0o21-972X/92/7501-0011$03.00/0 .Journal of Climcal Endocrinology and Metabolism Copyright(r) 1992 hy The Endocrine Society

Vol. 75, No. 1 Printed in U.S A

Sex Steroid Priming Effects Response to Pyridostigmine Cycle VERONICA Department

of

O’KEANE

AND

Psychiatry,

Trinity

TIMOTHY College Medical

on Growth throughout

Hormone the Menstrual

G. DINAN School, St. James’s Hospital,

Dublin

8, Ireland

ABSTRACT To explore the effect of estradiol and progesterone on the GH response to the indirect cholinergic agonist pyridostigmine nine healthy women were challenged with both active drug and placebo at three time points in two consecutive menstrual cycles: a total of six neuroendocrine tests. A randomized, double-blind, counterbalanced design was used. Subjects were tested in the early follicular, mid-cycle, and luteal phases of the cycle. A cannula was inserted in a forearm vein after an overnight fast and baseline GH, estradiol, and progesterone samples were drawn. After 120 mg oral pyridostigmine or placebo tablets further blood samples for GH analysis were drawn at intervals over 3 h. When expressed as maxium change from baseline (AGH) mean GH responses to pyridostigmine increased incrementally from early (8.4 &

2.7 fig/L) through mid (18 + 1.3 fig/L) to late (22.2 +- 1.9 pg/L) cycle. This represents a significant effect of cycle phase on the GH response to pyridostigmine (P < 0.001, as assessed by analysis of variance). Responses to placebo did not vary. Plasma estradiol values were significantly correlated with GH responsivity to active drug throughout the cycle (P < 0.02). Multiple regression analysis also revealed a significant positive correlation between progesterone levels and GH response to pyridostigmine (P < 0.02). Estrogens augment GH responses to other challenges but a priming effect of progesterone on GH responsivity has not previously been demonstrated. Various mechanisms are discussed including a possible sex steroid priming effect on acetylcholine neurotransmission. (J Clin Endocrinol Metab 75: 11-14, 1992)

T

metabolism (13) but has no consistent effect on the GH response to arginine. Possible progesterone effects on GH responses to other challenges have not been evaluated. Neither has any steroid effect on the GH response to pyridostigmine challenge been explored. Somatostatin is the main inhibitory hypothalamic peptide controlling GH secretion and is probably under tonic inhibitory cholinergic control (14). It is thought that pyridostigmine, an cholinesterase inhibitor, brings about an increase in GH secretion from the somatotrophs by inhibiting somatostatin release (15). In order to explore the effects of the gonadal steroids on the GH response to pyridostigmine we applied this challenge at three time points in the menstrual cycle: early follicullar phase when sex steroid levels are low; mid-cycle when estrogen levels are relatively high, and during the late luteal phase when progesterone levels are relatively high.

HERE IS a convincing body of evidence indicating that estrogens can augment GH secretion under certain physiological conditions: the increase in plasma concentration of estradiol during puberty is associated with an increase in the pulsatile secretion of GH (1, 2) and may be causally related to the accelerated growth during this period (3, 4); administration of sex steroids for hypogonadotropic hypogonadism (5) and for replacement therapy in postmenopausal women (6) is associated with increases in circulating GH levels and treatment that suppresses sex steroid production results in a decrease in GH secretion (7). Likewise GH response to a variety of probes is augmented by estrogens: GH responses to arginine are increased in women during high estrogen phases of the menstrual cycle and in men treated with estrogen (8); GH responses to clonidine are higher at peak estrogen compared to low estrogen phases of the cycle (9) and estrogen augments the exercise-induced rise in GH (10) and the GH response to GHRH (6,ll). Evans et al. (12), however, found no difference in GH responses to GHRH in 10 normal women at different sex-steroid phases of the menstrual cycle but it is probable that they used a supramaximal dose of GHRH (3.3 pg/kg). In a larger sample of 57 healthy females using a dose of 1 pg/kg a strong statistical association was established between serum estradiol values and GH responses (11). Progesterone administration decreases the GH response to insulin-induced hypoglycemia possibly by altering glucose Received Address Psychiatry, 8, Ireland.

