C'hronohro/og)' In/ernrr/ri~nul
Vol. 9. No. 5 , pp. 371-379
a 1992 International Society of Chronohiology
Rhythmic and Nonrhythmic Modes of Anterior Pituitary Gland Secretion
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Johannes D. Veldhuis, Michael L. Johnson, *German Lizarralde, and *Ali Iranmanesh Division o f Iindocrinology and Metuholism and the Interdisciplinary Graditate Biophy.sic..s Program, Deparimen 1.s of Internal Mcdicinc and Pharmacology. National Science Foiindation Center .Ibr Biological Timing, Univcrsitj?01' Virginia IIealth Scimcec. Center, Charlorriw~ille:and *Division of Endocrinology and Mc.tuholism, Deparimcni oj'Intprnal Mcdicinc., Sali>m Veterans Administration Midical CtwPr, Salem, Virginia, U.S.A.
Summary: Because of confounding effects of subject-specific and hormone-speci-
fic metabolic clearance, the nature of anterior pituitary secretory events in vivo is difficult to ascertain. We review an approach to this problem, in which deconvolution analysis is used to dissect the underlying secretory behavior of an endocrine gland quantitatively from available serial plasma hormone concentration measurements assuming one- or two-compartment elimination kinetics. This analytical tool allows one to ask the following physiological questions: (a) does the anterior pituitary gland secrete exclusively in randomly dispersed bursts, and/or does a tonic (constitutive) mode of interburst hormone secretion exist? and (b) what secretory mechanisms generate the circadian or nyctohemeral rhythms in blood concentrations of pituitary hormones? Waveform-independent deconvolution analysis of 24-h serum hormone concentration profiles of immunoreactive growth hormone (GH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and P-endorphin in normal men sampled every 10 min showed that (a) anterior pituitary gland secretion in vivo occurs in an exclusively burstlike mode for all hormones except TSH and prolactin (for the latter two, a mixed burst and basal mode pertains); (b) significant nyctohemeral regulation of secretory burst frequency alone is not demonstrable for any hormone; (c) prominent 24-h variations in secretory-burst amplitude alone are delineated for ACTH and LH; (d) TSH, G H , and @-endorphinare both frequency and amplitude controlled; (e) prolactin manifests 24-h rhythms in both secretory-burst amplitude and nadir secretory rates; (f) n o significant diurnal variations occur in FSH secretory parameters; and (8) a fixed hormone half-life yields good fits of the 24-h serum hormone concentration series, which indicates that there is no need to introduce diurnal variations in hormone half-lives. In summary, the normal human anterior pituitary gland appears t o release its various (g1yco)protein hormones via intermittent ~
~
Received September 3, 199 1; accepted with revisions December 13, 1991. Address correspondence and reprint requests to J. D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health SciencesCenter, Charlottesville,VA 22908, U.S.A. Presented in part at the 20th International Conference on Chronobiology, Tel Aviv, Israel, June 16-2 I , 1991.
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secretory episodes that are apparently unassociated with significant basal hormone secretion,except in the case of TSH and prolactin. Hormone-specific amplitude and/or frequency control of secretory burst activity over 24 h provides the mechanistic basis for the classically recognized nyctohemeral rhythms in plasma concentrations of adenohypophyseal hormones in the human. Key Words: Secretion-Clearance-Pulsatile-Rh ythm-Hormone.
Twenty-four-hour variations in plasma concentrations of various anterior pituitary hormones have been recognized for several decades (1-3). Implicit in the interpretation of such nyctohemeral and in some cases circadian variations is the view that pulses of hormone release are putatively superimposed on a baseline of tonic hormone secretion (4). An alternative model would require modulated ultradian secretory bursts with variable amplitude and frequency, so as to give rise to the 24-h variation in plasma hormone concentrations, even without the presence of, or variation in, any basal component of hormone release (5). In addition, most interpretations of 24-h serum hormone concentration rhythms assume that there is no systematic variation in hormone metabolic clearance as a function of time ofday. However, exactly how the 24-h rhythms in circulating amounts of hormones are generated mechanistically, or are assembled from and related to underlying ultradian secretory activity, has not been systematically investigated. Here, we have summarized some recent concepts of rhythmic and nonrhythmic modes of anterior pituitary hormone secretion, and enunciated a particular model in which 24-h rhythms in plasma concentrations of anterior pituitary hormones can be constructed by specific amplitude and/or frequency modulation of ultradian secretory bursts with or without basal (time-invariant) hormone secretion, and without any requirement to alter the halftime of hormone disappearance as a function of time of day. Mathematically explicit treatment of the problem of ultradian and nyctohemeral regulation of anterior pituitary hormone secretion can be accomplished by several techniques, including that of deconvolution analysis, in which underlying rates of hormone secretion are estimated from the serum hormone concentration profiles. Specifically, deconvolution analysis attempts to unmask underlying hormone secretory rates that give rise to the plasma hormone concentration changes (5-9). By dissecting the intrinsic secretion profile from the observed plasma hormone concentration patterns, and applying cosinor analysis to the secretory burst amplitudes and interpulse intervals so defined over 24 h, one can investigate nyctohemeral modulation of hormone secretory burst amplitude and/or frequency. Moreover, cosinor analysis of significant (nonzero) basal hormone secretion rates over 24 h can be used to investigate possible nyctohemeral variations in rates of basal hormone release.
