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Endocrinology Vol. 145, No. 7 3182-3189
Copyright © 2004 by The Endocrine Society

Homologous and Heterologous Regulation of Pituitary Receptors for Ghrelin and Growth Hormone-Releasing Hormone

Raúl M. Luque, Rhonda D. Kineman, Seungjoon Park, Xiao-Ding Peng, Francisco Gracia-Navarro, Justo P. Castaño and María M. Malagon

Department of Cell Biology, Physiology and Immunology (R.M.L., F.G.-N., J.P.C., M.M.M.), University of Córdoba, E-14014 Córdoba, Spain; and Department of Medicine, University of Illinois at Chicago (R.D.K., S.P., X.-D.P.), Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Dr. María M. Malagón, Department of Cell Biology, Physiology and Immunology, Edificio Severo Ochoa, Planta 3, Campus de Rabanales, University of Córdoba, E-14014 Córdoba, Spain. E-mail: bc1mapom{at}uco.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Secretion of GH by pituitary somatotropes is primarily stimulated by the hypothalamic GHRH through the activation of a specific G protein-coupled receptor, GHRH receptor (GHRH-R). GH is also released in response to ghrelin, a peptide produced in the stomach, hypothalamus, and pituitary that activates somatotropes via a distinct G protein-coupled receptor, referred to as the GH secretagogue receptor (GHS-R). Here, we have analyzed the expression of both GHRH-R and GHS-R (by multiplex RT-PCR) in porcine pituitary cell cultures, after acute (4 h) treatment with GHRH or ghrelin as well as with other regulators of somatotropes (somatostatin, dexamethasone). Exposure of cultures to GHRH decreased GHRH-R mRNA content and also diminished GHS-R transcript levels. Likewise, ghrelin down-regulated both GHS-R and GHRH-R expression. Interestingly, administration of the activator of adenylate cyclase, forskolin, decreased GHRH-R mRNA levels but had no effect on GHS-R, thus suggesting a distinct contribution of the various intracellular signals operating in somatotropes to the regulation of the expression of these receptors. Accordingly, an atypical activator of adenylate cyclase in the pig somatotrope is low-dose (10–13 M) somatostatin, which also suppressed GHRH-R mRNA levels without altering GHS-R expression. Finally, dexamethasone did not modify GHRH-R or GHS-R expression. In summary, our data show for the first time that ghrelin, as well as GHRH, mediates homologous and heterologous down-regulation of their own receptor synthesis. However, our results also indicate that the expression of porcine GHRH-R and GHS-R is regulated by distinct signals that may differ from those reported in other mammalian species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS COMMONLY ACCEPTED that the secretion of GH by pituitary somatotropes is primarily regulated through a complex interaction between the hypothalamic stimulator GHRH and the inhibitor somatostatin (SRIF) (1, 2, 3, 4). However, a new natural GH-releasing peptide of 28 amino acids, ghrelin, was recently cloned and isolated from rat and human gut on the basis of its ability to bind and activate the receptor for the family of synthetic GH secretagogues (GHSs) (5, 6, 7). Both ghrelin and the GHS receptor (GHS-R) are expressed in the pituitary (5, 8, 9, 10, 11, 12, 13) as well as in the hypothalamus (5, 6, 14, 15, 16, 17, 18). These findings, together with the reported ability of ghrelin to stimulate GH release in all the species tested to date (6, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31), including pig (32), strongly suggest that this novel peptide might play an important role in the control of GH secretion.

Both the receptor for GHRH (GHRH-R) and ghrelin (GHS-R) belong to the superfamily of seven-transmembrane helix, G protein-coupled receptors (5, 33, 34, 35). In the pituitary of most mammalian species, at least two molecular isoforms have been found for both GHRH-R and GHS-R, which are generated in each case by alternative splicing of a single precursor mRNA (5, 33, 34, 35, 36). These receptor isoforms differ in their relative abundance in the pituitary as well as in their respective ability to activate specific intracellular signaling pathways on binding of their ligands (reviewed in Refs. 35 , 37 , and 38). Thus, current information indicates that somatotropes express a predominant, short GHRH-R isoform (38) that is preferentially coupled with the adenylate cyclase (AC)/cAMP/protein kinase A pathway (1, 2, 3, 35). In contrast, of the two known GHS-R isoforms, only the long isoform, or type 1a GHS-R, is functional, activating primarily the phospholipase C (PLC)/inositol phosphate/protein kinase C (PKC) signaling route to promote GH release (5, 39). Despite these generalities, it is becoming increasingly evident that both the GHRH-R and GHS-R intracellular signaling is dependent on the ligand used and the animal species, and each ligand may simultaneously activate multiple and unique pathways (32, 40, 41, 42).

