J. Endocrinol. Invest. 14: 241-244,1991
COMMENT
Role of growth hormone-releasing hormone on pentagastrininduced growth hormone release in normal subjects J.F. Garcia-Rojas*, A. Mangas*, A. Barba*, J. Millan*, C. Dieguez**, and E. Zamora* *Department of Medicine, Faculty of Medicine, University of Cadiz, Cadiz and **Department of Physiology, Faculty of Medicine, University of Santiago, Santiago de Compostela Spain ABSTRACT. In order to investigate the mechanisms by which gastrin cause GH release in humans we measured the GH response to pentagastrin alone (1.5 /-tg/kg/hour from 120 to 210 min) and following pretreatment with GHRH (GHRH 1-29,250 /-tg, ivat o min) in normal male subjects. Prior GHRH administration abolished the GH response to the second bolus of GHRH (1 /-tg/kg) administered two hours lat-
er. Pentagastrin infusion induced a rise in GH levels maximal at 60 min (9.1 +0.6 ng/ml, mean+SE), but this rise was abolished by pretreatment with GHRH. Finally, we found that gastrin did not modify basal GH release or GH responses to GHRH by rat anterior pituitary cells in monolayer culture. Taken together, these data suggest that gastrin regulates GH secretion by acting at hypothalamic level.
INTRODUCTION
perimental model first suggested by the findings of Shibasaki et al. (7) showing that administration of 200 /-tg of GHRH to normal subjects completely abolishes the GH response to a second GHRH bolus given two hours later, while GH responses to insulin-induced hypoglycemia are maintained. This indicates that hypoglycemia release GH through a non GHRH-dependent mechanism and in the studies reported here, we have used pentagastrin infusion rather than insulin-induced hypoglycemia as the stimulus of GH release. Furthermore in order to rule out a direct pituitary effect of pentagastrin we have investigated the effect of different doses of gastrin on basal GH release and GH responses to GHRH by rat AP cells in monolayer culture.
Growth hormone secretion by the anterior pituitary (AP) gland is stimulated by growth hormone-releasing hormone (GHRH) and inhibited by somatostatin. In addition a complex network of neurotransmitters and neuropeptides are involved in the control of GH secretion, either by modulating GHRH and somatostatin release from the hypothalamus or by acting directly at the pituitary level (for review see 1-2). Recent studies have clearly shown that in addition to their gastrointestinal effects, gastrin may well be involved in the regulation of AP hormone secretion and the evidences are as follows. Gastrin is present at high concentrations in both, the hypothalamus and in the AP gland (3). Gastrin inhibits ACTH secretion in vitro and in vivo after intracerebroventricular administration (4). It also inhibits basal LH, prolactin and TSH while stimulates GH secretion in ovariectomized female rats (5). Furthermore, it has been reported that gastrin infusion elevates circulating GH levels in normal human subjects (6). The aim of this study was to investigate whether gastrin-induced GH release is exerted at the hypothalamic or pituitary level, and more specifically whether it is mediated through a GHRH-dependent mechanism. In this order, we have used the ex-
SUBJECTS AND METHODS Clinical studies Tests were performed in a group of seven normal subjects (aged 21-26 yr) in random order, and separated by at least one week. Approval from the Hospital Ethics Committee and subjects informed consent was obtained. A medically qualified observer was present on each occasion throughout the test. The subjects fasted from midnight and remained recumbent throughout the studies. In the first test, thirty minutes after the insertion of forearm cannulae, they received 250 /-tg iv of GHRH 1-29, Serono Spain) at 0 min, and the second GHRH bolus (1 /-tg/kg) at 120 min, with samples being obtained evey thirty minutes from -30 to 210 min. In
Key-words.' Gastrin, growth hormone, growth hormone-releasing hormone. Correspondence: C. Dieguez, MD., Department of Physiology, Faculty of Medicine, Santiago de Compostela. Spain.
Received January 15,1990; accepted December 4,1990.
