Life Sciences, Vol . 23, pp . 1351-1358 printed in the U .S .A .
Pergamon Press
EFFECTS OF CHOLINERGIC DRIIGS ON GROWTH HORMONE RELEASEI J .F . Bruai 2 and J . Meitea 3 Department of Physiology, Neuroeadocriae Research Laboratory, Michigan State IIniversity, East Lansing, Michigan 48824 (Received in Final £oz74 August 10, 1978) Summary Acetylcholine and the cholinergic agoaiets, pilocarpine and physoetigmine, increased GH release _in vivo . The increase in GH release by pilocarpine was reversed by concurrent administration of the cholinergic receptor Mocker, atropine, whereas atropine alone did not alter serum GH concentrations . Cholinergic stimulation of GH release appears to be partially mediated through a catecholaminergic ayatem since the response was partially inhibited by pimozide, a dopamine receptor Mocker, or phentolamine, as a-adrenergic receptor Mocker . The cholinergic ayatem may function physiologically to help regulate GH release . Secretion of anterior pituitary hormones has been shown to be influenced by hypothalamic catecholaminergic and serotonergic neurone (1,2) . Administration of L-dop~, apomorphine and peribedil, all dopaminergic drugs, were reported to induce release of GH in rats (3,4), whereas serotonin either stimulated (5) or inhibited (6) GH release . Acetylcholine, present in high conceatrationa in the hypothalamus (7), has been implicated in control of LH (8-12), and prolactin secretion (12-14) . A choline esterase inhibitor, paraoaon, was reported to increase pituitary GH concentration in rate (15), and a cholinergic agoaiet, ßmethyl-choline, increased serum GH concentration in human subjects (16) . The present study was designed to determine the effects of acetylcholiae and other cholinergic drugs on release of GH from the rat pituitary in vivo . Materiale and Methods Male Sprague Dawley rats, 225-250 g (Spartan Research Animals, Haslett, MI), were maintained in a temperature controlled room (25 ° +1 ° C) with 14h artificial illumination daily . Rate were provided with Purina Rat Chow (Ralston Purina, St . Louis, MO) and tap water ad libitum . In all experiments rats were decapitated following drug 1Published with the approval of the Michigan Agricultural Ezperiment Station as Journal Article No . 8468 . 2 A preliminary report of these results was presented by the âenior author at the 1977 FASEB meetings is Chicago, - Fed . Proc . _36 :323, 1977 . 3Aided in part by NIH research grant AM04784 from the National Institute of Arthritis, Metabolism sad Digestive Diseases . 0300-9653/78/1002-135102 .00/0 Copyright (c) 1978 Pergamon Press
1352
Acetylcholine and GH Release
Vol . 23, No . 13, 1978
treatment, trunk blood was collected between 1000 h and 1100 h, and serum was separated and stored at -20 ° C until assayed for GH . Rats were implanted with an indwelling polyethylene cannula in the lateral ventricle by the method of Verater _et _al . (17) . Rats were allowed to recover from surgery for 1 wk and injected with 10 ul 0 .87X NaCl intraventricularly for 3 days prior to drug treatment . A done of 25 or 50 ug acetylcholine bromide (K and R Labs, Plains View, N .Y .) was injected iatraventricularly . An additional control group of non-cannulated, uniajected rata was added to the eaperimeat . All animals were decapitated 30 min after injection of acetylcholine bromide (Ach) . In the second eaperiment, male rate were injected i .p . with the cholinergic receptor stimulator, pilocarpine nitrate (Nutrition Biochemical Co ., Cleveland, OH) (5 mg/kg) or the cholinesterase inhibitor, physostigmine sulfate (Merle and Co ., Rahway, N .J .) (0 .3 mg/kg) . Six animals in each group were decapitated at 30, 60, and 120 min after injection . An additional 6 animals were killed at 0 time to provide the initial control serum GH concentration . Ia the third experiment, pilocarpine nitrate in doses of 0, 2, 5, or 10 mg/kg, and physostigmine sulfate in dosse of 0, 0 .1, 0 .3, or 0 .5 mg/kg were injected i .p . and the animale were decapitated 1 h after injection . In the fourth eaperiment, male rata were injected i .p . with pilocarpine nitrate, physostigmine sulfate, atropine sulfate (Sigma Chemical Co ., St . Louis, MO) or with a combination of atropine and pilocarpine (for doaee see Fig . 