Regulation of the expression of membrane receptors for hypothalamic factors constitutes a key step in the control of hormone secretion from pituitary cells. Accordingly, a better understanding of the mechanisms underlying the response of somatotropes to their regulators would be facilitated by elucidating the factors that control the expression of such receptors. Previous studies have analyzed the expression of GHRH-R and GHS-R in response to major regulatory factors of the GH axis, including GHRH, SRIF, and glucocorticoids as well as to synthetic GHSs (43, 44, 45, 46, 47, 48, 49, 50), whereas the possible effects of ghrelin are as yet unknown. Furthermore, most studies have used normal and tumorous rat pituitary cells, whereas results may be species specific and not globally applicable. Therefore, in this series of studies, we used a multiplex RT-PCR methodology, which enables the simultaneous measurement of the mRNAs for GHRH-R and GHS-R, to evaluate the expression of these receptors in cultured porcine pituitary cells in response to their ligands, GHRH and ghrelin, as well as to other putative regulatory factors such as SRIF and glucocorticoids. The porcine somatotrope is an intriguing model to study in that the intracellular response to key GH regulators (GHRH, SRIF, and ghrelin) differ from that reported in the rat. Our results revealed that these receptors are subjected to both homologous and heterologous regulatory mechanisms that seem to involve common, as well as distinct, transduction signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents
Pituitary glands were obtained from prepubertal (4–6 months of age) female Large-White/Landrace pigs. Animals were killed according to the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals of the University of Illinois (Chicago, IL). After sacrifice, pituitaries were excised immediately and stored in sterile cold (4 C) culture medium ({alpha}MEM) containing 0.1% BSA, 100 µg/ml streptomycin, and 100 U/ml penicillin (S-MEM). At the laboratory, pituitaries were washed twice with fresh medium and the posterior lobes were discarded.

Unless indicated otherwise, chemical products were purchased from Sigma Chemical Co. (St. Louis, MO) and tissue culture products and molecular biology reagents were obtained from Life Technologies (Grand Island, NY).

Pituitary cell dispersion and culture
Porcine anterior pituitaries were dispersed into single cells after an enzymatic and mechanical dispersion protocol, as described previously (44). Dispersed cells were plated onto 12-well tissue culture plates at a density of 800,000 cells/well in 1 ml {alpha}MEM supplemented with 10% fetal bovine serum and antibiotics. After 3 d of culture, medium was removed and cells were preincubated with fresh, serum-free {alpha}MEM for 1 h at 37 C. The medium was then replaced with fresh {alpha}MEM containing the test substances at the appropriate concentrations or the corresponding control vehicle and incubated for an additional 4-h period at 37 C.

RNA extraction
Total RNA extraction was performed using the Tri Reagent protocol (Molecular Research Center, Cincinnati, OH). The resulting aqueous phase was further purified by extraction with phenol:chloroform:isoamyl alcohol (25:24:1, pH 5.2; Fisher Scientific, Pittsburgh, PA) to increase the efficiency of the reverse transcription (RT) reaction. Thereafter, RNA was precipitated with isopropanol, and the pellet washed with 70% ethanol, air dried, and resuspended in buffer containing 10 mM Tris-HCl (pH 7.6) and 1 mM EDTA. Concentration and purity of the RNA samples were determined by UV spectroscopy at 260/280 nm, and integrity was confirmed by electrophoresis through 1% agarose gels stained with ethidium bromide.

