Homologous and Heterologous in Vitro Regulation of Pituitary Receptors for Somatostatin, Growth Hormone (GH)-Releasing Hormone, and Ghrelin in a Nonhuman Primate (Papio anubis)

Abstract

Secretion of GH by pituitary somatotrophs is primarily stimulated by GHRH and ghrelin and inhibited by somatostatin through the activation of specific receptors [GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R) and somatostatin receptors (sst1–5), respectively]. However, we have shown that somatostatin, at low doses, can also stimulate GH release, directly and specifically, in primary pituitary cultures from a nonhuman primate (baboons, Papio anubis) and pigs. To determine whether somatostatin, GHRH, and ghrelin can also regulate the expression of their receptors in primates, pituitary cultures from baboons were treated for 4 h with GHRH or ghrelin (10⁻⁸ m) or with high (10⁻⁷ m) and low (10⁻¹⁵ m) doses of somatostatin, and GH release and expression levels of all receptors were measured. GHRH/ghrelin decreased the expression of their respective receptors (GHRH-R and GHS-R). Both peptides increased sst1, only GHRH decreased sst5 expression, whereas sst2 expression remained unchanged. The effects of GHRH/ghrelin were completely mimicked by forskolin (adenylate cyclase activator) and phorbol 12-myristate 13-acetate (protein kinase C activator), respectively, indicating the regulation of receptor subtype levels by GHRH and ghrelin involved distinct signaling pathways. In contrast, high-dose somatostatin did not alter GH release but increased sst1, sst2, and sst5 expression, whereas GHRH-R and GHS-R expression were unaffected. Interestingly, low-dose somatostatin increased GH release and sst1 mRNA but decreased sst5 and GHRH-R expression, similar to that observed for GHRH. Altogether, our data show for the first time in a primate model that the primary regulators of somatotroph function (GHRH/ghrelin/somatostatin) exert both homologous and heterologous regulation of receptor synthesis which is dose and subtype dependent and involves distinct signaling pathways.

To date, it is widely accepted that regulation of GH secretion is primarily exerted by the interaction of three peptide hormones, GHRH, ghrelin, and somatostatin (SST). GHRH and ghrelin bind to specific receptors [GHRH receptor (GHRH-R) and GH secretagogue receptor (GHS-R), respectively] located in the plasma membrane of pituitary somatotrophs to directly stimulate GH release, whereas SST binds to five different receptors (sst1–5) to inhibit basal and/or GHRH/ghrelin-stimulated GH release (1–11). However, our group has demonstrated that SST when delivered at low doses (10⁻¹⁷–10⁻¹³ m) can also function as a true GH-releasing factor in primary pituitary cell cultures from baboons (a nonhuman primate, Papio anubis) and pigs, where this paradoxical action has been attributed to activation of sst5 (9, 12–18). Also, it has been reported, but not emphasized in the literature, that some human GH-producing pituitary adenomas release GH in vitro in response to SST or SST receptor agonist challenge (19–23). These divergent effects have been attributed to SST activation of distinct receptors (sst1, sst2, and sst5) that have been identified as the most highly expressed sst within the pituitary of mammalian species (including humans, baboons, and rodents) (9, 14, 24, 25) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication) and are considered to be the primary mediators of the actions of SST on GH release (9, 14). Specifically, the inhibitory effect of SST is mediated primarily by activation of the sst1 and sst2, whereas the stimulatory effect is mediated by sst5 (9, 14) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication).

Previous studies have shown that GHRH-R, GHS-R, and the sst subtypes differ in their ability to activate specific intracellular signaling pathways upon ligand binding (3, 11, 26). Thus, data collected across species clearly indicate that GHRH/GHRH-R stimulates GH release by activating adenylate cyclase (AC), increasing cAMP production, which in turn leads to an increase in protein kinase A (PKA) activity. In contrast, ghrelin/GHS-R stimulates GH release by activating of phospholipase C, increasing phosphatidylinositol turnover, which in turn leads to an increase in PKC activity. Likewise, the GH-stimulatory effect of low-dose SST and sst5 agonist in baboons and pigs is mediated through AC/cAMP/PKA and, therefore, does not augment GH release induced by GHRH but does result in additive effects when combined with ghrelin on intracellular signaling pathways (14, 18) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication). On the other hand, the GH-inhibitory effect of high-dose SST (via sst2) requires intact AC and MAPK systems (18) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication). As recently reviewed (3), the dose-dependent, biphasic effect of SST on AC/cAMP is believed to be due to the ability of sst subtypes to couple to Gαi/o or Gαs, depending on the ligand-binding and receptor conformation.

