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Effects of Antagonists of Growth Hormone-Releasing Hormone (GHRH) on GH and Insulin-Like Growth Factor I Levels in Transgenic Mice Overexpressing the Human GHRH Gene, an Animal Model of Acromegaly¹

Endocrine, Polypeptide and Cancer Institute (A.V.S., K.G.), Veterans Administration Medical Center, New Orleans, Louisiana 70146; Department of Medicine (A.V.S., M.K., M.Z.), Tulane University School of Medicine, New Orleans, Louisiana 70146; and Department of Medicine (R.D.K., L.A.F.), University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Andrew V. Schally, Number 151, Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70146.

	Abstract

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Transgenic mice overexpressing the human GH-releasing hormone (hGHRH) gene, an animal model of acromegaly, were used to investigate the effects of potent GHRH antagonists MZ-4–71 and MZ-5–156 on the excessive GH and insulin-like growth factor I (IGF-I) secretion caused by overproduction of hGHRH. Because metallothionein (MT)-GHRH mice express the hGHRH transgene in various tissues, including the pituitary and hypothalamus, initial experiments focused on the effectiveness of the GHRH antagonists in blocking basal and stimulated GH secretion from pituitary cells in vitro. Both MZ-4–71 and MZ-5–156 suppressed basal release of GH from superfused MT-GHRH pituitary cells, apparently by blocking the action of endogenously produced hGHRH. In addition, these antagonists effectively eliminated the response to stimulatory action of exogenous hGHRH(1–29)NH₂ (30 and 100 nM). To ascertain whether MZ-4–71 and MZ-5–156 could antagonize the effect of hGHRH hyperstimulation in vivo, each antagonist was administered to MT-GHRH transgenic mice in a single iv dose of 10–200 µg. Both compounds decreased serum GH levels in transgenic mice by 39–72% at 1 h after injection. The inhibitory effect of 50 µg MZ-5–156 was maintained for 5 h. Twice daily ip administration of 100 µg MZ-5–156 for 3 days suppressed the highly elevated serum GH and IGF-I concentrations in transgenic mice by 56.8% and 39.0%, respectively. This treatment also reduced IGF-I messenger RNA levels in the liver by 21.8% but did not affect the level of GH messenger RNA in the pituitary. Our results demonstrate that GHRH antagonists MZ-4–71 and MZ-5–156 can inhibit elevated GH levels caused by overproduction of hGHRH. The suppression of circulating GH concentrations induced by the antagonists seems to be physiologically relevant, because both IGF-I secretion and synthesis also were reduced. Our findings, showing the suppression of GH and IGF-I secretion with GHRH antagonists, suggest that this class of analogs could be used for the diagnosis and therapy of disorders characterized by excessive GHRH secretion.

	Introduction

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MANY transgenic animal lines have been developed as useful models for human diseases (1, 2). A transgenic mouse, which has provided an insight into the pathophysiology of pituitary tumor formation, is the mouse metallothionein (MT)-human GH-releasing hormone (hGHRH) transgenic mouse (1, 3). In these mice, the human GHRH gene is expressed in several tissues, including the pituitary, pancreas, and arcuate nucleus of the hypothalamus, and the circulating levels of hGHRH are high. Overproduction of hGHRH results in a dramatic increase in somatotrope function, with markedly increased pituitary GH and GH messenger RNA (mRNA) content, coupled with increased GH and insulin-like growth factor I (IGF-I) secretion. Hyperstimulation of pituitary somatotropes results in hyperplasia, which leads to adenoma formation (4, 5). Many features of pituitary response to chronic GHRH stimulation in these mice parallel those observed in human patients with a rare type of acromegaly, resulting from ectopic expression of GHRH by the pituitary, pancreatic, or other tumors (6). This model of GHRH-induced somatotrope hyperactivity and proliferation was used in the present study to test the possible utility of two new potent GHRH antagonists to inhibit excessive GH and IGF-I synthesis and release. It was hoped that useful information might be obtained on the feasibility of using GHRH antagonists for the treatment of human acromegaly caused by GHRH overproduction.

