0021-972X/90/7002-0324$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 2 Printed in U.S.A.
Effect of Enhancement of Endogenous Cholinergic Tone with Pyridostigmine on the Dose-Response Relationships of Growth Hormone (GH)-Releasing Hormone-Induced GH Secretion in Normal Subjects* A. PENALVA, C. MURUAIS, F. F. CASANUEVA, AND C. DIEGUEZ Departments of Medicine (A.P., F.F.C.) and Physiology (CM., CD.), Facultad de Medicina, Santiago de Compostela, Spain
ABSTRACT. It is well known that GH responses to GHreleasing hormone (GHRH) show marked interindividual variations in normal subjects, which have been attributed to a variable somatostatinergic tone. Recently, it has been shown that enhancement of cholinergic tone with the acetylcholinesterase inhibitor pyridostigmine (PD), which presumably acts by inhibiting somatostatin release, stimulates basal GH secretion and GH responses to a maximal dose of GHRH. In this study we have investigated the effects of PD on the dose-response relationships of GHRH-induced GH secretion in normal subjects. Our data showed that PD (120 mg, orally, at -60 min) induced a clear-cut increase in basal GH levels, significantly different
from that after saline treatment, at 15, 30, 45, 60, 90, and 120 min. Moreover, PD administration markedly potentiated GH responses to GHRH at doses of 500, 100, 25, 10, and 3 fig/ subject, as assessed by either area under the curve or maximal peak GH levels. In fact, GH responses to pyridostigmine plus 3 ng GHRH were similar to those to the administration of 500 and 100 ng GHRH alone. Our findings of marked increases in GH response to GHRH after pyridostigmine administration show that with enhancement of cholinergic tone, the dose of GHRH needed to induce a similar increase in GH is reduced 30 times. (J Clin Endocrinol Metab 70: 324, 1990)
I
T IS NOW well established that cholinergic pathways play an important role in the control of GH secretion from the anterior pituitary. Muscarinic cholinergic agonists stimulate basal GH release and GH responses to GH-releasing hormone (GHRH) (1-5). Conversely, antagonism of endogenous cholinergic pathways with muscarinic receptor-blocking drugs, such as methscopolamine, atropine, or pirenzepine, causes a striking reduction in slow wave sleep-related GH release and GH responses to L-dopa, arginine, physical exercise, FK 33824, glucagon, clonidine, and GHRH (6-14). Since it has been shown that the inhibitory effect of atropine on GH responses to GHRH in normal rats is abolished by antisomatostatin antibodies (15), it has been proposed that acetylcholine regulates GH secretion by inhibiting somatostatin release from the hypothalamus. After the sequencing and synthesis of GHRH, several studies investigating the dose-response relationship between iv administered GHRH and GH secretion in normal subjects have been carried out (16-19). The available
evidence suggests that doses of GHRH as low as 3 subject can elicit a rise in plasma GH levels in a few normal subjects, with maximal GH release attained at doses above 20 /ug/subject. However, a large intersubject variability was noted in all studies, which has been attributed to a variable endogenous somatostatinergic tone. The aim of this study was to evaluate the dose-response effect of iv GHRH on GH secretion in normal subjects under low somatostatinergic tone. Hence, we have studied GH responses to GHRH in normal subjects treated with either placebo or pyridostigmine (PD).
Materials and Methods Studies were performed in a randomized fashion on healthy nonobese adult males (age range, 19-27 yr), mean (±SEM) body weight (69.8 ± 1.5 kg). Informed consent was given by each subject after local ethical approval was granted. All tests were performed after an overnight fast and were separated by at least 1 week. On the day of the study, an indwelling catheter was placed in a forearm vein for blood withdrawal and/or drug administration, and its patency was maintained by slow infusion of isotonic saline. Throughout the study, subjects remained recumbent and were not allowed to sleep. Blood samples were taken at -60, -30, 0, 15, 30, 45, 60, 90, and 120 min. Subjects participating in the PD-GHRH study underwent two tests on
Received March 3, 1989. Address all correspondence and requests for reprints to: C. Dieguez, P.O. Box 563, Santiago de Compostela, Spain. * This work was supported by Grants FISS9 88-0818 and 89-0765 and Xunta de Galicia Grant 609027-5380.
324 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:47 For personal use only. No other uses without permission. . All rights reserved.