In addition to differential receptor coupling to downstream signals, it is becoming evident that the relative expression level of each receptor within the target cell (i.e. pituitary) can differentially influence the sensitivity of the cells to their ligand. This may be important in the treatment of human pituitary adenomas with SST analogs because it has been demonstrated that the therapeutic response of GH-producing pituitary adenomas may be dependent on the relative expression pattern of the sst subtypes (27–29). It is also possible that ligand-mediated receptor expression may be important in short-term regulation of GH release under normal conditions. However, our knowledge regarding ligand regulation of pituitary receptor expression pattern is confusing and has been largely derived from nonprimate models (mainly rodent cell lines and rat and pig pituitary cell cultures) using different techniques (in situ hybridization, immunocytochemistry, and mRNA expression) (3, 9, 30–35), where it is not clear whether these data can be extrapolated to understand ligand-mediated regulation of receptor expression in humans. Therefore, the current study used primary pituitary cell cultures from normal female baboons (P. anubis), a primate species that closely models human physiology (36–39) to study, for the first time, the in vitro homologous and heterologous regulation of pituitary GHRH-R, GHS-R, and sst1-5 in response to their natural ligands (GHRH, ghrelin, and SST) and sst-receptor-specific agonists.

Materials and Methods

Culture reagents

Unless otherwise indicated, reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO). α-MEM, HEPES, horse serum, and penicillin-streptomycin were obtained from Invitrogen (Grand Island, NY). Ghrelin was purchased from Phoenix Pharmaceuticals (Burlingame, CA). SST-14 was purchased from Sigma-Aldrich and Phoenix Pharmaceuticals. Subtype-selective agonists for sst1 (L-797,591), sst2 (L-779,976), sst3 (L-796,778), sst4 (L-803,087), and sst5 (L-817,818) were generously provided by Merck & Co., Inc. (Whitehouse Station, NJ) (40).

Animals and pituitary collection

Pituitaries were obtained and dispersed into single cells for culture (see below) from random cycling female baboons (P. anubis, 7–14 yr of age) as previously described (24, 37, 38, 41, 42). These animals represent controls from studies conducted by other University of Illinois at Chicago investigators, where all studies were approved by the Institutional Animal Care and Use Committee.

Primary pituitary cell culture

Anterior pituitaries were dispersed into single cells by enzymatic and mechanical disruption, as previously described (24, 37, 38, 41, 42). To avoid fibroblast contamination, cultures of dispersed pituitary cells were filtered through a nylon gauze of 130-μm mesh, and d-valine-modified MEM (replaced for l-valine) was used to selectively inhibit fibroblast proliferation/overgrowth. Dispersed cells were plated onto 24-well tissue culture plates at a density of 200,000 cells per well in 0.5 ml medium containing 10% horse serum. After a 36- to 48-h incubation (37 C with 5% CO₂), medium was removed and cells were preincubated for 1 h in fresh, warm (37 C) serum-free medium to stabilize basal hormone secretion. Visual inspection of primary cell cultures showed no sign of cells displaying the typical fibroblast-like morphology. Then, medium were replaced for an additional 4 h (three to four wells per treatment per experiment) with serum-free medium alone (controls) or containing human SST-14 [at doses previously identified to exert a maximal inhibitory (10⁻⁷ m) or stimulatory (10⁻¹⁵ m) effect on GH release and/or GHRH/ghrelin-induced GH secretion (18)], GHRH (10⁻⁸ m), ghrelin (10⁻⁸ m), selective agonists for sst1-4 (10⁻⁷ m) and sst5 (10⁻⁷ and 10⁻¹¹ m), forskolin (a direct activator of AC; 10⁻⁶ m), or phorbol 12-myristate 13-acetate (TPA; a direct activator for PKC; 10⁻⁷ m). All these doses were selected based on previous reports and observations showing that those concentrations exerted a maximal effect on GH secretion (18, 37) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication). After peptide treatment, medium was removed and frozen for subsequent analysis of human GH levels and total cellular RNA recovered for determination of mRNA transcript levels, as described below.

