a commercially available cAMP RIA kit (Amersham Biosciences, Piscataway, NJ).
Statistical analysis
Significance between and within groups was determined by ANOVA with multiple comparison testing (Scheffe’s test). Significance was assigned to results that occurred with less than 5% probability (P < 0.05). Data are presented as the mean and SEM.
Results
IMD, in log molar concentrations ranging from 1.0 pM to 100 nM, failed to significantly alter basal ACTH, PRL, or GH release from dispersed, rat anterior pituitary cells. However, the mixture of releasing factors, as expected, stimulated significant secretion of all three hormones (Table 1). IMD pretreatment did not significantly alter TRH-stimulated PRL release or CRH-stimulated ACTH release (Table 1), but IMD concentration-dependently inhibited the release of GHRH-stimulated GH release (Fig. 1). Treatment of anterior pituitary cells with 1.0 and 10 nM GHRH caused a significant increase in the amount of GH hormone released during a 1-h incubation. Pretreatment of the cells with 100 or 1000 nM IMD significantly inhibited the GH secretion induced by 1.0 and 10 nM GHRH.
The effects of IMD to inhibit GHRH-stimulated GH release were similar to those of SRIF (Fig. 2A), although 100- to 1000-fold higher concentrations of IMD were required. GH release in response to 1.0 nM GHRH during a static 1-h incubation was significantly inhibited by SRIF pretreatment at concentrations of 0.1 nM or more. The minimum concentration of IMD necessary to inhibit GHRH-induced GH release was 10 nM (Fig. 2A), 100-fold higher than the minimum effective concentration of SRIF. The effects of IMD and SRIF on GHRH-stimulated GH release did not appear to be synergistic (Fig. 2, B and C). Addition of a subthreshold concentration of SRIF (0.01 nM) and IMD to pituitary cells treated with GHRH produced no greater inhibition of GH release than the addition of IMD alone (Fig. 2B). Similarly, the effect of a threshold concentration of SRIF (0.1 nM) was not potentiated by IMD (Fig. 2C).
Potential mechanisms for the effects of IMD on GHRH-stimulate GH release were examined. Pretreatment with PLC (U73122), PKC (calphostin C), or MAPK (PD98059) inhibitors or pretreatment with a potassium channel blocker (glyburide) did not significantly alter the inhibitory effect of IMD (Table 2). IMD, however, did inhibit GHRH-stimulated cAMP accumulation in dispersed anterior pituitary cells (Fig. 3A). IMD at a concentration of 1000 nM inhibited cAMP accumulation in cells pretreated with 1.0 and 10 nM GHRH. Lower concentrations of IMD had no significant effect on cAMP accumulation; however, there was a trend toward inhibition with both lower concentrations of IMD when the cells were treated in the presence of 10 nM GHRH. The cAMP inhibition resulting from IMD pretreatment was similar in magnitude to the inhibition of cAMP accumulation seen after treatment of the cells with concentrations of SRIF that produced equivalent inhibition of GHRH-stimulated GH release (data not shown). Pretreatment of the cells with pertussis toxin partially reversed the inhibitory effects of IMD on GHRH-stimulated GH release (Fig. 3B). The GHRH-stimulated GH release after IMD pretreatment was significantly less than that after treatment with GHRH alone, but was significantly greater than the release after IMD/GHRH treatment in the absence of pertussis toxin. As previously described (9), pertussis toxin also partially reversed the inhibitory action of SRIF on GH secretion in our study (Fig. 3B).
No significant effect of CGRP, AM, and AMY in concentrations similar to those employed for IMD was observed in either the basal or GHRH-stimulated GH release experiments (Fig. 4).