Subjects

and Methods

Nine healthy women gave informed consent to take part in this study (mean age 27.4 + 2.5 yr). They all had regular menstrual cycles with no alteration in mood premenstrually. Those on any medication, including oral contraceptives, were excluded. All were moderate consumers of alcohol and none were cigarette smokers. None had any significant medical or psychiatric history. All weighed within 15% of ideal body weight. Volunteers presented for testing on days 2-5, days 13-15, and days 24-26 of two consecutive cycles. The same procedure was employed on each occasion. An iv cannula was inserted in a forearm vein at 0900 h after an overnight fast and sealed with a heparin lock. After an adaptation period of 15 min two baseline GH samples were drawn at 15-min intervals. Either placebo or active medication (pyridostigmine, 120 mg)

July 9, 1991. requests for reprints to: Dr. Timothy Dinan, Department of Trinity College Medical School, St. James S. Hospital, Dublin

11

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O’KEANE

12

was given orally and plasma samples were subsequently drawn for GH estimation at +60, f90, +120, and +180 min. Samples for estradiol and progesterone determination were also drawn at baseline. Three subjects were commenced in the early, three in the mid, and three in the premenstrual phase of the cycle. Four were initially challenged with placebo and five with pyridostigmine, blind both to volunteers and testers, and subsequently were tested in an alternate pattern with either drug or placebo. All subjects were tested on six occasions. Plasma GH samples were analyzed in batch blind to subject status using a standard RIA technique (15) with a sensitivity of 0.27 pg/L; an intrassay precision of 1.6% and an interassay precision of 2.3% at 5 pg/ L. Estradiol and progesterone values were measured using commercial kits with sensitivities of 5.1 pmol/L and 0.159 nmol/L respectively and intrassay variations of 2.7 and 8.4%. Data was analyzed by means of Statgraphics (17). Endocrine responses over time to drug and placebo at the three phases of the cycles were compared by three-way ANOVA and Tukey’s post hoc comparisons were applied. Estradiol and progesterone levels throughout the cycle were analyzed by one-way analysis of variance (ANOVA). A GH values (difference between baseline and maxium subequent increase) were correlated with sex steroid levels using Pearson’s product moment correlation coefficient. Multiple regression analysis was applied to determine the separate effects of these variables on the response. Results are expressed as mean + SEM.

Results All subjects responded to pyridostigmine at the three test points with an increase in plasma GH concentration (range, 4.5-39.7 &g/L) ( see Table 1). The mean A GH response in the follicular phase was 8.4 f 1.3 pg/L; at ovulation was 18 + 1.3 pg/L and in the luteal phase was 22.2 + 1.9 pg/L (see Fig. 1). In contrast GH responses to placebo were not significant or different at the various test points (mean A GH: 1.1 P& I.5 pg/L, and 0.9 pg/L). Using a repeated measures three-way ANOVA to compare responses over time between active drug and placebo at the three test points in the cycles yields a significant time x drug effect (F(4,265) = 21.6, P < 0.001); a significant time X cyclephase effect (F(8,261) = 2.36, P < 0.02) and a significant drug X cycle-phase interaction (F(2,267) = 13.7, P < 0.001). Tukey’s post hoc analysis indicates that the GH responses at both mid-cycle and luteal time points are significantly differTABLE ovulatory,

DTNAN

JCE & M. 1992 Vol75.Nol

ent to those during the follicular phase (P < 0.05) and differences between mid- and late-cycle phases just fails to reach significance. Progesterone levels increased from early through to late cycle (F(2,26) = 50.7, P < 0.001) (see Table 1). Estradiol values peaked at mid-cycle, were lowest in the follicular phase, and intermediate between the two premenstrually (F(2,26) = 4.9, P < 0.02) (see Table 1). Estradiol plasma levels correlated significantly with GH responses to pyridostigmine throughout the cycle (r = 0.45, df = 27, P < 0.02). Progesterone levels were also significantly correlated with A GH values to active drug (r = 0.53, df = 27, P < 0.005). Multiplelinear regression analysis demonstrated an independent effect for progesterone on AGH (P < 0.02). Discussion The results of this study indicate that GH responses to pyridostigmine in normal females are profoundly influenced by the menstrual cycle phase, increasing incrementally from early through to late cycle. These findings also suggest that progesterone is an important modulator of this increased responsivity as well as estradiol. Our findings support the evidence as outlined in the introduction for a homeostatic interplay between estrogens and GH. We were unable to demonstrate increased GH secretion as baseline GH values did not alter at the three time points tested. Holst et al. (18) also found that GH secretion did not vary across the menstrual cycle. This is more likely to be a consequence of inadequate baseline assessment. Baseline values are only crude indicators of GH secretion since GH is secreted in a pulsatile fashion with the number of pulses detected increasing with increased sampling times (19). Also, 6 test days in two cycles is probably inadequate to detect subtle variations in GH secretion. Venous samples taken on a daily basis throughout normal menstrual cycles do indicate an increase in basal GH values during the periovulatory period when estradiol levels are also high (20).