Hormone analysis

Culture media was recovered, centrifuged (2000 × g for 5 min), and stored at −80 C for subsequent analysis of GH concentrations using a commercial human GH ELISA kit (Diagnostic Systems Laboratories, Webster, Texas, or DRG, Mountainside, NJ), as previously described (37, 38, 41, 42).

RNA isolation, reverse transcription (RT), and quantitative real-time RT-PCR (qrtRT-PCR)

Total RNA from primary pituitary cell cultures was extracted, quantified, and reverse transcribed, as previously described (37, 41, 42). The cDNA obtained was treated with ribonuclease H (1U; Fermentas, Hanover, MD) and duplicate aliquots (1 μl) were amplified by qrtRT-PCR using the Stratagene (La Jolla, CA) Brilliant SYBR green QPCR Master Mix. Details regarding the development, validation, and application of qrtRT-PCR to measure expression levels of different baboon transcripts have been recently reported by our laboratory (37, 42). Briefly, to determine the starting copy number of cDNA, RT samples were PCR amplified, and the signal (threshold cycle, Ct) of each sample was compared with that of a standard curve run in the same PCR plate. Standard curves consisted of 1, 10¹, 10², 10³, 10⁴, 10⁵, and 10⁶ copies of synthetic cDNA template for each of the transcripts of interest. In addition, total RNA samples that were not reversed transcribed and a no-DNA control were run on each plate to control for genomic DNA contamination and to monitor potential exogenous contamination, respectively. Specific sets of primers used in this study to measure expression levels of baboon sst1–sst5, GHRH-R, and GHS-R are shown in Supplemental Table 1 (published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). To control for variations in the amount of RNA used in the RT reaction and the efficiency of the RT reaction, mRNA copy number of the transcript of interest was adjusted by the mRNA copy number of cyclophilin A (used as housekeeping gene), where cyclophilin A mRNA levels did not significantly vary between experimental groups (data not shown).

Statistical analysis

To normalize mRNA values within each treatment and minimize intra-group variations (likely due to variations in age, body conditions, and/or reproductive status), the values obtained were compared with vehicle-treated controls (set at 100%), and the results are reported as the mean ± sem in all experiments. Each treatment group was tested in a minimum of three separate pituitary cultures, each prepared from a different animal, and within each pituitary cell preparation (experiment), treatments were replicated in at least three to four wells. Differences between treatment groups were assessed by Student's t test. P < 0.05 was considered significant. All statistical analyses were performed using GB-STAT software package (Dynamic Microsystems, Inc., Silver Spring, MD).

Results

Effects of SST on sst1–5, GHRH-R and GHS-R mRNA levels

The high dose of SST (10⁻⁷ m) had no effect on basal GH release, whereas the low dose of SST (10⁻¹⁵ m) increased basal GH release (Fig. 1A), as previously reported (18). It should be noted that the lack of effect of high doses of SST was not surprising based on the fact that several studies have revealed that the precise role of SST in the control of GH secretion is species dependent. Thus, in contrast with the ability of SST to decrease basal GH release both in vivo and in vitro in some species (e.g. rats and mice), the peptide seems unable to induce this effect in vivo or in vitro in other species (e.g. pigs and baboons) (1, 6, 9, 13, 17, 24 and reviewed in Ref.43).

Fig. 1.

Effects of 4 h treatment of SST 10⁻¹⁵ m and SST 10⁻⁷ m on GH release (A) and on sst1, sst2, sst5, GHRH-R, and GHS-R expression (B) in primary pituitary cell cultures from female baboons (P. anubis). A, GH release levels were determined by commercial ELISA; B, sst1, sst2, sst5, GHRH-R, and GHS-R expression was measured by qrtRT-PCR, and mRNA copy numbers were normalized with cyclophilin A mRNA copy number expression. Values are expressed as percentage of vehicle-treated controls (set at 100%) within the experiment and represent the mean ± sem of three to four independent experiments (three to four wells per treatment per experiment). Asterisks show significant differences between treatments compared with their controls assessed by Student's t test: **, P < 0.01; ***, P < 0.001.