Multiplex RT-PCR of porcine GHRH-R and GHS-R
mRNA levels of porcine GHRH-R and GHS-R were assessed using a semiquantitative RT-PCR method using hypoxanthine phosphoribosyltransferase (HPRT) mRNA levels as an endogenous control (51). Total RNA (1 µg) obtained from porcine pituitary cell cultures was used as a template for RT using random hexamer primers and the Superscript Preamplification System for First Strand Synthesis (Life Technologies, St. Louis, MO). The resultant cDNA was amplified by PCR using specific primers for porcine GHRH-R (0.2 µM), GHS-R (0.6 µM), and HPRT (0.2 µM). Primers were designed to provide comparable annealing temperatures and compatible product size: GHRH-R: sense 5'-CGGCTTTCTTCTCTCACTTC-3', antisense 5'-AGCACGCAACATCCTCAAAG-3' (GenBank accession no. L11869); GHS-R: sense 5'-ACTGTGCTCTATAGCCTCATCG-3', antisense 5'-CCGATACTTCTTGGACATGATG-3' (GenBank accession no. U60178); HPRT: sense 5'-TGAACGTCTTGCTCGAGATGTG-3', antisense 5'-TTATATCGCCCGTTGACTGGTC-3' (GenBank accession no. U69731). The expected sizes of PCR products were 582 bp for GHRH-R, 304 bp for GHS-R, and 206 bp for HPRT. The primers selected to analyze GHRH-R expression amplified the two isoforms of the porcine receptor (423 and 451 amino acids, respectively) (33, 37), whereas those used for GHS-R only detected the type 1a variant of the porcine receptor (5). The specificity of the fragments amplified by PCR was confirmed by direct sequencing.

PCRs were performed in a 50-µl volume containing 2 µl RT reaction, 2 U Taq Gold polymerase (PerkinElmer Corp., Branchburg, NJ), 1x PCR buffer, 0.2 mM deoxy-NTPs, 1.5 mM MgCl2, and 5 µCi [{alpha}-32P]deoxy-CTP (800 Ci/mmol). Primer concentrations were empirically determined to achieve a final signal that was comparable for all PCR products and that would provide noncompetitive and specific amplification for each PCR product. PCRs consisted of an initial denaturing step of 10 min at 95 C, followed by 25 cycles of 1-min denaturation at 95 C, 1-min annealing at 56 C, and 1-min extension at 72 C with a 10-min final extension at 72 C. Specificity of the PCR procedure was checked by omission of the cDNA template in the amplification reaction.

PCR products (20 µl) were separated on 5% polyacrylamide gels, which were then dried on chromatography paper and exposed to a phosphor image screen. The intensity of each band was analyzed using an image analysis software (Molecular Dynamics, Sunnyvale, CA). The intensity levels of the bands were expressed in pixels and corrected according to background levels. GHRH-R and GHS-R values were adjusted to the HPRT value obtained within the same reaction to normalize the amount of total RNA used in the RT reaction and the efficiency of conversion of RNA to cDNA. Data were expressed as a percentage of the values obtained in the corresponding control cultures (100%).

Statistical analysis
Data are expressed as means ± SEM from three independent experiments performed on different pituitary cell preparations. In each experiment, treatment groups were tested in triplicate. All comparisons were made between samples electrophoresed on the same gel. Statistical analysis was performed using a one-way ANOVA, followed by a statistical test for multiple comparisons (Duncan’s multiple range test and critical ranges) by use of the software package Statistica (StatSoft Inc., Tulsa, OK). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Validation of the multiplex RT-PCR for quantification of mRNA levels of pig GHRH-R and GHS-R
The multiplex PCR assay used in this study employs primer pairs for porcine GHRH-R, GHS-R, and HPRT transcripts from whole pituitary extracts in the same amplification reaction. To obtain the maximal amplification efficiency of the different primers, both the PCR cycle number and the amount of starting mRNA of the sample were optimized. Regarding the first parameter, PCR was performed initially over a range of 21–36 cycles to determine the number of cycles providing a parallel amplification of all PCR products within each reaction. As shown in Fig. 1Go, which corresponds to a representative example of the three separate experiments performed to assess the amplification kinetics of GHRH-R, GHS-R, and HPRT transcripts, all PCR products were amplified with similar efficiency between 21 and 27 cycles (Fig. 1Go, A and B). Accordingly, 25 cycles of PCR amplification, representing the midpoint of the linear portion of the curves, were chosen for subsequent PCR quantifications. Also, accuracy of the amplification was checked by varying the amount of RT product used in the PCR (Fig. 1CGo). The ratio between both GHRH-R/HPRT and GHS-R/HPRT remained constant when RT reaction volumes of 0.5–3 µl, equivalent to a total RNA concentration range of 0.025–0.15 µg, were used. Therefore, all subsequent PCR amplifications were performed with 2 µl of the RT reaction (equivalent to 0.1 µg total RNA).