METHODS Sampling Protocol
Healthy male volunteers 2 1-50 years of age underwent blood sampling at 10-min intervals for 24 h after overnight adaptation to the Clinical Research Unit. Blood withdrawal was performed via an indwelling venous catheter placed in a forearm vein
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2 1 h before 08:OO h, when sampling was begun. Eucaloric meals were served at 08:30,
12:30, and 17:OO h. Subjects had lights on at 07:OO and off at 23:OO h. Naps were not permitted during lights on. Volunteers were allowed to ambulate in the Clinical Research Center, but vigorous exercise, cigarette smoking, and caffeine ingestion were not permitted. Plasma or serum samples were then used for the subsequent assay of growth hormone (GH), adrenocorticotropic hormone (ACTH), P-endorphin, and thyroid-stimulating hormone (TSH) (immunoradiometric assay), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (radioimmunoassay). We studied six to eight men in each hormone group, after provision of written and informed consent approved by the Human Investigation Committee of the University of Virginia School of Medicine. These endocrine profiles have been reported previously (5). Deconvolution Analysis A waveform-independent deconvolution technique was used for most of the analysis reviewed here, in which sample hormone concentrations are assumed to arise from the combined effects of individual sample secretion rates and mono- or biexponential hormone disappearance kinetics (5,7). In this model, the amount of hormone secreted per unit time is estimated by nonlinear least-squares parameter estimation. Sample secretion rates were determined for each observation (145 samples distributed uniformly over 24 h in the 10-min sampling paradigm), with statistical confidence limits that combined wherever possible the experimental uncertainty in the immunoassay and the hormone half-life estimates (4,7). We used the following half-lives (and corresponding fractional amplitude for the second component) for each hormone: GH 3.5 and 2 1 min, fraction 0.63; LH 18 and 90 min, fraction 0.37; FSH 102 and 398 min, fraction 0.48; ACTH 3.5 and 14 min, fraction 0.33; and monocomponent half-lives for TSH, prolactin, and @-endorphinof 35, 25, and 45 min, respectively (5). Deconvolution analysis permitted us to (a) plot hormone secretory burst amplitude as a function of 24-h clocktime, where amplitude is defined as the maximal rate of hormone secretion attained within a burst; (b) relate interburst interval duration to 24-h clocktime (e.g., consecutive secretory bursts centered at 09:OO and 1O:OO h would have an interburst interval of 60 min centered at 09:30 h); and (c) delineate basal secretion rates as a function of time of day and night, in which basal secretion is defined as the hormone secretion rate in a nadir that precedes or follows a secretory burst (5,7). Cosinor analysis is then applied to evaluate whether there are 24-h rhythms in hormone secretory burst amplitude, interburst interval, or basal (nadir) secretory rates. We applied cosinor analysis to the set of all observations in the group of six to eight men for any given hormone, and developed joint statistical confidence limits for the fitted period, amplitude, and acrophase. Only amplitudes whose statistical confidence intervals exceeded zero by p < 0.05 were considered further here. In some illustrations, we used an alternative multiparameter waveform-specific method of deconvolution analysis (6,8,9). RESULTS AND DISCUSSION Deconvolution analysis provides a technique for evaluating underlying hormone secretion as a contributor to both circadian and ultradian rhythms in serum hormone
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FIG. I . Serum immunoradiometrically assayable LH concentrations (top, middle) and deconvolutioncalculated LH secretory rates (bottom) in blood collected at 5-min intervals for 24 h in a normal young man. Qualitatively similar results are obtained using a 10-min sampling interval. Vertical bars through the data points denote the dose-dependent intraasay standard deviations. The top panel gives the simple 24-h cosine fit of the serum LH concentrations. The middle panel depicts the deconvolution-specified fit of the data. Deconvolution by the multiparameter method (6) predicted the occurrence of individual punctuated LH secretory bursts with almost no tonic secretion (bottom). As discussed further in the text. cosinor analysis can he applied to not only the serum concentrations (top) but also the computer-estimated hormone secretory profiles (bottom) or their specific measures to assess 24-h variations in the frequency and/or amplitude of discrete hormone secretory bursts.
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concentrations. This concept is illustrated for the pituitary hormone LH using the multiparameter deconvolution technique (6) in Fig. 1. We have used deconvolution analysis to evaluate the experimental question, “How do ultradian rhythms in pituitary hormone secretion contribute to the 24-h variations in plasma hormone concentrations?’ To this end, we applied either a waveform-independent (5) or a model-specific muitiparameter (6) deconvolution technique to pituitary hormone concentration time series generated by sampling blood at 10-min intervals over 24 h. This sampling protocol yields inferences similar to those achieved by sampling blood every 5 min (8,9). Waveform-independent deconvolution analysis permitted quantitative estimates of the amplitudes (maximal rates of secretion attained within each secretory burst) and the temporal positions of hormone secretory episodes as well as the degree of concurrent interpulse secretion that statistically exceeds zero (5,7). By applying cosinor analysis to the plots of hormone secretory burst amplitude, intersecretory burst intervals, or basal (nadir) hormone secretory rates over 24 h, we could investigate the mechanisms by which nyctohemeral rhythms in plasma hormone concentrations are assembled. The results of waveform-specific multiparameter deconvolution analysis (6) are illustrated in Fig. 2, which can be used to show distinct mechanisms by which the individual anterior pituitary hormones achieve 24-h variations in concentration. For example, variations in plasma ACTH concentrations can be attributed to diurnally regulated ultradian ACTH secretory burst amplitude without any consistent 24-h variation in burst frequency (10). Our studies of seven anterior pituitary hormones in normal men using waveformindependent deconvolution analysis have indicated that the 24-h rhythms in the plasma concentration of three hormones are generated by both frequency and amplitude control of discrete secretory bursts (5). In particular, in the cases of GH, TSH, and &endorphin, there were significant 24-h variations in detectable hormone secretory burst number (inversely related to interburst interval), and secretory burst amplitude. In contrast, secretory burst amplitude alone was the modulated feature for both ACTH and LH release. Of interest, no hormone demonstrated significant nyctohemeral regulation of secretory burst frequency alone. Thus, amplitude modulation of hormone secretory bursts was observed for five of seven hormones (TSH, GH, P-endorphin, ACTH, and LH), and combined frequency and amplitude control for three (TSH, GH, and 0-endorphin). Deconvolution permitted us to infer that there was a mixed burst and constitutive mode of secretion for TSH and prolactin. This finding is of physiological interest, because both these hormones can be secreted in response to a common secretagogue, TSH-releasinghormone (TRH), and both are subject to tonic inhibition by the neurotransmitter dopamine. Indeed, other studies have demonstrated a relatively high cross-correlation between serum TSH and prolactin concentrations over 24 h (1 1). The basal secretion of TSH was not significantly regulated over 24 h. However, there were significant nyctohemeral variations in prolactin interpulse nadir secretory rates (5). The mechanisms that regulate interpeak nadir hormone secretion are not known, but the fact that prolactin is susceptible to tonic dopamine inhibition in normal physiology may offer a clue that nyctohemeral variations in dopaminergic tone contribute to the 24-h rhythm in nadir prolactin secretion rates.
