1 Center for Endocrinology, Metabolism and Molecular Medicine, Department of Medicine, Northwestern University Medical School, and Veterans Administration Chicago Health System, Lakeside Division, Chicago, Illinois 60611; 2 Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia 22908; and 3 Department of Endocrinology, Christie Hospital, Withington, Manchester M20 4BX, United Kingdom
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ABSTRACT |
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Growth hormone (GH) secretion is regulated by GH-releasing hormone (GHRH), somatostatin, and possibly ghrelin, but uncertainty remains about the relative contributions of these hypophysiotropic factors to GH pulsatility. Patients with genetic GHRH receptor (GHRH-R) deficiency present an opportunity to examine GH secretory dynamics in the selective absence of GHRH input. We studied circadian GH profiles in four young men homozygous for a null mutation in the GHRH-R gene by use of an ultrasensitive GH assay. Residual GH secretion was pulsatile, with normal pulse frequency, but severely reduced amplitude (<1% normal) and greater than normal process disorder (as assessed by approximate entropy). Nocturnal GH secretion, both basal and pulsatile, was enhanced compared with daytime. We conclude that rhythmic GH secretion persists in an amplitude-miniaturized version in the absence of a GHRH-R signal. The nocturnal enhancement of GH secretion is likely mediated by decreased somatostatin tone. Pulsatility of residual GH secretion may be caused by oscillations in somatostatin and/or ghrelin; it may also reflect intrinsic oscillations in somatotropes.
growth hormone-releasing hormone receptor; somatostatin; pituitary gland; short stature
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INTRODUCTION |
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GROWTH HORMONE (GH) IS SECRETED in a pulsatile or episodic manner. This secretion pattern is principally governed by hypothalamic growth hormone-releasing hormone (GHRH) and somatostatin (see Ref. 23 for review). Ghrelin (39) and/or a similar endogenous ligand for the GH secretagogue (GHS) receptor may also participate in GH pulse generation, although its role in physiological GH secretion is still largely unknown. GHRH and ghrelin stimulate pituitary GH release, whereas somatostatin inhibits it. In addition to its acute GH-releasing action, GHRH also stimulates GH synthesis (3) and somatotrope proliferation during pituitary ontogeny (41, 68).
The pulsatile pattern of GH secretion and its relation to GHRH and somatostatin rhythms has been evaluated extensively in a number of species (23), and a concept of GHRH-induced GH pulses, modulated by prevailing somatostatin tone and rapid somatostatin oscillations, has been formulated (65). A role of ghrelin in this interplay can be postulated but remains unproved. This model is supported by studies using immunoneutralization of GHRH and somatostatin (65, 80), GHRH, or somatostatin antagonists (5, 33, 34, 73), as well as direct pituitary portal sampling in experimental animals (18, 20, 55, 66). A reciprocal relationship between GHRH and somatostatin, where GHRH pulses coincide with somatostatin troughs, has been reported in rats (37, 55, 65), but this pattern is less clear in sheep or pigs, where more complex relationships between oscillations in GHRH, somatostatin, and GH prevail (8, 15, 20, 66). Thus important species as well as sex differences exist in the regulation of GH secretion. Additional complexities are imposed by nutritional status, sex steroid milieu, and feedback by insulin-like growth factor (IGF) I and GH. In humans, where hypophysial-portal sampling is not feasible, the roles of GHRH and somatostatin have been inferred from studies with continuous, presumably saturating infusions of GHRH, GHS, and somatostatin or its analogs (7, 13, 32, 35, 61, 72, 79) as well as by the use of a GHRH antagonist (33, 34). These studies have provided evidence for a predominant role of GHRH and an additive role of somatostatin in GH pattern regulation in humans. However, despite many years of ingenious investigations, the precise relative contributions of the hypophysiotropic factors to minute-to-minute GH secretion remain difficult to dissect in the intact organism.
One strategy used to gain a better understanding is to selectively remove one factor or its input from the system. This has been partially achieved by immunoneutralization studies (65, 80) or treatment with antagonists against GHRH (33, 34, 73) or somatostatin (5). However, pharmacological approaches often have limitations because of incomplete efficacy or lack of complete specificity. Genetic approaches frequently yield more definitive results because of their greater specificity. The recent identification of genetic GHRH receptor (GHRH-R) deficiency in humans presents an opportunity to examine GH secretion in the selective and complete absence of GHRH input.
We (4, 46) and others (50, 78) recently reported a syndrome of GH-deficient dwarfism resulting from a nonsense mutation in the GHRH-R gene (dwarfism of Sindh). This null mutation is predicted to truncate the GHRH-R near its amino terminus. In its homozygous state, the mutation causes severe, isolated GH deficiency with inability to respond to GHRH and several other GH provocative stimuli, including hexarelin (4, 45, 46, 50, 78). Affected patients also have pituitary hypoplasia, presumably due to lack of normal somatotrope development (49, 50). Other inactivating mutations in the GHRH-R gene exhibit a very similar phenotype (29, 31, 59, 60, 64). The murine homolog is the little (lit/lit) mouse, which harbors an inactivating missense mutation resulting in GH deficiency (14, 16, 24, 41). In all of these cases, GH production is inadequate to sustain normal somatic growth, thereby proving the crucial nature of GHRH for a functioning GH-IGF axis. However, little is known about the degree of residual GH secretion and its temporal pattern. We have employed this unique model of complete GHRH resistance at the GHRH-R to examine residual GH secretion in the absence of GHRH-R input, thereby gaining new insight into the roles of somatostatin and perhaps ghrelin (or its analogs) in generating pulsatile GH secretion.