Discussion
IMD has been reported to activate AM, CGRP, and AMY receptors in Chinese hamster ovary cells (1, 2). Like AM and CGRP, IMD has been reported to lower blood pressure and elevate heart rate when infused iv (1, 3, 4). In addition, when administered into the cerebroventricular cavity of the brain, IMD, like AM and CGRP, stimulates sympathetic tone, elevating blood pressure and heart rate (4). We have reported that the central and peripheral actions of IMD on cardiovascular function more closely mirror those of CGRP than those of AM (4). Within the brain, IMD acts to inhibit food and water intake in a fashion similar to CGRP and AM, respectively (4, 6, 7). Central administration of IMD also stimulates CRH release into hypophyseal portal blood as well as vasopressin and oxytocin secretion into the circulation (10) as has been reported for AM (11). To date, most of the reported in vivo actions of IMD could be explained by binding of the peptide to the already characterized AM, CGRP, or AMY receptors (1, 2, 3, 4). Those receptors (CRLR and CTR complexed with various RAMPs) have been localized to the anterior pituitary gland, and AM, CGRP, and AMY have been reported to exert actions within the gland to affect hormone secretion (12, 13, 14, 15, 16, 17, 18). AM and CGRP were demonstrated to increase basal GH secretion in cultured primary cells and pituitary adenomas (12, 14, 15). However, in our study, neither AM nor CGRP in log molar concentrations ranging from 1.0–100 nM significantly altered basal or GHRH-stimulated GH secretion in cultures of dispersed anterior pituitary cells. AMY was reported by others (16, 17) not to alter basal GH secretion in vitro, in agreement with our results, but was demonstrated to inhibit -endorphin-stimulated GH release (17), an action we did not examine in these studies. We demonstrate here that AMY did not alter GHRH-stimulated GH release in vitro.
If in anterior pituitary cells, IMD binds to the CRLR- or CTR-RAMP receptors, as has been reported in transformed cells (1, 2), we would hypothesize that IMD would either have no effect on GH release or would, in fact, stimulate hormone secretion (12, 14, 15, 16, 17). In addition, in the engineered Chinese hamster ovary cells, IMD exposure resulted in elevated intracellular cAMP levels (1, 2), again suggesting that the peptide would stimulate GH release. However, we observed quite the opposite. IMD had no effect on basal GH release, but, rather, significantly inhibited the response of those cells to GHRH. Furthermore, IMD coincubation significantly reduced GHRH-stimulated cAMP accumulation in our cell cultures, suggesting a unique action of the peptide that does not mirror the effects of the other members of its homologous peptide family. These findings strongly suggest that a receptor other than one of those described for AM, CGRP, or AMY mediates the effects of IMD in pituitary somatotrophs.
Although higher concentrations of IMD are required to inhibit GHRH-stimulated GH secretion, the same maximal inhibitions were attained with IMD as those observed in the presence of SRIF. Like SRIF, the maximal inhibitory concentrations of IMD also significantly reduced cAMP levels in our cell cultures. Additionally, like SRIF (9), IMD acts at least partially through a pertussis toxin-sensitive G protein, probably Gi/Go, because pertussis toxin was able to partially reverse the inhibitory effects of IMD on GH release. However, we do not believe that IMD exerts its GH inhibitory effects via the SRIF receptor, because IMD did not potentiate the actions of subthreshold or threshold concentrations of SRIF, although the possibility that IMD is a low potency SRIF receptor agonist still remains.
The major pathway for the inhibitory action of IMD in pituitary somatotrophs appears to be via inhibition of cAMP accumulation. It is clear that blockers of PLC, PKC, and MAPK pathways had no effect on the inhibition by IMD of GHRH-stimulated GH release. Although treatment with glyburide, a potassium channel inhibitor, did not significantly alter the effects of IMD in somatotrophs, there was a trend toward attenuation of the inhibitory action, suggesting the potential involvement of a potassium channel in the IMD signaling pathway. Additional studies will be needed to clarify this matter.