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FIG. 2. Illustrative profiles of 24-h variations in calculated ACTH secretory burst amplitudes (A) or ACTH intersecretory burst intervals (mint (B) in eight normal men. Each volunteer underwent blood sampling at 10-min intervals for 24 h. Plasma ACTH time series were assessed by multiparameter deconvolution analysis (6). The resultant deconvolution measures (ACTH secretory burst interpulse interval and amplitude) are shown for the group of eight men considered together. Adapted with permission (10).
One hormone, FSH as assessed by radioimmunoassay, exhibited no significant diurnal variations in its secretory parameters. This finding may reflect the true absence of diurnal variations in FSH release, and/or indicate the difficulty in accurately estimating in vivo FSH secretion and/or bioactivity by conventional immunological
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means. The latter possibility is suggested by occasionally (but not uniformly) divergent inferences made when FSH activity in plasma is measured by radioimmunoassay or in vitro bioassay (12). Therefore, further studies should be conducted of the 24-hour rhythms in plasma FSH bioactivity. Indeed, a recent study of plasma FSH concentrations assessed by two-site immunoradiometric assay, which correlates highly with in vitro FSH bioassay, has indicated a measurable 24-h rhythm in serum FSH immunoradiometric activity ( 13). This rhythm in plasma FSH concentrations was abolished by the infusion of either a potent androgen or estrogen in healthy men. Significant variations in hypothalamic neurotransmitter concentrations, turnover, and release rates have been described in experimental animals under different conditions of steroid hormone treatment, metabolic milieu, physiological state, and so forth. We hypothesize that changes in the frequency of anterior pituitary hormone release over 24 h result from diurnal control of relevant hypothalamic burst-generating systems. For example, in the case of LH, mediobasal hypothalamic release of the decapeptide gonadotropin releasing hormone (GnRH) occurs in discrete bursts, which presumably control the frequency of episodic LH release by the anterior pituitary gland ( 14,15).Thus, variations in LH secretory burst frequency can be assumed to mirror diurnal changes in hypothalamic GnRH “pulse generator” activity. This interpretation assumes that GnRH secretory episodes are coupled in a consistent manner to pituitary LH release, and that this coupling efficiency does not vary as a function of time of day. Although evidence is not available to disallow this assumption, limited studies are available that directly test it. Of interest, LH secretory burst frequency does vary as a function of time of day in women at certain stages of the menstrual cycle ( 16). In relation to GH release, control mechanisms include both withdrawal of the hypothalamic inhibitory tetradecapeptide somatostatin and activation of GH-releasing hormone (GHRH) secretion ( 17). For example, decreased somatostatin and increased GHRH release could account for augmented G H secretory burst frequency in association with stage three and four sleep ( 18). Indeed, the intravenous injection of GHRH at selected sleep stages in normal human volunteers shows enhanced responsiveness to GHRH in stage three and four sleep, which can be interpreted to signify reduced endogenous somatostatin inhibitory tone ( 19). Less is known about the regulation of TSH and &endorphin secretory burst frequency. Indeed, in the case of TSH, the presumptive in vivo secretagogue may include the tripeptide TRH, and one or more incompletely identified TSH releasing factors. The finding that 0-endorphin pulse frequency varies diurnally is consistent with some time-dependent regulation of pituitary corticotrope secretory burst activity, putatively by hypothalamic factors such as corticotropin-releasing hormone and/ or arginine vasopressin, or significant 24-h variations in pituitary corticotrope responsiveness to these secretagogues (20). To our knowledge, definitive in vivo information is not available to clarify the exact mechanisms that generate an apparent 24-h variation in the frequency of secretory bursts of TSH and 0-endorphin. Nyctohemeral regulation of pituitary hormone secretory burst amplitude might be accounted for by several plausible mechanisms. First, varying hypothalamic stimulus strength may elicit different amounts of hormone secretion per burst as a function of time of day, sleep stage, and so forth. Secondly, variations in pituitary target-cell
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responsiveness to the hypothalamic releasing factors may modulate the amount of hormone secreted at various times over the day and night. Such changes in pituitary responsiveness could reflect intrinsic paracrine and autocrine regulation of pituitary glandular function, as well as the important effects of systemically delivered secretagogues, inhibitors, trophic factors or feedback signals (e.g., steroid hormones, growth factors such as insulin-likegrowth factor or type I (IGF-I), etc.). Detailed information about variations in diurnal feedback signal strength is not generally available for most hormones. However, in the case of testosterone in the male, reduced plasma testosterone concentrations occur in the late afternoon and early evening hours (2 l), which mimics the general pattern for serum cortisol concentrations (22,23). The nadirs of such steroid hormone concentrations in general correspond with the nadirs in corresponding pituitary hormone concentrations (LH and ACTH). After several hours of delay, increased pituitary hormone secretion occurs with resultant higher concentrations of the steroid hormones. Although little information is available regarding the importance of time of day in the negative-feedback signal strength of sex steroid hormones, variations in corticotrope sensitivity to the feedback effects of cortisol in the morning and evening have been described, as well as significant diurnal variations in adrenal responsiveness to available ACTH (22). In summary, we have reviewed a model of anterior pituitary gland secretion in vivo, in which glycoprotein hormones are released via punctuated secretory episodes, which are typically unassociated with tonic basal (interpulse) hormone secretion, except in the case of TSH and prolactin (5). This model is based on deconvolution analysis of 24-h serum hormone concentration profiles. It predicts that 24-h variations in secretory burst amplitude contribute to the nyctohemeral rhythms in plasma ACTH and LH Concentrations. On the other hand, both amplitude and frequency control of episodic TSH, GH, and /?-endorphin secretion are responsible for the 24-h rhythms in the blood content of these hormones. Little diurnal variation occurs in immunoreactive FSH secretory parameters, whereas prolactin exhibits 24-h rhythms in both secretory burst amplitude and nadir secretory rates. Accordingly, we infer that hormone-specific mechanisms of amplitude and/or frequency control of discrete secretory burst activity can provide a basis for the classically recognized nyctohemeral rhythms in plasma concentrations of anterior pituitary hormones in normal men. Acknowledgment: We thank Patsy Craig for her skillful preparation of the manuscript, Paula P. Azimi for the artwork, Brenda Grisso for laboratory assistance, and Sandra Jackson and the expert nursing staff at the University of Virginia Clinical Research Center for conduct of the research protocols. This work was supported in part by National Institutes of Health (NIH) Grant No. RR 00847 to the Clinical Research Center of the University of Virginia, RCDA No. 1 KO4 H D 00634 (J.D.V.), Veterans Administration Medical Research Funds (A.I.), NIH Grant Nos. AM-30302 and GM-28928 (M.L.J.), Diabetes and Research Training Center Grant No. 5 P60 AM 22 125-05, NIH-supported Clinfo Data Reduction Systems, and the NSF Center for Biological Timing (J.D.V., M.L.J.).