Although high doses of SST had no effect on basal GH release, it increased sst1, sst2, and sst5 expression but did not alter GHRH-R and GHS-R expression (Fig. 1B). Interestingly, the low dose of SST also increased sst1 expression, inhibited sst5 and GHRH-R mRNA levels, but had no effect on sst2 or GHS-R mRNA levels. SST (low or high dose) had no effect on sst3 or sst4 expression (data not shown). These results suggest that SST, depending on the dose applied, exert both homologous regulation on the expression of its own receptors and heterologous regulation of the GHRH-R.

Effects of GHRH, ghrelin, forskolin, and TPA on sst1–5, GHRH-R, and GHS-R mRNA levels

As previously reported (37, 38), we observed that GHRH and ghrelin (10⁻⁸ m) markedly increased GH release in baboon pituitary cultures (Fig. 2A). As shown in Fig. 2B (top right panel), GHRH and ghrelin acutely decreased the expression of their own receptors, GHRH-R and GHS-R, whereas they did not alter the expression of the receptors for each other. Both GHRH and ghrelin up-regulated sst1 but did not alter sst2 expression. However, GHRH, but not ghrelin, decreased sst5 mRNA levels (Fig. 2B, top left panel). Interestingly, it should be noted that the effects of acute GHRH treatment on receptor expression were fully mimicked by an activator of AC (forskolin), whereas the effects of ghrelin treatment on the same receptors were fully mimicked by a PKC activator (TPA) (Fig. 2B, bottom panel), thereby suggesting that the homologous and heterologous regulation of receptor levels by GHRH and ghrelin involves distinct signaling pathways. Finally, expression of sst3 and sst4 was not altered by GHRH, ghrelin, forskolin, or TPA (data not shown).

Fig. 2.

Effects of 4 h treatment of GHRH and ghrelin on GH release (A); on sst1, sst2, sst5, GHRH-R, and GHS-R expression (B); and on effects of 4 h treatment of forskolin and TPA on sst1, sst2, sst5, GHRH-R, and GHS-R expression (C) in primary pituitary cell cultures from baboon (P. anubis). A, GH release levels were determined by commercial ELISA; B and C, sst1, sst2, sst5, GHRH-R, and GHS-R expression was measured by qrtRT-PCR and mRNA copy numbers were normalized with cyclophilin A mRNA copy number expression. Values are expressed as percentage of vehicle-treated controls (set at 100%) within the experiment and represent the mean ± sem of three to four independent experiments (three to four wells per treatment per experiment). Asterisks show significant differences between treatments compared with their controls assessed by Student's t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effect of selective sst1, sst2, and sst5 agonists on sst1–5 mRNA level

Because we have previously demonstrated that the inhibitory effect of SST at high dose is primarily mediated by activation of the sst1 and sst2, whereas the stimulatory effect of SST at low doses is mediated by sst5 (14, 18) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication), and based on the above results indicating that sst3 and sst4 expression was not altered by any treatment employed, we used selective agonist for sst1 and sst2 at a high (10⁻⁷ m) dose and sst5 at high or low (10⁻⁷ or 10⁻¹¹ m) doses to determine whether activation of specific sst receptor subtypes may influence the expression of other sst subtypes. As previously observed (Córdoba-Chacón, J., et al., submitted), we found that high doses of sst1 or sst5 agonists did not alter basal GH release (Fig. 3A, left panel). However, high doses of sst2 agonist inhibited basal GH secretion, whereas low doses of sst5 agonist markedly increased GH output (Fig. 3A, left panel). In term of regulation of transcript levels, we found that sst1 expression was not altered by any sst agonist tested (Fig. 3B); however, mRNA levels for sst2 were down-regulated by high doses of sst2 and sst5 agonist (10⁻⁷ m), whereas a small but significant increase in sst2 was observed with low doses of sst5 agonist (10⁻¹¹ m; Fig. 3B). On the other hand, sst5 expression was not altered by high doses of sst1 and sst2 agonists; however, sst5 mRNA levels were oppositely regulated by high and low doses of sst5 agonist (increased and decreased, respectively) (Fig. 3B).