Although the inhibitory actions of IMD required higher concentrations of the peptide than those of SRIF, IMD did achieve the same maximal efficacy. The concentrations of IMD needed for its inhibitory effects to be manifested (10–8 M) are similar to the concentrations of other well-recognized inhibitors and stimulators of anterior pituitary peptides. For instance, dopamine’s inhibition of PRL secretion from cultured anterior pituitary cells requires a concentration of 10–6 M (19); orexin A (10–9 M) and orexin B (10–7 M) require similar concentrations to inhibit the release of CRH-stimulated ACTH (20); the stimulation of ACTH by vasopressin (10–8 or 10–9 M) or oxytocin (10–8 M) is also seen at similar concentrations (21); and finally, vasoactive intestinal peptide-induced PRL release is not visible until concentrations of 10–6 M (natural peptide) or 10–9 M (synthetic peptide) are used (8). Although the exact concentration of IMD made in or reaching the anterior pituitary is not currently known, endogenous levels of IMD may be high enough to influence GH release in vivo (4). Indeed, IMD is produced in large amounts in anterior, intermediate, and posterior lobes of the pituitary gland (1–3 pg/μg total protein) (1, 4), suggesting that the peptide could have paracrine or autocrine actions in the gland. IMD is also present in hypothalamus in high concentrations (5.5 pg/μg total protein) (4) and thus may be released in the median eminence and thereby gain access to the portal vessels and the anterior lobe, where it may act as a classical release-inhibiting factor. It is imperative that the exact localization of IMD-producing neurons in brain, particularly the hypothalamus, be identified and the possibility that the peptide is present in nerve terminals of the external lamina of the median eminence be examined. Of course, the peptide also may gain access to the gland via the general circulation, where it is present in readily measurable amounts (17.5 ± 3.9 pg/ml in extracted plasma) (4). Interestingly, the highest amounts of immunoreactive IMD detected in rat tissue were found in stomach and kidney (1, 3, 4). Thus, like ghrelin and other GH regulatory factors (22, 23), IMD levels in the circulation may be regulated by nutritional state and may play a role in the metabolic regulation of GH secretion (e.g. postprandial GH suppression). However, we would hypothesize that transient elevations of circulating IMD, due to its hypotensive actions in the periphery (1, 3, 4), would actually result in increased GH secretion due to activation of the sympathetic nervous system in response to baroreceptor activation (23). Instead, we favor the neuroendocrine or paracrine action of IMD in the pituitary exerted by either hypothalamic or pituitary-derived peptide, and thus, we place higher priority on understanding the mechanisms controlling hypothalamic or pituitary peptide production and release. Based on the presence of IMD immunoreactivity in pituitary and hypothalamus as well as the fact that other anterior pituitary hormone modulators act at similar concentrations, it is possible that these in vitro effects of IMD may have physiological relevance. It will be important to develop tools with which to compromise intermedin production or actions in vivo and examine the effects on GH secretion.
We have demonstrated in this study that IMD acts in dispersed primary anterior pituitary cells in vitro to inhibit GHRH-stimulated GH release. Although the effects of IMD require higher concentrations than those of SRIF, the peptides may act, at least partially, via a common mechanism: activation of a pertussis toxin-sensitive G protein leading to inhibition of cAMP accumulation. The effects of IMD on GHRH-stimulated GH release are different from those described for AM, CGRP, and AMY, suggesting that there is a unique IMD receptor, separate from the CRLR or CTR-RAMP combinations. Somatotrophs (primary or GH3 cells) may represent a cell type in which this novel IMD receptor can be identified. The discovery of a unique IMD receptor and the development of selective IMD antagonists would facilitate studies of the physiological relevance of IMD in neuroendocrine function (GH regulation here and stress hormones) (10), fluid and electrolyte homeostasis (4), and cardiovascular physiology (1, 2, 3, 4).
Acknowledgments
The authors acknowledge the generous contribution of assay reagents by Dr. A. Parlow and the National Hormone and Pituitary Program (National Institute of Diabetes and Kidney Diseases, National Institutes of Health, Bethesda, MD).
Footnotes
This work was supported by National Institutes of Health Grants HL-66023 and HL-68652 from the National Heart, Lung, and Blood Institute.
M.M.T. and S.L.B. have nothing to disclose. W.K.S. has previously consulted for National Institutes of Health and receives royalties from Elsevier Publishing Co.
First Published Online November 3, 2005
Abbreviations: AM, Adrenomedullin; AMY, amylin; CGRP, calcitonin gene-related peptide; CRLR, CTR-like receptor; CTR, calcitonin receptor; IMD, intermedin; PKC, protein kinase C; PLC, phospholipase C; PRL, prolactin; RAMP, receptor activity-modifying protein; SRIF, somatostatin.