	Materials and Methods

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Peptides
GHRH antagonists [Ibu-Tyr¹,D-Arg²,Phe(4-Cl)⁶,Abu¹⁵,Nle²⁷,Agm²⁹]hGHRH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) (MZ-4–71) and [PhAc-Tyr¹,D-Arg²,Phe(4-Cl)⁶,Abu¹⁵,Nle²⁷,Agm²⁹]hGHRH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) (MZ-5–156) were synthesized and characterized in our laboratory, as reported previously (7, 8). hGHRH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)NH₂, used as a standard for experiments in vitro, was also produced in our laboratory.

Experimental procedure
The development and characterization of MT-hGHRH transgenic mouse line have been previously described (9, 10). Animals from the founder line 765–2 Tg(Mt-1, GHRF) Bri 11 were used for the present experiments. These transgenic males were mated with C57BL/6 females (Charles River, Wilmington, ME). Transgenic progeny were identified at 2 months of age by RIA for serum GH and hGHRH levels.

Administration of GHRH antagonists MZ-4–71 and MZ-5–156 in vitro.
Four transgenic hGHRH mice (Nos. 1, 2, 3, and 4) and five normal littermates, as controls, were used for the in vitro experiments in the superfused anterior pituitary cell system (11). Pituitaries from the mice were removed, cut into small pieces, incubated with collagenase (Type I, 0.5%, Worthington, Freehold, NJ) for 50 min in a metabolic shaker, dispersed, gently mixed with 1 ml swollen Sephadex G-10 [which had been equilibrated with oxygenated tissue culture medium (Medium 199, Sigma Chemical Co., St. Louis, MO), containing 0.1% BSA], and transferred into the superfusion chambers (1 pituitary in each chamber). To assure a stable baseline value, the cells were perfused with the enzyme-free Medium 199 overnight before collecting any fractions. The collection of fractions was started the next morning. At the beginning and the end of each experiment, a membrane-depolarizing dose of KCl (25 mM) was administered to assess the amount of releasable GH in the cells. Thirty minutes after the initial pulse of KCl, 1 nM GHRH was applied for 30 min in mice Nos. 1–3. Thirty minutes later, GHRH antagonists MZ-4–71 or MZ-5–156, at 30 or 100 nM concentrations, were perfused through the cells for 30 min, followed by a continuous 30-min administration of 1 nM GHRH, together with 30 nM or 100 nM of the antagonists using one cell-chamber for each dose. To check the duration of the inhibitory effects of the GHRH antagonists on stimulated GH release, 30 and 90 min after perfusion with the mixture of antagonists and GHRH, 1 nM GHRH was administered for 30 min (30- and 90-min response). To assess the effect of repeated administration of antagonist MZ-4–71 on basal GH secretion from the pituitary cells of mouse No. 4, the initial pulse of 25 mM KCl was followed by alternating administrations, for 30 min, of 30 and 100 nM MZ-4–71, at 30-min intervals. One-ml fractions of the superfusion medium were collected every 3 min, and GH concentration was determined by RIA. GH responses to GHRH after antagonist-treatment were compared with the initial response to 1 nM GHRH, which was used as reference. Inhibitory effects of the GHRH antagonists on the exogenous GHRH-stimulated GH release are expressed as percent inhibition of the reference response.

Acute, single-dose administration of GHRH antagonists MZ-4–71 and MZ-5–156 in vivo.
Six groups of 4–6 transgenic mice (30–40 g BW) at 3 months of age were used for these experiments. Because the changes in the pituitaries of female and male transgenic mice previously have been shown to be similar (10), the groups consisted of male and female mice in about equal ratio. The mice were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Inc., Mundelein, IL). Groups 1, 2, and 3 were injected iv with antagonist MZ-4–71 at doses of 10 µg, 50 µg, and 200 µg, respectively, dissolved in 0.9% saline. Groups 4 and 5 received 10 µg and 50 µg of antagonist MZ-5–156, respectively, and group 6 was treated with 0.9% saline. Immediately before and 1 and 5 h after the injection of the antagonists or saline, blood samples were taken from the jugular vein of all animals. The volume of blood taken (0.25 ml) was replaced by saline. Serum GH concentrations were determined by RIA.