241
J.F. Garcia-Rojas, A. Mangas, A. Barba, et al.
the second test, after saline administration at 0 min, they received a pentagastrin (ICI, Spain) infusion (1.5 /-1g/kg/hour) from 120 min to 210 min. Finally in the third test GHRH (250 /-1g iv bolus) was administered at 0 min, followed by a pentagastrin infusion from 120 min to 210 min as stated above. No side effects were reported in any of these tests. Human growth hormone levels were measured using commercial kits (Biomerieux, Spain) with a intrassay coefficient of variation of 7%. All samples of each subject were analyzed in the same assay.
30
®
E CI
15
:J:
10
+
+
20
c:
GHRH
GHRH
25
C)
5 0 -30
In vitro studies 30
Monolayer cultures of rat AP cells were prepared as described previously (8). Briefly, cells were isolated from AP glands using a collagenase based method and plated at a density of approximately 100,000/ml/weli. Limbro 96 multiwell plates were used, each having a growth area of 1.6 cm 2 (Flow Laboratories, Rockwell, Maryland, USA). The culture medium was semi-synthetic (9). After 4 days in culture, the cells were washed four times using Earles balanced salt solution. Thereafter the cells were incubated in serum free-medium with the appropriate concentrations of vehicle, gastrin or GHRH for 3 hours. At the end medium was collected and stored at -20 C until assayed. Rat GH was measured using materials kindly provided by the NIADDK as described previously (8).
-2'
::c C)
10
E
60
@
25 20
30
0
Pentagastrin ~~#'#'#'#'#~
GHRH
+
+
15
90 120 150 180 210
5 0 -30 30
25
E
20
01
c:
15
::c
10
0
30
60
®
90 120 150 180 210
Pentagastrin
Placebo
~#'..0W'/.-3
+
+
C)
RESULTS In vivo studies Following pentagastrin infusion, GH levels rose to a peak of (mean+SE) 9.1 +0.6 ng/ml at 60 min (p < 0.02). After administration of GHRH a marked increase in plasma GH levels was observed at 30,60, and 90 min (Fig. 1). Pentagastrin infusion preceded by GHRH administration did not induce any significant increase in circulating GH levels (Fig. 1).
5 0
-30
0
30
60
90 120 150 180 210
TIME (min) Figure 1 - Plasma GH levels (mean ± SE) in 7 normal subjects following administration of. a) GHRH, 250119 iv at 0 min, and 1 Ilg/kg, ivat 120 min; b) GHRH, 250 ug, iv at 0 min and pentagastrin (1. 5Ilg/kg/hour) from 120 to 210 min; c) placebo (saline) at 0 min and pentagastrin (1.5Ilg/kg/hour) from 120 to 210 min.
In vitro studies
No effect of gastrin (10- 5 to 10- 7 M) was observed on basal GH secretion. In contrast GHRH (10- 7 to 10- 11 M) induced a clear-cut dose-dependent increase in GH secretion. Administration of gastrin (1 0-5M) failed to modify somatotroph responsiveness to GHRH at any of the doses tested (Fig. 2).
Ius of GHRH given two hours later (7, 10-11). They are also in agreement with previous data showing that pentagastrin-infusion increases plasma GH levels in normal subjects (6). It is not possible at present to establish the physiological relevance of this finding since gastrin levels in hypothalamic-hypophyseal portal blood have not been reported. Furthermore it still remains to be established whether gastrin influence AP hormone secretion through endocrine or paracrine mechanims (3) or even if they are mediated by peripheral factors in response to their gastrointestinal effects.
DISCUSSION These data confirm previous reports, showing that pretreatment with GHRH completely abolishes the GH response to the administration of a second bo-
242
Pentagustrin-induced GH release in normal subjects
140
GH ng/ml
• ••
130
• ••
120 110 100
.•.
• ••
90 80
..... .
