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Failure of β-endorphin antiserum, naloxone, and naltrexone to alter physiologic growth hormone and insulin secretion
Life Sciences, Vol . 25, pp . 1983-1990 Printed in the U.S .A.
Pergamon Press
FAILURE OF ß-ENDORPHIN ANTISERUM, NALOXONE, AND NALTRERONE TO ALTER PHYSIOLOGIC GROWTH HORMONE AND INSULIN SECRETION) Gloria Shaffer Tannenbaum, Alberto E. Panerai and Henry G. Friesen Departments of Pediatrics, and Neurology and Neuroaurgery, McGill University (G .S .T .), Montreal, Quebec, and Department of Physiology, University of Manitoba (A .E .P ., H .G .F .), Winnipeg, Manitoba (Received in final form October 24, 1979) Summary The role of endogenous opiate-like peptides in physiologic regulation of growth hormone (GH) and insulin (IRI) secretion was assessed by passive immunization with ß-endorphin antiserum and by administration of the opiate antagonists naloxone and naltrexone . Six-hour secretioy profiles were obtained from 5 groups of freelymoving chronically cannulated male rats following the i .v . administration of (I) ß-endorphin antiserum, (II) normal rabbit serum, (III) naloxone (1 m8/kg), (IV) naltrexone (1 mg/kg), and (V) normal saline . The typical ultradun rhythm of GH secretion was evident in all groups with moat peak GH values >400 ng/ml . No disruption in amplitude or periodicity of the GH rhythm was observed and there was Plasma IRI no significant difference in mean 6-hr plasma GH levels . levels fluctuated minimally over the 6-hr sampling period . There was ao significant difference in mean 6-hr IRI levels between groups I and II, or between groups III, IV and V . These data do not support the view that endogenous opiate-like peptides play a physiologically important role in maintaining basal GH and IRI secretion. The normal physiologic role of endogenous opiate-like peptides in hormone regulation is unknown. It has been reported that ß-endorphin is a potent stimulator of growth hormone (GH) secretion in the rat when administered via the intravenous or intracerebral ventricular route (1-3) . These results have, in general, been obtained under a variety of unphyaiologic conditions including anesthesia, steroid, and anti-somatostatin pretreatment . Moreover, results obtained from experiments investigating the effects of the specific opiate antagonist naloxoae on basal GH secretion are inconsistent . Both a suppression (4,5) and ao significant effect (6) have been reported . In these studies, single samples of blood were obtained from groups of animals sacrificed at different times during the day . Since GH secretion in the rat is episodic in nature with major episodes of GH secretion occurring at 3-4 hr intervals
)Address requests for reprints : Dr . Gloria S . Tannenbaum, Department of Endocrinology, Montreal Children's Hospital, 2300 Tupper Street, Montreal, Quebec H3H 1P3 0024-3205/79/231983-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1984
Growth Hormone and Insulin Secretion
Vol . 25, No . 23, 1979
throughout a 24-hr period (7), it is important to obtain longitudinal GH profiles in individual animals in order to assess the effects of any manipulation The aim of the experiments described in this report was to on GH secretion. assess the role of ß-endorphin in physiologic regulation of GA secretion using chronically cannulated, freely-behaving rata . If circulating ß-endorphin plays a role is maintaining basal GH secretion we hypothesized that its effectif might be neutralized or blocked by the administration of a specific antiserum to ß-endorphin. In addition, the effects of opiate-receptor blockade on pulsatile GH secretion were examined . In view of the recent demonstration of endorphin-like immunoreactivity in the rat endocrine pancreas (8), together with the report that ß-endorphin stimulates the release of insulin from the perfused dog pancreas (9), plasma insulin levels were simultaneously monitored in all experiments in order to clarify the possible role of endogenous opiate-like peptides in physiologic regulation of insulin secretion . Materials and Methode Antibodies to camel ß-endorphin (Peninsula Antiserum to ß-endorphin. Labs, San Carlos, CA) were obtained in rabbits after conjugation of ßeadorphia to bovine serum albumin (10) . ß-endorphin was iodinated and the product purified using methods previously described for somatostatin (11) . The antiserum used in the present study binds 35% of 1251-labeled ß-endorphin at a final dilution of 1 :65,000 and can detect 100 pg ß-endorphin per assay tube . By Scatchard plot analysis (12), 1 ml ß-endorphin antiserum would be capable The antiserum cross reacts 50Z with ovine of binding 2 .5 ug ß-endorphin. ß-LPH on as equimolar basis but shows no cross-reactivity with any of the following substances : a-endorphin, y-endorphin, met-eakephalia, a-MSH, ß-MSH, ACTH, vasopreasia, insulin, glucagon, TRH, LH-RH, bombesin, myelin basic protein, GA, prolactin, morphine and naloxone . Adult male Sprague-Dawley rate (300Animals and experimental procedure . 