J. Endocrinol. Invest. 14: 41-45,1991
The growth hormone response to pyridostigmine plus growth hormone releasing hormone is not influenced by pubertal maturation M. Cappa*, S. Loche**, R. SaJvatori*, A. Faedda*, P. BorreJJi*, S.G. CeJJa***, C. Pintor**, and E.E. MuJJer***. *Oivisione di Endocrinologia, Ospedale Bambino Gesu, IRCCS, Roma, **Cattedra di Endocrinologia Pediatrica, Universita di Cagliari, ***Oipartimento di Farmacologia, Universita di Milano, Italy.
prepubertal children (16.0 ± 2.8,8.1 ± 1.3 and 51.1 ng/ml, mean ± SE, respectively) were not different from those observed in the early pubertal (18.4 ± 2.1 , 9.1 ± 1.9 and 41.2 ± 5.6 ng/ml, respectively) and in the late pubertal group (14.9 ± 2.3, 13.1 ± 2.4 and 42.6 ± 2.9 ng/ml, respectively). Evaluation of the area under the curve (AUG) also showed no difference in the GH response to GHRH, PO and PO + GHRH between the three groups studied. These results confirm that the combination PO + GHRH is a powerful test to study the GH secretory capacity of the pituitary, and show that pubertal maturation has no effect on the GH response to this test.
ABSTRACT. We have evaluated the effect of pubertal maturation on the GH response to growth hormone releasing hormone (GHRH), pyridostigmine (PO), and the combined administration of PO + GHRH in a group of short normal children. Fifteen were prepubertal (13 boys and 2 girls, age 5.0 - 12.5 yr), 10 were early pubertal (8 boys and 2 girls, age 11.5 -16.9 yr in Tanner stage 2-3 of pubertal maturation), and 6 were late pubertal (6 boys and 2 girls, age 13.6 - 17.1 yr in Tanner stage 4-5 of pubertal maturation). All subjects were tested on three occasions with GHRH 1-29 (1 /-lg/Kg iv), PO (60 mg po) and PO + GHRH (60 mg PO administered orally 60 min before GHRH). Peak GH levels after GHRH, PO, and PO + GHRH in the
± 5.5
in the GH response to the releasing hormone in relation to the pubertal status of the subjects (12-13). It has recently been reported (14) that the GH response to GHRH in children can be significantly enhanced by pretreatment with a cholinergic agonist, namely pyridostigmine (PO), which inhibits endogenous somatostatin release (15). Combined administration of PO and GHRH has been described as a highly suitable test to study the secretory capacity of the somatotropes in children (14). In this study we have evaluated the effect of pubertal maturation on the GH response to GHRH, PO and PO + GHRH in a group of short normal children.
INTRODUCTION Puberty is characterized by increased growth hormone (GH) and insulin-like growth factor I (IGFI) secretion, whose interaction with gonadal steroids is responsible for the pubertal growth spurt (1-4). An increased 24-h GH secretion associated with increased GH pulse amplitude has been demonstrated during spontaneous pubertal maturation as well as after sex steroids administration (3-5). Furthermore, the GH response to pharmacological stimuli such as insulin-hypoglycemia or arginine have been reported to be enhanced by androgens (6-8), or by the spontaneous onset of puberty (8-11). However, studies on the effect of GHRH on GH secretion during puberty have shown no changes
MATERIALS AND METHODS Thirty-one short children (25 boys and 6 girls) aged 5.0 - 17.1 yr [bone age (BA) 3.0 - 14.4 years] were studied. Fifteen [13 boys and 2 girls; chronologic age (CA) 5.0 - 12.5 years, BA 3.0 - 10.0 yr] were prepubertal (testicular volume < 2 ml and testosterone levels of 23.2 ± 1.8 ng/dl in boys; no breast or pubic
Key-words: GH, GHRH, somatostatin, pyridostigmine, puberty. Correspondence: Dr. Sandro Loche, Cattedra di Endocrinologia Pediatrica, Ospedale Microcitemico. via Jenner. 09100 Cagliari, Italy. Received November 27,1989; accepted October 15,1990.