REFERENCES 1. Weitzman ED, Boyar RM, Kapen S, Hellman L. The relationship ofsleep and sleep stages to neuroendocrine secretion and biological rhythms in man. Recent Prog Horm Res 1975;3 1:399-446.
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2. Daughaday WH. The anterior pituitary gland. In: Williams RH, ed. Textbook c~fendocrinology.Philadelphia: WB Saunders, 1981:73-86. 3. Brabant G, Brabant A, Ranft U, et al. Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration. J Clin Endocrinol Metab 1987;65:83-8. 4. Veldhuis JD. A parsimonious model of amplitude and frequency modulation of episodic hormone secretory bursts as a mechanism for ultradian signaling by endocrine glands. In: Wever R, Kleitman N, eds. Ultrudiun rhyihms in I@ processe.s: un inquiry into ,fnndumentul principles. London: SpringerVerlag, 1992 (in press). 5. Veldhuis JD. Iranmanesh A, Johnson ML, Lizarralde G. Twenty-four hour rhythms in plasma concentrations of adenohypophysial hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. J Clin Endocrinol Metub 1990;7 I : 16 16-23. 6. Veldhuis JD, Carlson ML, Johnson ML. The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proc Natl Acud Sci US.4 1987;84:7686-90. 7. Veldhuis JD, Johnson ML. Deconvolution analysis of hormone data. Methods Enzyrnol 1992;210:539-75. 8. Hartman ML, Faria ACS, Vance ML, Johnson ML, Thorner MO, Veldhuis JD. Temporal structure of in vivo growth hormone secretory events in man. Am J Phj)siol 199 I ;260:E 10I - 10. 9. Veldhuis JD, Johnson ML. In vivo dynamics of luteinizing hormone secretion and clearance in man: assessment by deconvolution mechanics. J Clin Endocrinol Metab 1988;66:I29 1-300. 10. Veldhuis JD, lranmanesh A, Johnson ML, Lizarralde G. Amplitude, but not frequency, modulation of ACTH secretory bursts gives rise to the nyctohemeral rhythm of the corticotropic axis in man. J Clin Endocrinol Metub 1990;7I :452-63. I 1. Samuels MH. Veldhuis JD, Henry P, Ridgway EC. Pathophysiology of pulsatile and co-pulsatile release of thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, and alpha subunit. J Clin Endocrinol Metah 1990;71:425-32. 12. Urban RJ, Padmanabhan V, Beitins I, Veldhuis JD. Metabolic clearance of human follicle-stimulating hormone in man as measured by radioimmunoassay, immunoradiometric, and in vitro Sertoli-cell bioassay. J Cllin EndocrinolMetuh 1991;73:818-23. 13. Urban RJ, Veldhuis JD. Specific regulatory actions of dihydrotestosterone and estradiol on the dynamics of FSH secretion and clearance in man. .I Androl 1991:12:27-35. 14. Marshall JC, Kelch RP. Gonadotropin-releasing hormone. Role of pulsatile gonadotropin secretion in the regulation of reproduction. N Bngl J Mcxl 1986;315:1459-67. 15. Clark IJ, Cummins JT. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 1982; I I 1: 1737-9. 16. Sollenberger ML, Carlson EC, Johnson ML, Veldhuis JD, Evans WS. Specific physiological regulation of LH secretory events throughout the human menstrual cycle: new insights into the pulsatile mode of gonadotropin release. J Newoendocrinol I990;2:845-52. 17. Plotsky PM, Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal circulation of the rat. Scicnce 1986:230:461-5. 18. Holl RW, Hartman ML, Veldhuis JD, Taylor WM, Thorner MO. Thirty-second sampling of plasma growth hormone (GH) in man: correlation with sleep stages. J Clin EndocrinolMcluh 1991;72:85461. 19. Thorner MO, Vance ML, Hartman ML, et al. Physiological role ofsomatostatin on growth hormone regulation in man. Mc,tuholism 1990;39:40-2. 20. Salata RA, Jarrett DB, Verbalis JG, Robinson AG. Vasopression stimulation of ACTH in humans: in vivo bioassay of corticotropin-releasing factor (CRF) which provides evidence for CRF mediation of the diurnal rhythm of ACTH. J Clin Invcst 1988;81:766-74. 2 I . Veldhuis JD, King JC, Urban RJ, et al. Operating characteristics of the male hypothalamo-pituitarygonadal axis: pulsatile release of testosterone and follicle-stimulating hormone and their temporal coupling with luteinizing hormone. J Clin Endocrinol Metub 1987;65:929-41. 22. Krieger DT. Rhythms in CRF, ACTH and corticosteroids. In: Krieger DT, ed. Endocrine rhy/hm.s. New York: Raven, 1979:123-42. 23. Veldhuis JD, Iranmanesh A, Lizarrdlde G, Johnson ML. Amplitude modulation o f a burst-like mode of cortisol secretion subserves the circadian glucocorticoid rhythm in man. A m J PIiy.siol 1989;257:E6- 14.
