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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 10 3578-3583
Copyright © 1998 by The Endocrine Society

Original Studies

Secretory Mechanisms of Growth Hormone (GH)-Releasing Peptide-, GH-Releasing Hormone-, and Thyrotropin-Releasing Hormone-Induced GH Release in Patients with Acromegaly

Kunihiko Hanew, Atushi Utsumi, Aki Tanaka, Hidetoshi Ikeda and Yutaka Yokogoshi

Hanew Endocrine Clinic (K.H.), Second Department of Internal Medicine (K.H., A.U., A.T.), and the Department of Neurosurgery (H.I.), Tohoku University School of Medicine, Sendai 980; and the First Department of Internal Medicine, Tokushima University School of Medicine (H.Y.), Tokushima 770, Japan

Address all correspondence and requests for reprints to: Kunihiko Hanew, M.D., Hanew Endocrine Clinic, 2–5 Hasekurachou, Aobaku, Sendai 980, Japan.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The GH secretory mechanism of GH-releasing hexapeptide (GHRP-6), GHRH, and TRH were studied in vivo and in vitro in seven patients with acromegaly. In an in vivo study, these patients showed clear GH responses to single administration of GHRP (four of four patients), GHRH (seven of seven patients), and TRH (seven of seven patients) and enhanced responses to GHRP plus GHRH (two of four patients) or TRH plus GHRH (six of six patients). In an in vitro dispersed cell study, the majority of patients examined also showed clear GH responses to GHRP (four of four patients), GHRH (six of six patients), and TRH (four of four patients) and an enhanced response to GHRP plus GHRH (three of three patients) or TRH plus GHRH (three of four patients). In one patient (no. 3), GHRP plus forskolin (adenylate cyclase activator), but not GHRP plus phorbol 12-myristate 13-acetate (protein kinase C activator), additively enhanced the GH response. Nordihydroguaiaretic acid (NDGA; inhibitor of arachidonic cascade) inhibited GH release induced by GHRP, TRH, GHRH, TRH plus GHRH, or GHRP plus GHRH, but did not inhibit basal GH secretion. In contrast, NDGA distinctly elevated intracellular cAMP levels in another patient (no. 7) when coadministered with GHRP, GHRH, or GHRP plus GHRH, whereas cAMP levels were not modified by single administration of GHRP and NDGA. The GH response to the combined administration of GHRP and GHRH was synergistic in this patient, but was additive in the other two patients.

It is concluded that GHRP, TRH, and GHRH directly stimulate in vivo and in vitro GH release from human somatotropinomas, and GHRP and TRH mainly exert their action through activation of the phosphatidylinositol-protein kinase C pathway, whereas GHRH exerts its action through the adenylate cyclase-protein kinase A pathway. These three agents seem to release GH via the arachidonic cascade.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GH-RELEASING hexapeptide (GHRP-6) is a very potent GH stimulator. It causes a greater GH response in vivo than in vitro and enhances the GH response when coadministered with GHRH (1, 2, 3, 4, 5, 6, 7). Many GHRP-related peptides and nonpeptidyl compounds have been successively synthesized, and these substances are generically termed GH secretagogues (GHS) (8). Recently, human and porcine GHS-specific receptor complementary DNAs were cloned and isolated (9). Therefore, the presence of putative endogenous ligand to such a receptor in human brain is highly probable, although the presence of GHS has not yet been identified. GHS acts on the pituitary gland and hypothalamus to stimulate and amplify pulsatile GH release (9).

It is known that GHRP, GHRH, and TRH stimulate GH secretion in patients with acromegaly (10, 11, 12). Although, GHRH, TRH, and GHRP act on the human pituitary gland directly, the exact intracellular transduction mechanisms induced by these secretagogues and comparisons between in vivo and in vitro GH responses to single or combined administration of these agents have not been well characterized (13). To clarify this, we investigated GH responses of monolayer cultured human pituitary GH secreting adenoma cells to single administration of GHRP, GHRH, and TRH and compared them with in vivo GH responses to these stimuli. Further, the roles of the adenylate cyclase-protein kinase A (PKA) system, the phosphatidylinositol-protein kinase C (PI-PKC) system, and the arachidonic cascade on GH secretion induced by the above agents were examined using forskolin (adenylate cyclase activator), phorbol myristate acetate (TPA; PKC activator) and nordihydroguaiaretic acid (NDGA; lipoxygenase inhibitor), respectively.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

Seven patients with active acromegaly (five men and two women, aged 24–56 yr) were examined in this study after obtaining approval from the ethical committee and informed consent from every subject. Each patient had elevated plasma GH and IGF-I levels, acral enlargement, hyperhidrosis, and pituitary macroadenoma confirmed by magnetic resonance imaging. All patients underwent selective transsphenoidal pituitary adenomectomy.