325
PD AND GHRH-INDUCED GH SECRETION separate occasions, serving as their own control on each occasion. PD (Mestinon, Roche, Madrid, Spain; 120 mg, orally) or placebo was administered at -60 min. GHRH was given iv 0 min after placebo or PD at the following doses: 500 jig/subject (n = 6), 100 Mg/subject (n = 8), 25 Mg/subject (n = 5), 10 /*g/ subject (n = 6), 3 /zg/subject (n = 6), and 0.5 /xg/subject (n = 7). Another group of normal subjects (n = 6) was studied after administration of PD (120 mg, orally) or placebo at —60 min and saline infusion thereafter without GHRH administration. GH was measured using commercial kits (Biomerieux, Madrid, Spain), with an intraassay coefficient of variation of 6.8%. All samples from each volunteer were assayed at the same time. Because of the relatively low numbers of subjects that underwent each study, statistical analysis was performed by a nonparametric test (Wilcoxon's paired test). The area under curve (AUC) of GH responses to GHRH alone, PD alone, or GHRH plus PD was calculated by a trapezoidal method. Comparisons between GHRH alone and GHRH plus PD were carried out using Wilcoxon's test as well.
80 70 60
* JGHRH 500 M9
2 50 5 40 °
20 10 0
100 GHRH 100 pg
9080 _
70-
^
60-
I 50I 403020100-
Results PD administration induced a clear-cut increase in basal plasma GH levels compared to those after placebo (Fig. 1), with significant differences observed at 15 (P < 0.05), 30, 45, 60, (P < 0.01), and 90 (P < 0.05) min. . PD administration (Fig. 1) also markedly increased GH responses to 500 Mg GHRH (P < 0.05 at 15 and 120 min and P < 0.01 at 30, 45, 60, and 90 min post-GHRH administration), 100 ng GHRH (P < 0.01 at 15, 30, 45, 60, 90, and 120 min), 25 jug GHRH (P < 0.05 at 30 and 45 min), 10 ixg GHRH (P < 0.05 at 30 and 45 min), and 3 fig GHRH (P < 0.05 at 15, 45, 60, 90, and 120 min and P < 0.01 at 30 min). GH responses to PD plus 0.5 ng GHRH were not different from those to PD alone at any time studied (Fig. 1). A similar pattern of responses was observed when the data were analyzed in terms of AUC, with GH responses to GHRH plus PD being greater than those to GHRH alone at doses of 3, 10, 25, 100 and 500 /ig/subject (Fig. 2). It is interesting to note that the AUC obtained after combined administration of PD and 3 ng GHRH was similar to that obtained after adding the AUC after PD alone and that after 100 or 500 /ug/subject GHRH alone.
Discussion IN recent studies it has been clearly shown that the cholinergic agonist drug PD stimulates basal GH levels and GH responses to GHRH (1-5) and counteracts, as well, the inhibitory effect exerted by repeated GHRH administration (2), exogenous GH infusion (3), oral glucose loading (4), or obesity (5) on GH responses to GHRH. Although direct proof of the mechanisms through which PD exerts its effects on GH secretion in normal subjects is still lacking, based on studies carried out in rats it has been proposed that PD inhibits hypothalamic somatostatin release (15).
6050 ^ S
s
GHRH 25 pg
40 30
20 10 0
50 ^ 40
GHRH 10 pg
j 30 — 20 o
10 0
50-
GHRH 3 pg
40-
"S 30-
20;
O
100-
~
30-
|
20-
~
10-
o
0-
GHRH 0.5 fjg *
*
*
*
SALINE
-60 0
15 30 45 60
90
120
TIME (minutes)
FIG. 1. Mean ± SEM plasma GH levels in response to different doses of GHRH (jig/subject) after administration of PD (120 mg, orally, at —60 min; O—O) or placebo (•—•). At the bottom is also shown the effect of either saline (•—•) and PD (O—O), but not GHRH, on plasma GH levels. Data are expressed as the mean ± SEM. *, P < 0.05; • • , P < 0.01.
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PENALVA ET AL.
326
FIG. 2. Effects of different doses of GHRH after administration of placebo (P) or PD on mean plasma GH levels, as assessed by the AUC. After PD administration, compared to placebo, greater GH responses to 3, 10, 25, 100, and 500 Hg GHRH/subject were observed (P < 0.01) in every case compared to the same dose of GHRH alone. Data are expressed as the mean ± SEM. *, P < 0.05; • • , P < 0.01.