Accepted for publication October 21, 2005.
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Abstract
Intermedin (IMD), a novel member of the adrenomedullin (AM), calcitonin gene-related peptide (CGRP), amylin (AMY) peptide family, has been reported to act promiscuously at all the known receptors for these peptides. Like AM and CGRP, IMD acts in the circulation to decrease blood pressure and in the brain to inhibit food intake, effects that could be explained by activation of the known CGRP, AM, or AMY receptors. Because AM, CGRP, and AMY have been reported to affect hormone secretion from the anterior pituitary gland, we examined the effects of IMD on GH, ACTH, and prolactin secretion from dispersed anterior pituitary cells harvested from adult male rats. IMD, in log molar concentrations ranging from 1.0 pM to 100 nM, failed to significantly alter basal release of the three hormones. Similarly, IMD failed to significantly alter CRH-stimulated ACTH or TRH-stimulated prolactin secretion in vitro. However, IMD concentration-dependently inhibited GHRH-stimulated GH release from these cell cultures. The effects of IMD, although requiring higher concentrations, were as efficacious as those of somatostatin and, like somatostatin, may be mediated, at least in part, by decreasing cAMP accumulation. These actions of IMD were not shared by other members of the AM-CGRP-AMY family of peptides, suggesting the presence of a novel, unique IMD receptor in the anterior pituitary gland and a potential neuroendocrine action of IMD to interact with the hypothalamic mechanisms controlling growth and metabolism.
Introduction
INTERMEDIN/ADRENOMEDULLIN-2 is a novel member of the adrenomedullin (AM) peptide family that has been reported to act promiscuously on all the receptors described to date for AM family peptides (1, 2). The peptide was independently cloned by two groups (1, 3). Roh and colleagues (1) discovered intermedin (IMD) through phylogenetic profiling and demonstrated that it was abundantly expressed in the intermediate lobe of the pituitary. Takei et al. (3) cloned mammalian AM-2 based on its homology to pufferfish AM-2. IMD (as it will be referred to here) is abundantly expressed in stomach, kidney, hypothalamus, and pituitary and can be found in the general circulation, suggesting that IMD could act as a local autocrine or paracrine factor or as a circulating hormone (1, 3, 4).
Initial reports concluded that IMD was able to activate multiple receptors, including the receptors for AM, calcitonin gene-related peptide (CGRP), and amylin (AMY) (1, 2). All these receptors are comprised of a seven-transmembrane-spanning, G protein-coupled receptor in association with a single transmembrane spanning receptor activity-modifying protein (RAMP) (reviewed in Ref.5). There are three members of the RAMP family, and each confers unique specificity to the G protein-coupled receptor by directly or indirectly influencing ligand binding. The calcitonin receptor (CTR)-like receptor (CRLR) in association with RAMP1 is a CGRP-preferring receptor, whereas the CRLR in association with RAMP2 or -3 is an AM receptor. The CRLR alone is a null receptor. The CTR alone binds calcitonin; however, the CTR in association with RAMP1, -2, or -3 comprises the AMY receptor family. Because IMD was reported to induce cAMP accumulation in vitro in cells engineered to express the CRLR or CTR in association with any RAMP (1, 2), it would be reasonable to assume that IMD would have physiological actions similar to those of AM, CGRP, and AMY.
IMD has indeed been reported to have many actions similar to those of AM and CGRP. When given iv, IMD lowers blood pressure (1, 3, 4). This action can be partially blocked with CGRP or AM antagonists. Like AM and CGRP, intracerebroventricular injection of IMD elevates blood pressure and heart rate via activation of the sympathetic nervous system (4). The central actions of IMD on blood pressure were most similar to those of CGRP and could be partially attenuated with the CGRP receptor antagonist, CGRP8–37 (4). Like CGRP (6), both ip and intracerebroventricular administration of IMD inhibited food intake (1, 4). Central administration of IMD also inhibited water intake (4), an action similar to that of AM (7). Because of the similarities in the actions of IMD, AM, and CGRP and the reports that these peptides share common receptor components, we examined the effects of IMD in cultured anterior pituitary cells, where AM, CGRP, and AMY have all been reported to have trophic actions.