Analysis of stimulatory GTP-binding protein (gsp) mutation

The presence of the gsp ({alpha}-subunit of stimulatory GTP-binding protein) mutation was studied in two patients. DNA was extracted from paraffin-embedded tumor specimens, and point mutations in codon 201 or 227 of the gsp gene were examined using PCR and the direct sequencing method, as reported previously (14).

In vivo effects of GHRH, TRH, and GHRP

Before surgery, plasma GH responses to single administration of GHRP-6 (Peninsula Laboratory, Belmont, CA; 100 µg, iv), GHRH-(1–44)NH2 (Sumitomo, Osaka, Japan; 100 µg, iv), and TRH (Tanabe, Osaka, Japan; 500 µg, iv) and to combined administration of GHRP plus GHRH, GHRP plus TRH, and GHRH plus TRH were studied in these patients.

Dispersed cell cultures

The pituitary adenoma tissues surgically obtained from seven patients with active acromegaly were used for the monolayer culture experiment. These patients had undergone selective transsphenoidal pituitary adenomectomy. Primary cultured monolayer cells were prepared according to methods previously reported (15). These cells were cultured in 1-mL plastic 48-well trays (no. 258301, Corning, Inc., Tokyo, Japan) at 37 C for 4 days in an atmosphere of 5% CO2 and 95% air. The number of cells per well ranged from 2 x 104 to 10 x 104, and cell viability, as determined by the trypan blue dye exclusion method, was more than 95% before and 4 days after cell culture.

In vitro effects of GHRH, TRH, GHRP, forskolin, TPA, and NDGA on GH release

After discarding the medium, GHRH (10-7 and 10-8 mol/L), TRH (10-7 and 10-8 mol/L), GHRP (10-7 and 10-8 mol/L), forskolin (Sigma Chemical Co., St. Louis, MO; 10-8 mol/L), TPA (Sigma; 10-8 mol/L), and NDGA (Aldrich Chemical Co., Inc., Milwaukee, WI; 50 µmol/L) were dissolved in serum-free MEM (Life Technologies, Grand Island, NY) and were added singly or in combination to the dispersed cell cultures. Cells cultured in serum-free MEM medium were used as controls. Five to 12 wells were used for the control cultures, and 5–6 wells were used for each agent. After 2 h in the presence of these agents, the media were collected. These samples were kept frozen at -20 C until GH assay, and in one patient (no. 7), 1 mL 0.1 N HCl was added to the remaining cells, which were frozen at -20 C until cAMP assay.

Determinations of the GH concentration in plasma or medium and of intracellular cAMP

Plasma and medium GH levels were measured in duplicate using a RIA kit (Dainabot, Tokyo, Japan), and intracellular cAMP was measured with a RIA kit (Yamasa, Tokyo, Japan). Intra- and interassay coefficients of variation were 4.1% and 4.7% for GH and 4.8% and 5.5% for cAMP, respectively.

Statistical analysis was performed by ANOVA followed by the Student-Newman-Keuls test. Results are expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Dose-response relationship between GH and GHRP, and GH responses to single administration of GHRH and GHRP and combined administration of GHRH and GHRP in patient 1

In vivo study. Case 1 showed a greater GH response to GHRP than to GHRH, and showed enhanced and additive responses to the combined administration of GHRH plus GHRP and GHRH plus TRH (Table 1Go).


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  		Table 1. Plasma GH responses to single or combined administrations of GHRP, GHRH, and TRH in seven patients with acromegaly

 
In vitro study. Pituitary adenoma cells of this patient showed dose-dependent GH release in response to GHRP administration from 10-9-10-6 mol/L. Further GH release was not observed with a much higher concentration (10-5 mol/L; Fig. 1aGo). As was observed in the in vivo study, the cells showed a greater GH increase to GHRP (10-7 mol/L) than to GHRH (10-7 mol/L), and the GH response was significantly enhanced when GHRP and GHRH were administered simultaneously (vs. GHRP, P < 0.01; vs. GHRH, P < 0.01); however, this response was additive and not synergistic (Fig. 1bGo).