41
M Cappa, S. Lache, R. Salvatori, et al.
hair development in girls), 10 (8 boys and 2 girls; CA 11.5 - 16.9 yr, BA 11.0 - 14A yr) were in Tanner stage 2 or 3 of pubertal maturation (testosterone levels in boys 160.2 ± 35.8 ng/dl) and 6 were in Tanner stage 4 or 5 (testosterone levels in boys 420 ± 50 ng/dl). All children referred to our Department for short stature; none had signs of malnutrition, dysmorphic syndromes, psychosocial deprivation and had taken long-term medication prior to enter the study. None had classical GH deficiency as assessed by nocturnal GH peak or by clonidine stimulation test; mean peak GH levels measured at 30 min intervals every 3 h after the onset of spontaneous sleep were 17.0 ± 3A ng/ml (mean ± SE, range 8.0 - 82.0 ng/ml), and peak GH levels after clonidine were always> 8.0 ng/ml. The mean growth velocity in the prepubertal and early pubertal group was 4.6 ± 0.3 (SE) and 6.5 ± 0.6 cm/yr, respectively (p < 0.01). In the four late pubertal boys, mean growth velocity was 6.7 ± 0.8 cm/yr, while the two late pubertal girls had completed their statural growth according to their bone age. Mean IGF-Ilevels in the prepubertal, early pubertal and late pubertal groups was 1.9 ± 0.2, 2.6 ± OA, and 1.5 ± 0.2 U/ml, respectively. The study was approved by the Ethical Commitee of the Department of Endocrinology of the Bambino Gesu Hospital, Rome, and informed consent was obtained from all children or from their legal guardians prior to the study.
After an overnight fast all children were tested three times in random order with GHRH (1-29, kindly provided by Pierrel-Kabivitrum, Milan) at the dose of 1.0 Ilg/Kg iv; with PD (Mestinon, Hoffman-La Roche, Nutley, NJ) at the dose of 60 mg orally, and with PD plus GHRH (60 mg PD administered orally 60 min before GHRH). Blood samples were taken 30 min and immediately before the administration of GHRH and then after 15, 30, 60, 90 and 120 min (GHRH and PD plus GHRH tests), and at times -60, -30, 0, 15, 30, 60, 90 and 120 min during the PD test. An interval of 3 to 7 days was left between each test. GH was measured by double antibody RIA using reagents provided by CEA-IRE Sorin (Saluggia, Italy). The sensitivity of the assay was 1.0 ng/ml with an intra and interassay coefficient of variation of 3.8 and 9.2 % respectively. IGF-I levels were measured by RIA using reagents provided by the Nichols Institute Diagnostic (S.Juan Capistrano, CA). Details on the IGF-I assay in our laboratory have been reported elsewhere (16). The GH secretory responses were expressed either as absolute peak values (ng/ml) or as areas under the curve (AUC) calculated by trapezoidal integration. The statistical significance of the differences was calculated by means of paired or unpaired t tests preceded by ANOVA. A P value less than 0.05 (two tailed) was considered to indicate a significant difference. All data are given as the mean + SE.
* ** ~." e:
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*
40 30 20 10
GHRH
PO
PO+GHlH
PREPUBERTY
GHRH
PD
P2-3
42
PD.GHRH
GHRH
PD
P4-5
PD.oHRH
Fig. 1 - Mean ± SE of the peak plasma GH responses to PO (60 mg orally), to a single bolus injection of GHRH (1 Ilg/kg ivY alone and after pretreatment with PO (60 mg po 60 min before the GHRH injection) in short prepubertal, early pubertal (P 2-3) and late pubertal (P 4-5) children. *p < 0.001 vs GHRH alone; **p < 0.005 vs GHRH alone.
Pyridostigmine and GH secretion
ng.min/ml, p < 0.005 vs GHRH alone; late pubertal= 2,395 ± 259 ng'min/ml, p < 0.005 vs GHRH alone) (Fig. 2). The GH response to PO plus GHRH was not significantly different between the three groups studied. Also after PO plus GHRH administration the peak GH levels showed a great interindividual variability, ranging from 18.0 to 97.0 ng/ml in the prepubertal children, from 20.0 to 72.0 ng/ml in the early pubertal, and from 33.4 to 50.0 ng/ml in the late pubertal children. Pretreatment with PO enhanced the GH response to GHRH to a similar extent in either group studied. There was no correlation between growth velocity or IGF-I levels and the degree of the plasma GH response to GHRH, PO and PO plus GHRH evaluated as either peak or AUC. PO administration was well tolerated and did not induce noticeable side effects in any of the children studied.