Fig. 3.

Effects of 4 h treatment of selective subtype somatostatin receptor agonists to sst1, sst2, and sst5 at 10⁻⁷ m (A-sst1, A-sst2, and A-sst5 H) and at 10⁻¹¹ m (A-sst5 L) on GH release (A) and on sst1, sst2, and sst5 expression (B) in primary pituitary cell culture from baboon (P. anubis). A, GH release levels were determined by commercial ELISA; B, sst1, sst2, and sst5 expression was measured by qrtRT-PCR, and mRNA copy numbers were normalized with cyclophilin A mRNA copy number expression. Values are expressed as percentage of vehicle-treated controls (set at 100%) within the experiment and represent the mean ± sem of three to four independent experiments (three to four wells per treatment per experiment). Asterisks show significant differences between treatments compared with their controls assessed by Student's t test: *, P < 0.05; **, P < 0.01.

Discussion

The bulk of our understanding regarding the direct effect of SST, GHRH, and ghrelin on the expression of their pituitary receptors is confusing and has been derived from studies conducted in nonprimate species (mainly rodent cell lines and rat pituitary) or human tumor preparations (1, 3, 7–11, 27). We now appreciate that the expression of specific sst subtypes, GHRH-R, and GHS-R can be regulated by their ligands involving receptor internalization and desensitization as well as regulation of gene expression, which, in the case of SST, is subtype specific and dose dependent (1, 3, 9, 10). However, the direct pituitary effect of SST, GHRH, and ghrelin on all sst subtypes, GHRH-R, and GHS-R in normal adult human remains unknown. Due to the close homology with humans at the genomic and physiological levels, over the last years, the baboons have been turned to as a nonhuman primate model to study different levels of human physiology (including pituitary function) (18, 37, 38, 42, 44). Therefore, the present study was conducted using primary pituitary cell cultures from normal adult female baboons (P. anubis) as a primate model to study the impact of these endogenous ligands on the expression of their own receptors (homologous regulation) and on each other's receptors (heterologous regulation).

Our results demonstrate that high doses of SST increased the expression of sst1, sst2, and sst5 expression in baboon primary pituitary cell cultures. These results are similar to that found in other pituitary in vitro models of nonprimate species (i.e. pigs and rats) (30, 31, 33, 34, 45) and also in an in vivo mouse model of enhanced hypothalamic SST expression (the MT-hGHRH) in which sst2 and sst5 mRNA levels were found to be up-regulated (35), therefore suggesting that the regulation of sst2 and sst5 (main receptors involved in GH release) (9) by high doses of SST is conserved across species. Interestingly, treatment of baboon pituitary cell cultures with low doses of SST partially evoked the effect observed in pig (45) in that it similarly increased sst1 and did not alter sst2 expression. However, low doses of SST markedly decrease sst5 expression in baboon, but not pig (45), pituitary cultures. We next examined whether SST could exert any regulatory effect on the expression of GHRH-R and GHS-R in the baboon pituitary. Specifically, we found that SST did not alter the expression of GHS-R at any of the doses tested, which is in clear contrast to the up-regulation in GHS-R mRNA levels previously shown in pig pituitary cultures (32). Interestingly, our results indicate that low, but not high, doses of SST potently decreased GHRH-R mRNA level, which is similar to that observed in pig (32) and suggest that heterologous regulation of GHRH-R by low doses of SST is conserved across species. Taken together, these results indicate that SST regulates not only its own receptor synthesis but also GHRH-R expression in the pituitary, and this regulation seems to be dose and species dependent as well as specific for the sst subtypes.