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SUBJECTS AND METHODS |
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Four adult males (patients 4, 5, 23, and 35 in Ref. 46) with a homozygous GHRH-R defect, aged 23, 28, 28, and 30 yr, respectively, participated in the study. Their genetic, physical, and endocrine characteristics have been described in detail previously (4, 45, 46, 49). None had ever been treated with GH or any other form of endocrine therapy. They traveled from Pakistan to Chicago and were admitted to the Northwestern University General Clinical Research Center (GCRC). After acclimatization for 3 days, the studies were initiated. The study protocol was approved by the Northwestern University Institutional Review Board, and the patients gave informed consent. An intravenous cannula was placed in a forearm vein at 7 AM, and starting at 8 AM, blood (1 ml) was drawn every 10 min over a 24-h period. Blood samples were immediately added to EDTA-containing 2-ml Vacutainer tubes, cooled on ice, and centrifuged within 30 min. The volume of the EDTA solution in the Vacutainers was measured as 36.2 ± 0.28 µl (mean ± SE; n = 9). Plasma was kept frozen until assayed. The patients remained ambulatory at the GCRC, ate their meals (Pakistani food) at regular times, and were allowed to sleep without disturbance during the night. All patient activities were recorded by the nurses on a continuing basis. Blood sampling during sleep was performed from outside the room via long tubing. Blood contained in the dead space of the tubing was reinjected. All patients tolerated the procedure well, and no adverse effects were observed.
GH was measured at the University of Virginia by a sensitive chemiluminescence assay (9) with a limit of detection of 0.01 µg/l and a coefficient of variation ranging from 3.9 to 8.8% in the measurement range relevant for this study. All samples derived from the four 24-h profiles were measured in a single assay to minimize variability due to technical reasons. No correction was made for the plasma dilution (~3%) by the EDTA solution derived from the Vacutainers.
To assess a potential contribution of ghrelin (or a similar endogenous
GHS receptor ligand) to fluctuations in plasma GH levels, we also
reassayed GH levels by chemiluminescence assay in plasma specimens
obtained during a previous study (45) that had examined the effect of hexarelin in the same four patients. Those samples had
been stored at 20°C since the original assay.
To determine the degree to which intrinsic assay variability (technical
variation) contributes to apparent pulsatility, we assayed a plasma
pool (GH level 0.038 µg/l) 136 times in duplicate, arranged the
results into a pseudo-time series akin to a circadian profile, and
subjected the pseudo-series to the statistical tests used for the
circadian profiles. Technical oscillations in this pseudo-series were
found to be random and were limited to 0.01 µg/l. The pseudo-series
also showed an absence of trends, such as assay drift, and absence of
features characteristic of hormone secretion such as progressive
increases/decreases in sequential samples.
Statistical analysis of the 24-h profiles was performed by cluster
analysis, an objective pulse detection program (74), deconvolution analysis, a modeling program designed to dissect various
aspects of hormonal time series such as secretion rate and clearance
(75, 76), and the approximate entropy (ApEn) statistic, an
estimate of underlying process irregularity (52, 54)
[deconvolution and ApEn (1, 20% SD) analyses were kindly performed by J. Y. Weltman and Dr. S. M. Pincus,
respectively]. The deconvolution method was used to derive pituitary
secretion parameters and GH half-life. Pulses with amplitudes of 0.01
µg/l (and derived parameters) were ignored, because they have been shown to represent primarily technical variation (12) (for
exceptions, see legend to Fig. 1). Minor pulsations within a larger
pulse were considered to be part of that composite pulse
(28). Differences between daytime and nighttime GH
secretion were assessed by comparing 1) mean overall GH
levels and 2) mean nadir GH levels between the first and
last 12 h of the sampling period (8 AM-8 PM vs. 8 PM-8 AM), or
between 8 AM and sleep onset and sleep onset and 8 AM. Areas under the
curve in GH stimulation tests were determined by the trapezoidal rule.
Summary data are expressed as means ± SD. Comparisons between
patient and normative data were made by t-test or by the
Mann-Whitney ranked sum test where data were not normally distributed
or variances were unequal.
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RESULTS |
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The four circadian plasma GH profiles are shown in Fig.
1A. All four patients had
extremely low overall GH levels compared with normal subjects. Their
24-h mean GH levels were 0.052, 0.020, 0.021, and 0.046 µg/l,
respectively (Table 1). This was
consistent with their phenotype of isolated GH deficiency and their
lack of response to several provocative stimuli for GH. However, it is
also apparent that plasma GH levels fluctuated in a manner qualitatively similar to that in normal subjects. This included peaks
occurring at 1- to 2-h intervals, higher overall levels at night, a
major nocturnal secretory pulse, and the composite nature of secretory
episodes consisting of sequential "volleys." Pulses were detected
by both the cluster and the deconvolution programs with a high degree
(89.5%) of concordance. There was no apparent relation of GH pulses to
eating or any other recorded activity (ambulation, talking to staff,
watching television, etc.). In all four patients, nocturnal overall
plasma GH levels were significantly higher (P < 0.001)
than daytime GH levels regardless of whether nocturnal was defined as
commencing at 8 PM or at sleep onset. Similarly, in all patients, the
nocturnal nadir GH levels were significantly higher (P < 0.001) than those during the day, with the use of either of the two
definitions for nocturnal. In contrast to the patients' circadian
profiles, neither cluster nor deconvolution analysis detected any
pulses in the pseudo-time series derived from the replicate assay of a
single plasma sample.
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Figure 1B depicts the deconvoluted GH secretion profiles. The higher secretory activity during the night is apparent. Two patients showed no detectable GH secretion during the daytime, and in one (patient 2), even the nocturnal secretion was so low that reliable quantification was questionable. We omitted those data/time periods from the overall analysis. Because of this limitation, pulse frequency data in three of the four patients are based on partial circadian (primarily nocturnal) profiles.