JCE & M • 1990 Vol70«No2
u 4000-
3000-
2000-
1000-
GHRH dose pg / subject
P PD !•— 500—H
Like other workers (16-19), we found that doses of GHRH between 25-500 jug/subject induced a maximal increase in plasma GH levels, as assessed by the AUC and maximal peaks. We also found that low doses of GHRH (3 jug/subject) can induce an increase in basal GH levels in a very few subjects, although with all doses tested there was a marked interindividual variation. Thus, it was of interest to find out whether after PD administration, and presumably low somatostatinergic tone, a greater homogeneity in GH responses to GHRH would be observed. We found that after PD administration there was a marked increase in GH responses to different doses of GHRH, although considerable interindividual variations persisted. Nevertheless, it was also clear that the number of low responders was very much reduced when PD was administered even with a low dose (3 ng) of GHRH. Whether the interindividual variations in GH responses to PD plus GHRH are due to differences in somatotroph sensitivity, cholinergic control of somatostatin, or the absorption of the drug among different subjects is not clear at present. The dose-response relationship of GHRH given in combination with PD is intriguing for several reasons: 1) Doses of GHRH as low as 3 ng produced a marked increase in plasma GH levels (mean maximal peak ± SEM, 40 ± 8.8 ng/mL; range, 11.9-65 ng/mL). This was due to both compounds exerting a synergistic effect, since the sum of the effect of each compound administered independently, as assessed by the AUC, was smaller, and clearly suggests that in vivo, the somatotroph is extremely sensitive to low doses of GHRH under conditions of low somatostatinergic tone. 2) Similar increases in plasma GH levels, as assessed by the AUC and maximal peak levels, were obtained after the administration of 3, 10, and 25 jug GHRH plus PD. This is in contrast to the
P PD \>— 100-H
data obtained after the administration of GHRH alone for which we and others (9, 16) found a clear doseresponse relationship. It is unclear at present why a plateau is reached in the GH responses to 3, 10, and 25 Hg GHRH plus PD. 3) Finally, while we found that with doses of or above 25 ng GHRH alone a maximal increase in plasma GH levels was obtained, we observed that after administration of PD there was a marked increase in GH responses to 100 and 500 ng GHRH compared to the response to 25 ng. Taking together this latter finding and the flattening of responses to 3, 10, and 25 ^g GHRH plus PD, it seems that while GH responses to GHRH alone follow a monophasic curve, when PD is administered the pattern more closely resembles a biphasic one. However, this assumption must be regarded as speculative, since the large interindividual variations in response and the difficulties in carrying out this sort of study on a larger number of subjects prevent us from reaching firmer conclusions in terms of the ED50 and binding capacity of the dose-responses curves under both experimental conditions. In any event, it is a very interesting finding that similar or even greater GH responses can be obtained by combined administration of PD plus 3 ^g GHRH compared to responses to 500 and 100 ng GHRH alone. Further experiments need to be carried out to ascertain whether chronic administration of PD combined with low doses of GHRH can increase somatic growth in both humans and animals in a similar fashion to the effects they exert on GH secretion.
Acknowledgments We are grateful to Ms. Dolores Vina and Ms. Mari Lage for expert technical assistance, and to Serono Ltd. (Spain) for the supply of GHRH-U-29).