400 g) maintained on a 12-hr light-dark cycle (lights on, 0600-1800 hr) in a temperature (22 _+ 1°C)- and humidity-controlled room were implanted with chronic iatracardiac venous cannulae and adapted to isolation test chambers, Purina rat chow and tap water were using methods previously described (7) . available ad libitum . After recovery of pre-operative body weight (usually one week), blood samples were withdrawn every 15 min for periods of 6 hr Group I (n=6) was administered 1 ml (1000-1600 hr) from 5 groups of rats . ß-endorphin antiserum i .v . through the cannula after removal of the first blood sample whereas a second group (n=5) nerved as controls and received 1 ml The effects of opiate receptor normal rabbit serum at the same time point . Naloxone hydrochloride blockade were studied in 3 additional groups of rate . (Narcan, Endo Laboratories) was administered i .v . in a dose of 1 mg/kg body weight . Because of its short biological half life (13), naloxone was injected at 3 different times - 1000 hr, 1230 hr, and 1400 hr - during the sampling period . Control rats received the normal saline vehicle at the same time The fifth group of rats was administered i.v . the longer-acting opiati' points . antagonist aaltrexone in a dose 1 mg/kg body weight at the onset of the sampling period . All rata were observed through a one~way viewing port in order Blood to monitor behavioral changes in response to the various treatments . samples were immediately centrifuged and the plasma separated sad stored at -20°C for subsequent assay .
Antibody titers in plasma of rate after administration of ß - endorphin antiserum . Antibody titers in plasma of ß-endorphin antiserum-treated rate
Vol. 25, No . 23, 1979
Growth Hormone and Insulin Secretion
1985
were assessed by determining the capacity of aliquots of rat plasma obtained 15 min, 3 hr, and 6 hr after antiserum administration to bind 1251 ß-endorphin. Plasma samples were diluted 1 :32 with 0.14 M sodium phosphate buffer and binding to tracer was determined under the conditions used for radioimmunoassay (RIA) of ß-endorphin (14) . Hormone assays . Plasma GH was measured in duplicate by a double antibody RIA using materials supplied by Dr . A. Parlow, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland . Values are expressed in terms of the rat GH reference preparation (GH-RP-1) . Plasma immunoreactive insulin (IRI) was measured by a deztran-coated charcoal method, utilizing guinea pig antiporciae insulin serum. Purified crystalline rat insulin (lot 615-1112B-295-C) was kindly provided by the Eli Lilly Co ., courtesy of Dr . R. Chance, and served ae a reference standard . Statistical analyses . Analysis of variance for repeated measures was used to compare data for significant differences between groups . Results _ß-endorphin antibody titers in plasma . The mesa binding capacity of a 1 :32 dilution of plasma at 15 min, 3 hr, sad 6 hr post ß-endorphin antiserum treatment was found to be 71 .0 _+ 3x, 69 .5 _+ 4Z, 62 .5 _+ 4 .5x, respectively . When compared to titers of antiserum used in the RIA, the binding capacities of these plasma samples were equivalent to 1 :1500, 1:1760, and 1:3000, respectively . Effects of ß-endorphin antiserum on GH and IRI secretiry patterns . Animas treated with normal rabbit serum (n~5) ezhibited the typical ultradun rhythm of GH secretion . Two major episodes of GH secretion were evident during the 6-hr sampling period with moat peak GH values >400 ng/ml and trough values <6 .2 ng/ml (Fig . lA) . Plasma IRI levels fluctuated minimally over the 6-hr sampling period, with values ranging from 0 .4-2 .6 ag/ml. Prominent GH aecretory bursts were still evident is ß-endorphin antiserum-treated rate (Fig . 1B) . No disruption in amplitude or periodicity of the GH rhythm was observed and there was no significant difference in mean 6-hr plasma GH levels between the 2 groups of rats (Table I) . Similarly, ao significant difference was observed in mean 6-hr plasma IRI level of ß-endorphin antiserum-treated rats when compared to normal rabbit serum-treated control animals (Table I) . Although passive immunization with ß-endorphin antiserum did not alter basal hormone secretion, immunized animals appeared behaviorally flaccid, lying with limbs extended, for a period of 15-30 min following antiserum administration . Effects of aaloaone and naltrexone on GH and IRI profiles . The i.v . administration of both naloaoae and naltrexone failed to significantly alter either the amplitude or periodicity of the GH secretiry bursts and plasma IRI levels were unchanged when compared to saline-treated controls . A rapreseatative profile of a naltreaone-treated animal is illustrated is Fig. 2 . No significant differences in mean 6-hr plasma GH or IRI levels were observed between the 3 groups of rate, although the mean 6-hr GH levels in the 2 groups treated with opiate antagonists were slightly higher than that of saliaeiajectad control rate (Table I) . No behavioral changes were observed in these animals .