Table 1 summarizes the numerical data descriptive of these circadian profiles and the deconvolution and ApEn results. In one patient (patient 2), plasma GH values were near the detection limit throughout the day, a fact that rendered deconvolution difficult. In another (patient 3), levels were similarly low during the daytime, imposing the same limitation for the first half of the diurnal profile. Nevertheless, the remaining information showed that there are peaks occurring on average every 94 min, similar (P = 0.231) to that in normal young (20-28 yr old) male subjects (every 76 min) when examined by the same ultrasensitive assay (M. O. Thorner, unpublished data) or in middle-aged (47 ± 3.5 yr old) males (every 72 min) when an equally sensitive immunofluorometric assay (detection limit 0.0115 µg/l) was used (71). GH plasma half-life (15.5 ± 0.8 min) was also similar to that in normal subjects (17, 28). The GH production per pulse was 45- to 450-fold lower than normal, and the daily GH production rates were 1.8-6.2% of the average GH production rate for normal males in the same age group (28). Basal (i.e., apulsatile) secretion accounted for a high proportion (57-83%) of the total GH secretion, in contrast to normal men, where ~95% of daily GH production is derived from secretory pulses (71). It should be recognized that the numerical values listed here and in Table 1 are based on only three deconvolution profiles and are partly derived from GH measurements near the assay detection limit. They should, therefore, be considered approximations.
ApEn values were intermediate between zero and maximal (~1.844 for this data series length), implying GH dynamics that were neither flat nor random (patient 2 approached maximal ApEn values, implying a high degree of randomicity). Furthermore, ApEn values were higher in all four subjects than in normal males, indicating a greater degree of disorderliness, or lack of quiescence between peaks.
The observed pulsatility in the absence of a GHRH signal raises the
question of what drives this pulsatility. To gain information about the
potential involvement of ghrelin or a related factor, we reexamined by
ultrasensitive chemiluminescence assay the response of these same
patients to hexarelin, previously determined only by conventional GH
assay (45). All four patients showed some, albeit very
minor, GH responses to hexarelin (Fig.
2). This finding is consistent with the
responses to GH-releasing peptide-2 reported for patients affected by
other inactivating mutations in the GHRH-R gene (25,
58). The responses, based on area under the curve, were
0.03-0.73% of what is typically seen in normal subjects (2, 22). There was a significant correlation (r = 0.973, P = 0.027) between mean plasma GH in a given
patient and his response to hexarelin (area under the curve). There was
no correlation between pituitary volume (49) and either
mean plasma GH or hexarelin response (P ~ 0.4).
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DISCUSSION |
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The present study was designed to evaluate GH secretion patterns in vivo in the selective absence of GHRH input to the somatotrope, secondary to genetic GHRH-R deficiency. The study demonstrates that, under those circumstances, some pulsatile GH secretion persists, albeit at greatly decreased amplitudes. Also preserved is another feature of GH secretion, namely enhanced GH secretion during the night, with a dominant sleep-related GH pulse. Rhythmicity of GH secretion dynamics is not only visually apparent but was also detected by three objective, independent methods: cluster, deconvolution, and ApEn. Without GHRH input, the amplitude of GH secretory spikes is two to three orders of magnitude lower than normal, resulting in severe clinical and biochemical GH deficiency (1, 4, 29, 46, 50, 78). The sensitive GH assay employed permitted robust GH measurements in two patients throughout the day and in an additional patient at night (100% of the GH values exceeded two times the minimal detectable concentration in patients 1 and 4, and 100% of nocturnal values exceeded twice the detection limit in patient 3). Of interest, the four patients we studied varied over a threefold range in their GH production and peak amplitude despite the fact that they carried the same genetic defect. However, there was no correlation between GH production and statural height or serum IGF-I level. Other parameters of GH pulsatility, such as pulse frequency, half-duration of peak width, and plasma half-life, were similar to those in normal subjects (28, 71). ApEn analysis confirmed that GH dynamics in these patients are both real and nonrandom and that there is a greater degree of process disorder than in normal men. Our findings are in substantial agreement with another report on GH secretion in two patients with a different inactivating mutation in the GHRH-R gene (58).
The possibility must be considered that residual GH secretion was due to incomplete resistance to GHRH, either because low levels of GHRH-Rs were present or through interaction of GHRH with other, related receptors [e.g., vasoactive intestinal peptide (VIP) and/or pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors]. Theoretically, GHRH-R expression, despite the mutation, could result from an alternative translational start site downstream from the mutation or from low-level readthrough through the stop codon. Scrutiny of the entire GHRH-R gene (Ref. 44 and GenBank accession no. AC005155) reveals no downstream start site capable of encoding a functional GHRH-R [this includes reported splice variants (57)]. Furthermore, on the basis of the strength of the mutant stop codon and its RNA context as a chain terminator, as well as information about expression levels of mutant proteins resulting from similar nonsense mutations in other human diseases, we consider it highly unlikely that readthrough occurs at a level sufficient to explain our findings. GHRH binds with low affinity to VIP/PACAP receptors (26, 40, 77), and activation of type I PACAP receptors occurs at GHRH concentrations of 10-100 nM, levels at least 100-fold higher than those needed for GHRH-R activation (21). Because the GHRH concentration in the hypophysial-portal blood of our patients is unknown, it is impossible to ascertain directly whether residual GHRH action through PACAP/VIP receptors is responsible for GH pulsatility. It is reasonable to postulate that, in GHRH-R deficiency, portal GHRH levels are increased because of a lack of GH and IGF-I feedback on hypothalamic GHRH neurons. However, hypothalamic GHRH expression in the little mouse is only about threefold above normal (19); hence, the magnitude of the postulated GHRH increase in portal blood would appear to be modest. Furthermore, Roelfsma et al. (58) found no GH response to a pharmacological dose of GHRH in two patients with GHRH-R deficiency, although those same patients were able to respond to GH-releasing peptide-2. In the aggregate, these observations do not support a significant role for GHRH in GH pulse generation in our patients. Thus, whereas we cannot formally exclude residual GHRH action mediated through alternate receptors, we consider this a remote possibility.