70
••
60
••
SO 40 30 20 10 0
C
GAS
to- ' M
GAS
GAS
GHRH
GHRH
GHRH
GHRH
GHRH
10-' hi
10-'104
10- 11 104
10- ' '''
10- 11 '"
10-' M
10-' M
• GAS
+GAS
-GAS
Figure 2 - Effect of different concentrations of gastrin (10- 7 to to-SM) on (mean ± SE) basal GH release and GH responses to GHRH (10- 11 , to- 9 and to- 7M) by rat anterior pituitary cells in monolayer cultures. The data is representative of three different experiments. ** ** P < 0.05 *** P < 0.01
REFERENCES
Our data showing that pretreatment with GHRH abolishes GH responses to a subsequent pentagastrin-infusion seem to suggest that gastrin effects on GH secretion are GHRH-dependent. It is unlikely that the lack of response to gastrin is due to exhaustion of the G H-releasable pool, since it has been shown using a similar experimental design that GH responses to either , clonidine , adrenalin plus propanolol, arginine or insulin-induced hypoglycemia are maintained or even increased after previous GHRH administration (7,10-11). In fact our data resemble more to that obtained with GH responses to L-dopa, where it was also found that pretreatment with GHRH abolishes the GH response to L-dopa (11), thus suggesting that GH responses to pentagastrin are GHRH-mediated, though direct proof for this hypothesis is lacking at present. Also it may be possible that pentagastrin could not inhibit somatostatin release caused by prior administration of GHRH since GHRH is reported to stimulate somatostatin release from the hypothalamus (12). In any event, since we found that gastrin did not affect in vitro basal GH release or GH responses to GHRH, this suggest, though not proved, that is acting at hypothalamic level.
1. Mueller EE Neural control of somatotrophic function. Physio!. Rev. 67: 962, 1987. 2. Dieguez C., Page MD., Scanlon M.F. Neuroregulation of GH secretion and its alterations in disease states. Clin. Endocrino!. (Oxf.) 28: 109,1988.
3. Rehfeld J.F. The expression of progastrin, procholecystokinin and their hormonal products in pituitary cells. J. Mo!. Endocrino!. 1: 87,1988. 4. Itoh S., Hirota R., Katsmura G., Odaguchi K. Suppressive effect of pentagastrin on pituitary adrenocortical secretion. Endocrino!. Jpn. 26: 741, 1979.
5. Vijayan E., Samson W.K., McCann S.M. Effects of intraventricular injection of gastrin on release of LH, Prolactin, TSH and GH in conscious ovariectomized rats . Life Sci. 23: 2225, 1978. 6. Domschke W., Lux G ., Domschke S. Growth hormone secretion and pentagastrin. N. Eng!. J . Med. 303: 458, 1980.
ACKNOWLEDGEMENTS
7. Shibasaki T., Hotta M. , Masuda A., Imaki T., Obara N., Demura H., Ling N. , Shizume K. Plasma GH responses to GHRH and insulin-induced hypoglycaemia in man. J. Clin . Endocrino!. Metab. 60: 1265, 1985.
This work was supported by the XUNTA DE GALICIA. We would like to thank the NIDDK for kind supply of rGH kits.
8. Dieguez C, Foord S.M., Peters J.R. , Hall R., Scanlon M.F.
243
JF Garcia-Rojas, A. Mangas, A. Barba, et al.
The effects of thyroid hormone deprivation in vivo and in vitro on GH responses to human pancreatic (tumour) GH-releasing factor (1-40) by dispersed rat anterior pituitary cells. Endocrinology 116: 1066, 1985. 9. Brazeau P. , Ling N., Bohlen P Growth hormone releasing factor, somatocrinin, releases pituitary growth hormone in vitro. Proceedings of the national Academy of Sciences (U.SA) 79: 7909,1982. 10. Valcavi R., Dieguez C., Page MD., Zini M., Casoli P. , Portioli I. , Scanlon M.F. Alpha-2-adrenergic pathways release growth hor-
mone via a non-GRF dependent mechanism in normal human subjects. Clin. Endocrinol. (Oxf.) 29: 309,1988. 11. Page MD., Dieguez C, Valcavi R., Edwards C., Hall R., Scanlon M.F. Growth hormone (GH) responses to arginine and L-dopa alone and after GHRH pretreament. Clin. Endocrinol. (Oxf.) 28:551,1988. 12. Aguila M.C, McCann S.M. Stimulation of somatostatin release in vitro by synthetic growth hormone-releasing hormone by a nondopaminergic mechanism. Endocrinology 117: 762, 1985.
