Life Sciences, Vol . 25, pp . 1201-1206 Printed in the U .S .A .
Pergamon Press
CHOLECYSTOKININ OCTAPEPTIDE RELEASES GROWTH HORMONE FROM THE PITUITARY IN VITRO John E . Morley', Shlomo Melmed', Jacqueline Briggs', Harold E . Carlson', Jerome M . Hershman', Travis E . Solomon 2, Cornelis Lamers 2 and David A . Damassa 2 'Endocrine Research Laboratory, Wadsworth VA Medical Center and Department of Medicine, University of California Los Angeles, CA 90073 2 Center for Ulcer Research and Education, University of California, Los Angeles, CA 90024 9 Brain Research Institute, University of California, Los Angeles, CA 90024 (Received in final form August 20, 1979) Summary Cholecyso kinin-octapeptide (CCK-8)(10-6 to 10 -8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH3 pituitary tumor cells . Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion in vitro . In the GH3 cell'line, CCK-8 (10 - 7M) reversed theT i fbitory effect of somatostatin (10-5M) on GH release . As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor .
n
Several peptides including somatostatin (1), thyrotropin-releasing hormone (2), substance P (3), bombesin (4), neurotensin (5), the enkephalins (6), vasoactive intestinal peptide (7) and cholecystokinin (8) have been found in the brain and the gastrointestinal tract . A number of these hormones influence both gastrointestinal function and pituitary hormone release (9-13) . For this reason we assessed the effects of five peptides originally isolated from the gastrointestinal tract on the secretion of hormones from incubated rat anterior pituitary quarters . We report here that cholecystokinin-octapeptide (CCK-8) produced a marked increase in growth hormone release in this system . Several analogues of CCK-8 also produced GH release . CCK-8 prevented the inhibitory effect of somatostatin on GH release from cultured GH3 rat pituitary tumor cells . Methods Acute anterior pituitary cultures : Anterior pituitaries were removed from male Sprague-Dawley rats weighing approximately 150 g . The pituitaries were quartered and the quarters randomized . Three quarters were placed in 10 ml stoppered Erlenmeyer flasks containing 1 ml modified Gey and Gey medium (14) which had been equilibrated to the culture conditions . The tissue portions were individually preincubated for 2 hours in a gyrotory water-bath shaker at 370C under 5% C02-95% 02 . The preincubation medium was then
0024-3205/79/141201-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1202
Cholecystokinin Releases Growth Hormone
Vol . 25, No . 14, 1979
replaced by 1 ml gassed medium containing either no additions (control) or one of the gastrointestinal hormones at varying concentrations . After 1 hour further incubation at 37oC the media were collected and stored frozen until assayed for growth hormone (GH), prolactin (PRL), thyrotropin (TSH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) . At the end of each experiment the pituitary tissue from each flask was weighed ; all results were calculated per mg wet weight of tissue and results are expressed as % of the mean control value . All the anterior pituitary hormones were measured by radioimmunoassay using materials and reference preparations supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethseda, Maryland . There was no cross-reaction of any of the gastrointestinal peptides used in any of the radioimmunoassays . Comparisons were made by using the Student's t-test . GH3 pituitary tumor cell cultures : Cells were supplied by ATCC and were cultured as monolayers in plastic multiwell 35 mm tissue culture plates (LUX), using Ham's F10 medium supplemented with 15% horse serum and 2 .5% fetal calf serum and containing penicillin (50 U/ml) and streptomycin (50 ug/ml) . Cells were maintained as described (15) . Experimental procedure : When cells were in late logarithmic phase of growth, medium was aspirated from each well and cells washed with serum-free Ham's F10 medium . Wells were then incubated with serum-free Ham's F10 medium containing appropriate dilutions of CCK-8 . Control wells contained medium alone . Incubations were in a humidified atmosphere, 95% air - 5% C02 at 37oC . At the end of each incubation, medium was aspirated for hormone assay . Monolayer of cells was harvested in saline suspension and counted in a Coulter counter . Medium and CCK solutions in Ham's F10 were equilibrated with 95% air - 5% C02 at 37oC before use . Results The results of the experiments using cultured pituitary quarters are given in Table I . In addition to the stimulatory effect of CCK-8 on GH release, tetragastrin (10-6M) also produced GH release . None of the other hor mones tested altered secretion of growth hormone . Bombesin, secretin, CCK-8 and pancreatic polypeptide all increased gonadotropin release at high concentrations . Tetragastrin and CCK-8 partially inhibited thyrotropin . Cholecystokinin-39 was also a potent releaser of GH (10-6 M, 253±60%, SEM and 10-7M, 160±20% of controls, p
Pergamon Press
CHOLECYSTOKININ OCTAPEPTIDE RELEASES GROWTH HORMONE FROM THE PITUITARY IN VITRO John E . Morley', Shlomo Melmed', Jacqueline Briggs', Harold E . Carlson', Jerome M . Hershman', Travis E . Solomon 2, Cornelis Lamers 2 and David A . Damassa 2 'Endocrine Research Laboratory, Wadsworth VA Medical Center and Department of Medicine, University of California Los Angeles, CA 90073 2 Center for Ulcer Research and Education, University of California, Los Angeles, CA 90024 9 Brain Research Institute, University of California, Los Angeles, CA 90024 (Received in final form August 20, 1979) Summary Cholecyso kinin-octapeptide (CCK-8)(10-6 to 10 -8M) produced a marked increase in growth hormone (GH) release from incubated rat anterior pituitary quarters and from cultured GH3 pituitary tumor cells . Although several CCK-8 analogues also caused GH release, bombesin, secretin and pancreatic polypeptide had no effect on GH secretion in vitro . In the GH3 cell'line, CCK-8 (10 - 7M) reversed theT i fbitory effect of somatostatin (10-5M) on GH release . As CCK immunoreactivity has been demonstrated to be present in the hypothalamus, these results suggest that CCK-8 may be a physiologically important growth hormone releasing factor .