It is generally believed that, in humans, GH secretory spikes result primarily from GHRH pulses and that their amplitude, timing, and interpulse nadirs are modulated by somatostatin (see introductory section). Whether or not ghrelin plays a role in pulsatility is not yet known. Oscillations in somatostatin may also induce GH pulses such as those seen in the presence of constant, high levels of GHRH (72, 79) or GHS (32, 35). The paramount importance of GHRH for GH pulses of normal amplitude is obvious from the present study, in agreement with previous studies with a GHRH antagonist. Administration of the competitive GHRH antagonist (N-Ac-Tyr1,D-Arg2)-GHRH-(1-29) largely suppressed spontaneous GH secretion as well as the response to provocative pharmacological stimuli (33, 34, 51). However, in those studies, GH secretion was not completely abolished, a finding that was attributed to incomplete pharmacological blockade. The present study shows that, in men, the fundamental GH rhythm persists in a miniaturized version even in the absence of a GHRH-R signal. Therefore, the incomplete abolition of GH secretion by GHRH antagonist treatment may in part be explained by this GHRH-independent GH rhythm. Ours is a more severe paradigm of GHRH resistance than is GHRH antagonist treatment of normal subjects because of its chronicity, somatotrope hypoplasia, and impact on GH synthesis. GHRH can therefore be viewed to play a dual role in GH secretion. First, it acts as a pacesetter for GH secretory episodes through its own pulsatile pattern in the pituitary portal system. This has been directly shown in sheep, where there is a high concordance between hypophysial-portal GHRH pulses and GH pulses in the peripheral circulation (8, 20). It appears likely that the same obtains in humans, although no direct information is available. Second, GHRH acts as an important amplifier of an underlying GH rhythm, as revealed in the present study. Among the three biological actions of GHRH, somatotrope proliferation (6), GH synthesis (3), and GH release (11), it is primarily the first and second that subserve this amplifier role.
The present study also indicates that factors other than GHRH contribute to the augmented nocturnal GH secretion. Both baseline and pulse amplitudes were higher at night than during the day in our patients, a phenomenon that clearly cannot be attributed to GHRH. This is in agreement with other, more circumstantial evidence that enhanced nocturnal GH secretion is not fully attributable to GHRH (36, 48, 62, 69) and supports the concept that decreased somatostatin tone contributes to enhanced nocturnal GH secretion (70). In the absence of GHRH action, it is either somatostatin withdrawal, ghrelin pulses, or another, unknown oscillator that must be responsible for residual nocturnal pulse generation. The magnitude of the GH response to hexarelin in our patients is comparable to the spontaneous pulse amplitudes observed; hence, ghrelin would be a theoretical possibility. However, in the absence of any information about the physiological role of ghrelin in GH secretion, somatostatin would appear the most likely candidate at present. Other neuropeptides to be considered here are PACAP, VIP, galanin, and perhaps other hypophysiotropic factors acting through their own receptors. PACAP can act as a GH secretagogue in the rat but appears to lack this property in humans (see Ref. 56 for review). Similarly, VIP does not stimulate GH release in normal humans (10, 38). Galanin affects GH secretion primarily at the hypothalamic level by acting on neurons to release GHRH (47); direct pituitary effects are modest and seen only at pharmacological levels (42, 43). Thus none of these neuropeptides appears to be a compelling candidate for driving residual GH dynamics in our patients.
Pulsatility of GH secretion has been observed, albeit at varying amplitudes, under most, if not all, experimental conditions, including infusions with GHRH (72, 79), GHS (27, 32, 35), and somatostatinergic agents (7, 13, 61) alone or in combination (61), GHRH antagonist treatment (34), and now, genetic GHRH resistance. Thus, pulsatility appears as a very robust feature of GH secretion that persists despite attempts to disrupt hypothalamic input. This phenomenon suggests either that there is redundancy among hypothalamic factors in driving pulsatility or that a fundamental secretory rhythm is inherent in the somatotrope. Indeed, both possibilities may be true. The coordinate GHRH-somatostatin rhythm in the rat is an example of the former. Rat somatotropes incubated in vitro without hypothalamic peptides secrete GH in an oscillatory manner, a pattern that is related to oscillating transmembrane calcium fluxes (30). The amplitude, but not the frequency, of these oscillations is modulated by GHRH and somatostatin (67). Furthermore, monkey hemipituitary explants in a perifusion chamber secrete GH in a pulsatile pattern (63). These are examples of episodic secretory activity intrinsic to the somatotrope. These in vitro oscillations occur with a higher frequency (2-13/min and 12-15/h, respectively) than what is observed in vivo. However, the relatively short observation times of these studies (5 and 75-150 min, respectively) preclude conclusions about the potential existence of longer periodicities. Thus, it is unclear how much intrinsic somatotrope pulsatility contributes to the residual GH pulse pattern observed in the present study.
In summary, we report that patients with genetic, complete GHRH resistance exhibit residual pulsatile GH secretion with a greatly diminished amplitude yet normal frequency. The diurnal profile is qualitatively similar to normal, with enhanced GH secretion at night, including a raised baseline and higher peaks. These findings confirm the critical need for GHRH as a principal pacesetter and amplifier of GH secretion but also illustrate that other factors contribute to the pulsatile and diurnal pattern of GH secretion in men. We speculate that lowered somatostatin tone and rhythmic withdrawal are likely responsible for the nocturnally enhanced GH secretion, but we cannot exclude a role for ghrelin or a similar endogenous GHS in pulse generation. The intrinsically oscillatory nature of GH secretion by somatotropes in the absence of hypothalamic input may also contribute to the observed pulsatility.
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ACKNOWLEDGEMENTS |
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We thank Judy Y. Weltman for assistance with the deconvolution analysis and Dr. Steven M. Pincus for help with the ApEn analysis and interpretation.
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FOOTNOTES |
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This work was supported by National Institutes of Health (NIH) Grants RR-00048, RR-00847 (General Clinical Research Centers of Northwestern University and University of Virginia, respectively), a travel grant from Pharmacia & Upjohn, grants from the National Science Foundation, the Department of Veterans Affairs, and the Northwestern Memorial Foundation (G. Baumann), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32632 (M. O. Thorner).
This study was presented in part at the 82nd Meeting of the Endocrine Society, Toronto, Canada, June 2000, and reported in abstract form (Program 82nd Meeting of the Endocrine Society, p. 483).