Materials and Methods
Peptides and cell signaling blockers
All peptides [rat IMD1–47, GHRH, somatostatin (SRIF), CRH, TRH, AM, CGRP, and AMY] were purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). A MAPK inhibitor (PD98059) and the inactive analog (SB20274), a potassium channel blocker (glyburide), the protein kinase C (PKC) inhibitor (calphostin C), the protein kinase A inhibitor (H-89), and pertussis toxin were purchased from Calbiochem (La Jolla, CA). The phospholipase C (PLC) inhibitor and its inactive analog (U73122 and U73343, respectively) were purchased from Sigma-Aldrich Corp. (St. Louis, MO).
Cell culture
Procedures were approved by the Saint Louis University animal care committee. Male Sprague Dawley rats (Harlan, Indianapolis, IN) were killed by rapid decapitation. Anterior pituitary glands were collected as previously described (8) into MEM containing 20 mM HEPES, 1% penicillin-streptomycin (all obtained from Invitrogen Life Technologies, Inc., Carlsbad, CA), 0.1% BSA (Sigma-Aldrich Corp.), and 0.1% trypsin (1:250; Difco, Detroit, MI), and mechanically dispersed until a single-cell suspension was obtained (37 C). Single-cell suspensions were aliquoted into 12 x 75-mm test tubes (200,000 cells/tube) and incubated for 72 h at 37 C (room air) in medium 199 (Sigma-Aldrich Corp.) containing 20 mM HEPES, 10% horse serum, and 1% penicillin-streptomycin (both obtained from Invitrogen Life Technologies, Inc.). On the day of experimentation, cells were pelleted by centrifugation (600 x g, 10 min, room temperature), and medium was removed and replaced with test medium [medium 199, 0.1% BSA, 20 mM HEPES, 1% penicillin-streptomycin, and 2.5 mM bacitracin (Sigma-Aldrich Corp.)] alone or medium containing test substances in a final volume of 0.5 ml. Cells were treated with control medium or medium containing IMD and/or SRIF 10 min before the addition of GHRH. Incubations lasted a total of 60 min and were terminated by centrifugation and collection of medium for subsequent determination of prolactin (PRL), GH, and ACTH contents by RIA. For experiments involving pertussis toxin, cells were pretreated with 10–300 ng/ml pertussis toxin for 24 h before experimentation. On the day of the experiment, cells were pelleted and placed in treatment medium without pertussis toxin (control, IMD, or SRIF), followed by the addition of GHRH 10 min later. Medium was collected as before for determination of GH levels by RIA. For cAMP determinations, after 30 min of incubation, an equal volume of ice-cold 95% ethanol was added to each tube, and the entire mixture was sonicated, then dried in a rotary evaporator before reconstitution in assay buffer. The total protein content was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). All experiments were repeated in three separate cell harvests. In each harvest, group size for each treatment was minimally six. Similar results were obtained in all three harvests.
RIAs
GH levels in incubation medium were measured using the material provided by the National Hormone and Pituitary Program (rGH-RP-2 standard). The minimum detectable hormone level was 0.5 ng/ml (defined as <90% bound/free ratio), and the inter- and intraassay coefficients of variation were 4.3% and 4.8% respectively. PRL levels were similarly determined using the National Institutes of Health kit materials (rPRL-RP-3 standard; minimum detectable hormone level, 0.5 ng/ml; all samples were run in one RIA; 7.6% intraassay coefficient of variation). The ACTH content of the incubation medium was determined using a single commercial RIA kit (rat ACTH, Phoenix Pharmaceuticals, Inc.). The ACTH assay has a minimum detectable hormone level of 1 pg/tube (intraassay coefficient of variation, 2.3%). Total cAMP levels in cells and medium were determined using a commercially available cAMP RIA kit (Amersham Biosciences, Piscataway, NJ).