4) and killed 1 h after injection . Ia the fifth eaperimeat, pilocarpine (5 mg/kg) was injected alone and in combination with pimozide (McNeil Labs, Ft . Washington, PA) a dopamine receptor Mocker, phentolamine (CIBA Pharmaceutical Co ., Summitt, N .J .), an a-adrenergic Mocker, and propraaolol (Ayeret Labs, New York, N .Y .) a ß-adrenergic Mocker, to determine if the effects of acetylcholine are mediated through a dopaminergic or adrenergic mechanism . Growth hormone (GH) was assayed with a NIAMDD-GH kit, kindly provided by Dr . A .F . Parlow, NIAMDD . Values for serum and incubation media are expressed in terms of GH-RP-1 . In vivo assay results were analyzed by analysis of variance (ANOVA) followed by the Student-Newmaa-Reuls (SNR) test for multiple comparison (18) . Results The effects of injections of acetylcholine into the lateral ventricle are shown in Fig . 1 . Intraventricular injections of 10 ul of 0 .87% NaCl of 25 ug of Ach failed to significantly alter serum GH concentrations, whereas a 50 yg dose of Ach increased serum GH concentrations significantly, by approximately 50z . The effects of pilocarpine, a cholinergic receptor stimulator, and physostigmine, as aaticholineaterase, are shown in Fig . 2 and Table I . Intraperitoneal injection or 0 .3 mg/kg physostigmine maximally increased serum GH concentrations 1 h after injection (Fig . 2) . This appeared to be the optimal dose employed is the
Acetylcholine and GH Release
Vol . 23, No . 13, 1978
1353
FIG . 1 . Serum GH concentrations 30 min after injection of acetylchonine bromide . The vertical bars represent standard error of the mean (SEM) . Number of rats per group (n) m 7 or 8 . 2ô0-
n g ßH ~~~ p~r ml SE R U1A CONTROLS
PHYSOST Iß 1A 1 N E, 0.3mg/kg PILOCARPINE NOs,Smg/kg
0
0
.b
1 HOURS FIG . 2 .
2
Serum GH concentrations after physostigmine, as aaticholinesterase, and pilocarpine, a cholinergic receptor stimulator, injections over a 2 b experimental period . Vertical bars represent SEM . a ~ 6 per determination . Stare indicate P
Pergamon Press
EFFECTS OF CHOLINERGIC DRIIGS ON GROWTH HORMONE RELEASEI J .F . Bruai 2 and J . Meitea 3 Department of Physiology, Neuroeadocriae Research Laboratory, Michigan State IIniversity, East Lansing, Michigan 48824 (Received in Final £oz74 August 10, 1978) Summary Acetylcholine and the cholinergic agoaiets, pilocarpine and physoetigmine, increased GH release _in vivo . The increase in GH release by pilocarpine was reversed by concurrent administration of the cholinergic receptor Mocker, atropine, whereas atropine alone did not alter serum GH concentrations . Cholinergic stimulation of GH release appears to be partially mediated through a catecholaminergic ayatem since the response was partially inhibited by pimozide, a dopamine receptor Mocker, or phentolamine, as a-adrenergic receptor Mocker . The cholinergic ayatem may function physiologically to help regulate GH release . Secretion of anterior pituitary hormones has been shown to be influenced by hypothalamic catecholaminergic and serotonergic neurone (1,2) . Administration of L-dop~, apomorphine and peribedil, all dopaminergic drugs, were reported to induce release of GH in rats (3,4), whereas serotonin either stimulated (5) or inhibited (6) GH release . Acetylcholine, present in high conceatrationa in the hypothalamus (7), has been implicated in control of LH (8-12), and prolactin secretion (12-14) . A choline esterase inhibitor, paraoaon, was reported to increase pituitary GH concentration in rate (15), and a cholinergic agoaiet, ßmethyl-choline, increased serum GH concentration in human subjects (16) . The present study was designed to determine the effects of acetylcholiae and other cholinergic drugs on release of GH from the rat pituitary in vivo . Materiale and Methods Male Sprague Dawley rats, 225-250 g (Spartan Research Animals, Haslett, MI), were maintained in a temperature controlled room (25 ° +1 ° C) with 14h artificial illumination daily . Rate were provided with Purina Rat Chow (Ralston Purina, St . Louis, MO) and tap water ad libitum . In all experiments rats were decapitated following drug 1Published with the approval of the Michigan Agricultural Ezperiment Station as Journal Article No . 8468 . 2 A preliminary report of these results was presented by the âenior author at the 1977 FASEB meetings is Chicago, - Fed . Proc . _36 :323, 1977 . 3Aided in part by NIH research grant AM04784 from the National Institute of Arthritis, Metabolism sad Digestive Diseases . 0300-9653/78/1002-135102 .00/0 Copyright (c) 1978 Pergamon Press