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  		Figure 1. Dose-response relationship between GH and GHRP (10-9-10-5 mol/L; a) and GH responses to single administration of GHRH (10-7 mol/L) and GHRP (10-7 mol/L) and combined administration of GHRH (10-7 mol/L) plus GHRP (10-7 mol/L) in cultured adenoma cells in patient 1 (b). *, P < 0.05; **, P < 0.01.

 
GH responses to single administration of GHRP, GHRH, and TRH and combined administration of GHRP plus GHRH, GHRH plus TRH, and GHRP plus TRH in patient 2

In vivo study. Case 2, who had no gsp mutation, showed a clear GH increase in response to GHRP and TRH and a slight increase in response to GHRH. The GH response was distinctly enhanced by the combined administration of GHRH plus TRH, but not by GHRP plus GHRH (Table 1Go).

In vitro study. Single administration of GHRP (10-8 mol/L), GHRH (10-8 mol/L), and TRH (10-8 mol/L) significantly stimulated GH secretion from cultured cells of this patient compared to that in the control. As with the in vivo study, the magnitude of the GH response was greatest in the GHRP test. The combined administration of GHRP plus GHRH or GHRH plus TRH significantly enhanced the response additively compared to the single administration of these agents (Fig. 2Go), although the combined administration of GHRP and GHRH in vivo did not enhance the GH response. The combined administration of GHRP and TRH slightly exceeded the response to single administration of each agent (vs. GHRP alone, P < 0.05).



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  		Figure 2. GH responses to single administration of GHRP (10-8 mol/L), GHRH (10-8 mol/L), and TRH (10-8 mol/L) and combined administration of GHRP (10-8 mol/L) plus GHRH (10-8 mol/L), GHRH (10-8 mol/L) plus TRH (10-8 mol/L), and GHRH (10-8 mol/L) plus TRH (10-8 mol/L) in cultured adenoma cells in patient 2. *, P < 0.05; **, P < 0.01.

 
GH responses to single administration of GHRP, forskolin, and TPA and combined administration of GHRP plus forskolin and GHRP plus TPA in patient 3

In vivo study. Case 3 showed clear GH increases in response to GHRP, GHRH, and TRH and an enhanced response to simultaneous administration of GHRP plus GHRH and GHRH plus TRH (Table 1Go).

In vitro study. In cultured cells of this patient, GH release was increased by 10-7 mol/L GHRP, as was observed in vivo, but was not affected by the single administration of forskolin (10-8 mol/L) or TPA (10-8 mol/L). Although the combined administration of GHRP plus forskolin or TPA plus forskolin significantly exceeded the response to the single administration of these agents, TPA did not modify GH response to GHRP (Fig. 3Go).



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  		Figure 3. GH responses to single administration of GHRP (10-7 mol/L), forskolin (10-8 mol/L), and TPA (10-8 mol/L) and combined administration of GHRP (10-7 mol/L) plus forskolin (10-8 mol/L) and GHRP (10-7 mol/L) plus TPA (10-8 mol/L) in cultured adenoma cells in patient 3. **, P < 0.01.

 
GH responses to GHRH, TRH, and TPA and combined administration of GHRH plus TRH and TRH plus TPA in patient 4

In vivo study. In patient 4, GHRH and TRH induced clear GH increases, and the GH response to GHRH plus TRH was distinctly greater than that to single administration of each agent (Table 1Go).

In vitro study. Similar to in vivo studies, single administration of GHRH (10-7 mol/L) and TRH (10-7 mol/L) significantly increased GH release compared to the control value. The combined administration of GHRH and TRH significantly enhanced the GH response compared to single administration of each agent. However, the combined administration of TRH and TPA (10-8 mol/L) did not enhance the response induced by single administration of each agent (Fig. 4Go).



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  		Figure 4. GH responses to single administration of GHRH (10-7 mol/L), TRH (10-7 mol/L), and TPA (10-8 mol/L) and combined administration of GHRH (10-7 mol/L) plus TRH (10-7 mol/L) and TRH (10-7 mol/L) plus TPA (10-8 mol/L) in cultured adenoma cells in patient 4. *, P < 0.05; **, P < 0.01.

 
GH responses to single administration of GHRH, TRH, and NDGA and combined administration of GHRH plus TRH, GHRH plus NDGA, TRH plus NDGA, and GHRH plus TRH plus NDGA in patient 5

In vivo study. This patient showed a clear GH increase in response to GHRH or TRH, and the response to simultaneous administration of these agents slightly exceeded the response to single administration of each agent (Table 1Go).