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  		FIG. 1. Amplification kinetics of cDNA for pig pituitary GHRH-R, GHS-R, and an endogenous internal standard (HPRT) using multiplex RT-PCR. Total pituitary RNA was reverse transcribed using random hexamer priming. cDNA was amplified by PCR in a single sample, in the presence of radiolabeled [{alpha}-32P] deoxy-CTP, using specific primers for GHRH-R, GHS-R, and HPRT. The radiolabeled PCR products were separated on a 5% polyacrylamide gel, and the gel was dried on chromatography paper and exposed to a phosphor image screen. The signal intensities (pixel densities) of the PCR products were measured by image analysis software. All PCR products were of the expected size. A, A phosphor image of radiolabeled GHRH-R, GHS-R, and HPRT PCR products at 21, 24, 27, 30, 33, and 36 cycles of PCR amplification from a representative kinetic experiment. B, Graphical representation of the signal intensity for each PCR product, showing parallel amplification of GHRH-R, GHS-R, and HPRT between 21 and 27 cycles; similar results were obtained in two additional, separate experiments. Accordingly, all subsequent PCR amplifications were performed at 25 cycles. C, Amplification of varying concentrations of total pituitary RNA (0.025–0.15 µg) under the PCR conditions mentioned above yielded constant GHRH-R/HPRT and GHS-R/HPRT ratios.

 
No amplification products were obtained when the cDNA template was omitted in the PCR (data not shown). It should also be noted that HPRT levels remained constant regardless of the experimental conditions tested.

Effect of GHRH and ghrelin on GHRH-R and GHS-R mRNA levels
Once the multiplex RT-PCR methodology was validated, we tested whether GHRH and ghrelin could mediate acute homologous regulation of GHRH-R and GHS-R expression. To this end, porcine pituitary cells were treated with a single dose of GHRH (10–8 M) or ghrelin (10–6 M), in which the doses chosen represent the level previously demonstrated to induce maximal GH release in porcine somatotrope cultures (32, 52). As shown in Fig. 2Go, treatment of porcine pituitary cell cultures for a 4-h period with GHRH markedly decreased GHRH-R mRNA levels (89% of control value; P < 0.05, n = 3). Likewise, ghrelin significantly reduced the expression of its own receptor with respect to that found in vehicle-treated cultures (Fig. 2Go; 62% of control value; P < 0.05, n = 3). Interestingly, both GHRH-R and GHS-R were also responsive to heterologous down-regulation by ghrelin and GHRH, respectively. Specifically, ghrelin caused an 86% reduction in GHRH-R transcript levels (Fig. 2Go; P < 0.05, n = 3) whereas GHRH reduced by half GHS-R mRNA content (Fig. 2Go; P < 0.05, n = 3), with respect to their corresponding controls. Taken together, these results suggest that GHRH and ghrelin exert both homologous and heterologous down-regulation on the expression of their corresponding receptors in somatotropes.



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  		FIG. 2. Effects of GHRH and ghrelin on mRNA levels of pig pituitary GHRH-R and GHS-R in vitro. After 3 d of culture, dispersed pig pituitary cells were incubated in medium alone (Control) or in the presence of GHRH (10–8 M) or ghrelin (10–6 M) for 4 h, and both GHRH-R and GHS-R mRNA levels were determined by multiplex RT-PCR. Receptor-specific band intensities were determined and adjusted by the signal intensity for HPRT. The averaged results were then calculated and expressed as a percentage of vehicle-treated control levels (means ± SEM; n = 3 separate experiment each containing three wells per group per experiment). *, P < 0.01 vs. corresponding control.