1. AGH after pyridostigmine administration (difference between basal and maximal GH concentration) during and luteal phases of the menstrual cycle with corresponding estradiol (est) and progesterone (prog) levels Follicular Subject no.

Mean +SEM

AGH (PdL)

Est (PmwL)

values

phase

Mid-cycle Prog (nmol/L)

AGH b%lL)

Est (Pmol/L)

797

11.7 16 8.2 7.5 7.2 6.2 4.7 9.2

581 74 108 216 174 283 298 400

0.5 2.8 1.5 0.8 1.7 1.1 1.4 0.9

16.7 22.7 20.2 18 22 14 19 17

4.5

118

0.8

12

224 556

8.4

250 f 54

1.3

18 + 1.3

591 zk 106

+ 1.3

Mean

AND

(+SEM)

+ 0.3

222 187 650 470 500 800 l-

the follicular,

Luteal phase Prog (nmol/L)

AGH km

4.7 2.1 1.6 3.1 2 1.2 2.1 1.8

Est

Prog

(pmol/L)

(nmol/L)

39.7 19.2 18.7 23.1 23.2 14.2 27.2 15.5

326 100 578 569 698 460 787 797

19.4 34 38 40 18.3 15.1 47 26

0.8

19.7

300

21

2.15 +0.4

22.2 +

512 f

1.9

79

28.8 f 3.7

are provided.

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SEX e-. 6-A o--o

20-

0

STEROIDS

AND

tats Luteal Phase Mid - cycle Early Follicular Phase

90

60 TIME

120

180

(minutes)

FIG. 1. GH responses to pyridostigmine (120 mg orally) in nine healthy women during the follicular, mid-cycle, and luteal phases of the menstrual cycle. Results are expressed as mean + SEM.

What is more apparent is that estrogens modulate GH responsivity in healthy adult females to a variety of stimuli; some physiological and some pharmacological. Estradiol increasesinsulin-like growth factor-l via genomic mechanisms in certain selective target tissues in the rat (21, 22) and it may be that it is exerting a similar effect on GH synthesis at the somatotrophs in a manner analagous to the estradiolinduced PRL secretion at the lactotrophs (23). Another important aspect of steroid hormone action involves their effects on neurotransmitter function. Estrogen treatment of female rats induces profound increasesin choline acetyltransferase activity in the basal forebrain area (24) and acetylcholine levels vary throughout the estrous cycle in the anterior pituitary of rats (25). Acute estrogen treatment results in an up-regulation and chronic treatment an increasedresponsivity of muscarinic receptors in rabbit uterus (26). In humans pyridostigmine potentiates the GH response to GHRH in males but not in females and Barbarino et al. (27) suggest as an explanation that females may have a higher cholinergic GH control. The secretory capacity of the somatotrophs is therefore maximally stimulated by the addition of GHRH stimulus in females but not in males. Increased cholinergic activity in females would be compatible with a hypothesis of a sex steroid-induced increase in acetylcholine function and provides a possible explanation for our current findings. These results suggest that progesterone too has a significant priming effect on GH secretion. Progesterone often modifies the physiological effects of estrogens (28) and this may represent an amplification of an estrogen-induced response. Supporting this is the absence of significant differences in GH responses between the mid-cycle and luteal phase indicating that estradiol may be the major modulator.