RESULTS
Mean peak plasma GH levels (Fig. 1) and mean AUC (Fig . 2) after GHRH in the prepubertal children (16.0 ± 2.8 ng/ml and 785 ± 141 ng.min/ml) were not significantly different from those observed in the early pubertal (18.4 ± 2.1 ng/ml and 968 ± 78 ng'min/ml) and in the late pubertal group (14.9 ± 2.3 ng/ml and 725 ± 118 ng·min/ml). The GH peak after GHRH showed a great variability, ranging from 3.0 to 45.0 ng/ml in the prepubertal children, from 12.0 to 32.0 ng/ml in the ear!y pubertal, and from 5.6 to 21.0 ng/ml in the late pubertal. Similarly, the mean peak plasma GH levels and mean AUC after PO alone were not different between prepubertal (8.1 ± 1.3 ng/ml and 366 ± 77 ng 'min/ml) , early pubertal (9.1 ± 1.9 ng/ml and 457 ± 94 ng'min/ml) and late pubertal children (13.1 ± 2.4 ng/ml and 659 ± 41 ng.min/ml) (Figs. 1 and 2). Peak GH levels after PO alone ranged from 2 .5 to 21.0 ng/ml in the prepubertal children, from 5.0 to 21.0 ng/ml in the pubertal group, and from 5.4 to 21.8 ng/ml in the late pubertal group. PO administration greatly enhanced the GH response to GHRH evaluated as either peak (prepubertal= 51.1 ± 5.5 ng/ml, p < 0.001 vs GHRH alone ; early pubertal= 41 .2 ± 5.6 ng/ml, p < 0.005 vs GHRH alone; late pubertal= 42.6 ± 2.9 ng/ml, p < 0.001 vs GHRH alone) (Fig. 1) or AUC (prepubertal= 2,840 ± 372 ng'min/ml, p < 0.001 vs GHRH alone; early pubertal= 2,004 ± 254
.... ...~. ~c
DISCUSSION
Our results confirm the ability of PO to enhance the GH response to a single bolus injection of GHRH in short children (14). As previously reported (12,13), we found no difference in the GH response to GHRH between prepubertal and pubertal children . Moreover, no difference related to the state of puberty in the GH response to GHRH was found after the children were administered PO, i.e. after the endogenous SRIF influence on the pituitary had been
*
30
** **
20
.~
r 10
0
:l
et
8
GHRH
PO
POtGHIH
PREPUBERTY
CiHRH
PO
P2-3
43
PI>tGH!H
GHRH
PO
P4-5
PD+GHRH
Fig. 2 - Mean ± SE of AUC after PO (60 mg orally), and after a single bolus injection of GHRH (1 Ilg/kg ivy alone and after pretreatment with PO (60 mg po 60 min before the GHRH injection) in short prepubertal, early pubertal (P 2-3) and late pubertal (P 4-5) children. *p < 0.001 VS GHRH alone; **p < 0.005 vs GHRH alone.