1986
Growth Hormone and Insulin Secretion
2(2)
Vol . 25, No . 23, 1979
Normal Rabbit Serum
A
400
3 .6
6
y r
A~
100
b,~~d
0.4 1000
1100
1200
1900
1400
W 36 ß-Endorphin Antiserum
1500
1600
ô
~(460)
400
300 OI C
3 .6
200
3s
P loo ~~`\tf ~ 0.~ d `~.4 r-t 0 1000
y r
1100
1200
i 1300
hb -
1 ~
n
1400
1300
1
0 .4 1600
TIME (hours)
FIG . 1 Six-hour GH and IRI secretioy patterns in 2 rats administered 1 ml of either normal rabbit serum (A) or ß-endorphin antiserum (B) i .v . Arrows indicate ß-endorphin antiserum did not significantly alter GH or time of injection . IRI secretion .
Growth Hormone and Insulin Secretion
Vol . 25, No . 23, 1979
1987
2 (3) Normal Saline
3 .6
s N 3 s
s.o
0 0 .4 1000
2
1100
iR00
1]00
1400
1500
3
1600
Naltrexone (lm~/kp)
400
E
0 ~00 e ~ .6
aoo N t G
s N
100
0.4
3
FIG . 2 Plasllla GH sad IRI levels after i .v . administration of saline (upper) or naltreaone (lower . Arrows indicate time of injection . No significant effect of naltrexoae eves observed .
Growth Hormone and Insulin Secretion
1988
Vol . 25, No . 23, 1979
TABLE I Effects of 8-Endorphin Antiserum, Naloxone, and Naltrexone on Mean 6-hour Plasma GA and IRI Levels
Experimental group
n
Meaa 6-hr GH level (ng/ml)
Mean 6-hr IRI level (ng/ml)
~-Endorphin Antiserum
6
113 .8 + 10 .8a
1 .13 + 0 .03
Normal Rabbit Serum
5
97 .7 + 11 .1
1 .27 + 0 .05
Naloxone
5
136.0 + 22 .4
1 .68 + 0 .05
Naltrexone
4
144 .0 + 23 .9
1 .43 + 0 .07
Saline
5
118 .2 + 20 .4
1 .85 + 0 .08
aMean + SEM Discuseion The availability of an antiserum to ~endorphia provides a useful experimental tool for assessing the possible involvement of endogenous ß-endorphin in hormone regulation . Basal B-endorphin concentrations in the peripheral plasma of the rat have been reported to range from 0.25-1 .5 ng/ml (15,16) . Although the concentration of ß-endorphin in the hypophyseal portal circulatim of the rat is unknown, values of 3 .4-3 .8 ag/ml have been documented in the monkey (17) . Assuming that ßrendorphin concentrations in the rat hypophyseal portal circulation are similar, 1 ml of endorphin antiserum used in the present experiment would be more than sufficient to neutralize endogenous ß-endorphin circulating in both the peripheral and hypophyseal portal plasmas . The finding that neither the amplitude nor the periodicity of the GH pulses was altered following B-endorphin antiserum administration, together with the lack of effect on basal plasma IRI levels, suggests that circulating B-endorphin does not play a physiologically important role in maintaining basal GH and IRI secretion. Of interest is the fact that the animals exhibited a behavioral response following antiserum administration . Previous reports have documented that exogenous administration of endorphin results in a variety of behavioral changes including generalized muscular rigidity (18), marked hyperactivity (19), and wet-dog shaken (20) . The finding that immunized animals were rendered flaccid for a 15-30 min period suggests that circulating ß-endorphin may play a role in modulating behavioral excitability . The administration of the opiate antagonists naloxone and naltrexone also failed to significantly alter the GH rhythm or plasma IRI levels . These findings are in contrast to previous reports (4,5) that aaloxone significantly suppressed basal GH levels in the rat, although the dose of naloxone (0 .2 mg/ kg) employed in those studies was different from the present one, and only single samples of blood were obtained . The present findings are in agreement with those of Cocchi et al (6) and Martin and co-workers (21) . In the latter study, various doses of aaloxone ranging from 0 .4-20 mg/kg were employed and