The studies using sst-subtype-specific agonists (40, 46) revealed that high doses of sst2 agonist (that inhibited basal GH release) down-regulated the expression of sst2, which is in clear contrast to that exerted by high doses of SST. The discrepancies between the effects observed after treatment with a high concentration of SST and the sst2 agonist on basal GH release and receptor expression regulation might be related to the most obvious fact that SST binds to all sst subtypes, where each is known to differentially couple to G proteins and activate distinct signaling pathways (1–3, 10). Moreover, sst can form homo- or heterodimers in a ligand-dependent manner, and this in turn can modify their association with intracellular signaling cascades (47). In line with this notion, studies using SST receptor-specific agonists in pituitary cells from different species have revealed that only sst2 consistently confers the inhibitory actions of SST on GH release, whereas the effect on GH release after the activation of the other SST receptor subtypes are variable depending on the concentration of agonist supplied and the model tested (reviewed in Refs. 6 and 9). One alternate, nonexcluding explanation for the discrepant effects of SST and the sst2 agonist on basal GH release and sst2 expression might be related to the differential ligand-induced (SST vs. sst2 agonist) activation of sst2 because it has been previously indicated that different SST analogs may induce distinct subcellular expression pattern of sst subtypes and/or different conformations of the receptor/ligand complex, preferentially coupled to either receptor signaling or receptor endocytosis (48, 49). In fact, SST and sst2 agonist may activate distinct signaling mechanism or molecular events (i.e. homo- or heterodimerization between sst subtypes) (47), where the differential activation of these intracellular signaling pathways and/or association between sst subtypes would have variable effects on sst2 expression. In support of this hypothesis is the fact that 1) we have recently shown an intact MAPK activity is required for sst2 agonist but not SST-mediated regulation of GH release in baboon primary pituitary cell cultures (18) (Córdoba-Chacón, J., et al., submitted); 2) it has been shown that treatment with SST and sst2 agonist promotes the formation of sst2–sst5 heterodimer complexes in a heterologous cellular system (HEK293 and CHO-K1 cells), although the subsequent MAPK signaling and AC inhibition was significantly higher with sst2 agonist than with SST treatment (50, 51); and 3) it has been demonstrated sst2 is retained (not internalized) at the plasma membrane in response to sst2 agonist challenge in cells that coexpress sst2 and sst5 (as occurs in somatotrophs) (52, 53), whereas endogenous SST potently internalizes and desensitizes sst2 (50, 51). Although caution should be taken in the interpretation of these data generated by the use of SST agonists, these results are novel and intriguing and set the stage for future investigations.

Interestingly, we observed that treatment with high or low doses of sst5 agonist (which do not alter or increase basal GH release, respectively) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication, and present study) induced opposite effects on sst2 and sst5 expression. Specifically, high doses of sst5 agonist reduced sst2 and increased sst5 expression, whereas low doses of sst5 agonist increased sst2 and decreased sst5 mRNA levels. These observations may not be atypical and could be of physiological relevance for the somatotrophs, in that the activation of sst5 by low doses of SST or sst5 agonists markedly increase GH release in baboons and pigs (14, 18) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication, and present study), and therefore, the somatotroph cells may respond to this challenge by 1) increasing the main receptor involved in the inhibition of somatotroph function (sst2) and 2) by decreasing the stimulatory receptor (sst5) to suppress further stimulation of GH release (9, 14, 18). On the other hand, an activation of sst5 by high doses of SST may activate distinct signaling mechanisms or molecular events (i.e. heterodimerization of sst5 and sst2) (50–53) that transduce an overall inhibitory signal that may be compensated by increasing the expression of the sst5 and decreasing sst2 mRNA levels to maintain GH release. In support of this idea, our laboratory has reported that low doses of the sst5 agonist (L-817,818) used in the present study, as well as low doses of SST, signal through AC/cAMP/PKA to increase basal GH release, whereas this signaling pathway is not required for the actions of high-dose SST or sst5 agonist (18) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication). It is also noteworthy that high-dose sst5 agonist increased sst5 mRNA levels despite its lack of effect on GH release, because this underscores the complex ability of this receptor to exert markedly divergent, concentration-dependent actions to differentially, and separately, regulate GH secretion and SST receptor gene expression. Taken together, our results suggest that this opposite regulation of sst2 and sst5 in response to different doses of sst5 agonist might mediate an inner counterbalance of somatotroph cells to facilitate or avoid excessive GH release. Moreover, these results may be (patho)physiologically relevant in humans in that we have shown that the amount of and ratio between pituitary sst2 and sst5 expression in GH-secreting adenomas might be a key factor for the hormonal control of these patients in response to octreotide (a preferential agonist for sst2 and sst5) treatment (28, 29).