n
Several peptides including somatostatin (1), thyrotropin-releasing hormone (2), substance P (3), bombesin (4), neurotensin (5), the enkephalins (6), vasoactive intestinal peptide (7) and cholecystokinin (8) have been found in the brain and the gastrointestinal tract . A number of these hormones influence both gastrointestinal function and pituitary hormone release (9-13) . For this reason we assessed the effects of five peptides originally isolated from the gastrointestinal tract on the secretion of hormones from incubated rat anterior pituitary quarters . We report here that cholecystokinin-octapeptide (CCK-8) produced a marked increase in growth hormone release in this system . Several analogues of CCK-8 also produced GH release . CCK-8 prevented the inhibitory effect of somatostatin on GH release from cultured GH3 rat pituitary tumor cells . Methods Acute anterior pituitary cultures : Anterior pituitaries were removed from male Sprague-Dawley rats weighing approximately 150 g . The pituitaries were quartered and the quarters randomized . Three quarters were placed in 10 ml stoppered Erlenmeyer flasks containing 1 ml modified Gey and Gey medium (14) which had been equilibrated to the culture conditions . The tissue portions were individually preincubated for 2 hours in a gyrotory water-bath shaker at 370C under 5% C02-95% 02 . The preincubation medium was then
0024-3205/79/141201-05$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1202
Cholecystokinin Releases Growth Hormone
Vol . 25, No . 14, 1979
replaced by 1 ml gassed medium containing either no additions (control) or one of the gastrointestinal hormones at varying concentrations . After 1 hour further incubation at 37oC the media were collected and stored frozen until assayed for growth hormone (GH), prolactin (PRL), thyrotropin (TSH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) . At the end of each experiment the pituitary tissue from each flask was weighed ; all results were calculated per mg wet weight of tissue and results are expressed as % of the mean control value . All the anterior pituitary hormones were measured by radioimmunoassay using materials and reference preparations supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethseda, Maryland . There was no cross-reaction of any of the gastrointestinal peptides used in any of the radioimmunoassays . Comparisons were made by using the Student's t-test . GH3 pituitary tumor cell cultures : Cells were supplied by ATCC and were cultured as monolayers in plastic multiwell 35 mm tissue culture plates (LUX), using Ham's F10 medium supplemented with 15% horse serum and 2 .5% fetal calf serum and containing penicillin (50 U/ml) and streptomycin (50 ug/ml) . Cells were maintained as described (15) . Experimental procedure : When cells were in late logarithmic phase of growth, medium was aspirated from each well and cells washed with serum-free Ham's F10 medium . Wells were then incubated with serum-free Ham's F10 medium containing appropriate dilutions of CCK-8 . Control wells contained medium alone . Incubations were in a humidified atmosphere, 95% air - 5% C02 at 37oC . At the end of each incubation, medium was aspirated for hormone assay . Monolayer of cells was harvested in saline suspension and counted in a Coulter counter . Medium and CCK solutions in Ham's F10 were equilibrated with 95% air - 5% C02 at 37oC before use . Results The results of the experiments using cultured pituitary quarters are given in Table I . In addition to the stimulatory effect of CCK-8 on GH release, tetragastrin (10-6M) also produced GH release . None of the other hor mones tested altered secretion of growth hormone . Bombesin, secretin, CCK-8 and pancreatic polypeptide all increased gonadotropin release at high concentrations . Tetragastrin and CCK-8 partially inhibited thyrotropin . Cholecystokinin-39 was also a potent releaser of GH (10-6 M, 253±60%, SEM and 10-7M, 160±20% of controls, p