C%rOn/JhlO/Int. Vol. Y, No 5. 1992
Vol. 9. No. 5 , pp. 371-379
a 1992 International Society of Chronohiology
Rhythmic and Nonrhythmic Modes of Anterior Pituitary Gland Secretion
Chronobiol Int Downloaded from informahealthcare.com by University of Newcastle on 12/28/14 For personal use only.
Johannes D. Veldhuis, Michael L. Johnson, *German Lizarralde, and *Ali Iranmanesh Division o f Iindocrinology and Metuholism and the Interdisciplinary Graditate Biophy.sic..s Program, Deparimen 1.s of Internal Mcdicinc and Pharmacology. National Science Foiindation Center .Ibr Biological Timing, Univcrsitj?01' Virginia IIealth Scimcec. Center, Charlorriw~ille:and *Division of Endocrinology and Mc.tuholism, Deparimcni oj'Intprnal Mcdicinc., Sali>m Veterans Administration Midical CtwPr, Salem, Virginia, U.S.A.
Summary: Because of confounding effects of subject-specific and hormone-speci-
fic metabolic clearance, the nature of anterior pituitary secretory events in vivo is difficult to ascertain. We review an approach to this problem, in which deconvolution analysis is used to dissect the underlying secretory behavior of an endocrine gland quantitatively from available serial plasma hormone concentration measurements assuming one- or two-compartment elimination kinetics. This analytical tool allows one to ask the following physiological questions: (a) does the anterior pituitary gland secrete exclusively in randomly dispersed bursts, and/or does a tonic (constitutive) mode of interburst hormone secretion exist? and (b) what secretory mechanisms generate the circadian or nyctohemeral rhythms in blood concentrations of pituitary hormones? Waveform-independent deconvolution analysis of 24-h serum hormone concentration profiles of immunoreactive growth hormone (GH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and P-endorphin in normal men sampled every 10 min showed that (a) anterior pituitary gland secretion in vivo occurs in an exclusively burstlike mode for all hormones except TSH and prolactin (for the latter two, a mixed burst and basal mode pertains); (b) significant nyctohemeral regulation of secretory burst frequency alone is not demonstrable for any hormone; (c) prominent 24-h variations in secretory-burst amplitude alone are delineated for ACTH and LH; (d) TSH, G H , and @-endorphinare both frequency and amplitude controlled; (e) prolactin manifests 24-h rhythms in both secretory-burst amplitude and nadir secretory rates; (f) n o significant diurnal variations occur in FSH secretory parameters; and (8) a fixed hormone half-life yields good fits of the 24-h serum hormone concentration series, which indicates that there is no need to introduce diurnal variations in hormone half-lives. In summary, the normal human anterior pituitary gland appears t o release its various (g1yco)protein hormones via intermittent ~
~
Received September 3, 199 1; accepted with revisions December 13, 1991. Address correspondence and reprint requests to J. D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health SciencesCenter, Charlottesville,VA 22908, U.S.A. Presented in part at the 20th International Conference on Chronobiology, Tel Aviv, Israel, June 16-2 I , 1991.
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secretory episodes that are apparently unassociated with significant basal hormone secretion,except in the case of TSH and prolactin. Hormone-specific amplitude and/or frequency control of secretory burst activity over 24 h provides the mechanistic basis for the classically recognized nyctohemeral rhythms in plasma concentrations of adenohypophyseal hormones in the human. Key Words: Secretion-Clearance-Pulsatile-Rh ythm-Hormone.
Twenty-four-hour variations in plasma concentrations of various anterior pituitary hormones have been recognized for several decades (1-3). Implicit in the interpretation of such nyctohemeral and in some cases circadian variations is the view that pulses of hormone release are putatively superimposed on a baseline of tonic hormone secretion (4). An alternative model would require modulated ultradian secretory bursts with variable amplitude and frequency, so as to give rise to the 24-h variation in plasma hormone concentrations, even without the presence of, or variation in, any basal component of hormone release (5). In addition, most interpretations of 24-h serum hormone concentration rhythms assume that there is no systematic variation in hormone metabolic clearance as a function of time ofday. However, exactly how the 24-h rhythms in circulating amounts of hormones are generated mechanistically, or are assembled from and related to underlying ultradian secretory activity, has not been systematically investigated. Here, we have summarized some recent concepts of rhythmic and nonrhythmic modes of anterior pituitary hormone secretion, and enunciated a particular model in which 24-h rhythms in plasma concentrations of anterior pituitary hormones can be constructed by specific amplitude and/or frequency modulation of ultradian secretory bursts with or without basal (time-invariant) hormone secretion, and without any requirement to alter the halftime of hormone disappearance as a function of time of day. Mathematically explicit treatment of the problem of ultradian and nyctohemeral regulation of anterior pituitary hormone secretion can be accomplished by several techniques, including that of deconvolution analysis, in which underlying rates of hormone secretion are estimated from the serum hormone concentration profiles. Specifically, deconvolution analysis attempts to unmask underlying hormone secretory rates that give rise to the plasma hormone concentration changes (5-9). By dissecting the intrinsic secretion profile from the observed plasma hormone concentration patterns, and applying cosinor analysis to the secretory burst amplitudes and interpulse intervals so defined over 24 h, one can investigate nyctohemeral modulation of hormone secretory burst amplitude and/or frequency. Moreover, cosinor analysis of significant (nonzero) basal hormone secretion rates over 24 h can be used to investigate possible nyctohemeral variations in rates of basal hormone release.