244
COMMENT
Role of growth hormone-releasing hormone on pentagastrininduced growth hormone release in normal subjects J.F. Garcia-Rojas*, A. Mangas*, A. Barba*, J. Millan*, C. Dieguez**, and E. Zamora* *Department of Medicine, Faculty of Medicine, University of Cadiz, Cadiz and **Department of Physiology, Faculty of Medicine, University of Santiago, Santiago de Compostela Spain ABSTRACT. In order to investigate the mechanisms by which gastrin cause GH release in humans we measured the GH response to pentagastrin alone (1.5 /-tg/kg/hour from 120 to 210 min) and following pretreatment with GHRH (GHRH 1-29,250 /-tg, ivat o min) in normal male subjects. Prior GHRH administration abolished the GH response to the second bolus of GHRH (1 /-tg/kg) administered two hours lat-
er. Pentagastrin infusion induced a rise in GH levels maximal at 60 min (9.1 +0.6 ng/ml, mean+SE), but this rise was abolished by pretreatment with GHRH. Finally, we found that gastrin did not modify basal GH release or GH responses to GHRH by rat anterior pituitary cells in monolayer culture. Taken together, these data suggest that gastrin regulates GH secretion by acting at hypothalamic level.
INTRODUCTION
perimental model first suggested by the findings of Shibasaki et al. (7) showing that administration of 200 /-tg of GHRH to normal subjects completely abolishes the GH response to a second GHRH bolus given two hours later, while GH responses to insulin-induced hypoglycemia are maintained. This indicates that hypoglycemia release GH through a non GHRH-dependent mechanism and in the studies reported here, we have used pentagastrin infusion rather than insulin-induced hypoglycemia as the stimulus of GH release. Furthermore in order to rule out a direct pituitary effect of pentagastrin we have investigated the effect of different doses of gastrin on basal GH release and GH responses to GHRH by rat AP cells in monolayer culture.
Growth hormone secretion by the anterior pituitary (AP) gland is stimulated by growth hormone-releasing hormone (GHRH) and inhibited by somatostatin. In addition a complex network of neurotransmitters and neuropeptides are involved in the control of GH secretion, either by modulating GHRH and somatostatin release from the hypothalamus or by acting directly at the pituitary level (for review see 1-2). Recent studies have clearly shown that in addition to their gastrointestinal effects, gastrin may well be involved in the regulation of AP hormone secretion and the evidences are as follows. Gastrin is present at high concentrations in both, the hypothalamus and in the AP gland (3). Gastrin inhibits ACTH secretion in vitro and in vivo after intracerebroventricular administration (4). It also inhibits basal LH, prolactin and TSH while stimulates GH secretion in ovariectomized female rats (5). Furthermore, it has been reported that gastrin infusion elevates circulating GH levels in normal human subjects (6). The aim of this study was to investigate whether gastrin-induced GH release is exerted at the hypothalamic or pituitary level, and more specifically whether it is mediated through a GHRH-dependent mechanism. In this order, we have used the ex-
SUBJECTS AND METHODS Clinical studies Tests were performed in a group of seven normal subjects (aged 21-26 yr) in random order, and separated by at least one week. Approval from the Hospital Ethics Committee and subjects informed consent was obtained. A medically qualified observer was present on each occasion throughout the test. The subjects fasted from midnight and remained recumbent throughout the studies. In the first test, thirty minutes after the insertion of forearm cannulae, they received 250 /-tg iv of GHRH 1-29, Serono Spain) at 0 min, and the second GHRH bolus (1 /-tg/kg) at 120 min, with samples being obtained evey thirty minutes from -30 to 210 min. In
Key-words.' Gastrin, growth hormone, growth hormone-releasing hormone. Correspondence: C. Dieguez, MD., Department of Physiology, Faculty of Medicine, Santiago de Compostela. Spain.
Received January 15,1990; accepted December 4,1990.