Chronic administration of GHRH antagonist MZ-5–156 to transgenic mice.
Because MZ-5–156 showed a more prolonged inhibitory effect on GH release than did MZ-4–71 in the single-dose test, we used MZ-5–156 for chronic administration. Four adult transgenic mice (three males and one female, 30–40 g BW) were treated ip twice daily for 3 days with 100 µg of antagonist MZ-5–156 in saline. Five control mice (three males and two females) received saline. Before the first injection and 1 h after the last injection of the antagonist, blood samples were taken from the jugular vein under Metofane anesthesia, and the serum was used for GH and IGF-I determination. The animals were killed by decapitation, and pituitaries and portions of liver were quickly removed and frozen in liquid nitrogen. Pituitary GH mRNA and liver IGF-I mRNA were determined by Northern blot hybridization.

RIAs
Mouse GH and IGF-I.
GH was determined by using materials provided by Dr. A. F. Parlow (Pituitary Hormones and Antisera Center, Torrance, CA; mouse GH reference preparation AFP10783B, mouse GH antigen AFP10783B, and antirat GH-RIA-5/AFP-411S). For determination of IGF-I, all serum samples were extracted by a modified acid-ethanol cryoprecipitation method described earlier (13). This method eliminates most of the binding proteins, which can interfere in the RIA. The extracted IGF-I was measured by RIA using IGF-I (88-G4 from Genentech, San Francisco, CA) as a standard in the range of 2–500 pg/tube and also for iodination by the standard chloramine-T method. Antibody UB2–495 (a gift from Dr. Underwood and J. Van Wyk), obtained from NIDDK, was used at the final dilution of 1:14,000 in the RIA.

Human GHRH.
GHRH was measured as described previously (14). The hGHRH (1–40) antiserum (SV-95), which cross-reacts with hGHRH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29), was generated in our laboratory and used at a final dilution of 1:70,000, corresponding to 10 nL/tube of original antiserum. I¹²⁵ hGHRH(1–40) was used as the labeled hormone. The range of standard curve was 0.05–50 ng/tube of hGHRH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29), and B/B_o and nonspecific binding were 46.6% and 2.8%, respectively.

Northern blot analysis for GH and IGF-I mRNA
Pituitary and liver total RNA was isolated using TriReagent according to the manufacturers instructions (Molecular Research Center, Cincinnati, OH). RNA from whole pituitaries or 10 µg of liver RNA was separated on a 1% agarose gel containing 1.2% formaldehyde and 0.5% mg/ml ethidium bromide. Consistency of gel loading was confirmed by the intensity of ribosomal bands. RNA was transferred to a nylon membrane (Nytran Plus; Schleicher and Schuell, Inc., Keene, NH), and pituitary and liver RNA were hybridized with a ³²P-labeled rat GH complementary DNA (cDNA) (Dr. J. D. Baxter, University of California, San Francisco, CA) or rat IGF-I complementary DNA (Dr. C. T. Roberts, NIH, Bethesda, MD), respectively. Hybridization was performed with a total of 16 x 106 dpm [³²P]deoxy-CTP-labeled probe at 42 C for 24 h. The membrane was washed with 1 x saline-sodium citrate buffer (SSC)/1% sodium dodecyl sulfate at 25 C for 30 min, at 65 C for 30 min, and with 0.1 x SSC/1% SDS at 65 C for 30 min. The Northern blots were developed using a phosphorimager (Molecular Dynamics, Sunnyvale, CA), and the relative intensity of each band was evaluated by the image analysis software package, ImageQuant (Molecular Dynamics), where band intensity is expressed in pixels.

Statistical analysis of data
The superfusion data were analyzed with a computer program developed in our institute (11). Using this program, we analyzed the peaks and the baseline and calculated the net integral value of the area under the peak (the difference between the total area under the peak and the area under the baseline along the peak), representing the net amount of GH secreted in the response to stimulus. Baseline data are expressed as mean ± SEM of GH release before and between administration of the test samples.

mRNA data were evaluated by ANOVA, followed by the t test. Because GH and IGF-I levels in MT-hGHRH mice are variable, each animal was used as its own control (pretreatment levels), and repeated-measures ANOVA (followed by the all pair-wise multiple-comparison procedure of Tukey) was applied for the analysis of serum GH and IGF-I data. A P value less than 0.05 was considered significant.

	Results

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Effects of GHRH antagonists MZ-4–71 and MZ-5–156 on the GH release in vitro
The variability in serum hGHRH and GH levels of the transgenic mice was high. Serum hGHRH of transgenic mice ranged from 133–978 ng/ml and GH, from 37–93 ng/ml, whereas those of their normal littermates were less than 5 ng/ml and 8.13 ± 1.89 ng/ml, respectively. The pituitaries of these mice were used for the in vitro experiments.