 
Effect of forskolin on GHRH-R and GHS-R mRNA levels
As found for other species, GHRH has been shown to stimulate GH release in porcine somatotropes by increasing cAMP production (42). Moreover, we recently demonstrated that ghrelin increases GH release from porcine somatotropes, among other systems, via an increase in intracellular cAMP and that this response can be blocked by an inhibitor of AC (32). Hence, it was of interest to ascertain whether GHRH- or ghrelin-induced inhibition of GHRH-R and GHS-R mRNA was mediated through the activation of the AC system. To this end, porcine pituitary cells were treated with the receptor-independent activator of AC, forskolin (10 µM). Quantification of mRNA levels by multiplex RT-PCR revealed that a 4-h treatment with forskolin decreased GHRH-R mRNA levels, whereas it had no effect on GHS-R expression (Fig. 3Go; P < 0.05, n = 3). These results support the view that the AC/cAMP signaling pathway could mediate the inhibitory action of GHRH or ghrelin on the expression of the GHRH-R, whereas this pathway would not be involved in the down-regulation of GHS-R by its own ligand (ghrelin) or GHRH.



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  		FIG. 3. Effects of the AC activator forskolin on mRNA levels of GHRH-R and GHS-R in pig pituitary cell cultures. Dispersed pituitary cells were cultured for 4 h in the absence (Control) or presence of forskolin (10 µM), and receptor mRNA levels were determined by multiplex RT-PCR. Receptor-specific band intensities were adjusted by the signal intensity for HPRT. Results are expressed as a percentage of vehicle-treated control levels and represent the means ± SEM (n = 3 separate experiment each containing three wells per group per experiment). *, P < 0.01 vs. corresponding control.

 
Effect of SRIF on GHRH-R and GHS-R mRNA levels
We have previously shown that SRIF induces a dual stimulatory/inhibitory effect on GH release from porcine somatotropes depending on its concentration (53, 54, 55, 56). Specifically, high-dose SRIF (10–7 M) inhibits GHRH- and ghrelin-stimulated GH release (32, 54). However, low-dose SRIF (10–15 M) stimulates basal GH release via AC-initiated increase in intracellular cAMP (53, 55, 56). Therefore, it was of interest to determine whether SRIF also exerts any regulatory effect on the expression of GHRH-R and GHS-R in the porcine pituitary. Acute, 4-h treatment with a high dose of SRIF (10–7 M) did not modify GHRH-R mRNA levels whereas it increased GHS-R expression (Fig. 4Go). However, comparable with the actions of GHRH, ghrelin, and forskolin, a low SRIF dose (10–13 M) decreased GHRH-R expression, although this inhibitory action did not extend to the GHS-R (Fig. 4Go).



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  		FIG. 4. Effect of two SRIF doses on GHRH-R and GHS-R expression levels in pig pituitary cells in culture. Cultures were exposed for a 4-h period with two distinct SRIF doses (high, 10–7 M; low, 10–13 M), and receptor mRNA levels were determined by multiplex RT-PCR. Receptor-specific band intensities were determined and adjusted by the signal intensity for HPRT. The averaged results were then calculated and expressed as a percentage of vehicle-treated control levels (means ± SEM; n = 3 separate experiment each containing three wells per group per experiment). *, P < 0.01 vs. corresponding controls.

 
Effect of dexamethasone on GHRH-R and GHS-R mRNA levels
Glucocorticoids are known to modulate GH secretion in several species by regulating, among other functions, GHRH-R and GHS-R gene expression at both the hypothalamic and pituitary levels (43, 47, 50, 57). To determine the possible role of glucocorticoids on the regulation of porcine GHRH-R and GHS-R, cultures of pituitary cells were treated with 10 nM dexamethasone. This showed that neither GHRH-R nor GHS-R mRNA levels were modified after 4 h of exposure to dexamethasone (Fig. 5Go).



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  		FIG. 5. Expression of pig pituitary GHRH-R and GHS-R in response to 10 nM dexamethasone treatment. Receptor-specific band intensities were adjusted by the signal intensity for HPRT. Results are expressed as a percentage of vehicle-treated control levels and represent the means ± SEM (n = 3 separate experiment each containing three wells per group per experiment).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is well recognized that ligands of G protein-coupled receptors can regulate the expression level of their own receptors (58, 59). A number of reports have demonstrated that GHRH causes a marked short-term decrease in rat GHRH-R transcript levels both in vivo and in vitro (44, 48, 60), which is consistent with the decrease in GHRH-binding sites observed after exposure of cultures of rat anterior pituitary cells to the peptide (61). The present results also demonstrate that GHRH is capable of down-regulating pig GHRH-R expression in vitro, thereby suggesting that this phenomenon is common among mammalian species. Notwithstanding, long-term exposure (i.e. 12 h) of rat pituitary cells to GHRH either in vitro (62) or in vivo (63, 64, 65) up-regulates GHRH-R mRNA levels, thus indicating that GHRH may exert a dual time-dependent effect on GHRH-R expression.