241
J.F. Garcia-Rojas, A. Mangas, A. Barba, et al.
the second test, after saline administration at 0 min, they received a pentagastrin (ICI, Spain) infusion (1.5 /-1g/kg/hour) from 120 min to 210 min. Finally in the third test GHRH (250 /-1g iv bolus) was administered at 0 min, followed by a pentagastrin infusion from 120 min to 210 min as stated above. No side effects were reported in any of these tests. Human growth hormone levels were measured using commercial kits (Biomerieux, Spain) with a intrassay coefficient of variation of 7%. All samples of each subject were analyzed in the same assay.
30
®
E CI
15
:J:
10
+
+
20
c:
GHRH
GHRH
25
C)
5 0 -30
In vitro studies 30
Monolayer cultures of rat AP cells were prepared as described previously (8). Briefly, cells were isolated from AP glands using a collagenase based method and plated at a density of approximately 100,000/ml/weli. Limbro 96 multiwell plates were used, each having a growth area of 1.6 cm 2 (Flow Laboratories, Rockwell, Maryland, USA). The culture medium was semi-synthetic (9). After 4 days in culture, the cells were washed four times using Earles balanced salt solution. Thereafter the cells were incubated in serum free-medium with the appropriate concentrations of vehicle, gastrin or GHRH for 3 hours. At the end medium was collected and stored at -20 C until assayed. Rat GH was measured using materials kindly provided by the NIADDK as described previously (8).
-2'
::c C)
10
E
60
@
25 20
30
0
Pentagastrin ~~#'#'#'#'#~
GHRH
+
+
15
90 120 150 180 210
5 0 -30 30
25
E
20
01
c:
15
::c
10
0
30
60
®
90 120 150 180 210
Pentagastrin
Placebo
~#'..0W'/.-3
+
+
C)
RESULTS In vivo studies Following pentagastrin infusion, GH levels rose to a peak of (mean+SE) 9.1 +0.6 ng/ml at 60 min (p < 0.02). After administration of GHRH a marked increase in plasma GH levels was observed at 30,60, and 90 min (Fig. 1). Pentagastrin infusion preceded by GHRH administration did not induce any significant increase in circulating GH levels (Fig. 1).
5 0
-30
0
30
60
90 120 150 180 210
TIME (min) Figure 1 - Plasma GH levels (mean ± SE) in 7 normal subjects following administration of. a) GHRH, 250119 iv at 0 min, and 1 Ilg/kg, ivat 120 min; b) GHRH, 250 ug, iv at 0 min and pentagastrin (1. 5Ilg/kg/hour) from 120 to 210 min; c) placebo (saline) at 0 min and pentagastrin (1.5Ilg/kg/hour) from 120 to 210 min.
In vitro studies
No effect of gastrin (10- 5 to 10- 7 M) was observed on basal GH secretion. In contrast GHRH (10- 7 to 10- 11 M) induced a clear-cut dose-dependent increase in GH secretion. Administration of gastrin (1 0-5M) failed to modify somatotroph responsiveness to GHRH at any of the doses tested (Fig. 2).
Ius of GHRH given two hours later (7, 10-11). They are also in agreement with previous data showing that pentagastrin-infusion increases plasma GH levels in normal subjects (6). It is not possible at present to establish the physiological relevance of this finding since gastrin levels in hypothalamic-hypophyseal portal blood have not been reported. Furthermore it still remains to be established whether gastrin influence AP hormone secretion through endocrine or paracrine mechanims (3) or even if they are mediated by peripheral factors in response to their gastrointestinal effects.
DISCUSSION These data confirm previous reports, showing that pretreatment with GHRH completely abolishes the GH response to the administration of a second bo-
242
Pentagustrin-induced GH release in normal subjects
140
GH ng/ml
• ••
130
• ••
120 110 100
.•.
• ••
90 80
..... .