Statistical analysis
Significance between and within groups was determined by ANOVA with multiple comparison testing (Scheffe’s test). Significance was assigned to results that occurred with less than 5% probability (P < 0.05). Data are presented as the mean and SEM.
Results
IMD, in log molar concentrations ranging from 1.0 pM to 100 nM, failed to significantly alter basal ACTH, PRL, or GH release from dispersed, rat anterior pituitary cells. However, the mixture of releasing factors, as expected, stimulated significant secretion of all three hormones (Table 1). IMD pretreatment did not significantly alter TRH-stimulated PRL release or CRH-stimulated ACTH release (Table 1), but IMD concentration-dependently inhibited the release of GHRH-stimulated GH release (Fig. 1). Treatment of anterior pituitary cells with 1.0 and 10 nM GHRH caused a significant increase in the amount of GH hormone released during a 1-h incubation. Pretreatment of the cells with 100 or 1000 nM IMD significantly inhibited the GH secretion induced by 1.0 and 10 nM GHRH.
The effects of IMD to inhibit GHRH-stimulated GH release were similar to those of SRIF (Fig. 2A), although 100- to 1000-fold higher concentrations of IMD were required. GH release in response to 1.0 nM GHRH during a static 1-h incubation was significantly inhibited by SRIF pretreatment at concentrations of 0.1 nM or more. The minimum concentration of IMD necessary to inhibit GHRH-induced GH release was 10 nM (Fig. 2A), 100-fold higher than the minimum effective concentration of SRIF. The effects of IMD and SRIF on GHRH-stimulated GH release did not appear to be synergistic (Fig. 2, B and C). Addition of a subthreshold concentration of SRIF (0.01 nM) and IMD to pituitary cells treated with GHRH produced no greater inhibition of GH release than the addition of IMD alone (Fig. 2B). Similarly, the effect of a threshold concentration of SRIF (0.1 nM) was not potentiated by IMD (Fig. 2C).
Potential mechanisms for the effects of IMD on GHRH-stimulate GH release were examined. Pretreatment with PLC (U73122), PKC (calphostin C), or MAPK (PD98059) inhibitors or pretreatment with a potassium channel blocker (glyburide) did not significantly alter the inhibitory effect of IMD (Table 2). IMD, however, did inhibit GHRH-stimulated cAMP accumulation in dispersed anterior pituitary cells (Fig. 3A). IMD at a concentration of 1000 nM inhibited cAMP accumulation in cells pretreated with 1.0 and 10 nM GHRH. Lower concentrations of IMD had no significant effect on cAMP accumulation; however, there was a trend toward inhibition with both lower concentrations of IMD when the cells were treated in the presence of 10 nM GHRH. The cAMP inhibition resulting from IMD pretreatment was similar in magnitude to the inhibition of cAMP accumulation seen after treatment of the cells with concentrations of SRIF that produced equivalent inhibition of GHRH-stimulated GH release (data not shown). Pretreatment of the cells with pertussis toxin partially reversed the inhibitory effects of IMD on GHRH-stimulated GH release (Fig. 3B). The GHRH-stimulated GH release after IMD pretreatment was significantly less than that after treatment with GHRH alone, but was significantly greater than the release after IMD/GHRH treatment in the absence of pertussis toxin. As previously described (9), pertussis toxin also partially reversed the inhibitory action of SRIF on GH secretion in our study (Fig. 3B).
No significant effect of CGRP, AM, and AMY in concentrations similar to those employed for IMD was observed in either the basal or GHRH-stimulated GH release experiments (Fig. 4).