GHRH antagonists MZ-5–156 and MZ-4–71 suppressed both the stimulated and basal GH release from superfused transgenic mouse pituitary cells. Figure 1 illustrates the effect of antagonists MZ-4–71 and MZ-5–156 on basal and stimulated-GH release from superfused pituitary cells of four transgenic hGHRH mice and a control mouse. Table 1 shows the basal GH values and GH responses to GHRH before and during treatment with the antagonists. These values were obtained by analyzing the superfusion data in Fig. 1. Antagonist MZ-5–156, at 30–100 nM doses, inhibited basal GH secretion by 45–49% and entirely prevented the GH response to 1 nM GHRH. MZ-4–71, at 30 nM dose, caused a 38% reduction in the basal release and 85% decrease in the stimulated GH release. At 90 min after administration of antagonists, GH responses to GHRH still did not entirely recover (Fig. 1, a, b, and c). Repeated administration of MZ-4–71, at alternating doses of 30 and 100 nM, caused a degree of inhibition of basal GH secretion similar to that of a single administration (Fig. 1d). In nontransgenic control cells, antagonist MZ-5–156, in 30- to 100-fold molar excess, produced a dose-related inhibition of GHRH-stimulated GH release. At 30-fold molar excess, this antagonist did not cause a significant suppression of stimulated GH release; but at doses 100 times greater, it induced a 77% decrease (Table 1).

View larger version (38K): [in this window] [in a new window]   Figure 1. Effects of GHRH antagonists MZ-5–156 (a, c, and e) and MZ-4–71 (b and d) at 30–100 nM concentrations on the basal and stimulated GH release in vitro in superfused pituitary cells of four transgenic hGHRH mice (Tg) (a–d) and a control mouse (e). After the initial pulse of 25 mM KCl (K), 1 nM hGHRH(1–29)NH2 (G) was administered to the cells of mice Nos. 1–3 for 30 min. Thirty minutes later, these cells were perfused with the GHRH antagonist (A) for 30 min, followed by a continuous 30-min administration of the mixture of 1 nM GHRH and 30 nM or 100 nM of the antagonists (A+G). GHRH, 1 nM (G), was then administered twice for 30 min (30 and 90 min response). (a–c, and e) In mouse No. 4, the initial pulse of K was followed by alternating administrations for 30 min of 30 and 100 nM MZ-4–71, at 30-min intervals (d). At the end of each experiment, 25 mM K was applied again. One-milliliter fractions of the superfusion medium were collected every 3 min. Numbers below the horizontal lines indicate nM concentrations of the antagonists.

In transgenic hGHRH mouse pituitary cells, 1 nM GHRH (pretreatment response), induced net integrated GH responses of 77.4 ± 4.82%, compared with the response to nonspecific stimulation of the somatotropes with the membrane-depolarizing agent KCl (25 mM for 3 min), applied at the beginning of the experiments (Fig. 1, a–c). However, in nontransgenic cells, the responsiveness of somatotropes to a specific receptor-mediated stimulus was found to be 700 ± 101% of the GH response to KCl (Fig. 1e). A negative correlation was found between the responsiveness of somatotrope cells to specific receptor-mediated stimuli and the basal GH levels; a higher GH baseline was coupled with a lower responsiveness to GHRH (Fig. 1, a–c, and e).

Effects of acute, single-dose administration of GHRH antagonists MZ-4–71 and MZ-5–156 in vivo
GHRH antagonist MZ-4–71, at 50 µg and 200 µg doses, decreased the serum GH levels in transgenic mice by 72.1% and 46.1%, respectively, at 1 h after administration (P < 0.05 vs. pretreatment mean) (Fig. 2a). A 10-µg dose of this antagonist did not significantly lower the serum GH levels, although a 40.8% reduction of mean serum GH was measured at 1 h after the injection. No dose-response correlation was found at the doses used. All three doses of antagonist suppressed the serum GH concentration to a similar 113–138 ng/ml level. At 5 h after the injection of MZ-4–71, GH levels of all treated groups returned to the pretreatment level (Fig. 2a).