METHODS Sampling Protocol
Healthy male volunteers 2 1-50 years of age underwent blood sampling at 10-min intervals for 24 h after overnight adaptation to the Clinical Research Unit. Blood withdrawal was performed via an indwelling venous catheter placed in a forearm vein
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2 1 h before 08:OO h, when sampling was begun. Eucaloric meals were served at 08:30,
12:30, and 17:OO h. Subjects had lights on at 07:OO and off at 23:OO h. Naps were not permitted during lights on. Volunteers were allowed to ambulate in the Clinical Research Center, but vigorous exercise, cigarette smoking, and caffeine ingestion were not permitted. Plasma or serum samples were then used for the subsequent assay of growth hormone (GH), adrenocorticotropic hormone (ACTH), P-endorphin, and thyroid-stimulating hormone (TSH) (immunoradiometric assay), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (radioimmunoassay). We studied six to eight men in each hormone group, after provision of written and informed consent approved by the Human Investigation Committee of the University of Virginia School of Medicine. These endocrine profiles have been reported previously (5). Deconvolution Analysis A waveform-independent deconvolution technique was used for most of the analysis reviewed here, in which sample hormone concentrations are assumed to arise from the combined effects of individual sample secretion rates and mono- or biexponential hormone disappearance kinetics (5,7). In this model, the amount of hormone secreted per unit time is estimated by nonlinear least-squares parameter estimation. Sample secretion rates were determined for each observation (145 samples distributed uniformly over 24 h in the 10-min sampling paradigm), with statistical confidence limits that combined wherever possible the experimental uncertainty in the immunoassay and the hormone half-life estimates (4,7). We used the following half-lives (and corresponding fractional amplitude for the second component) for each hormone: GH 3.5 and 2 1 min, fraction 0.63; LH 18 and 90 min, fraction 0.37; FSH 102 and 398 min, fraction 0.48; ACTH 3.5 and 14 min, fraction 0.33; and monocomponent half-lives for TSH, prolactin, and @-endorphinof 35, 25, and 45 min, respectively (5). Deconvolution analysis permitted us to (a) plot hormone secretory burst amplitude as a function of 24-h clocktime, where amplitude is defined as the maximal rate of hormone secretion attained within a burst; (b) relate interburst interval duration to 24-h clocktime (e.g., consecutive secretory bursts centered at 09:OO and 1O:OO h would have an interburst interval of 60 min centered at 09:30 h); and (c) delineate basal secretion rates as a function of time of day and night, in which basal secretion is defined as the hormone secretion rate in a nadir that precedes or follows a secretory burst (5,7). Cosinor analysis is then applied to evaluate whether there are 24-h rhythms in hormone secretory burst amplitude, interburst interval, or basal (nadir) secretory rates. We applied cosinor analysis to the set of all observations in the group of six to eight men for any given hormone, and developed joint statistical confidence limits for the fitted period, amplitude, and acrophase. Only amplitudes whose statistical confidence intervals exceeded zero by p < 0.05 were considered further here. In some illustrations, we used an alternative multiparameter waveform-specific method of deconvolution analysis (6,8,9). RESULTS AND DISCUSSION Deconvolution analysis provides a technique for evaluating underlying hormone secretion as a contributor to both circadian and ultradian rhythms in serum hormone
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FIG. I . Serum immunoradiometrically assayable LH concentrations (top, middle) and deconvolutioncalculated LH secretory rates (bottom) in blood collected at 5-min intervals for 24 h in a normal young man. Qualitatively similar results are obtained using a 10-min sampling interval. Vertical bars through the data points denote the dose-dependent intraasay standard deviations. The top panel gives the simple 24-h cosine fit of the serum LH concentrations. The middle panel depicts the deconvolution-specified fit of the data. Deconvolution by the multiparameter method (6) predicted the occurrence of individual punctuated LH secretory bursts with almost no tonic secretion (bottom). As discussed further in the text. cosinor analysis can he applied to not only the serum concentrations (top) but also the computer-estimated hormone secretory profiles (bottom) or their specific measures to assess 24-h variations in the frequency and/or amplitude of discrete hormone secretory bursts.
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concentrations. This concept is illustrated for the pituitary hormone LH using the multiparameter deconvolution technique (6) in Fig. 1. We have used deconvolution analysis to evaluate the experimental question, “How do ultradian rhythms in pituitary hormone secretion contribute to the 24-h variations in plasma hormone concentrations?’ To this end, we applied either a waveform-independent (5) or a model-specific muitiparameter (6) deconvolution technique to pituitary hormone concentration time series generated by sampling blood at 10-min intervals over 24 h. This sampling protocol yields inferences similar to those achieved by sampling blood every 5 min (8,9). Waveform-independent deconvolution analysis permitted quantitative estimates of the amplitudes (maximal rates of secretion attained within each secretory burst) and the temporal positions of hormone secretory episodes as well as the degree of concurrent interpulse secretion that statistically exceeds zero (5,7). By applying cosinor analysis to the plots of hormone secretory burst amplitude, intersecretory burst intervals, or basal (nadir) hormone secretory rates over 24 h, we could investigate the mechanisms by which nyctohemeral rhythms in plasma hormone concentrations are assembled. The results of waveform-specific multiparameter deconvolution analysis (6) are illustrated in Fig. 2, which can be used to show distinct mechanisms by which the individual anterior pituitary hormones achieve 24-h variations in concentration. For example, variations in plasma ACTH concentrations can be attributed to diurnally regulated ultradian ACTH secretory burst amplitude without any consistent 24-h variation in burst frequency (10). Our studies of seven anterior pituitary hormones in normal men using waveformindependent deconvolution analysis have indicated that the 24-h rhythms in the plasma concentration of three hormones are generated by both frequency and amplitude control of discrete secretory bursts (5). In particular, in the cases of GH, TSH, and &endorphin, there were significant 24-h variations in detectable hormone secretory burst number (inversely related to interburst interval), and secretory burst amplitude. In contrast, secretory burst amplitude alone was the modulated feature for both ACTH and LH release. Of interest, no hormone demonstrated significant nyctohemeral regulation of secretory burst frequency alone. Thus, amplitude modulation of hormone secretory bursts was observed for five of seven hormones (TSH, GH, P-endorphin, ACTH, and LH), and combined frequency and amplitude control for three (TSH, GH, and 0-endorphin). Deconvolution permitted us to infer that there was a mixed burst and constitutive mode of secretion for TSH and prolactin. This finding is of physiological interest, because both these hormones can be secreted in response to a common secretagogue, TSH-releasinghormone (TRH), and both are subject to tonic inhibition by the neurotransmitter dopamine. Indeed, other studies have demonstrated a relatively high cross-correlation between serum TSH and prolactin concentrations over 24 h (1 1). The basal secretion of TSH was not significantly regulated over 24 h. However, there were significant nyctohemeral variations in prolactin interpulse nadir secretory rates (5). The mechanisms that regulate interpeak nadir hormone secretion are not known, but the fact that prolactin is susceptible to tonic dopamine inhibition in normal physiology may offer a clue that nyctohemeral variations in dopaminergic tone contribute to the 24-h rhythm in nadir prolactin secretion rates.