Discussion
IMD has been reported to activate AM, CGRP, and AMY receptors in Chinese hamster ovary cells (1, 2). Like AM and CGRP, IMD has been reported to lower blood pressure and elevate heart rate when infused iv (1, 3, 4). In addition, when administered into the cerebroventricular cavity of the brain, IMD, like AM and CGRP, stimulates sympathetic tone, elevating blood pressure and heart rate (4). We have reported that the central and peripheral actions of IMD on cardiovascular function more closely mirror those of CGRP than those of AM (4). Within the brain, IMD acts to inhibit food and water intake in a fashion similar to CGRP and AM, respectively (4, 6, 7). Central administration of IMD also stimulates CRH release into hypophyseal portal blood as well as vasopressin and oxytocin secretion into the circulation (10) as has been reported for AM (11). To date, most of the reported in vivo actions of IMD could be explained by binding of the peptide to the already characterized AM, CGRP, or AMY receptors (1, 2, 3, 4). Those receptors (CRLR and CTR complexed with various RAMPs) have been localized to the anterior pituitary gland, and AM, CGRP, and AMY have been reported to exert actions within the gland to affect hormone secretion (12, 13, 14, 15, 16, 17, 18). AM and CGRP were demonstrated to increase basal GH secretion in cultured primary cells and pituitary adenomas (12, 14, 15). However, in our study, neither AM nor CGRP in log molar concentrations ranging from 1.0–100 nM significantly altered basal or GHRH-stimulated GH secretion in cultures of dispersed anterior pituitary cells. AMY was reported by others (16, 17) not to alter basal GH secretion in vitro, in agreement with our results, but was demonstrated to inhibit -endorphin-stimulated GH release (17), an action we did not examine in these studies. We demonstrate here that AMY did not alter GHRH-stimulated GH release in vitro.
If in anterior pituitary cells, IMD binds to the CRLR- or CTR-RAMP receptors, as has been reported in transformed cells (1, 2), we would hypothesize that IMD would either have no effect on GH release or would, in fact, stimulate hormone secretion (12, 14, 15, 16, 17). In addition, in the engineered Chinese hamster ovary cells, IMD exposure resulted in elevated intracellular cAMP levels (1, 2), again suggesting that the peptide would stimulate GH release. However, we observed quite the opposite. IMD had no effect on basal GH release, but, rather, significantly inhibited the response of those cells to GHRH. Furthermore, IMD coincubation significantly reduced GHRH-stimulated cAMP accumulation in our cell cultures, suggesting a unique action of the peptide that does not mirror the effects of the other members of its homologous peptide family. These findings strongly suggest that a receptor other than one of those described for AM, CGRP, or AMY mediates the effects of IMD in pituitary somatotrophs.
Although higher concentrations of IMD are required to inhibit GHRH-stimulated GH secretion, the same maximal inhibitions were attained with IMD as those observed in the presence of SRIF. Like SRIF, the maximal inhibitory concentrations of IMD also significantly reduced cAMP levels in our cell cultures. Additionally, like SRIF (9), IMD acts at least partially through a pertussis toxin-sensitive G protein, probably Gi/Go, because pertussis toxin was able to partially reverse the inhibitory effects of IMD on GH release. However, we do not believe that IMD exerts its GH inhibitory effects via the SRIF receptor, because IMD did not potentiate the actions of subthreshold or threshold concentrations of SRIF, although the possibility that IMD is a low potency SRIF receptor agonist still remains.
The major pathway for the inhibitory action of IMD in pituitary somatotrophs appears to be via inhibition of cAMP accumulation. It is clear that blockers of PLC, PKC, and MAPK pathways had no effect on the inhibition by IMD of GHRH-stimulated GH release. Although treatment with glyburide, a potassium channel inhibitor, did not significantly alter the effects of IMD in somatotrophs, there was a trend toward attenuation of the inhibitory action, suggesting the potential involvement of a potassium channel in the IMD signaling pathway. Additional studies will be needed to clarify this matter.