GH

RESPONSIVITY

13

Responseswere enhanced in the higher progesterone phase however; though not significantly, and given the small sample size this may represent a type 2 error. Also there was a positive statistical correlation between progesterone levels and GH response independent of fluctuations in estradiol level. Bhatia and workers (13) observed that progesterone diminished the GH response to insulin but not arginine; on which it exerted both inhibitory and stimulatory influences. The apparently paradoxical effects exerted by progesterone on GH responsivity to different challenge agents may be explained by their divergent GH-releasing mechanisms.Hypoglycemia increasesGH secretion probably by stimulating GHRH and reducing somatostatin discharge whereas arginine may act solely via the latter mechanism (29). Pyridostigmine stimulates GH secretion by increasing the inhibitory cholinergic control of somatostatin release. It may be that progesterone influences these pathways in different or even antagonistic ways. Finally, PRL responses to a variety of neuroendocrine probes including TRH (30), dopamine antagonists (31), buspirone (32), o-fenfluramine (33), and GnRH (34) are altered in a similar sex steroid-induced manner to GH responses suggestingperhaps a nonspecific priming effect operating at a more basic physiological level. This might involve a sex steroid modulation of membrane excitability operating, for example, via calcium mechanisms (35). Neither can one assumethat substancesother than the sex hormones; such as PRL gonadotrophins or follicular fluid factors, that also demonstrate cyclical variations in secretory patterns are not influencing the GH response. References 1. Evens WS, Ho KY, Feuria ACS. 1988 Age and sex-related neurosecretion of growth hormone. In: Bereu BB, ed. Basic and clinical aspects of growth hormone. New York: Plenum Press; 157-72. 2. Maurus N, Blizzard RM, Linh K, Johnson ML, Rogol AD, Veldhuis JD. 1987 Augmentation of growth hormone secretion during puberty: evidence for a pulse amplitude-modulated phenomenon. J Clin Endocrinol Metab 64:596-601. 3. Rosenfield RI, Furlanette R, Bock D. 1983 Relationship of somatomedin-C concentrations to pubertal changes. J Pediatr 103:723-8. 4. Ho KY, Evans WS, Blizzard RM, et al. 1987 Effects of sex and age on 24.hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J Clin Endocrinol Metab 64:51 l-8. 5. Liu L, Merriam GR, Sherins RJ. 1987 Chronic sex steroid exposure increases mean plasma growth hormone concentration and pulse amplitude in men with isolated hypogonadotropic hypogonadism J Clin Endocrinol Metab 64:651-6. 6. Hughes-Dawson B, Stern D, Goldman J, Reichlin S. 1986 Regulation of growth hormone and somatomedin-C secretion in postmenopausal women: effect of physiological estrogen replacement. J Clin Endocrinol Metab 63:424-9. 7. Mansfield MJ, Rudlin JF, Crigler Jr JF, et al. 1988 Changes in growth and serum growth hormone and plasma somatomedin-C levels during suppression of gonadal sex secretion in girls with central precocious puberty. J Clin Endocrin Metab 66:3-9. 8. Merimee TJ, Fineberg ES. 1971 Studies of the sex based variation of human growth hormone secretion. J Clin Endocrinol Metab 33:896-902. 9. Matussek N, Ackenheil M, Hertz M. 1984 The dependence of the clonidine growth hormone test on alchol drinking habits and the

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14

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menstrual cycle. Psychoneuroendocrinology 9:173-7. 10. Frantz AG, Rabkin MJ. 1965 Effects of estrogen and sex difference on secretion of human growth hormone. J Clin Endocrinol26:147080. 11. Lang I, Schernthaner G, Pietschmann I’, Kurz R, Stephenson JM, Temnl H. 1987 Effects of age and sex on growth hormone resuonse to growth hormone-releasiig hormone inhealthy individuals.! Clin Endocrinol Metab 65:535-40. 12. Evans WS, Faria ACS, Christiansen E, et al. 1987 Impact of intensive venous sampling on the characterization of pulsatile GH release. Am J Physiol 252:E549-56. 13. Bhatia SK, Moore D, Kalkhoff RK. 1972 Progesterone suppression of the growth hormone response. J Clin Endocrinol Metab 35:3649. 14. Locatelli V, Torsello A, Redaelli E, Ghigo E, Massara F, Muller EE. 1986 Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone-releasing hormone response in the rat: evidence for mediation by somatostatin. J Endocrinol111:271-8. 15. Ross RJ, Tsagarakis S, Grossman A, et al. 1987 GH feedback occurs through modulation of hypothalamic somatostatin under choloinergic control: studies with pyridostigmine and GHRH. Clin Endocrinol (Oxf) 27:727-33. 16. Raite S. 1983 The standards for human growth hormone assays. In: Laron Z, Butenandt 0, eds. Evaluation of growth hormone secretion. Basel: Karger; 162-9. 17. Statistical Graphics Corporation. Statgraphics, Version 2.7. New York: Author; 1987.. 18. Holst N, Jenssen TG, Barhal PG, Haug E, Forsdahl F. 1989 Plasma gastrointestinal hormones during sport and induced menstrual cycles. J Clin Endocrinol Metab 68:1160-6. 19. Evans WS, gorges JLC, Vance ML, et al. 1984 Effects of human pancreatic growth hormone-releasing factor-40 on serum growth hormone, prolactin, luteinizing hormone, follicle-stimulating hormone, and somatomedin-C concentrations in normal women throughout the menstrual cycle. J Clin Endocrinol Metab 59:100610. 20. Genazzani AR, Lemarchand-Beraud T, Aubert ML. 1975 Pattern of plasma ACTH, hGH, and cortisol during menstrual cycle. J Clin Endocrinol Metab 41:431-7. 21. Ernst M, Heath JK, Rodan GA. 1989 Estradiol effects on proliferation, messenger ribonucleic acid for collagen and insulin-like growth factor-l and parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells from calvariae and long bones. Endocrinology 125:825-33. 22. Murphy LJ, Friesen HG. 1988 Differential effects of estrogen and