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FIG. 2. Illustrative profiles of 24-h variations in calculated ACTH secretory burst amplitudes (A) or ACTH intersecretory burst intervals (mint (B) in eight normal men. Each volunteer underwent blood sampling at 10-min intervals for 24 h. Plasma ACTH time series were assessed by multiparameter deconvolution analysis (6). The resultant deconvolution measures (ACTH secretory burst interpulse interval and amplitude) are shown for the group of eight men considered together. Adapted with permission (10).
One hormone, FSH as assessed by radioimmunoassay, exhibited no significant diurnal variations in its secretory parameters. This finding may reflect the true absence of diurnal variations in FSH release, and/or indicate the difficulty in accurately estimating in vivo FSH secretion and/or bioactivity by conventional immunological
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means. The latter possibility is suggested by occasionally (but not uniformly) divergent inferences made when FSH activity in plasma is measured by radioimmunoassay or in vitro bioassay (12). Therefore, further studies should be conducted of the 24-hour rhythms in plasma FSH bioactivity. Indeed, a recent study of plasma FSH concentrations assessed by two-site immunoradiometric assay, which correlates highly with in vitro FSH bioassay, has indicated a measurable 24-h rhythm in serum FSH immunoradiometric activity ( 13). This rhythm in plasma FSH concentrations was abolished by the infusion of either a potent androgen or estrogen in healthy men. Significant variations in hypothalamic neurotransmitter concentrations, turnover, and release rates have been described in experimental animals under different conditions of steroid hormone treatment, metabolic milieu, physiological state, and so forth. We hypothesize that changes in the frequency of anterior pituitary hormone release over 24 h result from diurnal control of relevant hypothalamic burst-generating systems. For example, in the case of LH, mediobasal hypothalamic release of the decapeptide gonadotropin releasing hormone (GnRH) occurs in discrete bursts, which presumably control the frequency of episodic LH release by the anterior pituitary gland ( 14,15).Thus, variations in LH secretory burst frequency can be assumed to mirror diurnal changes in hypothalamic GnRH “pulse generator” activity. This interpretation assumes that GnRH secretory episodes are coupled in a consistent manner to pituitary LH release, and that this coupling efficiency does not vary as a function of time of day. Although evidence is not available to disallow this assumption, limited studies are available that directly test it. Of interest, LH secretory burst frequency does vary as a function of time of day in women at certain stages of the menstrual cycle ( 16). In relation to GH release, control mechanisms include both withdrawal of the hypothalamic inhibitory tetradecapeptide somatostatin and activation of GH-releasing hormone (GHRH) secretion ( 17). For example, decreased somatostatin and increased GHRH release could account for augmented G H secretory burst frequency in association with stage three and four sleep ( 18). Indeed, the intravenous injection of GHRH at selected sleep stages in normal human volunteers shows enhanced responsiveness to GHRH in stage three and four sleep, which can be interpreted to signify reduced endogenous somatostatin inhibitory tone ( 19). Less is known about the regulation of TSH and &endorphin secretory burst frequency. Indeed, in the case of TSH, the presumptive in vivo secretagogue may include the tripeptide TRH, and one or more incompletely identified TSH releasing factors. The finding that 0-endorphin pulse frequency varies diurnally is consistent with some time-dependent regulation of pituitary corticotrope secretory burst activity, putatively by hypothalamic factors such as corticotropin-releasing hormone and/ or arginine vasopressin, or significant 24-h variations in pituitary corticotrope responsiveness to these secretagogues (20). To our knowledge, definitive in vivo information is not available to clarify the exact mechanisms that generate an apparent 24-h variation in the frequency of secretory bursts of TSH and 0-endorphin. Nyctohemeral regulation of pituitary hormone secretory burst amplitude might be accounted for by several plausible mechanisms. First, varying hypothalamic stimulus strength may elicit different amounts of hormone secretion per burst as a function of time of day, sleep stage, and so forth. Secondly, variations in pituitary target-cell
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responsiveness to the hypothalamic releasing factors may modulate the amount of hormone secreted at various times over the day and night. Such changes in pituitary responsiveness could reflect intrinsic paracrine and autocrine regulation of pituitary glandular function, as well as the important effects of systemically delivered secretagogues, inhibitors, trophic factors or feedback signals (e.g., steroid hormones, growth factors such as insulin-likegrowth factor or type I (IGF-I), etc.). Detailed information about variations in diurnal feedback signal strength is not generally available for most hormones. However, in the case of testosterone in the male, reduced plasma testosterone concentrations occur in the late afternoon and early evening hours (2 l), which mimics the general pattern for serum cortisol concentrations (22,23). The nadirs of such steroid hormone concentrations in general correspond with the nadirs in corresponding pituitary hormone concentrations (LH and ACTH). After several hours of delay, increased pituitary hormone secretion occurs with resultant higher concentrations of the steroid hormones. Although little information is available regarding the importance of time of day in the negative-feedback signal strength of sex steroid hormones, variations in corticotrope sensitivity to the feedback effects of cortisol in the morning and evening have been described, as well as significant diurnal variations in adrenal responsiveness to available ACTH (22). In summary, we have reviewed a model of anterior pituitary gland secretion in vivo, in which glycoprotein hormones are released via punctuated secretory episodes, which are typically unassociated with tonic basal (interpulse) hormone secretion, except in the case of TSH and prolactin (5). This model is based on deconvolution analysis of 24-h serum hormone concentration profiles. It predicts that 24-h variations in secretory burst amplitude contribute to the nyctohemeral rhythms in plasma ACTH and LH Concentrations. On the other hand, both amplitude and frequency control of episodic TSH, GH, and /?-endorphin secretion are responsible for the 24-h rhythms in the blood content of these hormones. Little diurnal variation occurs in immunoreactive FSH secretory parameters, whereas prolactin exhibits 24-h rhythms in both secretory burst amplitude and nadir secretory rates. Accordingly, we infer that hormone-specific mechanisms of amplitude and/or frequency control of discrete secretory burst activity can provide a basis for the classically recognized nyctohemeral rhythms in plasma concentrations of anterior pituitary hormones in normal men. Acknowledgment: We thank Patsy Craig for her skillful preparation of the manuscript, Paula P. Azimi for the artwork, Brenda Grisso for laboratory assistance, and Sandra Jackson and the expert nursing staff at the University of Virginia Clinical Research Center for conduct of the research protocols. This work was supported in part by National Institutes of Health (NIH) Grant No. RR 00847 to the Clinical Research Center of the University of Virginia, RCDA No. 1 KO4 H D 00634 (J.D.V.), Veterans Administration Medical Research Funds (A.I.), NIH Grant Nos. AM-30302 and GM-28928 (M.L.J.), Diabetes and Research Training Center Grant No. 5 P60 AM 22 125-05, NIH-supported Clinfo Data Reduction Systems, and the NSF Center for Biological Timing (J.D.V., M.L.J.).