M. Cappa, S. Lache, R. Salvatori, et 81.
removed. The GH response to GHRH showed a great variability, which was not abolished by pretreating the children with PD. This indicates that such variability is not solely due to the endogenous SRIF tone (17), since PO administration would have been able to inhibit hypothalamic SRIF release (15). GH secretion is episodic in mammals (18); in the rat, each GH secretory episode is triggered by a peak of GHRH release, which is preceded by a reduction of hypothalamic SRIF release (19). These findings suggest that the interrelationship between these two hypophysiotropic peptides is crucial to maintain a normal pulsatile GH secretion. However, the respective roles of GHRH and SRIF in determining the endogenous GH secretory pattern is not fully understood. Based on elegant in vitro studies, Kraicer et al. (20) have recently suggested that SRIF withdrawal is the primary factor in setting the timing and duration of the bursts of GH secretion, while GHRH is the primary factor in determining the amplitude of these bursts. Interestingly, the pattern of GH secretion during puberty is characterized by an increased GH pulse amplitude, but not frequency, which accounts for the increased daily GH secretion (4). Viewed in this light, our findings would support the hypothesis that an increased release of hypothalamic GHRH is responsible for the pubertal increase in GH secretion, since PO, which affects GH secretion by inhibition of endogenous SRIF release (15) was unable to elicit different GH secretory patterns either alone or in combination with GHRH in our prepubertal and pubertal children. Abundant evidence indicate that the effect of sex steroids on GH secretion is mediated by their interaction with brain neurotransmitters. In particular, sex steroids are known to affect the turnover of brain catecholamines (21), the same neurotransmitters that regulate the release of GHRH and SRIF from the hypothalamic neurones (22). Interestingly, the GH response to clonidine, which stimulates GH release via GHRH (22), is drastically reduced in the male rat after castration, and is restored to normal by testosterone replacement therapy (23). In children, pretreatment with androgens (6-8), or the spontaneous onset of puberty (8-11) induces an increase in the GH response to eNS-acting GH secretagogues such as insulin-hypoglycemia or arginine. These changes of GH secretion occurring during spontaneous puberty or after sex steroid administration might not be reflected in a modification of the somatotrope sensitivity to the action of a direct GH secretagogue, thus explaining the lack of any difference in the GH response to GHRH or to PO plus GHRH throughout pubertal maturation.
In conclusion, we have confirmed that the combination of PO plus GHRH is a powerful test to study the secretory capacity of the somatotropes (14), and shown that puberty does not affect the GH response to this test.
REFERENCES 1. Finkelstein J.w., Roffwarg H.P, Boyar R.M., Kream J., Hellman L. Age-related change in the twenty-four-hour spontaneous secretion of growth hormone. J. Clin. Endocrinol. Metab. 35: 665, 1972. 2. Zadik Z., Chalew SA, McCarter M.J., Meistas M, Kowarski AA The influence of age on the 24-hour integrated concentration of growth hormone in normal individuals. J. Clin. Endocrinol. Metab. 60: 513, 1985 3. Costin G., Kauffman F.R. Growth hormone secretory patterns in children with short stature. J. Pediatr. 110: 362,1987. 4. Mauras N., Blizzard R.M., Link K., Johnson M.L., Rogol A.D., Veldhuis J.D. Augmentation of growth hormone secretion during puberty: evidence for a pulse amplitude-modulated phenomenon. J. Clin. Endocrinol. Metab. 64: 596, 1987. 5. Liu L., Merriam G.R., Sherins R.J. 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, 1987. 6. Illig R., Prader A. Effect of testosterone on growth hormone secretion in patients with anorchia and delayed puberty. J. Clin. Endocrinol. Metab. 30: 615, 1970. 7. Martin L.G., Clark J.w., Connor T.B. Growth hormone secretion enhanced by androgens J. Clin. Endocrinol. Metab. 28: 425, 1968 8. Martin L.G., Grossman L.S, Connor T.B, Levitsky L.L., Clark JW, Camitta FD. Effect of androgen on growth hormone secretion and growth in boys with short stature. Acta Endocrinol. 91: 201,1979. 9. Frasier S.D., Hilburn B.S., Smith FG. Effect of adolescence on the serum growth hormone response to hypoglycemia. J. Pediatr. 77: 465,1970. 10. Gourmelen R., Phaam-Huu-Trung M.T., Girard F Transient partial hGH deficiency in prepubertal children with delay of growth. Pediatr. Res. 13: 221, 1979. 11. Penny M., Blizzard R.M. The possible influence of puberty in the release of growth hormone in three males with apparent growth hormone deficiency. J. Clin. Endocrinol. Metab. 34: 82, 1972.
44
Pyridostigmine and GH secretion
12. Chalew SA , Armour K.M ., Levin PA, Thorner M.O., Kowarski AA Growth hormone (GH) response to GH-releasing hormone in children with subnormal integrated concentrations of GH. J. Clin. Endocrinol. Metab. 62 1110, 1986.
18.
13. Gelato M.C., Malozowski S. , Caruso-Nicoletti M. , Ross J.L., Pescovitz O.H., Rose S., Loriaux D.L. , Cassorla F, Merriam G.R. Growth hormone (GH) response to GH-releasing hormone during pubertal development in normal boys and girls: comparison to idiopathic short stature and GH deficiency. J. Clin. Endocrinol. Metab. 63: 174, 1986.