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growth hormone on uterine and hepatic insulin-like growth factor1 gene expression in the ovariectomized hypophysectiomized rat. Endocrinology 122:325-32. Bertrand P, Drouva S, Krantic I’, Epelbaum J, Kordon C, Enjalbert A 1988 Modulation of receptivity of lactotroph cells by oestradiol. In: Genazzani AR, ed. The brain and female reproductive function. United Kingdom: Partheon; 137-45. Lucine VN, McEwan BS. 1983 Sex differences in cholinergic diagonal band nuclei in the rat preoptic area. Neuroendocrinology 36~475-82. Eaozi Y, Klooe; Y. Flemineer G. Sokolovskv M. 1988 Acetvlcholine in-the rat pituzary: a possyble humoral factor. Brain Res 475:376-9. Batra S. 1990 Influence of chronic oestrogen treatment on the density of muscarinic cholinergic receptors and calcium channels in the rabbit uterus. J Endocrinol 125:185-9. Barbarino A, Corsello SM, Tofani A, et al. 1991 Sexual dimorphism of pyridostigmine potentiation of growth hormone (GH)-releasing hormone-induced GH release in humans. J Clin Endocrinol Metab 73:75-8. McEwan BS. 1988 Basic research perspective: ovarian hormone influence on brain neurochemical functions. In: Hartly Gise L, ed. Contemporary issues in obstetrics and gynaecology, vol 2: the premenstrual syndromes. New York: Churchill Livingstone; 21-33. Cordido F, Dieguez C, Casanueva FF. 1990 Effect of central cholinergic neurotransmission enhancement by pyridostigmine on the growth hormone secretion elicited by clonidine, arginine, or hypoglycaemia in normal and obese subjects. J Clin Endocrinol Metab 70:1361-70. Boyd AE, Sanchez-Franc0 E. 1976 Changes in prolactin responses to thyrotrophin releasing hormone during the menstrual cycle of normal women. J Clin Endocrinol Metab 44:985-9. Buckman M, Peake GT, Srivastava LS. 1976 Endogenous oestrogen modulates phenothiazine-stimulated orolactin secretion. I Clin Endocrinol Metab 43:901-6. Dinan TG, Barrv S. 1990 The reoroducibilitv of the orolactin response to buspiione: relationship tb the men&al cycle.’ Int Clin Psychopharmacol5:119-23. O’Keane V, O’Hanlon M, Webb M, Dinan TG. 1991 D-Fenfluramine/prolactin responses throughout the menstrual cycle: evidence for an oestrogen-induced alteration. Clin Endocrinol (Oxf) 34:28992.. Tan YM, Steele PA, Judd SJ. 1986 The effect of physiological changes in ovarian steroids on the prolactin response to gonadotrophic releasing factor. Clin Endocrinol (Oxf) 24:71-8. Batra S. 1987 Increase by oestrogen of calcium entry and calcium channel densitv in uterine smooth muscle. Br I Pharmacol 92:38992.

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Sex steroid priming effects on growth hormone response to pyridostigmine throughout the menstrual cycle.

To explore the effect of estradiol and progesterone on the GH response to the indirect cholinergic agonist pyridostigmine nine healthy women were chal...
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