Although the inhibitory actions of IMD required higher concentrations of the peptide than those of SRIF, IMD did achieve the same maximal efficacy. The concentrations of IMD needed for its inhibitory effects to be manifested (10–8 M) are similar to the concentrations of other well-recognized inhibitors and stimulators of anterior pituitary peptides. For instance, dopamine’s inhibition of PRL secretion from cultured anterior pituitary cells requires a concentration of 10–6 M (19); orexin A (10–9 M) and orexin B (10–7 M) require similar concentrations to inhibit the release of CRH-stimulated ACTH (20); the stimulation of ACTH by vasopressin (10–8 or 10–9 M) or oxytocin (10–8 M) is also seen at similar concentrations (21); and finally, vasoactive intestinal peptide-induced PRL release is not visible until concentrations of 10–6 M (natural peptide) or 10–9 M (synthetic peptide) are used (8). Although the exact concentration of IMD made in or reaching the anterior pituitary is not currently known, endogenous levels of IMD may be high enough to influence GH release in vivo (4). Indeed, IMD is produced in large amounts in anterior, intermediate, and posterior lobes of the pituitary gland (1–3 pg/μg total protein) (1, 4), suggesting that the peptide could have paracrine or autocrine actions in the gland. IMD is also present in hypothalamus in high concentrations (5.5 pg/μg total protein) (4) and thus may be released in the median eminence and thereby gain access to the portal vessels and the anterior lobe, where it may act as a classical release-inhibiting factor. It is imperative that the exact localization of IMD-producing neurons in brain, particularly the hypothalamus, be identified and the possibility that the peptide is present in nerve terminals of the external lamina of the median eminence be examined. Of course, the peptide also may gain access to the gland via the general circulation, where it is present in readily measurable amounts (17.5 ± 3.9 pg/ml in extracted plasma) (4). Interestingly, the highest amounts of immunoreactive IMD detected in rat tissue were found in stomach and kidney (1, 3, 4). Thus, like ghrelin and other GH regulatory factors (22, 23), IMD levels in the circulation may be regulated by nutritional state and may play a role in the metabolic regulation of GH secretion (e.g. postprandial GH suppression). However, we would hypothesize that transient elevations of circulating IMD, due to its hypotensive actions in the periphery (1, 3, 4), would actually result in increased GH secretion due to activation of the sympathetic nervous system in response to baroreceptor activation (23). Instead, we favor the neuroendocrine or paracrine action of IMD in the pituitary exerted by either hypothalamic or pituitary-derived peptide, and thus, we place higher priority on understanding the mechanisms controlling hypothalamic or pituitary peptide production and release. Based on the presence of IMD immunoreactivity in pituitary and hypothalamus as well as the fact that other anterior pituitary hormone modulators act at similar concentrations, it is possible that these in vitro effects of IMD may have physiological relevance. It will be important to develop tools with which to compromise intermedin production or actions in vivo and examine the effects on GH secretion.
We have demonstrated in this study that IMD acts in dispersed primary anterior pituitary cells in vitro to inhibit GHRH-stimulated GH release. Although the effects of IMD require higher concentrations than those of SRIF, the peptides may act, at least partially, via a common mechanism: activation of a pertussis toxin-sensitive G protein leading to inhibition of cAMP accumulation. The effects of IMD on GHRH-stimulated GH release are different from those described for AM, CGRP, and AMY, suggesting that there is a unique IMD receptor, separate from the CRLR or CTR-RAMP combinations. Somatotrophs (primary or GH3 cells) may represent a cell type in which this novel IMD receptor can be identified. The discovery of a unique IMD receptor and the development of selective IMD antagonists would facilitate studies of the physiological relevance of IMD in neuroendocrine function (GH regulation here and stress hormones) (10), fluid and electrolyte homeostasis (4), and cardiovascular physiology (1, 2, 3, 4).
Acknowledgments
The authors acknowledge the generous contribution of assay reagents by Dr. A. Parlow and the National Hormone and Pituitary Program (National Institute of Diabetes and Kidney Diseases, National Institutes of Health, Bethesda, MD).
Footnotes
This work was supported by National Institutes of Health Grants HL-66023 and HL-68652 from the National Heart, Lung, and Blood Institute.
M.M.T. and S.L.B. have nothing to disclose. W.K.S. has previously consulted for National Institutes of Health and receives royalties from Elsevier Publishing Co.
First Published Online November 3, 2005
Abbreviations: AM, Adrenomedullin; AMY, amylin; CGRP, calcitonin gene-related peptide; CRLR, CTR-like receptor; CTR, calcitonin receptor; IMD, intermedin; PKC, protein kinase C; PLC, phospholipase C; PRL, prolactin; RAMP, receptor activity-modifying protein; SRIF, somatostatin.
Accepted for publication October 21, 2005.
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