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PD AND GHRH-INDUCED GH SECRETION
References 1. Massara F, Ghigo E, Demislis K, et al. Cholinergic involvement in the growth hormone releasing hormone-induced growth hormone release studies in normal and acromegalic subjects. Neuroendocrinology 1986;43:670-5. 2. Massara F, Ghigo E, Molinatti P, et al. Potentiation of cholinergic tone by pyridostigmine bromide re-instates and potentiates the growth hormone responsiveness to intermittent administration of growth hormone releasing factor in man. Acta Endocrinol (Copenh). 1986;113:12-6. 3. Ross RJM, Tsagarakis S, Grossman A, et al. GH feedback occurs through modulation of hypothalamic somatostatin under cholinergic control: studies with pyridostigmine and GHRH. Clin Endocrinol (Oxf). 1987;27:727-34. 4. Pefialva A, Burguera B, Casabiell X, Tresguerres JAF, Dieguez C, Casanueva FF. Activation of cholinergic neurotransmission by pyridostigmine reverses the inhibitory effect of hyperglycaemia on growth hormone releasing hormone induced growth hormone secretion in man: suggesting that glucose acts through hypothalamic release of somatostatin. Neuroendocrinology 1989;49:551-4. 5. Cordido F, Casanueva FF, Dieguez C. Cholinergic receptor activation by pyridostigmine restored growth hormone responsiveness after growth hormone-releasing hormone administration in obese subjects: evidence for hypothalamic somatostatinergic participation in the blunted GH release of obesity. J Clin Endocrinol Metab. 1989;68:290-3. 6. Massara F, Ghigo E, Golfi S, Molinatti MG, Miiller EE, Camanni F. Blockage of hp-GRF 40 induced GH release in normal men by a cholinergic muscarinic antagonist. J Clin Endocrinol Metab. 1984;59:1025-6. 7. Jordan V, Dieguez C, Lafaffian I, et al. Influence of dopaminergic, adrenergic and cholinergic blockade and TRH administration on GH responses to GRF 1-29. Clin Endocrinol (Oxf). 1986;24:291-8. 8. Casanueva FF, Villanueva L, Dieguez C, et al. Atropine blockade of growth hormone releasing hormone-induced GH secretion is not exerted at pituitary level. J Clin Endocrinol Metab. 1986;62:18691. 9. Delitala G, Palermo H, Ross R, Coy D, Besser M, Grossman A. Dopaminergic and cholinergic influences on the growth hormone response to growth hormone-releasing hormone in man. Neuroen-
327
docrinology. 1987;45:243-6. 10. Casanueva FF, Villanueva L, Cabranes JA, Cabezas-Cerrato J, Fernandez-Cruz A. Cholinergic mediation of GH secretion elicited by arginine, clonidine and physical exercise in man. J Clin Endocrinol Metab. 1984;59:526-30. 11. Delitala G, Maoili M, Pacifico A, Brianda SA, Palermo M, Manelli M. Cholinergic receptor control mechanism for L-dopa, apomorphine and clonidine-induced growth hormone secretion in man. J Clin Endocrinol Metab. 1983;57:1145-9. 12. Pefialva A, Villanueva L, Casanueva FF, Cavagnini F, Gomez-Pan A, Miiller EE. Cholinergic and histaminergic involvement in the growth hormone releasing effect of an enkephalin analog FK 33824 in man. Psychopharmacology 1983;80:120-4. 13. Peters JR, Evans PI, Page MD, et al. Cholinergic muscarinic blockade with piranzepine abolishes low wave sleep-related growth hormone release in normal adult males. Clin Endocrinol (Oxf). 1986;25:213-7. 14. Piestchmann P, Schernthaner G, Luger A. Effect of cholinergic muscarinic receptor blockade on human growth hormone (GH)releasing hormone-(l-44)-induced GH secretion in acromegalic and type I diabetes mellitus. J Clin Endocrinol Metab. 1986;63:38992. 15. Locatelli V, Torsello A, Redaelli M, Ghigo E, Massara F, Miiller EE. Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone releasing hormone in the rat: evidence for mediation by somatostatin. J Endocrinol. 1986;lll:271-8. 16. Vance ML, Borges JLC, Kaiser DL, et al. Human pancreatic tumor growth hormone-releasing factor: dose-response relationships in normal man. J Clin Endocrinol Metab. 1984;58:838-42. 17. Gelato MC, Perscovitz OH, Cassorla F, Lynn Loriaux D, Merriam GR. Dose-response relationships for the effects of growth hormonereleasing factor-(l-44)-NH2 in young adult men and women. J Clin Endocrinol Metab. 1984;59:197-2O1. 18. Wood SM, Ch'ng JLC, Adams EF, et al. Abnormalities of growth hormone release in response to human growth hormone releasing factor (GRF 1-44) in acromegaly and hypopituitarism. Br Med J. 1983;286:1687-90. 19. Boissel JP, Cohen R, Biot-Laporte S, et al. Synthetic human growth hormone releasing factor (h-GRF 1-44 NH2) dose-response effect on growth hormone and prolactin secretion in healthy adult men..Eur J Clin Pharmacol. 1986;29:609-13.
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Vol. 70, No. 2 Printed in U.S.A.