Vol . 25, No . 23, 1979
Grawth Hormone and Insulin Secretion
1989
ao significant effect on pulsatile GH secretion was demonstrated . Moreover, studies is other species including goat (22) and man (23,24) have failed to demonstrate a significant effect of naloxone on basal GH secretion . In conclusion, the failure of B-endorphin antiserum, aaloaone, and naltrexone to influence basal GH and insulin secretion argues against a direct tonic participation of endogenous opioid peptides in GH and insulin regulation is the normal physiologic state . While these findings do not support the view that endogenous opiate-like peptides play a physiologically important role in maintaining basal GH and insulin secretion, they do not exclude the possibility that they are involved is mediating neuroendocrine responses to pain and stress . Açknowledgements This research was supported by grants from the McGill University-Montreal Children's Hospital Research Institute and the Medical Research Council of Canada . The stimulating contribution of Dr . Eleanor Colle is gratefully acknowledged . The technical assistance of Carol Fascia and James MacLean, and the secretarial assistance of Mary Youakim are appreciated . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
A . DUPONT, L . CUSAN, M . CARON, F . LABRIE and C .H . LI, Proc . Natl . Acad . Sci . US A _74 358-359 (1977) . C . RIVIER, W . VALE, N . LING, M . BROWN and R . GUILLEMIN, Endocrinology 100 238-241 (1977) . R . CHIHARA, A . ARIMURA, D .H . COY and A .V . SCHALLY, Endocrinology 102 281-290 (1978) . J .F . BRUNI, D . VAN VUGT, S . MARSHALL and J . MEITES, Life Sci . _21 461-466 (1977) . C .J . SHAAR, R .C .A . FREDERICKSON, N .B . DININGER and L . JACKSON, Life Sci . _21 853-860 (1977) . D . COCCHI, A . SANTAGOSTINO, I . GIL-AD, S . FERRI and E .E . MULLER, Life Sci . 20 2041-2046 (1977) . G .S . TANNENBAUM and J .B . MARTIN, Endocrinology _98 562-570 (1976) . D . GRUBE, R.H . VOIGT and B . WEBER, Histochem . 59 75-79 (1978) . E . IPP, R . DOBBS and R .H UNGER, Nature _276 190-191 (1978) . M . REICHLIN, J .J . SCHMUZE and V .R . VANCE, Proc . Soc . Exptl . Biol . Med . _128 347-366 (1968) . N . OGAWA, T . THOMPSON, H .G . FRIESEN, J .B . MARTIN and P . BRAZEAU, Biochem . J . 165 269-277 (1977) . G . SCATCHARD, Ann .N .Y . Acad . Sci . 5 1 660672 (1949) . B .A . BERROWITZ, S .H . NGAI, J . HEMPSTEAD and S .J . SPECTOR, J . Pharmac . Exper . Therap . _195 499-504 (1975) . N . OGAWA, A .E . PANERAI, J . THLIVERIS and H .G . FRIESEN, Life Sci .(.in press) R . GUILLEMLN, T . VARGO, J . BOSSIER, S . MINICK, N . LING, C . RIVIER, W . VALE and F . BLOOM, Science _179 1367-1369 (1977) . V . HOLLT, R . PRZEWLOCRI and A . HERZ, Life Sci . _23 1057-1066 (1978) . S .L . WARDLAW, M . FERIN, P .W . CARMEL and A .G . FRANTZ, Program 61st Annual Meeting Endocrine Society, Anaheim, CA . A443 (1979) . F . BLOOM, D . SEGAL, N . LING and R . GUILLEMIN, Science _194 630-632 (1976) . D .S . SEGAL, R .G . BROWNE, F . BLOOM, N . LING and R . GUILLEMIN, Science 198 411-414 (1977) . J .W . HOLADAY, H .H . LOH and C .H . LI, Life Sci . 22 1525-1536 (1978) .