To date, few studies have addressed the effects of GHSs on GHS-R mRNA levels at the pituitary level. In fact, our results constitute the first evidence supporting a major role of the endogenous ligand of the GHS-R, ghrelin, in the regulation of the expression of its own receptor in the mammalian pituitary. We clearly demonstrate that short-term ghrelin treatment decreases GHS-R transcript content in porcine pituitary cells. Consistent with our observation, a recent report on the effects of ghrelin on chicken pituitary cells in culture has shown that the peptide down-regulates chicken GHS-R mRNA within 15 min (66). Also, significant suppression of pituitary GHS-R mRNA was observed in rats infused for 4 h with the nonpeptidyl GHS L-692,585 (48). Hence, when viewed as a whole, our results and those described in other species indicate that GHS-R synthesis seems to be rapidly down-regulated after binding of its ligand(s).

In addition to the inhibitory effect induced by both GHRH and ghrelin on the expression of their respective receptors in pig pituitary cells, we observed that these peptides also evoked a heterologous down-regulation of porcine GHRH-R and GHS-R. This acute cross-inhibition between the GHRH/GHRH-R and ghrelin/GHS-R systems seems to be specific for swine because in rat it has been shown that GHRH does not alter GHS-R mRNA pituitary levels in vitro and actually increases GHS-R expression in vivo (48). Similarly, the nonpeptidyl GHS L-692,585 did not modify rat pituitary GHRH-R mRNA levels in vivo (48). These results suggest that the regulation of the expression of GHRH-R and GHS-R by heterologous signals differs between species. Indeed, this may also be the case for glucocorticoids, because an acute treatment with dexamethasone had no effect on the expression of either GHRH-R or GHS-R in porcine pituitary cells, whereas both short- and long-term treatments with corticosterone or synthetic glucocorticoids up-regulate pituitary mRNA levels of these receptors both in vivo and in vitro in the rat (57, 67, 68, 69). Although it is conceivable that longer exposures to dexamethasone could reveal a similar effect in pig pituitary cells, our results clearly show that this synthetic glucocorticoid is not as efficient as GHRH or ghrelin in regulating the expression of GHRH-R and GHS-R in pig pituitary cells.

In all mammals tested to date, including pig, GHRH increases cAMP production as a necessary step to augment GH release (2, 42), and this same pathway appears to mediate homologous down-regulation of GHRH-R mRNA levels in the rat (44, 48). Accordingly, we found forskolin mimicked the inhibitory effect of GHRH on porcine GHRH-R expression, implicating a cAMP-dependent event. It remains to be determined in the pig whether this action is direct or indirect; however, in the rat, a consensus cAMP-response element (CRE) is located within the GHRH-R promoter, suggesting a direct action of cAMP/CRE-binding protein in this species (38). Interestingly, recent data from our laboratory have shown that ghrelin activates cAMP/protein kinase A signaling in pig pituitary somatotropes as well as the PLC/PKC intracellular pathway, in which both pathways are required for ghrelin-stimulated GH release (32). Therefore, ghrelin-induced cAMP increase could be responsible for the reduction in porcine GHRH-R mRNA levels observed after treatment with the peptide. Furthermore, cAMP-mediated regulation of the GHRH-R may also account for the results obtained in the present study with the two doses of SRIF tested. We have previously demonstrated that high doses of SRIF do not modify basal GH release but inhibit both GHRH- and ghrelin-induced GH release (32, 53, 54, 56). However, at very low concentrations, SRIF activates AC, raises intracellular cAMP levels, and stimulates GH release in pig somatotrope cultures (53, 55, 56). Interestingly, when the effects of these distinct doses of SRIF on the expression of porcine GHRH-R were tested, we observed that a high (10–7 M) dose of SRIF did not alter GHRH-R mRNA levels but low-dose SRIF suppressed GHRH-R transcripts to levels comparable with that observed for GHRH, ghrelin, and forskolin. Therefore, in the porcine somatotrope, activation of AC by multiple extracellular signals acutely suppresses GHRH-R expression.