Effect of Enhancement of Endogenous Cholinergic Tone with Pyridostigmine on the Dose-Response Relationships of Growth Hormone (GH)-Releasing Hormone-Induced GH Secretion in Normal Subjects* A. PENALVA, C. MURUAIS, F. F. CASANUEVA, AND C. DIEGUEZ Departments of Medicine (A.P., F.F.C.) and Physiology (CM., CD.), Facultad de Medicina, Santiago de Compostela, Spain
ABSTRACT. It is well known that GH responses to GHreleasing hormone (GHRH) show marked interindividual variations in normal subjects, which have been attributed to a variable somatostatinergic tone. Recently, it has been shown that enhancement of cholinergic tone with the acetylcholinesterase inhibitor pyridostigmine (PD), which presumably acts by inhibiting somatostatin release, stimulates basal GH secretion and GH responses to a maximal dose of GHRH. In this study we have investigated the effects of PD on the dose-response relationships of GHRH-induced GH secretion in normal subjects. Our data showed that PD (120 mg, orally, at -60 min) induced a clear-cut increase in basal GH levels, significantly different
from that after saline treatment, at 15, 30, 45, 60, 90, and 120 min. Moreover, PD administration markedly potentiated GH responses to GHRH at doses of 500, 100, 25, 10, and 3 fig/ subject, as assessed by either area under the curve or maximal peak GH levels. In fact, GH responses to pyridostigmine plus 3 ng GHRH were similar to those to the administration of 500 and 100 ng GHRH alone. Our findings of marked increases in GH response to GHRH after pyridostigmine administration show that with enhancement of cholinergic tone, the dose of GHRH needed to induce a similar increase in GH is reduced 30 times. (J Clin Endocrinol Metab 70: 324, 1990)
I
T IS NOW well established that cholinergic pathways play an important role in the control of GH secretion from the anterior pituitary. Muscarinic cholinergic agonists stimulate basal GH release and GH responses to GH-releasing hormone (GHRH) (1-5). Conversely, antagonism of endogenous cholinergic pathways with muscarinic receptor-blocking drugs, such as methscopolamine, atropine, or pirenzepine, causes a striking reduction in slow wave sleep-related GH release and GH responses to L-dopa, arginine, physical exercise, FK 33824, glucagon, clonidine, and GHRH (6-14). Since it has been shown that the inhibitory effect of atropine on GH responses to GHRH in normal rats is abolished by antisomatostatin antibodies (15), it has been proposed that acetylcholine regulates GH secretion by inhibiting somatostatin release from the hypothalamus. After the sequencing and synthesis of GHRH, several studies investigating the dose-response relationship between iv administered GHRH and GH secretion in normal subjects have been carried out (16-19). The available
evidence suggests that doses of GHRH as low as 3 subject can elicit a rise in plasma GH levels in a few normal subjects, with maximal GH release attained at doses above 20 /ug/subject. However, a large intersubject variability was noted in all studies, which has been attributed to a variable endogenous somatostatinergic tone. The aim of this study was to evaluate the dose-response effect of iv GHRH on GH secretion in normal subjects under low somatostatinergic tone. Hence, we have studied GH responses to GHRH in normal subjects treated with either placebo or pyridostigmine (PD).
Materials and Methods Studies were performed in a randomized fashion on healthy nonobese adult males (age range, 19-27 yr), mean (±SEM) body weight (69.8 ± 1.5 kg). Informed consent was given by each subject after local ethical approval was granted. All tests were performed after an overnight fast and were separated by at least 1 week. On the day of the study, an indwelling catheter was placed in a forearm vein for blood withdrawal and/or drug administration, and its patency was maintained by slow infusion of isotonic saline. Throughout the study, subjects remained recumbent and were not allowed to sleep. Blood samples were taken at -60, -30, 0, 15, 30, 45, 60, 90, and 120 min. Subjects participating in the PD-GHRH study underwent two tests on
Received March 3, 1989. Address all correspondence and requests for reprints to: C. Dieguez, P.O. Box 563, Santiago de Compostela, Spain. * This work was supported by Grants FISS9 88-0818 and 89-0765 and Xunta de Galicia Grant 609027-5380.
324 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 08:47 For personal use only. No other uses without permission. . All rights reserved.