1990
21 . 22 . 23 . 24 .
Growth Hormone and Insulin Secretion
Vol . 25, No . 23, 1979
J .B . MARTIN, G . TOUS, I . WOODS and H . GUYDA, Brain Res . 168 210-215, (1979) . I .C . HART and A .T . COWIE, J . Endocr . _77 16P-17P (1977) . D . JANOWSRY, L . JUDD, L . HUEY, N . ROITMAN, D . PARKER and D . SEGAL, Lancet _II 320 (1978) . S . LAL, P .V . NAIR, P . CERVANTES, J .PUIalAN and H . GUYDA, Acta Psychiat . Scand.59 173-179 (1979) .
Pergamon Press
FAILURE OF ß-ENDORPHIN ANTISERUM, NALOXONE, AND NALTRERONE TO ALTER PHYSIOLOGIC GROWTH HORMONE AND INSULIN SECRETION) Gloria Shaffer Tannenbaum, Alberto E. Panerai and Henry G. Friesen Departments of Pediatrics, and Neurology and Neuroaurgery, McGill University (G .S .T .), Montreal, Quebec, and Department of Physiology, University of Manitoba (A .E .P ., H .G .F .), Winnipeg, Manitoba (Received in final form October 24, 1979) Summary The role of endogenous opiate-like peptides in physiologic regulation of growth hormone (GH) and insulin (IRI) secretion was assessed by passive immunization with ß-endorphin antiserum and by administration of the opiate antagonists naloxone and naltrexone . Six-hour secretioy profiles were obtained from 5 groups of freelymoving chronically cannulated male rats following the i .v . administration of (I) ß-endorphin antiserum, (II) normal rabbit serum, (III) naloxone (1 m8/kg), (IV) naltrexone (1 mg/kg), and (V) normal saline . The typical ultradun rhythm of GH secretion was evident in all groups with moat peak GH values >400 ng/ml . No disruption in amplitude or periodicity of the GH rhythm was observed and there was Plasma IRI no significant difference in mean 6-hr plasma GH levels . levels fluctuated minimally over the 6-hr sampling period . There was ao significant difference in mean 6-hr IRI levels between groups I and II, or between groups III, IV and V . These data do not support the view that endogenous opiate-like peptides play a physiologically important role in maintaining basal GH and IRI secretion. The normal physiologic role of endogenous opiate-like peptides in hormone regulation is unknown. It has been reported that ß-endorphin is a potent stimulator of growth hormone (GH) secretion in the rat when administered via the intravenous or intracerebral ventricular route (1-3) . These results have, in general, been obtained under a variety of unphyaiologic conditions including anesthesia, steroid, and anti-somatostatin pretreatment . Moreover, results obtained from experiments investigating the effects of the specific opiate antagonist naloxoae on basal GH secretion are inconsistent . Both a suppression (4,5) and ao significant effect (6) have been reported . In these studies, single samples of blood were obtained from groups of animals sacrificed at different times during the day . Since GH secretion in the rat is episodic in nature with major episodes of GH secretion occurring at 3-4 hr intervals
)Address requests for reprints : Dr . Gloria S . Tannenbaum, Department of Endocrinology, Montreal Children's Hospital, 2300 Tupper Street, Montreal, Quebec H3H 1P3 0024-3205/79/231983-08$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1984
Growth Hormone and Insulin Secretion
Vol . 25, No . 23, 1979
throughout a 24-hr period (7), it is important to obtain longitudinal GH profiles in individual animals in order to assess the effects of any manipulation The aim of the experiments described in this report was to on GH secretion. assess the role of ß-endorphin in physiologic regulation of GA secretion using chronically cannulated, freely-behaving rata . If circulating ß-endorphin plays a role is maintaining basal GH secretion we hypothesized that its effectif might be neutralized or blocked by the administration of a specific antiserum to ß-endorphin. In addition, the effects of opiate-receptor blockade on pulsatile GH secretion were examined . In view of the recent demonstration of endorphin-like immunoreactivity in the rat endocrine pancreas (8), together with the report that ß-endorphin stimulates the release of insulin from the perfused dog pancreas (9), plasma insulin levels were simultaneously monitored in all experiments