19.
14. Ghigo E., Mazza E., Imperiale E., Rizzi G., Benso L., Muller E.E., Camanni F. , Massara F The enhancement of the cholinergic tone by pyridostigmine promotes both basal and growth hormone (GH) releasing hormone-induced GH secretion in children with short stature. J. Clin. Endocrinol. Metab. 60: 452,1987.
20.
21.
15. Locatelli V , Torsello A, Redaelli M. , Ghigo E. , Massara F , Muller E.E. Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormonereleasing hormone in the rat. Evidence for mediation by somatostatin. J. Endocrinol. 111: 271 , 1986.
22.
16. Loche S., Cappa M., Borrelli P., Faedda A, Crino A , Cella S.G ., Corda R. , Muller E.E., Pintor C. Reduced growth hormone response to growth hormone releasing hormone in children with simple obesity: evidence for somatomedin C mediated inhibition. Clin. Endocrinol. (Oxf.) 27: 145, 1987.
23 .
17. Martha P.M. Jr, Blizzard R.M., McDonald JA , Thorner M.O. , Rogol AD. A persistent pattern of varying pituitary responsivity
45
to exogenous growth hormone (GH)-releasing hormone in GH deficient children: evidence supporting periodic somatostatin secretion. J. Clin. Endocrinol. Metab. 67: 449,1988. Quabbe H.J. Growth hormone. In: Lightman S.L., Everitt B.J. (Eds.), Neuroendocrinology. Blackwell Scientific Pubblications, Ed inburg, 1986, p.409. Plotsky P.M. , Vale W. Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophysial portal circulation of the rat. Science 230: 461 , 1985. Kraicer J., Sheppard M.S., Luke J, Lussier B., Moor B.C., Cowan J.S. Effect of withdrawal of somatostatin and growth hormone (GH)-releasing factor on GH release in vitro. Endocrinology 122: 1810, 1988. Engel J., Ahlenius S., Almgren 0., Carlsson A., Larsson K., Sodersten P. Effect of gonadectomy and hormone replacement on brain monoamine synthesis in the male rat. Pharmacol. Biochem. Behav. 10: 149, 1979. Muller E.E. , Locatelli V, Ghigo E. , Loche S. , De Gennaro Colonna V , Cocchi D., Pintor C., Camanni F. Involvement of brain neurotransmitters in the control of growth hormone secretion . In: Frish H., Thorner M.O. (Eds.), Hormonal Regulation of Growth. Serono Symposia, Raven Press, New York, vol 58 , 1989, p. 49. Jansson J.O ., Eriksson E. , Eden S. , Modigh K. Effect of gonadectomy and testosterone replacement on growth hormone response to alpha 2-adrenergic stimulation in the male rat. Psychoneuroendocrinology 7: 245, 1982.
The growth hormone response to pyridostigmine plus growth hormone releasing hormone is not influenced by pubertal maturation M. Cappa*, S. Loche**, R. SaJvatori*, A. Faedda*, P. BorreJJi*, S.G. CeJJa***, C. Pintor**, and E.E. MuJJer***. *Oivisione di Endocrinologia, Ospedale Bambino Gesu, IRCCS, Roma, **Cattedra di Endocrinologia Pediatrica, Universita di Cagliari, ***Oipartimento di Farmacologia, Universita di Milano, Italy.
prepubertal children (16.0 ± 2.8,8.1 ± 1.3 and 51.1 ng/ml, mean ± SE, respectively) were not different from those observed in the early pubertal (18.4 ± 2.1 , 9.1 ± 1.9 and 41.2 ± 5.6 ng/ml, respectively) and in the late pubertal group (14.9 ± 2.3, 13.1 ± 2.4 and 42.6 ± 2.9 ng/ml, respectively). Evaluation of the area under the curve (AUG) also showed no difference in the GH response to GHRH, PO and PO + GHRH between the three groups studied. These results confirm that the combination PO + GHRH is a powerful test to study the GH secretory capacity of the pituitary, and show that pubertal maturation has no effect on the GH response to this test.