In vitro study. GH release was slightly, but significantly, stimulated by single administration of GHRH (10-7 mol/L) or TRH (10-7 mol/L). Combined administration of GHRH and TRH caused greater and additive GH release compared to that caused by the single administration of each agent. The GH-releasing effects of GHRH or TRH and the enhanced GH response induced by GHRH plus TRH were inhibited by coadministration of NDGA (Fig. 5Go). The single administration of NDGA had no effect on GH release.



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  		Figure 5. GH responses to single administration of GHRH (10-7 mol/L), TRH (10-7 mol/L), and NDGA (50 µmol/L) and combined administration of GHRH (10-7 mol/L) plus TRH (10-7 mol/L), GHRH (10-7 mol/L) plus NDGA (50 µmol/L), and GHRH (10-7 mol/L) plus TRH (10-7 mol/L) plus NDGA (50 µmol/L) in cultured adenoma cells in patient 5. *, P < 0.05; **, P < 0.01.

 
GH responses to single administration of GHRH or TRH and combined administrations of GHRH plus TRH and GHRH plus TRH plus NDGA in patient 6

In vivo study. Case 6 showed a clear GH response to single administrations of GHRH and TRH, and the response to simultaneous administration of GHRH plus TRH slightly exceeded that to single administration of each agent (Table 1Go).

In vitro study. Similar to in vivo studies, single administration of GHRH (10-8 mol/L) or TRH (10-8 mol/L) significantly stimulated GH secretion, and combined administration of these agents enhanced the response additively compared to the response to single administration of each agent. The GH releases induced by GHRH and GHRH plus TRH were significantly inhibited by coadministration of NDGA (Fig. 6Go). The GH response to NDGA plus TRH was slightly lower than that to single TRH administration, but it did not reach statistical significance.



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  		Figure 6. GH responses to single administration of GHRH (10-8 mol/L) and TRH (10-8 mol/L) and combined administration of GHRH (10-8 mol/L) plus TRH (10-8 mol/L), and GHRH (10-8 mol/L) plus TRH (10-8 mol/L) plus NDGA (50 µmol/L) in cultured adenoma cells in patient 6. *, P < 0.05; **, P < 0.01.

 
GH responses to single administration of GHRH, GHRP, and NDGA and combined administration of GHRH plus GHRP, GHRH plus NDGA, GHRP plus NDGA, and GHRH plus GHRP plus NDGA in patient 7

In vivo study. In patient 7, who has no mutation of gsp, plasma GH secretion was stimulated by TRH, GHRH, or GHRP. However, simultaneous administration of GHRH and GHRP did not exceed the response to single administration of each agent (Table 1Go).

In vitro study. Single administration of GHRH (10-8 mol/L) or GHRP (10-8 mol/L) significantly stimulated GH secretion, as was observed in vivo. In contrast, combined administration of GHRH and GHRP enhanced the response synergistically compared to the responses to single administration of each agent (P < 0.05). Although, single administration of NDGA did not affect GH secretion, coadministration of NDGA significantly inhibited the GH response to GHRH, GHRP, or GHRH plus GHRP (Fig. 7aGo). cAMP production was significantly stimulated only by GHRH and was not modulated by GHRP or NDGA alone. GHRP did not enhance the cAMP response to GHRH. However, coadministration of NDGA distinctly enhanced cAMP responses not only to GHRH and GHRH plus GHRP, but also to GHRP (Fig. 7bGo).



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  		Figure 7. GH (a) and cAMP (b) responses to single administration of GHRH (10-8 mol/L), GHRP (10-8 mol/L), and NDGA (50 µmol/L) and combined administration of GHRH (10-8 mol/L) plus GHRP (10-8 mol/L), GHRH (10-8 mol/L) plus NDGA (50 µmol/L), GHRP (10-8 mol/L) plus NDGA (50 µmol/L), and GHRH (10-8 mol/L) plus GHRP (10-8 mol/L) plus NDGA (50 µmol/L) in cultured adenoma cells in patient 7. *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study, monolayer cultured human GH-producing tumor showed clear GH responses to single administration of GHRP, GHRH, or TRH and enhanced responses to GHRP plus GHRH, GHRP plus forskolin, and GHRH plus TRH. Further, the in vivo GH responses to these secretagogues were similar to the in vitro responses.