in order to clarify the possible role of endogenous opiate-like peptides in physiologic regulation of insulin secretion . Materials and Methode Antibodies to camel ß-endorphin (Peninsula Antiserum to ß-endorphin. Labs, San Carlos, CA) were obtained in rabbits after conjugation of ßeadorphia to bovine serum albumin (10) . ß-endorphin was iodinated and the product purified using methods previously described for somatostatin (11) . The antiserum used in the present study binds 35% of 1251-labeled ß-endorphin at a final dilution of 1 :65,000 and can detect 100 pg ß-endorphin per assay tube . By Scatchard plot analysis (12), 1 ml ß-endorphin antiserum would be capable The antiserum cross reacts 50Z with ovine of binding 2 .5 ug ß-endorphin. ß-LPH on as equimolar basis but shows no cross-reactivity with any of the following substances : a-endorphin, y-endorphin, met-eakephalia, a-MSH, ß-MSH, ACTH, vasopreasia, insulin, glucagon, TRH, LH-RH, bombesin, myelin basic protein, GA, prolactin, morphine and naloxone . Adult male Sprague-Dawley rate (300Animals and experimental procedure . 400 g) maintained on a 12-hr light-dark cycle (lights on, 0600-1800 hr) in a temperature (22 _+ 1°C)- and humidity-controlled room were implanted with chronic iatracardiac venous cannulae and adapted to isolation test chambers, Purina rat chow and tap water were using methods previously described (7) . available ad libitum . After recovery of pre-operative body weight (usually one week), blood samples were withdrawn every 15 min for periods of 6 hr Group I (n=6) was administered 1 ml (1000-1600 hr) from 5 groups of rats . ß-endorphin antiserum i .v . through the cannula after removal of the first blood sample whereas a second group (n=5) nerved as controls and received 1 ml The effects of opiate receptor normal rabbit serum at the same time point . Naloxone hydrochloride blockade were studied in 3 additional groups of rate . (Narcan, Endo Laboratories) was administered i .v . in a dose of 1 mg/kg body weight . Because of its short biological half life (13), naloxone was injected at 3 different times - 1000 hr, 1230 hr, and 1400 hr - during the sampling period . Control rats received the normal saline vehicle at the same time The fifth group of rats was administered i.v . the longer-acting opiati' points . antagonist aaltrexone in a dose 1 mg/kg body weight at the onset of the sampling period . All rata were observed through a one~way viewing port in order Blood to monitor behavioral changes in response to the various treatments . samples were immediately centrifuged and the plasma separated sad stored at -20°C for subsequent assay .
Antibody titers in plasma of rate after administration of ß - endorphin antiserum . Antibody titers in plasma of ß-endorphin antiserum-treated rate
Vol. 25, No . 23, 1979
Growth Hormone and Insulin Secretion
1985
were assessed by determining the capacity of aliquots of rat plasma obtained 15 min, 3 hr, and 6 hr after antiserum administration to bind 1251 ß-endorphin. Plasma samples were diluted 1 :32 with 0.14 M sodium phosphate buffer and binding to tracer was determined under the conditions used for radioimmunoassay (RIA) of ß-endorphin (14) . Hormone assays . Plasma GH was measured in duplicate by a double antibody RIA using materials supplied by Dr . A. Parlow, National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland . Values are expressed in terms of the rat GH reference preparation (GH-RP-1) . Plasma immunoreactive insulin (IRI) was measured by a deztran-coated charcoal method, utilizing guinea pig antiporciae insulin serum. Purified crystalline rat insulin (lot 615-1112B-295-C) was kindly provided by the Eli Lilly Co ., courtesy of Dr . R. Chance, and served ae a reference standard . Statistical analyses . Analysis of variance for repeated measures was used to compare data for significant differences between groups . Results _ß-endorphin antibody titers in plasma . The mesa binding capacity of a 1 :32 dilution of plasma at 15 min, 3 hr, sad 6 hr post ß-endorphin antiserum treatment was found to be 71 .0 _+ 3x, 69 .5 _+ 4Z, 62 .5 _+ 4 .5x, respectively . When compared to titers of antiserum