ABSTRACT. We have evaluated the effect of pubertal maturation on the GH response to growth hormone releasing hormone (GHRH), pyridostigmine (PO), and the combined administration of PO + GHRH in a group of short normal children. Fifteen were prepubertal (13 boys and 2 girls, age 5.0 - 12.5 yr), 10 were early pubertal (8 boys and 2 girls, age 11.5 -16.9 yr in Tanner stage 2-3 of pubertal maturation), and 6 were late pubertal (6 boys and 2 girls, age 13.6 - 17.1 yr in Tanner stage 4-5 of pubertal maturation). All subjects were tested on three occasions with GHRH 1-29 (1 /-lg/Kg iv), PO (60 mg po) and PO + GHRH (60 mg PO administered orally 60 min before GHRH). Peak GH levels after GHRH, PO, and PO + GHRH in the
± 5.5
in the GH response to the releasing hormone in relation to the pubertal status of the subjects (12-13). It has recently been reported (14) that the GH response to GHRH in children can be significantly enhanced by pretreatment with a cholinergic agonist, namely pyridostigmine (PO), which inhibits endogenous somatostatin release (15). Combined administration of PO and GHRH has been described as a highly suitable test to study the secretory capacity of the somatotropes in children (14). In this study we have evaluated the effect of pubertal maturation on the GH response to GHRH, PO and PO + GHRH in a group of short normal children.
INTRODUCTION Puberty is characterized by increased growth hormone (GH) and insulin-like growth factor I (IGFI) secretion, whose interaction with gonadal steroids is responsible for the pubertal growth spurt (1-4). An increased 24-h GH secretion associated with increased GH pulse amplitude has been demonstrated during spontaneous pubertal maturation as well as after sex steroids administration (3-5). Furthermore, the GH response to pharmacological stimuli such as insulin-hypoglycemia or arginine have been reported to be enhanced by androgens (6-8), or by the spontaneous onset of puberty (8-11). However, studies on the effect of GHRH on GH secretion during puberty have shown no changes
MATERIALS AND METHODS Thirty-one short children (25 boys and 6 girls) aged 5.0 - 17.1 yr [bone age (BA) 3.0 - 14.4 years] were studied. Fifteen [13 boys and 2 girls; chronologic age (CA) 5.0 - 12.5 years, BA 3.0 - 10.0 yr] were prepubertal (testicular volume < 2 ml and testosterone levels of 23.2 ± 1.8 ng/dl in boys; no breast or pubic
Key-words: GH, GHRH, somatostatin, pyridostigmine, puberty. Correspondence: Dr. Sandro Loche, Cattedra di Endocrinologia Pediatrica, Ospedale Microcitemico. via Jenner. 09100 Cagliari, Italy. Received November 27,1989; accepted October 15,1990.
41
M Cappa, S. Lache, R. Salvatori, et al.
hair development in girls), 10 (8 boys and 2 girls; CA 11.5 - 16.9 yr, BA 11.0 - 14A yr) were in Tanner stage 2 or 3 of pubertal maturation (testosterone levels in boys 160.2 ± 35.8 ng/dl) and 6 were in Tanner stage 4 or 5 (testosterone levels in boys 420 ± 50 ng/dl). All children referred to our Department for short stature; none had signs of malnutrition, dysmorphic syndromes, psychosocial deprivation and had taken long-term medication prior to enter the study. None had classical GH deficiency as assessed by nocturnal GH peak or by clonidine stimulation test; mean peak GH levels measured at 30 min intervals every 3 h after the onset of spontaneous sleep were 17.0 ± 3A ng/ml (mean ± SE, range 8.0 - 82.0 ng/ml), and peak GH levels after clonidine were always> 8.0 ng/ml. The mean growth velocity in the prepubertal and early pubertal group was 4.6 ± 0.3 (SE) and 6.5 ± 0.6 cm/yr, respectively (p < 0.01). In the four late pubertal boys, mean growth velocity was 6.7 ± 0.8 cm/yr, while the two late pubertal girls had completed their statural growth according to their bone age. Mean IGF-Ilevels in the prepubertal, early pubertal and late pubertal groups was 1.9 ± 0.2, 2.6 ± OA, and 1.5 ± 0.2 U/ml, respectively. The study was approved by the Ethical Commitee of the Department of Endocrinology of the Bambino Gesu Hospital, Rome, and informed consent was obtained from all children or from their legal guardians prior to the study.