As the GH response to GHRP was enhanced by the coadministration of forskolin (PKA activator), but not by TPA (PKC activator), GHRP seems to modulate the PI-PKC system, as was reported in human and animal studies in vitro (13, 16, 17, 18, 19). GHRH and TRH are known to stimulate the adenylate cyclase-cAMP-PKA and PI-PKC systems, respectively (20, 21, 22, 23). The fact that coadministration of GHRP plus GHRH and GHRH plus TRH, but not GHRP plus TRH, caused a greater GH response than the single administration of these agents supports the above explanation. Namely, the stimulation of different transduction mechanisms enhanced the GH response, and the stimulation of similar transduction mechanisms did not enhance the response.

We and others have previously reported that normal subjects (2, 3) and acromegalic patients (10, 24) show a synergistic GH responses to the simultaneous administration of GHRP plus GHRH and TRH plus GHRH in vivo. In this in vitro study, only one patient slightly showed a synergistic response to the combined administration of GHRP and GHRH. Such synergistic responses might be caused by the interaction between distinct transduction pathways (so-called cross-talk phenomenon). In the other patients, the GH responses to GHRP plus GHRH and TRH plus GHRH were additive and not synergistic. In relation to this, the GH-releasing actions of GHRP and GHRH in maximal amounts are additive or slightly synergistic in vitro in rat and human pituitary cell culture systems (5, 6, 8, 17). The reason for the reduced responsiveness in vitro is not clear. However, as hypothalamic influences are totally removed for 4 days in the in vitro studies, it is possible that such disconnection between hypothalamus and pituitary blunted the interaction between second messengers.

There is increasing evidence that the arachidonic cascade plays an important role in hormonal signal transduction (25). Lipoxygenase, which converts arachidonic acid to hydroxyeicosatetraenoic acids and leukotrienes, is part of the arachidonic cascade and is involved in the regulation of hormone secretion from the anterior pituitary gland (26). As far as we know, these are the first experiments using NDGA, which is a lipoxygenase inhibitor, for human somatotropinomas. Coadministration of NDGA distinctly inhibited GH responses induced by GHRP, GHRH, and TRH. However, single administration of NDGA did not affect basal GH secretion. This implies that the arachidonic cascade (especially lipoxygenase pathway) has an important role in stimulated GH release.

In this study, the cAMP responses to GHRP, GHRH, and NDGA were examined in one patient. Single administration of GHRH and combined administration of GHRP plus GHRH increased GH and cAMP levels in this patient, whereas single administration of GHRP only increased GH levels. With coadministration of GHRP, the cAMP response to GHRH was not modified, whereas the GH response to GHRH was synergistically enhanced. These results suggest that an intracellular cross-talk phenomenon occurred at a level other than that of cAMP. Relating to GHRP action, intracellular transduction pathways other than the PI-PKC system are proposed, because the GH-releasing activity of GHRP in rat or human pituitaries does not completely disappear even after the depletion of PKC in TPA-pretreated cells or inhibition of PKC activity with staurosporin or phloretin (16, 17).

After NDGA administration, cAMP responses to GHRH and GHRP plus GHRH were increased despite the significant suppression of GH responses to these stimuli. It is noteworthy that cAMP levels were also clearly increased after combined administration of GHRP and NDGA, whereas the levels were not modified by the single administration of each agent. From these observations, it is conceivable that both adenylate cyclase-PKA and PI-PKC systems participate in GH secretion through the arachidonic cascade, and the former systems are stimulated or accumulated when both systems are blocked at the level of arachidonic cascade.

It was reported that about 40% of human somatotropinomas possess the gsp mutation (27, 28). In this study, two of the seven patients had gene analysis and did not have the gsp mutation. However, as all patients showed GH responses to GHRH, it is unlikely that the remaining patients possessed the mutation.

In summary, GHRP, TRH, and GHRH directly stimulate GH release from human GH-secreting adenoma cells in vivo and in vitro, and GHRP plus TRH or GHRH exert their action mainly through the activation of the PI-PKC or adenylate cyclase-cAMP-PKA system, respectively. These three agents seem to modulate intracellular GH secretory processes via the arachidonic cascade, and this cascade may play an important role in the secretory processes pertinent to the final GH release.


   Acknowledgments
 
We are grateful to Dr. Gordon B. Coutts for his careful review of our manuscript. We thank Miss Kumi Kikuchi for her secretarial assistance.

Received April 9, 1998.

Revised June 25, 1998.

Accepted July 2, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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R. D. Kineman and R. M. Luque
Evidence that Ghrelin Is as Potent as Growth Hormone (GH)-Releasing Hormone (GHRH) in Releasing GH from Primary Pituitary Cell Cultures of a Nonhuman Primate (Papio anubis), Acting through Intracellular Signaling Pathways Distinct from GHRH
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