We also examined the impact of acute (4 h) treatment of GHRH and ghrelin on regulating receptor synthesis in adult baboon pituitary cell cultures. Our results revealed that GHRH-R and GHS-R were down-regulated by its endogenous ligand, which is consistent with previous reports in other species (including the baboons, pigs, and rodents) (32, 37, 54–57), showing that the synthesis of these receptors can be rapidly down-regulated after ligand binding. However, it should be noted that the homologous regulation of GHRH-R and GHS-R by its own ligands may be dependent on the time of incubation, culture conditions, species, and age studied (32, 54, 57–61).

In addition to the inhibitory effect induced by both GHRH and ghrelin on the expression of their respective receptors in baboon pituitary cell cultures, we observed that these peptides exerted a heterologous regulation of sst subtypes. Specifically, both GHRH and ghrelin increased sst1 and did not alter sst2 expression, whereas only GHRH was able to down-regulate sst5 expression. Interestingly, we noticed that the regulation of all sst subtypes, GHRH-R, and GHS-R was completely similar/parallel in response to GHRH and low doses of SST, suggesting that these events may be mediated through a common signaling pathway. In fact, we have recently reported that the effects of GHRH and low doses of SST on stimulating baboon GH release were mainly mediated through an increase in intracellular cAMP levels (18, 37) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication). Therefore, in an attempt to better understand the relative contribution of elevated intracellular cAMP levels in the synthesis of pituitary receptors, we sought to determine whether forskolin could exert the same effect as GHRH or low-dose SST on sst1, sst2, sst5, GHRH-R, and GHS-R. Our results clearly indicate that an increase in intracellular cAMP levels seems to be responsible for the changes observed in the expression of all these receptors after GHRH and low-dose SST challenge. It is also interesting to note that the effects of ghrelin on the synthesis of all the pituitary receptors analyzed in this study were mimicked by TPA (phorbol esters, an activator of PKC), suggesting that this signaling pathway is required not only for the GH-releasing effect of ghrelin (37) but also for the regulation of the synthesis of these pituitary receptors in response to ghrelin. However, it should be mentioned that we have found some species differences in the regulation of pituitary receptor synthesis in response to SST, GHRH, and ghrelin, which may be probably due to the differences in the intracellular signaling pathways activated by these endogenous ligands in pigs (16, 32, 45, 62–66) and baboons (18, 37) (Córdoba-Chacón, J., M. D. Gahete, M. D. Culler, J. P. Castaño, R. D. Kineman, and R. M. Luque, submitted for publication).

Finally, our results suggest that changes in locally produced GH levels in the culture system do not seem to be directly involved in sst mRNA modulation based on the fact that a distinct pattern in the regulation of sst expression was observed in response to different treatments that evoke an overall increase in basal GH levels (low doses of SST or sst5 agonist and ghrelin) and different treatments that do not alter basal GH levels (high doses of SST, sst5 agonist, or sst1 agonist) (Supplemental Table 2). In fact, of all the treatments used in this study, only SST at low doses and GHRH, which share a common signaling route to increase basal GH release (the AC/cAMP/PKA route) (18, 37), evoked a similar pattern in the regulation of pituitary receptor expression, suggesting that sst mRNA modulation may be directly linked to the activation of specific second messenger routes and not to changes in basal GH levels. In support of this hypothesis is the fact that forskolin, an AC activator that increases cAMP levels, exerts a similar pattern of regulation of sst1, sst2, and sst5 as well as GHRH-R and GHS-R expression than low doses of SST and GHRH. In addition, the effects of ghrelin treatment, acting through the phospholipase C/phosphatidylinositol/PKC pathway (37), on the same receptors were fully mimicked by a PKC activator (TPA), thereby supporting the idea that sst mRNA modulation may be linked to the activation of specific signaling pathways or molecular events independent of changes in locally produced GH secretion.

In summary, our data show for the first time in a primate model that in addition to the well-defined role of SST, GHRH, and ghrelin in modulating GH release, these primary regulators of somatotroph function act through distinct signaling pathways to exert both homologous and heterologous regulation of receptor expression, a mechanism that may contribute significantly to modulate the somatotroph response to acute ligand stimulation and, thus, provide an additional layer of complexity to the regulatory mechanisms required to maintain circulating GH levels in a normal range.