325
PD AND GHRH-INDUCED GH SECRETION separate occasions, serving as their own control on each occasion. PD (Mestinon, Roche, Madrid, Spain; 120 mg, orally) or placebo was administered at -60 min. GHRH was given iv 0 min after placebo or PD at the following doses: 500 jig/subject (n = 6), 100 Mg/subject (n = 8), 25 Mg/subject (n = 5), 10 /*g/ subject (n = 6), 3 /zg/subject (n = 6), and 0.5 /xg/subject (n = 7). Another group of normal subjects (n = 6) was studied after administration of PD (120 mg, orally) or placebo at —60 min and saline infusion thereafter without GHRH administration. GH was measured using commercial kits (Biomerieux, Madrid, Spain), with an intraassay coefficient of variation of 6.8%. All samples from each volunteer were assayed at the same time. Because of the relatively low numbers of subjects that underwent each study, statistical analysis was performed by a nonparametric test (Wilcoxon's paired test). The area under curve (AUC) of GH responses to GHRH alone, PD alone, or GHRH plus PD was calculated by a trapezoidal method. Comparisons between GHRH alone and GHRH plus PD were carried out using Wilcoxon's test as well.
80 70 60
* JGHRH 500 M9
2 50 5 40 °
20 10 0
100 GHRH 100 pg
9080 _
70-
^
60-
I 50I 403020100-
Results PD administration induced a clear-cut increase in basal plasma GH levels compared to those after placebo (Fig. 1), with significant differences observed at 15 (P < 0.05), 30, 45, 60, (P < 0.01), and 90 (P < 0.05) min. . PD administration (Fig. 1) also markedly increased GH responses to 500 Mg GHRH (P < 0.05 at 15 and 120 min and P < 0.01 at 30, 45, 60, and 90 min post-GHRH administration), 100 ng GHRH (P < 0.01 at 15, 30, 45, 60, 90, and 120 min), 25 jug GHRH (P < 0.05 at 30 and 45 min), 10 ixg GHRH (P < 0.05 at 30 and 45 min), and 3 fig GHRH (P < 0.05 at 15, 45, 60, 90, and 120 min and P < 0.01 at 30 min). GH responses to PD plus 0.5 ng GHRH were not different from those to PD alone at any time studied (Fig. 1). A similar pattern of responses was observed when the data were analyzed in terms of AUC, with GH responses to GHRH plus PD being greater than those to GHRH alone at doses of 3, 10, 25, 100 and 500 /ig/subject (Fig. 2). It is interesting to note that the AUC obtained after combined administration of PD and 3 ng GHRH was similar to that obtained after adding the AUC after PD alone and that after 100 or 500 /ug/subject GHRH alone.
Discussion IN recent studies it has been clearly shown that the cholinergic agonist drug PD stimulates basal GH levels and GH responses to GHRH (1-5) and counteracts, as well, the inhibitory effect exerted by repeated GHRH administration (2), exogenous GH infusion (3), oral glucose loading (4), or obesity (5) on GH responses to GHRH. Although direct proof of the mechanisms through which PD exerts its effects on GH secretion in normal subjects is still lacking, based on studies carried out in rats it has been proposed that PD inhibits hypothalamic somatostatin release (15).
6050 ^ S
s
GHRH 25 pg
40 30
20 10 0
50 ^ 40
GHRH 10 pg
j 30 — 20 o
10 0
50-
GHRH 3 pg
40-
"S 30-
20;
O
100-
~
30-
|
20-
~
10-
o
0-
GHRH 0.5 fjg *
*
*
*
SALINE
-60 0
15 30 45 60
90
120
TIME (minutes)
FIG. 1. Mean ± SEM plasma GH levels in response to different doses of GHRH (jig/subject) after administration of PD (120 mg, orally, at —60 min; O—O) or placebo (•—•). At the bottom is also shown the effect of either saline (•—•) and PD (O—O), but not GHRH, on plasma GH levels. Data are expressed as the mean ± SEM. *, P < 0.05; • • , P < 0.01.
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PENALVA ET AL.
326
FIG. 2. Effects of different doses of GHRH after administration of placebo (P) or PD on mean plasma GH levels, as assessed by the AUC. After PD administration, compared to placebo, greater GH responses to 3, 10, 25, 100, and 500 Hg GHRH/subject were observed (P < 0.01) in every case compared to the same dose of GHRH alone. Data are expressed as the mean ± SEM. *, P < 0.05; • • , P < 0.01.