View larger version (20K): [in this window] [in a new window]   Figure 2. Time-course effect of single iv administration of GHRH antagonists MZ-4–71 (a) and MZ-5–156 (b) at different doses on the serum GH level in hGHRH transgenic mice. Data points represent mean ± SEM. **, P < 0.01; *, P < 0.05 vs. pretreatment level.

Effects of chronic treatment of transgenic mice with GHRH antagonist MZ-5–156
Serum hGHRH, GH, and IGF-I.
Serum hGHRH concentrations measured before the treatment of the transgenic mice with GHRH antagonist MZ-5–156 and serum GH and IGF-I levels, before and after the therapy, are given in Table 2. Chronic administration of MZ-5–156 to transgenic mice at doses of 100 µg, twice daily for 3 days, caused a significant 56.8% decrease in the mean serum GH concentration (P < 0.05 vs. pretreatment mean) and a 39.0% reduction in the serum IGF-I level (P < 0.05 vs. pretreatment mean). Treatment of the control transgenic mice with saline did not cause significant changes in the serum GH or the IGF-I levels (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effects of chronic ip administration of GHRH antagonist MZ-5–156 at a dose of 100 µg, twice daily, for 3 days on serum GH and IGF-I levels in hGHRH transgenic mice. Control mice were treated with saline. Data points represent mean ± SEM. *, P < 0.05 vs. pretreatment level.

	Discussion

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Our results show that GHRH antagonists, such as MZ-4–71 and MZ-5–156, can inhibit GH release in transgenic mice overexpressing hGHRH. Administration of single or repeated doses of these antagonists reduced the highly elevated serum GH concentrations of transgenic mice by 40–70%, although these reduced GH levels were still much higher than the normal GH values of nontransgenic littermates. The lack of a complete suppression is likely caused by the short treatment time (single dose or 3-day treatment), which was not adequate to overcome the markedly increased GH and GH mRNA content of the pituitary caused by the prolonged stimulation of the somatotropes by hGHRH (1). To achieve a complete suppression of serum GH and IGF-I levels, a more prolonged treatment may be needed that would cause a suppression of the highly elevated pituitary GH and GH mRNA content. Additional experiments, using an extended treatment with GHRH antagonists, should be performed to determine whether this is the case.

The results of our in vitro study, using pituitary cells from transgenic mice, show that basal GH secretion from these cells is about 3–10 times higher than that from the nontransgenic type. Not only the stimulated release of GH, but also a part of basal GH secretion, can be inhibited by GHRH antagonists. Because endogenous mouse GH and the human GHRH transgene product are found within the same secretory granules of the pituitary somatotrope expressing the human GHRH transgene (6, 10), that portion of the basal GH release that can be inhibited by GHRH antagonist might be induced by human GHRH generated in the pituitary. Human GHRH is constantly released from the pituitary cells, and thus, it can exert a paracrine effect on local GHRH receptors. The other portion of the basal GH secretion, which cannot be inhibited by GHRH antagonists, might result from a certain degree of autonomy in the hyperplastic somatotropes caused by the increased GH production. This view is supported by some of our present findings, namely, that no dose-response correlation for the antagonists was found in the transgenic mice either in vivo or in vitro. It is likely that the constantly stimulated portion of the greatly increased GH secretion may be inhibited entirely, even by the low doses of the antagonists, and that this represents a maximal response. The lack of a greater suppression of basal GH secretion indicates that the residual secretion is constitutive and independent of GHRH. In contrast, MZ-5–156 showed a dose-effect correlation in normal control mouse pituitary cells in vitro.

Our in vitro results, demonstrating that the responsiveness of somatotrope cells to specific receptor-mediated stimuli is about 9 times lower in transgenic than in nontransgenic mouse pituitaries, suggest that a desensitization of the GH secretory response was induced by the excessive hGHRH production. The observation that the GHRH antagonists, used in our experiments, showed weaker inhibitory effects in normal than in the transgenic mouse pituitary cells in vitro also supports the view that desensitization of GH secretion might occur because of the long exposure of pituitary somatotropes to GHRH. It is possible that the reduced responsiveness of the somatotropes might be associated with a decreased number of GHRH binding sites on the transgenic pituitary cells, which could be saturated by lower doses of the antagonists. A recent study in rat pituitary cell cultures demonstrates that exposure of the cells to GHRH for 4 h inhibits the production of GHRH receptor by a receptor-mediated down-regulation of GHRH receptor mRNA level (see Ref.21). However, in the transgenic mouse, hGHRH levels are extremely elevated, and the duration of exposure of pituitary cells to GHRH is prolonged, so that our results cannot be compared with findings obtained from short-term experiments. Because our in vitro results were obtained from experiments on cells from three transgenic and five nontransgenic mice only, further studies should be performed in transgenic hGHRH mice to determine the mechanism of desensitization of GH secretion caused by the long-term effect of GHRH.