After an overnight fast all children were tested three times in random order with GHRH (1-29, kindly provided by Pierrel-Kabivitrum, Milan) at the dose of 1.0 Ilg/Kg iv; with PD (Mestinon, Hoffman-La Roche, Nutley, NJ) at the dose of 60 mg orally, and with PD plus GHRH (60 mg PD administered orally 60 min before GHRH). Blood samples were taken 30 min and immediately before the administration of GHRH and then after 15, 30, 60, 90 and 120 min (GHRH and PD plus GHRH tests), and at times -60, -30, 0, 15, 30, 60, 90 and 120 min during the PD test. An interval of 3 to 7 days was left between each test. GH was measured by double antibody RIA using reagents provided by CEA-IRE Sorin (Saluggia, Italy). The sensitivity of the assay was 1.0 ng/ml with an intra and interassay coefficient of variation of 3.8 and 9.2 % respectively. IGF-I levels were measured by RIA using reagents provided by the Nichols Institute Diagnostic (S.Juan Capistrano, CA). Details on the IGF-I assay in our laboratory have been reported elsewhere (16). The GH secretory responses were expressed either as absolute peak values (ng/ml) or as areas under the curve (AUC) calculated by trapezoidal integration. The statistical significance of the differences was calculated by means of paired or unpaired t tests preceded by ANOVA. A P value less than 0.05 (two tailed) was considered to indicate a significant difference. All data are given as the mean + SE.
* ** ~." e:
J:
e!)
*
40 30 20 10
GHRH
PO
PO+GHlH
PREPUBERTY
GHRH
PD
P2-3
42
PD.GHRH
GHRH
PD
P4-5
PD.oHRH
Fig. 1 - Mean ± SE of the peak plasma GH responses to PO (60 mg orally), to a single bolus injection of GHRH (1 Ilg/kg ivY alone and after pretreatment with PO (60 mg po 60 min before the GHRH injection) in short prepubertal, early pubertal (P 2-3) and late pubertal (P 4-5) children. *p < 0.001 vs GHRH alone; **p < 0.005 vs GHRH alone.
Pyridostigmine and GH secretion
ng.min/ml, p < 0.005 vs GHRH alone; late pubertal= 2,395 ± 259 ng'min/ml, p < 0.005 vs GHRH alone) (Fig. 2). The GH response to PO plus GHRH was not significantly different between the three groups studied. Also after PO plus GHRH administration the peak GH levels showed a great interindividual variability, ranging from 18.0 to 97.0 ng/ml in the prepubertal children, from 20.0 to 72.0 ng/ml in the early pubertal, and from 33.4 to 50.0 ng/ml in the late pubertal children. Pretreatment with PO enhanced the GH response to GHRH to a similar extent in either group studied. There was no correlation between growth velocity or IGF-I levels and the degree of the plasma GH response to GHRH, PO and PO plus GHRH evaluated as either peak or AUC. PO administration was well tolerated and did not induce noticeable side effects in any of the children studied.