Address for reprint requests and other correspondence: G. Baumann, Northwestern Univ. Medical School, 303 East Chicago Ave., Chicago, IL 60611, (E-mail: gbaumann{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00537.2001
Received 3 December 2001; accepted in final form 31 December 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aguiar-Oliveira, MH,
Gill MS,
de Barretto AES,
Alcantara MR,
Miraki-Moud F,
Menezes CA,
Souza AH,
Martinelli CE,
Pereira FA,
Salvatori R,
Levine MA,
Shalet SM,
Camacho-Hübner C,
and
Clayton PE.
Effect of severe growth hormone (GH) deficiency due to a mutation in the GH-releasing hormone receptor on insulin-like growth factors (IGFs), IGF-binding proteins, and ternary complex formation throughout life.
J Clin Endocrinol Metab
84:
4118-4126,
1999
2.
Arvat, E,
Gianotti L,
Grottoli S,
Imbimbo BP,
Lenaerts V,
Deghenghi R,
Camanni F,
and
Ghigo E.
Arginine and growth hormone-releasing hormone restore the blunted growth hormone-releasing activity of hexarelin in elderly subjects.
J Clin Endocrinol Metab
79:
1440-1443,
1994[Abstract].
3.
Barinaga, M,
Yamonoto G,
Rivier C,
Vale W,
Evans R,
and
Rosenfeld MG.
Transcriptional regulation of growth hormone gene expression by growth hormone-releasing factor.
Nature
306:
84-85,
1983[ISI][Medline].
4.
Baumann, G,
and
Maheshwari H.
The Dwarfs of Sindh: severe growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene.
Acta Paediatr Suppl
423:
33-38,
1997[Medline].
5.
Baumbach, WR,
Carrick TA,
Pausch MH,
Bingham B,
Carmignac D,
Robinson IC,
Houghten R,
Eppler CM,
Price LA,
and
Zysk JR.
A linear hexapeptide somatostatin antagonist blocks somatostatin activity in vitro and influences growth hormone release in rats.
Mol Pharmacol
54:
864-873,
1998
6.
Billestrup, N,
Swanson LW,
and
Vale W.
Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro.
Proc Natl Acad Sci USA
83:
6854-6857,
1986[Abstract].
7.
Calabresi, E,
Ishikawa E,
Bartolini L,
Delitala G,
Fanciulli G,
Oliva O,
Veldhuis JD,
and
Serio M.
Somatostatin infusion suppresses GH secretory burst frequency and mass in normal men.
Am J Physiol Endocrinol Metab
270:
E975-E979,
1996[ISI][Medline].
8.
Cataldi, M,
Magnan E,
Guillaume V,
Dutour A,
Conte-Devolx B,
Lombardi G,
and
Oliver C.
Relationship between hypophyseal portal GHRH and somatostatin and peripheral GH levels in the conscious sheep.
J Endocrinol Invest
17:
717-722,
1994[ISI][Medline].
9.
Chapman, IM,
Hartman ML,
Straume M,
Johnson ML,
Veldhuis JD,
and
Thorner MO.
Enhanced sensitivity growth hormone (GH) chemiluminescence assay reveals lower postglucose nadir GH concentrations in men than women.
J Clin Endocrinol Metab
78:
1312-1319,
1994[Abstract].
10.
Chihara, K,
Kaji H,
Minamitani N,
Kodama H,
Kita T,
Goto B,
Chiba T,
Coy DH,
and
Fujita T.
Stimulation of growth hormone by vasoactive intestinal polypeptide in acromegaly.
J Clin Endocrinol Metab
58:
81-86,
1984[Abstract].
11.
Cronin, MJ,
Hewlett EL,
Evans WS,
Thorner MO,
and
Rogol AD.
Human pancreatic tumor growth hormone (GH)-releasing factor and cyclic adenosine 3',5'-monophosphate evoke GH release from anterior pituitary cells: the effects of pertussis toxin, cholera toxin, forskolin, and cycloheximide.
Endocrinology
114:
904-913,
1984[Abstract].
12.
Dimaraki EV, Jaffe CA, DeMott-Friberg R, Pan W, Brown MB, and Barkan
AL. Analysis of pulsatile hormone secretion: relative importance
of biological versus technical variability (Abstract). Prog Ann
Mtg Endocrine Soc 82nd Toronto 2000, p. 487.
13.
Dimaraki, EV,
Jaffe CA,
Demott-Friberg R,
Russell-Aulet M,
Bowers CY,
Marbach P,
and
Barkan AL.
Generation of growth hormone pulsatility in women: evidence against somatostatin withdrawal as pulse initiator.
Am J Physiol Endocrinol Metab
280:
E489-E495,
2001
14.
Donahue, LR,
and
Beamer WG.
Growth hormone deficiency in `little' mice results in aberrant body composition, reduced insulin-like growth factor-I and insulin-like growth factor-binding protein-3 (IGFBP-3), but does not affect IGFBP-2, -1 or -4.
J Endocrinol
136:
91-104,
1993[Abstract].
15.
Drisko, JE,
Faidley TD,
Chang CH,
Zhang D,
Nicolich S,
Hora DF,
McNamara L,
Rickes E,
Abribat T,
Smith RG,
and
Hickey GJ.
Hypophyseal-portal concentrations of growth hormone-releasing factor and somatostatin in conscious pigs: relationship to production of spontaneous growth hormone pulses.
Proc Soc Exp Biol Med
217:
188-196,
1998[Abstract].
16.
Eicher, EM,
and
Beamer WG.
Inherited ateliotic dwarfism in mice. Characteristics of the mutation, little, on chromosome 6.
J Hered
67:
87-91,
1976[ISI][Medline].
17.
Faria, AC,
Veldhuis JD,
Thorner MO,
and
Vance ML.
Half-time of endogenous growth hormone (GH) disappearance in normal man after stimulation of GH secretion by GH-releasing hormone and suppression with somatostatin.
J Clin Endocrinol Metab
68:
535-541,
1989[Abstract].
18.
Fletcher, TP,
Thomas GB,
and
Clarke IJ.
Growth hormone-releasing hormone and somatostatin concentrations in the hypophysial portal blood of conscious sheep during the infusion of growth hormone-releasing peptide-6.
Domest Anim Endocrinol
13:
251-258,
1996[ISI][Medline].