1352
Acetylcholine and GH Release
Vol . 23, No . 13, 1978
treatment, trunk blood was collected between 1000 h and 1100 h, and serum was separated and stored at -20 ° C until assayed for GH . Rats were implanted with an indwelling polyethylene cannula in the lateral ventricle by the method of Verater _et _al . (17) . Rats were allowed to recover from surgery for 1 wk and injected with 10 ul 0 .87X NaCl intraventricularly for 3 days prior to drug treatment . A done of 25 or 50 ug acetylcholine bromide (K and R Labs, Plains View, N .Y .) was injected iatraventricularly . An additional control group of non-cannulated, uniajected rata was added to the eaperimeat . All animals were decapitated 30 min after injection of acetylcholine bromide (Ach) . In the second eaperiment, male rate were injected i .p . with the cholinergic receptor stimulator, pilocarpine nitrate (Nutrition Biochemical Co ., Cleveland, OH) (5 mg/kg) or the cholinesterase inhibitor, physostigmine sulfate (Merle and Co ., Rahway, N .J .) (0 .3 mg/kg) . Six animals in each group were decapitated at 30, 60, and 120 min after injection . An additional 6 animals were killed at 0 time to provide the initial control serum GH concentration . Ia the third experiment, pilocarpine nitrate in doses of 0, 2, 5, or 10 mg/kg, and physostigmine sulfate in dosse of 0, 0 .1, 0 .3, or 0 .5 mg/kg were injected i .p . and the animale were decapitated 1 h after injection . In the fourth eaperiment, male rata were injected i .p . with pilocarpine nitrate, physostigmine sulfate, atropine sulfate (Sigma Chemical Co ., St . Louis, MO) or with a combination of atropine and pilocarpine (for doaee see Fig . 4) and killed 1 h after injection . Ia the fifth eaperimeat, pilocarpine (5 mg/kg) was injected alone and in combination with pimozide (McNeil Labs, Ft . Washington, PA) a dopamine receptor Mocker, phentolamine (CIBA Pharmaceutical Co ., Summitt, N .J .), an a-adrenergic Mocker, and propraaolol (Ayeret Labs, New York, N .Y .) a ß-adrenergic Mocker, to determine if the effects of acetylcholine are mediated through a dopaminergic or adrenergic mechanism . Growth hormone (GH) was assayed with a NIAMDD-GH kit, kindly provided by Dr . A .F . Parlow, NIAMDD . Values for serum and incubation media are expressed in terms of GH-RP-1 . In vivo assay results were analyzed by analysis of variance (ANOVA) followed by the Student-Newmaa-Reuls (SNR) test for multiple comparison (18) . Results The effects of injections of acetylcholine into the lateral ventricle are shown in Fig . 1 . Intraventricular injections of 10 ul of 0 .87% NaCl of 25 ug of Ach failed to significantly alter serum GH concentrations, whereas a 50 yg dose of Ach increased serum GH concentrations significantly, by approximately 50z . The effects of pilocarpine, a cholinergic receptor stimulator, and physostigmine, as aaticholineaterase, are shown in Fig . 2 and Table I . Intraperitoneal injection or 0 .3 mg/kg physostigmine maximally increased serum GH concentrations 1 h after injection (Fig . 2) . This appeared to be the optimal dose employed is the
Acetylcholine and GH Release
Vol . 23, No . 13, 1978
1353
FIG . 1 . Serum GH concentrations 30 min after injection of acetylchonine bromide . The vertical bars represent standard error of the mean (SEM) . Number of rats per group (n) m 7 or 8 . 2ô0-
n g ßH ~~~ p~r ml SE R U1A CONTROLS
PHYSOST Iß 1A 1 N E, 0.3mg/kg PILOCARPINE NOs,Smg/kg
0
0
.b
1 HOURS FIG . 2 .
2
Serum GH concentrations after physostigmine, as aaticholinesterase, and pilocarpine, a cholinergic receptor stimulator, injections over a 2 b experimental period . Vertical bars represent SEM . a ~ 6 per determination . Stare indicate P