Chronic administration of antagonist MZ-5–156 in vivo caused a suppression of serum GH concentration but did not affect the highly elevated GH mRNA levels in mice bearing the hGHRH transgene. A longer and/or more frequent application of the GHRH antagonist might be necessary to inhibit the greatly increased GH mRNA levels of the transgenic mice. However, the suppression of serum GH by the antagonist resulted in a decrease in both IGF-I release and liver IGF-I mRNA. These findings indicate that IGF-I secretion and gene transcription are very sensitive to changes of serum GH.

It has been well documented in human beings (22, 23, 24) and in rats (25, 26), by using GHRH antagonists, that GH pulsatility is controlled by GHRH. In our previous study, using GHRH antagonist MZ-4–71 in normal rats (26), we demonstrated that a prolonged suppression of the episodic GH pulses, even with maintained basal GH levels, or a shorter but marked suppression of the basal serum GH concentration are both associated with a suppression of serum IGF-I levels. This effect was again demonstrated in the present work in hGHRH transgenic mice. Prior studies also showed a positive correlation between basal serum IGF-I and spontaneous GH secretion in young and old men (27, 28). Thus, it seems that circulating IGF-I levels are related more to the magnitude of GHRH-stimulated GH pulses than to basal GH levels, in both human (29) and rats (30).

The regulation of IGF-I gene expression by GHRH in experimental animals or humans has not been previously shown. The present work is the first to demonstrate that potent GHRH antagonists, like MZ-5–156, are capable of inhibiting IGF-I production at the mRNA level in transgenic mice overexpressing the human GHRH gene.

It recently has been reported that GHRH antagonist (N-Ac-Tyr¹,D-Arg²)GHRH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)NH₂ suppressed GH hypersecretion in a patient with acromegaly caused by ectopic GHRH production (22). Results in this patient, and our present study in the transgenic animal model of human acromegaly caused by excessive GHRH secretion, support the use of GHRH antagonists to probe the potential involvement of endogenous GHRH in patients with acromegaly.

GHRH antagonist (N-Ac-Tyr¹,D-Arg²)GHRH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)NH₂, which was used as a standard in numerous experimental studies (7, 8, 31), was at least 17 times less potent in vivo in normal rats and 100–200 times less active in vitro on inhibition of GH release than the antagonist MZ-5–156 (8, 31). The lower activity of this antagonist can be explained, in part, by its much lower binding affinity to pituitary GHRH receptors (8).

In conclusion, our study demonstrates that GHRH antagonist MZ-5–156 is capable of inhibiting GH and IGF-I release in the transgenic hGHRH mouse, which can be considered an animal model of human acromegaly caused by excessive GHRH secretion. The reduction in circulating GH induced by this antagonist is sufficient to suppress IGF-I gene expression. Our findings in vivo indicate that potent GHRH antagonists, such as MZ-4–71 and MZ-5–156, possibly could be useful for the diagnosis and treatment of disorders characterized by excessive GHRH secretion (32, 33).

	Acknowledgments

 
We thank Ms. Katalin C. Halmos, Ms. Elena Glotser, and Michael Butz for technical assistance. The gifts of materials for RIA from the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases are greatly appreciated.

	Footnotes

 
¹ This work was supported by grants to Tulane University School of Medicine and the Medical Research Service of the Veterans Affairs Department (to A.V.S.) and by Grant DK-30667 from NIH (to L.A.F.).

² On leave from the Department of Human Anatomy, University Medical School of Pecs, H-7643, Hungary.

³ On leave from the Department of Medical Chemistry, Albert Szent-Gyorgyi Medical University, Szeged H-6720, Hungary.

Received May 19, 1997.

	References

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