19.
Frohman, MA,
Downs TR,
Chomczynski P,
and
Frohman LA.
Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse.
Mol Endocrinol
3:
1529-1536,
1989[Abstract].
20.
Frohman, LA,
Downs TR,
Clarke IJ,
and
Thomas GB.
Measurement of growth hormone-releasing hormone and somatostatin in hypothalamic-portal plasma of unanesthetized sheep. Spontaneous secretion and response to insulin-induced hypoglycemia.
J Clin Invest
86:
17-24,
1990[ISI][Medline].
21.
Gaylinn, BD,
DeAlmeida VI,
Lyons CE, Jr,
Wu KC,
Mayo KE,
and
Thorner MO.
The mutant growth hormone-releasing hormone (GHRH) receptor of the little mouse does not bind GHRH.
Endocrinology
140:
5066-5074,
1999
22.
Ghigo, E,
Arvat E,
Gianotti L,
Imbimbo BP,
Lenaerts V,
Deghenghi R,
and
Camanni F.
Growth hormone-releasing activity of hexarelin, a new synthetic hexapeptide, after intravenous, subcutaneous, intranasal, and oral administration in man.
J Clin Endocrinol Metab
78:
693-698,
1994[Abstract].
23.
Giustina, A,
and
Veldhuis JD.
Pathophysiology of the neuroregulation of growth hormone secretion in experimental animals and the human.
Endocr Rev
19:
717-797,
1998
24.
Godfrey, P,
Rahal JO,
Beamer WG,
Copeland NG,
Jenkins NA,
and
Mayo KE.
GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function.
Nat Genet
4:
227-232,
1993[ISI][Medline].
25.
Gondo, RG,
Aguiar-Oliveira MH,
Hayashida CY,
Toledo SP,
Abelin N,
Levine MA,
Bowers CY,
Souza AH,
Pereira RM,
Santos NL,
and
Salvatori R.
Growth hormone-releasing peptide-2 stimulates GH secretion in GH-deficient patients with mutated GH-releasing hormone receptor.
J Clin Endocrinol Metab
86:
3279-3283,
2001
26.
Gourlet, P,
Vandermeers A,
Van Rampelbergh J,
De Neef P,
Cnudde J,
Waelbroeck M,
and
Robberecht P.
Analogues of VIP, helodermin, and PACAP discriminate between rat and human VIP1 and VIP2 receptors.
Ann NY Acad Sci
865:
247-252,
1998
27.
Granda-Ayala, R,
Bowers CY,
Parulkar A,
Anand M,
Baker V,
and
Veldhuis JD.
Continuous infusion of GHRP-2 for 30 days in elderly GH deficient subjects (Abstract).
J Investig Med
48:
114A,
2000.
28.
Hartman, ML,
Faria AC,
Vance ML,
Johnson ML,
Thorner MO,
and
Veldhuis JD.
Temporal structure of in vivo growth hormone secretory events in humans.
Am J Physiol Endocrinol Metab
260:
E101-E110,
1991
29.
Hayashida, CY,
Gondo RG,
Ferrari C,
Toledo SP,
Salvatori R,
Levine MA,
Ezabella MC,
Abelin N,
Gianella-Neto D,
and
Wajchenberg BL.
Familial growth hormone deficiency with mutated GHRH receptor gene: clinical and hormonal findings in homozygous and heterozygous individuals from Itabaianinha.
Eur J Endocrinol
142:
557-563,
2000[ISI][Medline].
30.
Holl, RW,
Thorner MO,
Mandell GL,
Sullivan JA,
Sinha YN,
and
Leong DA.
Spontaneous oscillations of intracellular calcium and growth hormone secretion.
J Biol Chem
263:
9682-9685,
1988
31.
Horikawa R, Fujita K, Nakajima R, Gaylinn B, and Tanaka T. A novel
growth hormone-releasing hormone (GHRH) receptor mutation as a cause
for isolated GH deficiency in a Japanese boy with severe short stature
(Abstract). Prog 82nd Ann Mtg Endocrine Soc 82nd Toronto
2000, p. 482.
32.
Huhn, WC,
Hartman ML,
Pezzoli SS,
and
Thorner MO.
Twenty-four-hour growth hormone (GH)-releasing peptide (GHRP) infusion enhances pulsatile GH secretion and specifically attenuates the response to a subsequent GHRP bolus.
J Clin Endocrinol Metab
76:
1202-1208,
1993[Abstract].
33.
Jaffe, CA,
DeMott-Friberg R,
and
Barkan AL.
Endogenous growth hormone (GH)-releasing hormone is required for GH responses to pharmacological stimuli.
J Clin Invest
97:
934-940,
1996
34.
Jaffe, CA,
Friberg RD,
and
Barkan AL.
Suppression of growth hormone (GH) secretion by a selective GH-releasing hormone (GHRH) antagonist. Direct evidence for involvement of endogenous GHRH in the generation of GH pulses.
J Clin Invest
92:
695-701,
1993[ISI][Medline].
35.
Jaffe, CA,
Ho PJ,
Demott-Friberg R,
Bowers CY,
and
Barkan AL.
Effects of a prolonged growth hormone (GH)-releasing peptide infusion on pulsatile GH secretion in normal men.
J Clin Endocrinol Metab
77:
1641-1647,
1993[Abstract].
36.
Jaffe, CA,
Turgeon DK,
Friberg RD,
Watkins PB,
and
Barkan AL.
Nocturnal augmentation of growth hormone (GH) secretion is preserved during repetitive bolus administration of GH-releasing hormone: potential involvement of endogenous somatostatina clinical research center study.
J Clin Endocrinol Metab
80:
3321-3326,
1995[Abstract].
37.
Kasting, NW,
Martin JB,
and
Arnold MA.
Pulsatile somatostatin release from the median eminence of the unanesthetized rat and its relationship to plasma growth hormone levels.
Endocrinology
109:
1739-1745,
1981[Abstract].
38.
Kato, Y,
Shimatsu A,
Matsushita N,
Ohta H,
and
Imura H.