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2. Daughaday WH. The anterior pituitary gland. In: Williams RH, ed. Textbook c~fendocrinology.Philadelphia: WB Saunders, 1981:73-86. 3. Brabant G, Brabant A, Ranft U, et al. Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration. J Clin Endocrinol Metab 1987;65:83-8. 4. Veldhuis JD. A parsimonious model of amplitude and frequency modulation of episodic hormone secretory bursts as a mechanism for ultradian signaling by endocrine glands. In: Wever R, Kleitman N, eds. Ultrudiun rhyihms in I@ processe.s: un inquiry into ,fnndumentul principles. London: SpringerVerlag, 1992 (in press). 5. Veldhuis JD. Iranmanesh A, Johnson ML, Lizarralde G. Twenty-four hour rhythms in plasma concentrations of adenohypophysial hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. J Clin Endocrinol Metub 1990;7 I : 16 16-23. 6. Veldhuis JD, Carlson ML, Johnson ML. The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proc Natl Acud Sci US.4 1987;84:7686-90. 7. Veldhuis JD, Johnson ML. Deconvolution analysis of hormone data. Methods Enzyrnol 1992;210:539-75. 8. Hartman ML, Faria ACS, Vance ML, Johnson ML, Thorner MO, Veldhuis JD. Temporal structure of in vivo growth hormone secretory events in man. Am J Phj)siol 199 I ;260:E 10I - 10. 9. Veldhuis JD, Johnson ML. In vivo dynamics of luteinizing hormone secretion and clearance in man: assessment by deconvolution mechanics. J Clin Endocrinol Metab 1988;66:I29 1-300. 10. Veldhuis JD, lranmanesh A, Johnson ML, Lizarralde G. Amplitude, but not frequency, modulation of ACTH secretory bursts gives rise to the nyctohemeral rhythm of the corticotropic axis in man. J Clin Endocrinol Metub 1990;7I :452-63. I 1. Samuels MH. Veldhuis JD, Henry P, Ridgway EC. Pathophysiology of pulsatile and co-pulsatile release of thyroid stimulating hormone, luteinizing hormone, follicle stimulating hormone, and alpha subunit. J Clin Endocrinol Metah 1990;71:425-32. 12. Urban RJ, Padmanabhan V, Beitins I, Veldhuis JD. Metabolic clearance of human follicle-stimulating hormone in man as measured by radioimmunoassay, immunoradiometric, and in vitro Sertoli-cell bioassay. J Cllin EndocrinolMetuh 1991;73:818-23. 13. Urban RJ, Veldhuis JD. Specific regulatory actions of dihydrotestosterone and estradiol on the dynamics of FSH secretion and clearance in man. .I Androl 1991:12:27-35. 14. Marshall JC, Kelch RP. Gonadotropin-releasing hormone. Role of pulsatile gonadotropin secretion in the regulation of reproduction. N Bngl J Mcxl 1986;315:1459-67. 15. Clark IJ, Cummins JT. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 1982; I I 1: 1737-9. 16. Sollenberger ML, Carlson EC, Johnson ML, Veldhuis JD, Evans WS. Specific physiological regulation of LH secretory events throughout the human menstrual cycle: new insights into the pulsatile mode of gonadotropin release. J Newoendocrinol I990;2:845-52. 17. Plotsky PM, Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial-portal circulation of the rat. Scicnce 1986:230:461-5. 18. Holl RW, Hartman ML, Veldhuis JD, Taylor WM, Thorner MO. Thirty-second sampling of plasma growth hormone (GH) in man: correlation with sleep stages. J Clin EndocrinolMcluh 1991;72:85461. 19. Thorner MO, Vance ML, Hartman ML, et al. Physiological role ofsomatostatin on growth hormone regulation in man. Mc,tuholism 1990;39:40-2. 20. Salata RA, Jarrett DB, Verbalis JG, Robinson AG. Vasopression stimulation of ACTH in humans: in vivo bioassay of corticotropin-releasing factor (CRF) which provides evidence for CRF mediation of the diurnal rhythm of ACTH. J Clin Invcst 1988;81:766-74. 2 I . Veldhuis JD, King JC, Urban RJ, et al. Operating characteristics of the male hypothalamo-pituitarygonadal axis: pulsatile release of testosterone and follicle-stimulating hormone and their temporal coupling with luteinizing hormone. J Clin Endocrinol Metub 1987;65:929-41. 22. Krieger DT. Rhythms in CRF, ACTH and corticosteroids. In: Krieger DT, ed. Endocrine rhy/hm.s. New York: Raven, 1979:123-42. 23. Veldhuis JD, Iranmanesh A, Lizarrdlde G, Johnson ML. Amplitude modulation o f a burst-like mode of cortisol secretion subserves the circadian glucocorticoid rhythm in man. A m J PIiy.siol 1989;257:E6- 14.
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