RESULTS
Mean peak plasma GH levels (Fig. 1) and mean AUC (Fig . 2) after GHRH in the prepubertal children (16.0 ± 2.8 ng/ml and 785 ± 141 ng.min/ml) were not significantly different from those observed in the early pubertal (18.4 ± 2.1 ng/ml and 968 ± 78 ng'min/ml) and in the late pubertal group (14.9 ± 2.3 ng/ml and 725 ± 118 ng·min/ml). The GH peak after GHRH showed a great variability, ranging from 3.0 to 45.0 ng/ml in the prepubertal children, from 12.0 to 32.0 ng/ml in the ear!y pubertal, and from 5.6 to 21.0 ng/ml in the late pubertal. Similarly, the mean peak plasma GH levels and mean AUC after PO alone were not different between prepubertal (8.1 ± 1.3 ng/ml and 366 ± 77 ng 'min/ml) , early pubertal (9.1 ± 1.9 ng/ml and 457 ± 94 ng'min/ml) and late pubertal children (13.1 ± 2.4 ng/ml and 659 ± 41 ng.min/ml) (Figs. 1 and 2). Peak GH levels after PO alone ranged from 2 .5 to 21.0 ng/ml in the prepubertal children, from 5.0 to 21.0 ng/ml in the pubertal group, and from 5.4 to 21.8 ng/ml in the late pubertal group. PO administration greatly enhanced the GH response to GHRH evaluated as either peak (prepubertal= 51.1 ± 5.5 ng/ml, p < 0.001 vs GHRH alone ; early pubertal= 41 .2 ± 5.6 ng/ml, p < 0.005 vs GHRH alone; late pubertal= 42.6 ± 2.9 ng/ml, p < 0.001 vs GHRH alone) (Fig. 1) or AUC (prepubertal= 2,840 ± 372 ng'min/ml, p < 0.001 vs GHRH alone; early pubertal= 2,004 ± 254
.... ...~. ~c
DISCUSSION
Our results confirm the ability of PO to enhance the GH response to a single bolus injection of GHRH in short children (14). As previously reported (12,13), we found no difference in the GH response to GHRH between prepubertal and pubertal children . Moreover, no difference related to the state of puberty in the GH response to GHRH was found after the children were administered PO, i.e. after the endogenous SRIF influence on the pituitary had been
*
30
** **
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r 10
0
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et
8
GHRH
PO
POtGHIH
PREPUBERTY
CiHRH
PO
P2-3
43
PI>tGH!H
GHRH
PO
P4-5
PD+GHRH
Fig. 2 - Mean ± SE of AUC after PO (60 mg orally), and after a single bolus injection of GHRH (1 Ilg/kg ivy alone and after pretreatment with PO (60 mg po 60 min before the GHRH injection) in short prepubertal, early pubertal (P 2-3) and late pubertal (P 4-5) children. *p < 0.001 VS GHRH alone; **p < 0.005 vs GHRH alone.
M. Cappa, S. Lache, R. Salvatori, et 81.
removed. The GH response to GHRH showed a great variability, which was not abolished by pretreating the children with PD. This indicates that such variability is not solely due to the endogenous SRIF tone (17), since PO administration would have been able to inhibit hypothalamic SRIF release (15). GH secretion is episodic in mammals (18); in the rat, each GH secretory episode is triggered by a peak of GHRH release, which is preceded by a reduction of hypothalamic SRIF release (19). These findings suggest that the interrelationship between these two hypophysiotropic peptides is crucial to maintain a normal pulsatile GH secretion. However, the respective roles of GHRH and SRIF in determining the endogenous GH secretory pattern is not fully understood. Based on elegant in vitro studies, Kraicer et al. (20) have recently suggested that SRIF withdrawal is the primary factor in setting the timing and duration of the bursts of GH secretion, while GHRH is the primary factor in determining the amplitude of these bursts. Interestingly, the pattern of GH secretion during puberty is characterized by an increased GH pulse amplitude, but not frequency, which accounts for the increased daily GH secretion (4). Viewed in this light, our findings would support the hypothesis that an increased release of hypothalamic GHRH is responsible for the pubertal increase in GH secretion, since PO, which affects GH secretion by inhibition of endogenous SRIF release (15) was unable to elicit different GH secretory patterns either alone or in combination with GHRH in our prepubertal and pubertal children. Abundant evidence indicate that the effect of sex steroids on GH secretion is mediated by their interaction with brain neurotransmitters. In particular, sex steroids are known to affect the turnover of brain catecholamines (21), the same neurotransmitters that regulate the release of GHRH and SRIF from the hypothalamic neurones (22). Interestingly, the GH response to clonidine, which stimulates GH release via GHRH (22), is drastically reduced in the male rat after castration, and is restored to normal by testosterone replacement therapy (23). In children, pretreatment with androgens (6-8), or the spontaneous onset of puberty (8-11) induces an increase in the GH response to eNS-acting GH secretagogues such as insulin-hypoglycemia or arginine. These changes of GH secretion occurring during spontaneous puberty or after sex steroid administration might not be reflected in a modification of the somatotrope sensitivity to the action of a direct GH secretagogue, thus explaining the lack of any difference in the GH response to GHRH or to PO plus GHRH throughout pubertal maturation.
In conclusion, we have confirmed that the combination of PO plus GHRH is a powerful test to study the secretory capacity of the somatotropes (14), and shown that puberty does not affect the GH response to this test.
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