70
••
60
••
SO 40 30 20 10 0
C
GAS
to- ' M
GAS
GAS
GHRH
GHRH
GHRH
GHRH
GHRH
10-' hi
10-'104
10- 11 104
10- ' '''
10- 11 '"
10-' M
10-' M
• GAS
+GAS
-GAS
Figure 2 - Effect of different concentrations of gastrin (10- 7 to to-SM) on (mean ± SE) basal GH release and GH responses to GHRH (10- 11 , to- 9 and to- 7M) by rat anterior pituitary cells in monolayer cultures. The data is representative of three different experiments. ** ** P < 0.05 *** P < 0.01
REFERENCES
Our data showing that pretreatment with GHRH abolishes GH responses to a subsequent pentagastrin-infusion seem to suggest that gastrin effects on GH secretion are GHRH-dependent. It is unlikely that the lack of response to gastrin is due to exhaustion of the G H-releasable pool, since it has been shown using a similar experimental design that GH responses to either , clonidine , adrenalin plus propanolol, arginine or insulin-induced hypoglycemia are maintained or even increased after previous GHRH administration (7,10-11). In fact our data resemble more to that obtained with GH responses to L-dopa, where it was also found that pretreatment with GHRH abolishes the GH response to L-dopa (11), thus suggesting that GH responses to pentagastrin are GHRH-mediated, though direct proof for this hypothesis is lacking at present. Also it may be possible that pentagastrin could not inhibit somatostatin release caused by prior administration of GHRH since GHRH is reported to stimulate somatostatin release from the hypothalamus (12). In any event, since we found that gastrin did not affect in vitro basal GH release or GH responses to GHRH, this suggest, though not proved, that is acting at hypothalamic level.
1. Mueller EE Neural control of somatotrophic function. Physio!. Rev. 67: 962, 1987. 2. Dieguez C., Page MD., Scanlon M.F. Neuroregulation of GH secretion and its alterations in disease states. Clin. Endocrino!. (Oxf.) 28: 109,1988.
3. Rehfeld J.F. The expression of progastrin, procholecystokinin and their hormonal products in pituitary cells. J. Mo!. Endocrino!. 1: 87,1988. 4. Itoh S., Hirota R., Katsmura G., Odaguchi K. Suppressive effect of pentagastrin on pituitary adrenocortical secretion. Endocrino!. Jpn. 26: 741, 1979.
5. Vijayan E., Samson W.K., McCann S.M. Effects of intraventricular injection of gastrin on release of LH, Prolactin, TSH and GH in conscious ovariectomized rats . Life Sci. 23: 2225, 1978. 6. Domschke W., Lux G ., Domschke S. Growth hormone secretion and pentagastrin. N. Eng!. J . Med. 303: 458, 1980.
ACKNOWLEDGEMENTS
7. Shibasaki T., Hotta M. , Masuda A., Imaki T., Obara N., Demura H., Ling N. , Shizume K. Plasma GH responses to GHRH and insulin-induced hypoglycaemia in man. J. Clin . Endocrino!. Metab. 60: 1265, 1985.
This work was supported by the XUNTA DE GALICIA. We would like to thank the NIDDK for kind supply of rGH kits.
8. Dieguez C, Foord S.M., Peters J.R. , Hall R., Scanlon M.F.
243
JF Garcia-Rojas, A. Mangas, A. Barba, et al.
The effects of thyroid hormone deprivation in vivo and in vitro on GH responses to human pancreatic (tumour) GH-releasing factor (1-40) by dispersed rat anterior pituitary cells. Endocrinology 116: 1066, 1985. 9. Brazeau P. , Ling N., Bohlen P Growth hormone releasing factor, somatocrinin, releases pituitary growth hormone in vitro. Proceedings of the national Academy of Sciences (U.SA) 79: 7909,1982. 10. Valcavi R., Dieguez C., Page MD., Zini M., Casoli P. , Portioli I. , Scanlon M.F. Alpha-2-adrenergic pathways release growth hor-
mone via a non-GRF dependent mechanism in normal human subjects. Clin. Endocrinol. (Oxf.) 29: 309,1988. 11. Page MD., Dieguez C, Valcavi R., Edwards C., Hall R., Scanlon M.F. Growth hormone (GH) responses to arginine and L-dopa alone and after GHRH pretreament. Clin. Endocrinol. (Oxf.) 28:551,1988. 12. Aguila M.C, McCann S.M. Stimulation of somatostatin release in vitro by synthetic growth hormone-releasing hormone by a nondopaminergic mechanism. Endocrinology 117: 762, 1985.
244