JCE & M • 1990 Vol70«No2
u 4000-
3000-
2000-
1000-
GHRH dose pg / subject
P PD !•— 500—H
Like other workers (16-19), we found that doses of GHRH between 25-500 jug/subject induced a maximal increase in plasma GH levels, as assessed by the AUC and maximal peaks. We also found that low doses of GHRH (3 jug/subject) can induce an increase in basal GH levels in a very few subjects, although with all doses tested there was a marked interindividual variation. Thus, it was of interest to find out whether after PD administration, and presumably low somatostatinergic tone, a greater homogeneity in GH responses to GHRH would be observed. We found that after PD administration there was a marked increase in GH responses to different doses of GHRH, although considerable interindividual variations persisted. Nevertheless, it was also clear that the number of low responders was very much reduced when PD was administered even with a low dose (3 ng) of GHRH. Whether the interindividual variations in GH responses to PD plus GHRH are due to differences in somatotroph sensitivity, cholinergic control of somatostatin, or the absorption of the drug among different subjects is not clear at present. The dose-response relationship of GHRH given in combination with PD is intriguing for several reasons: 1) Doses of GHRH as low as 3 ng produced a marked increase in plasma GH levels (mean maximal peak ± SEM, 40 ± 8.8 ng/mL; range, 11.9-65 ng/mL). This was due to both compounds exerting a synergistic effect, since the sum of the effect of each compound administered independently, as assessed by the AUC, was smaller, and clearly suggests that in vivo, the somatotroph is extremely sensitive to low doses of GHRH under conditions of low somatostatinergic tone. 2) Similar increases in plasma GH levels, as assessed by the AUC and maximal peak levels, were obtained after the administration of 3, 10, and 25 jug GHRH plus PD. This is in contrast to the
P PD \>— 100-H
data obtained after the administration of GHRH alone for which we and others (9, 16) found a clear doseresponse relationship. It is unclear at present why a plateau is reached in the GH responses to 3, 10, and 25 Hg GHRH plus PD. 3) Finally, while we found that with doses of or above 25 ng GHRH alone a maximal increase in plasma GH levels was obtained, we observed that after administration of PD there was a marked increase in GH responses to 100 and 500 ng GHRH compared to the response to 25 ng. Taking together this latter finding and the flattening of responses to 3, 10, and 25 ^g GHRH plus PD, it seems that while GH responses to GHRH alone follow a monophasic curve, when PD is administered the pattern more closely resembles a biphasic one. However, this assumption must be regarded as speculative, since the large interindividual variations in response and the difficulties in carrying out this sort of study on a larger number of subjects prevent us from reaching firmer conclusions in terms of the ED50 and binding capacity of the dose-responses curves under both experimental conditions. In any event, it is a very interesting finding that similar or even greater GH responses can be obtained by combined administration of PD plus 3 ^g GHRH compared to responses to 500 and 100 ng GHRH alone. Further experiments need to be carried out to ascertain whether chronic administration of PD combined with low doses of GHRH can increase somatic growth in both humans and animals in a similar fashion to the effects they exert on GH secretion.
Acknowledgments We are grateful to Ms. Dolores Vina and Ms. Mari Lage for expert technical assistance, and to Serono Ltd. (Spain) for the supply of GHRH-U-29).