Role of vasoactive intestinal polypeptide (VIP) in regulating the pituitary function in man.
Peptides
5:
389-394,
1984[ISI][Medline].
39.
Kojima, M,
Hosoda H,
Date Y,
Nakazato M,
Matsuo H,
and
Kangawa K.
Ghrelin is a growth-hormone-releasing acylated peptide from stomach.
Nature
402:
656-660,
1999[ISI][Medline].
40.
Laburthe, M,
Amiranoff B,
Boige N,
Rouyer-Fessard C,
Tatemoto K,
and
Moroder L.
Interaction of GRF with VIP receptors and stimulation of adenylate cyclase in rat and human intestinal epithelial membranes. Comparison with PHI and secretin.
FEBS Lett
159:
89-92,
1983[ISI][Medline].
41.
Lin, SC,
Lin CR,
Gukovsky I,
Lusis AJ,
Sawchenko PE,
and
Rosenfeld MG.
Molecular basis of the little mouse phenotype and implications for cell type-specific growth.
Nature
364:
208-213,
1993[ISI][Medline].
42.
Lindström, P,
and
Savendahl L.
Effects of galanin on growth hormone release in isolated cultured rat somatotrophs.
Acta Endocrinol
129:
268-272,
1993[ISI][Medline].
43.
Lopez, FJ,
Meade EH, Jr,
and
Negro-Vilar A.
Development and characterization of a specific and sensitive radioimmunoassay for rat galanin: measurement in brain tissue, hypophyseal portal and peripheral serum.
Brain Res Bull
24:
395-399,
1990[ISI][Medline].
44.
Maheshwari HG and Baumann G. Genomic organization and structure of
the human growth hormone releasing hormone receptor gene (Abstract).
Prog Ann Mtg Endocrine Soc 79th Minneapolis 1997, p. 156.
45.
Maheshwari, HG,
Rahim A,
Shalet SM,
and
Baumann G.
Selective lack of growth hormone (GH) response to the GH-releasing peptide hexarelin in patients with GH-releasing hormone receptor deficiency.
J Clin Endocrinol Metab
84:
956-959,
1999
46.
Maheshwari, HG,
Silverman BL,
Dupuis J,
and
Baumann G.
Phenotype and genetic analysis of a syndrome caused by an inactivating mutation in the growth hormone-releasing hormone receptor: dwarfism of Sindh.
J Clin Endocrinol Metab
83:
4065-4074,
1998
47.
Maiter, DM,
Hooi SC,
Koenig JI,
and
Martin JB.
Galanin is a physiological regulator of spontaneous pulsatile secretion of growth hormone in the male rat.
Endocrinology
126:
1216-1222,
1990[Abstract].
48.
Martha, PM,
Blizzard RM,
McDonald JA,
Thorner MO,
and
Rogol AD.
A persistent pattern of varying pituitary responsivity to exogenous growth hormone (GH)-releasing hormone in GH-deficient children: evidence supporting periodic somatostatin secretion.
J Clin Endocrinol Metab
67:
449-454,
1988[Abstract].
49.
Murray, RA,
Maheshwari HG,
Russell EJ,
and
Baumann G.
Pituitary hypoplasia in patients with a mutation in the growth hormone-releasing hormone receptor gene.
ANJR Am J Neuroradiol
21:
685-689,
2000
50.
Netchine, I,
Talon P,
Dastot F,
Vitaux F,
Goossens M,
and
Amselem S.
Extensive phenotypic analysis of a family with growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene.
J Clin Endocrinol Metab
83:
432-436,
1998
51.
Ocampo-Lim, B,
Guo W,
DeMott-Friberg R,
Barkan AL,
and
Jaffe CA.
Nocturnal growth hormone (GH) secretion is eliminated by infusion of GH-releasing hormone antagonist.
J Clin Endocrinol Metab
81:
4396-4399,
1996[Abstract].
52.
Pincus, SM.
Approximate entropy as a measure of system complexity.
Proc Natl Acad Sci USA
88:
2297-2301,
1991[Abstract].
53.
Pincus, SM,
Gevers EF,
Robinson IC,
van den Berg G,
Roelfsema F,
Hartman ML,
and
Veldhuis JD.
Females secrete growth hormone with more process irregularity than males in both humans and rats.
Am J Physiol Endocrinol Metab
270:
E107-E115,
1996
54.
Pincus, SM,
and
Keefe DL.
Quantification of hormone pulsatility via an approximate entropy algorithm.
Am J Physiol Endocrinol Metab
262:
E741-E754,
1992
55.
Plotsky, PM,
and
Vale W.
Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal circulation of the rat.
Science
230:
461-463,
1985[ISI][Medline].
56.
Rawlings, SR,
and
Hezareh M.
Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland.
Endocr Rev
17:
4-29,
1996[ISI][Medline].
57.
Rekasi, Z,
Czompoly T,
Schally AV,
and
Halmos G.
Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers.
Proc Natl Acad Sci USA
97:
10561-10566,
2000
58.
Roelfsma, F,
Biermasz NR,
Veldman RG,
Veldhuis JD,
Frolich M,
Stokvis-Brantsma WH,
and
Wit JM.
Growth hormone (GH) secretion in patients with an inactivating defect of the GH-releasing hormone (GHRH) receptor is pulsatile: evidence for a role for non-GHRH inputs into the generation of GH pulses.
J Clin Endocrinol Metab
86:
2459-2464,
2001
59.
Salvatori, R,
Fan X,
Phillips JA, III,
Espigares-Martin R,
Martin de Lara I,
Freeman KL,
Plotnick L,
Al-Ashwal A,
and
Levine MA.
Three new mutations in the gene for the growth hormone (GH)-releasing hormone receptor in familial isolated GH deficiency type IB.
J Clin Endocrinol Metab
86:
273-279,
2001
60.
Salvatori, R,
Hayashida CY,
Aguiar-Oliveira MH,
Phillips JA, III,
Souza AH,
Gondo RG,
Toledo SP,
Conceicao MM,
Prince M,
Maheshwari HG,
Baumann G,
and
Levine MA.
Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene.
J Clin Endocrinol Metab
84:
917-923,
1999
61.
Sassolas, G,
Khalfallah Y,
Chayvialle JA,
Cohen R,
Merabet S,
Casez JP,
Calvet P,
and
Cabrera P.
Effects of the somatostatin analog BIM 23014 on the secretion of growth hormone, thyrotropin, and digestive peptides in normal men.
J Clin Endocrinol Metab
68:
239-246,
1989[Abstract].
62.
Schriock, EA,
Hulse JA,
Harris DA,
Kaplan SL,
and
Grumbach MM.
Evaluation of hypothalamic dysfunction in growth hormone (GH)-deficient patients using single versus multiple doses of GH-releasing hormone (GHRH-44) and evidence for diurnal variation in somatotroph responsiveness to GHRH in GH-deficient patients.
J Clin Endocrinol Metab
65:
1177-1182,
1987[Abstract].
63.
Stewart, JK,
Clifton DK,
Koerker DJ,
Rogol AD,
Jaffe T,
and
Goodner CJ.
Pulsatile release of growth hormone and prolactin from the primate pituitary in vitro.
Endocrinology
116:
1-5,
1985[Abstract].
64.
Stokvis-Brantsma, WH,
Blankenstein O,
Oostdijk W,
Roelfsma F,
Pfäffle R,
and
Wit JM.
Endocrine findings and growth response to combined GH and GnRH agonist treatment in 2 siblings with a novel mutation of the GHRH receptor (Abstract).
Horm Res
53, Suppl2:
57,
2000[ISI][Medline].
65.
Tannenbaum, GS,
and
Ling N.
The interrelationship of growth hormone (GH)-releasing factor and somatostatin in generation of the ultradian rhythm of GH secretion.
Endocrinology
115:
1952-1957,
1984[Abstract].
66.
Thomas, GB,
Cummins JT,
Francis H,
Sudbury AW,
McCloud PI,
and
Clarke IJ.
Effect of restricted feeding on the relationship between hypophysial portal concentrations of growth hormone (GH)-releasing factor and somatostatin, and jugular concentrations of GH in ovariectomized ewes.
Endocrinology
128:
1151-1158,
1991[Abstract].
67.
Thorner, MO,
Holl RW,
and
Leong DA.
The somatotrope: an endocrine cell with functional calcium transients.
J Exp Biol
139:
169-179,
1988[Abstract].
68.
Treier, M,
Gleiberman AS,
O'Connell SM,
Szeto DP,
McMahon JA,
McMahon AP,
and
Rosenfeld MG.
Multistep signaling requirements for pituitary organogenesis in vivo.
Genes Dev
12:
1691-1704,
1998
69.
Van Cauter, E,
Caufriez A,
Kerkhofs M,
Van Onderbergen A,
Thorner MO,
and
Copinschi G.
Sleep, awakenings, and insulin-like growth factor-I modulate the growth hormone (GH) secretory response to GH-releasing hormone.
J Clin Endocrinol Metab
74:
1451-1459,
1992[Abstract].
70.
Van Cauter, E,
Plat L,
and
Copinschi G.
Interrelations between sleep and the somatotropic axis.
Sleep
21:
553-566,
1998[ISI][Medline].
71.
Van den Berg, G,
Frolich M,
Veldhuis JD,
and
Roelfsma F.
Growth hormone secretion in recently operated acromegalic patients.
J Clin Endocrinol Metab
79:
1706-1715,
1994[Abstract].
72.
Vance, ML,
Kaiser DL,
Evans WS,
Furlanetto R,
Vale W,
Rivier J,
and
Thorner MO.
Pulsatile growth hormone secretion in normal man during a continuous 24-hour infusion of human growth hormone releasing factor (1-40). Evidence for intermittent somatostatin secretion.
J Clin Invest
75:
1584-1590,
1985[ISI][Medline].
73.
Varga, JL,
Schally AV,
Csernus VJ,
Zarandi M,
Halmos G,
Groot K,
and
Rekasi Z.
Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted in vivo activities.
Proc Natl Acad Sci USA
96:
692-697,
1999
74.
Veldhuis, JD,
and
Johnson ML.
Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection.
Am J Physiol Endocrinol Metab
250:
E486-E493,
1986
75.
Veldhuis, JD,
and
Johnson ML.
Deconvolution analysis of hormone data.
Methods Enzymol
210:
539-575,
1992[ISI][Medline].
76.
Veldhuis, JD,
and
Johnson ML.
Specific methodological approaches to selected contemporary issues in deconvolution analysis of pulsatile neuroendocrine data.
Methods Neurosci
28:
25-92,
1995.
77.
Waelbroeck, M,
Robberecht P,
Coy DH,
Camus JC,
De Neef P,
and
Christophe J.
Interaction of growth hormone-releasing factor (GRF) and 14 GRF analogs with vasoactive intestinal peptide (VIP) receptors of rat pancreas. Discovery of (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 as a VIP antagonist.
Endocrinology
116:
2643-2649,
1985[Abstract].
78.
Wajnrajch, MP,
Gertner JM,
Harbison MD,
Chua SC, Jr,
and
Leibel RL.
Nonsense mutation in the human growth hormone-releasing hormone receptor causes growth failure analogous to the little (lit) mouse.
Nat Genet
12:
88-90,
1996[ISI][Medline].
79.
Webb, CB,
Vance ML,
Thorner MO,
Perisutti G,
Thominet J,
Rivier J,
Vale W,
and
Frohman LA.
Plasma growth hormone responses to constant infusions of human pancreatic growth hormone releasing factor. Intermittent secretion or response attenuation.
J Clin Invest
74:
96-103,
1984[ISI][Medline].
80.
Wehrenberg, WB,
Brazeau P,
Luben R,
Bohlen P,
and
Guillemin R.
Inhibition of the pulsatile secretion of growth hormone by monoclonal antibodies to the hypothalamic growth hormone releasing factor (GRF).
Endocrinology
111:
2147-2148,
1982[ISI][Medline].