In contrast to that found for GHRH and ghrelin, forskolin did not modify the expression of GHS-R in pig pituitary cells. In agreement with this, forskolin did not alter the expression of GHS-R in cultures of rat pituitary cells (48) and was also without effect on a reporter system driven by the human GHS-R promoter in GH4 cells (47). These findings indicate that the inhibitory action of GHRH and ghrelin on GHS-R expression would be mediated via other intracellular signaling mechanisms, different from those activated by cAMP. In fact, the 5'-flanking region of the human GHS-R promoter has no CRE-binding sites but contains other potential regulatory elements, including activator protein-1, activator protein-2, and Sp-1 sites (47, 70), that have been proposed to be involved in the transcriptional regulation of several genes by PKC (71, 72, 73, 74). Inasmuch as ghrelin, as well as synthetic GHSs, activate the PLC/PKC signaling pathway to induce GH release (32, 39), this same pathway may be responsible for ghrelin-mediated suppression of the GHS-R observed in the present study. In support of this notion, Kaji et al. (75) observed a significant reduction in GHS-R expression after treating GH3 cells transfected with the 5'-flanking region of the human GHS-R gene with substances that activate PLC/PKC intracellular signaling pathways (12-O-tetradecanoylphorbol-13-acetate or Bay K8644). A similar signaling process may operate in the case of GHRH. Indeed, although the prevailing signaling cascade used by GHRH in porcine somatotropes is the AC/cAMP system, this peptide also requires activation of the PLC/inositol phosphate pathway to exert its full effect, at least in a significant subpopulation of somatotropes (42). Hence, it is tempting to propose that a PKC-related mechanism may regulate GHS-R expression in response to GHRH and/or to ghrelin. Obviously, additional research will be required to unequivocally establish the possible contribution of this and other alternative intracellular signals activated by these peptides (76, 77) in the regulation of somatotrope receptors. Notwithstanding this, it seems clear that the distinct effects of forskolin on the expression of GHRH-R and GHS-R provide compelling evidence that the intracellular mechanisms regulating the expression of these two types of receptors are, at least in part, different. Also in support of this hypothesis are our results on the differential effect of a high dose of SRIF on GHRH-R and GHS-R expression in pig pituitary cell cultures. It is noteworthy that, among all the substances tested in the present study, only 10–7 M SRIF increased porcine receptor expression levels, in particular that of GHS-R. The transduction systems that are regulated by high-dose SRIF in this cell type are not completely known yet. Nevertheless, microfluorimetric recording of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes has shown that, in contrast to that found for either low-dose SRIF (55, 56) or GHRH (42) and ghrelin (32), treatment of pituitary cultures with high-dose SRIF significantly decreases this parameter in a considerable proportion of somatotropes (55, 56). Whether changes in [Ca2+]i or in other alternative messengers activated by SRIF or by other extracellular signals preferentially regulates one or both stimulatory receptors in somatotropes remains to be elucidated.

In conclusion, this study demonstrated that the expression of the two major stimulatory receptors operating in somatotropes, GHRH-R and GHS-R, is down-regulated in conditions of increased GH release, as those induced by treatment of pituitary cells with their respective ligands, GHRH and ghrelin, and, at least in pigs, by low SRIF concentrations. These factors may thus act in a coordinated and integrated manner to finely tune the response of somatotropes to multiple stimulatory signals by activating different, precise signaling pathways that might be both distinct or common for each of these receptors.


   Acknowledgments
 
We thank Geraldine A. Amargo for excellent technical assistance.


   Footnotes
 
This work was supported by Grants CVI-0139 (Plan Andaluz de Investigación, Junta de Andalucía, Spain), BFI-2000-0872 and BFI-2001-2007 (Ministerio de Ciencia y Tecnología, Spain; to M.M.M. and J.P.C.), and DK30667 (National Institutes of Health; to R.D.K.).

Abbreviations: AC, Adenylate cyclase; CRE, cAMP-response element; GHRH-R, GHRH receptor; GHS, GH secretagogue; GHS-R, GHS receptor; HPRT, hypoxanthine phosphoribosyltransferase; PKC, protein kinase C; PLC, phospholipase C; RT, reverse transcription; SRIF, somatostatin.

Received December 1, 2003.

Accepted for publication March 15, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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