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PD AND GHRH-INDUCED GH SECRETION
References 1. Massara F, Ghigo E, Demislis K, et al. Cholinergic involvement in the growth hormone releasing hormone-induced growth hormone release studies in normal and acromegalic subjects. Neuroendocrinology 1986;43:670-5. 2. Massara F, Ghigo E, Molinatti P, et al. Potentiation of cholinergic tone by pyridostigmine bromide re-instates and potentiates the growth hormone responsiveness to intermittent administration of growth hormone releasing factor in man. Acta Endocrinol (Copenh). 1986;113:12-6. 3. Ross RJM, Tsagarakis S, Grossman A, et al. GH feedback occurs through modulation of hypothalamic somatostatin under cholinergic control: studies with pyridostigmine and GHRH. Clin Endocrinol (Oxf). 1987;27:727-34. 4. Pefialva A, Burguera B, Casabiell X, Tresguerres JAF, Dieguez C, Casanueva FF. Activation of cholinergic neurotransmission by pyridostigmine reverses the inhibitory effect of hyperglycaemia on growth hormone releasing hormone induced growth hormone secretion in man: suggesting that glucose acts through hypothalamic release of somatostatin. Neuroendocrinology 1989;49:551-4. 5. Cordido F, Casanueva FF, Dieguez C. Cholinergic receptor activation by pyridostigmine restored growth hormone responsiveness after growth hormone-releasing hormone administration in obese subjects: evidence for hypothalamic somatostatinergic participation in the blunted GH release of obesity. J Clin Endocrinol Metab. 1989;68:290-3. 6. Massara F, Ghigo E, Golfi S, Molinatti MG, Miiller EE, Camanni F. Blockage of hp-GRF 40 induced GH release in normal men by a cholinergic muscarinic antagonist. J Clin Endocrinol Metab. 1984;59:1025-6. 7. Jordan V, Dieguez C, Lafaffian I, et al. Influence of dopaminergic, adrenergic and cholinergic blockade and TRH administration on GH responses to GRF 1-29. Clin Endocrinol (Oxf). 1986;24:291-8. 8. Casanueva FF, Villanueva L, Dieguez C, et al. Atropine blockade of growth hormone releasing hormone-induced GH secretion is not exerted at pituitary level. J Clin Endocrinol Metab. 1986;62:18691. 9. Delitala G, Palermo H, Ross R, Coy D, Besser M, Grossman A. Dopaminergic and cholinergic influences on the growth hormone response to growth hormone-releasing hormone in man. Neuroen-
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docrinology. 1987;45:243-6. 10. Casanueva FF, Villanueva L, Cabranes JA, Cabezas-Cerrato J, Fernandez-Cruz A. Cholinergic mediation of GH secretion elicited by arginine, clonidine and physical exercise in man. J Clin Endocrinol Metab. 1984;59:526-30. 11. Delitala G, Maoili M, Pacifico A, Brianda SA, Palermo M, Manelli M. Cholinergic receptor control mechanism for L-dopa, apomorphine and clonidine-induced growth hormone secretion in man. J Clin Endocrinol Metab. 1983;57:1145-9. 12. Pefialva A, Villanueva L, Casanueva FF, Cavagnini F, Gomez-Pan A, Miiller EE. Cholinergic and histaminergic involvement in the growth hormone releasing effect of an enkephalin analog FK 33824 in man. Psychopharmacology 1983;80:120-4. 13. Peters JR, Evans PI, Page MD, et al. Cholinergic muscarinic blockade with piranzepine abolishes low wave sleep-related growth hormone release in normal adult males. Clin Endocrinol (Oxf). 1986;25:213-7. 14. Piestchmann P, Schernthaner G, Luger A. Effect of cholinergic muscarinic receptor blockade on human growth hormone (GH)releasing hormone-(l-44)-induced GH secretion in acromegalic and type I diabetes mellitus. J Clin Endocrinol Metab. 1986;63:38992. 15. Locatelli V, Torsello A, Redaelli M, Ghigo E, Massara F, Miiller EE. Cholinergic agonist and antagonist drugs modulate the growth hormone response to growth hormone releasing hormone in the rat: evidence for mediation by somatostatin. J Endocrinol. 1986;lll:271-8. 16. Vance ML, Borges JLC, Kaiser DL, et al. Human pancreatic tumor growth hormone-releasing factor: dose-response relationships in normal man. J Clin Endocrinol Metab. 1984;58:838-42. 17. Gelato MC, Perscovitz OH, Cassorla F, Lynn Loriaux D, Merriam GR. Dose-response relationships for the effects of growth hormonereleasing factor-(l-44)-NH2 in young adult men and women. J Clin Endocrinol Metab. 1984;59:197-2O1. 18. Wood SM, Ch'ng JLC, Adams EF, et al. Abnormalities of growth hormone release in response to human growth hormone releasing factor (GRF 1-44) in acromegaly and hypopituitarism. Br Med J. 1983;286:1687-90. 19. Boissel JP, Cohen R, Biot-Laporte S, et al. Synthetic human growth hormone releasing factor (h-GRF 1-44 NH2) dose-response effect on growth hormone and prolactin secretion in healthy adult men..Eur J Clin Pharmacol. 1986;29:609-13.
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