used in the RIA, the binding capacities of these plasma samples were equivalent to 1 :1500, 1:1760, and 1:3000, respectively . Effects of ß-endorphin antiserum on GH and IRI secretiry patterns . Animas treated with normal rabbit serum (n~5) ezhibited the typical ultradun rhythm of GH secretion . Two major episodes of GH secretion were evident during the 6-hr sampling period with moat peak GH values >400 ng/ml and trough values <6 .2 ng/ml (Fig . lA) . Plasma IRI levels fluctuated minimally over the 6-hr sampling period, with values ranging from 0 .4-2 .6 ag/ml. Prominent GH aecretory bursts were still evident is ß-endorphin antiserum-treated rate (Fig . 1B) . No disruption in amplitude or periodicity of the GH rhythm was observed and there was no significant difference in mean 6-hr plasma GH levels between the 2 groups of rats (Table I) . Similarly, ao significant difference was observed in mean 6-hr plasma IRI level of ß-endorphin antiserum-treated rats when compared to normal rabbit serum-treated control animals (Table I) . Although passive immunization with ß-endorphin antiserum did not alter basal hormone secretion, immunized animals appeared behaviorally flaccid, lying with limbs extended, for a period of 15-30 min following antiserum administration . Effects of aaloaone and naltrexone on GH and IRI profiles . The i.v . administration of both naloaoae and naltrexone failed to significantly alter either the amplitude or periodicity of the GH secretiry bursts and plasma IRI levels were unchanged when compared to saline-treated controls . A rapreseatative profile of a naltreaone-treated animal is illustrated is Fig. 2 . No significant differences in mean 6-hr plasma GH or IRI levels were observed between the 3 groups of rate, although the mean 6-hr GH levels in the 2 groups treated with opiate antagonists were slightly higher than that of saliaeiajectad control rate (Table I) . No behavioral changes were observed in these animals .
1986
Growth Hormone and Insulin Secretion
2(2)
Vol . 25, No . 23, 1979
Normal Rabbit Serum
A
400
3 .6
6
y r
A~
100
b,~~d
0.4 1000
1100
1200
1900
1400
W 36 ß-Endorphin Antiserum
1500
1600
ô
~(460)
400
300 OI C
3 .6
200
3s
P loo ~~`\tf ~ 0.~ d `~.4 r-t 0 1000
y r
1100
1200
i 1300
hb -
1 ~
n
1400
1300
1
0 .4 1600
TIME (hours)
FIG . 1 Six-hour GH and IRI secretioy patterns in 2 rats administered 1 ml of either normal rabbit serum (A) or ß-endorphin antiserum (B) i .v . Arrows indicate ß-endorphin antiserum did not significantly alter GH or time of injection . IRI secretion .
Growth Hormone and Insulin Secretion
Vol . 25, No . 23, 1979
1987
2 (3) Normal Saline
3 .6
s N 3 s
s.o
0 0 .4 1000
2
1100
iR00
1]00
1400
1500
3
1600
Naltrexone (lm~/kp)
400
E
0 ~00 e ~ .6
aoo N t G
s N
100
0.4
3
FIG . 2 Plasllla GH sad IRI levels after i .v . administration of saline (upper) or naltreaone (lower . Arrows indicate time of injection . No significant effect of naltrexoae eves observed .
Growth Hormone and Insulin Secretion
1988
Vol . 25, No . 23, 1979
TABLE I Effects of 8-Endorphin Antiserum, Naloxone, and Naltrexone on Mean 6-hour Plasma GA and IRI Levels
Experimental group
n
Meaa 6-hr GH level (ng/ml)
Mean 6-hr IRI level (ng/ml)
~-Endorphin Antiserum
6
113 .8 + 10 .8a
1 .13 + 0 .03
Normal Rabbit Serum
5
97 .7 + 11 .1
1 .27 + 0 .05
Naloxone
5
136.0 + 22 .4
1 .68 + 0 .05
Naltrexone
4
144 .0 + 23 .9
1 .43 + 0 .07
Saline
5
118 .2 + 20 .4
1 .85 + 0 .08
aMean + SEM Discuseion The availability of an antiserum to ~endorphia provides a useful experimental tool for assessing the possible involvement of endogenous ß-endorphin in hormone regulation . Basal B-endorphin concentrations in the peripheral plasma of the rat have been reported to range from 0.25-1 .5 ng/ml (15,16) . Although the concentration of ß-endorphin in the hypophyseal portal circulatim of the rat is unknown, values of 3 .4-3 .8 ag/ml have been documented in the monkey (17) . Assuming that ßrendorphin concentrations in the rat hypophyseal portal circulation are similar, 1 ml of endorphin antiserum used in the present experiment would be more than sufficient to neutralize endogenous ß-endorphin circulating in both the peripheral and hypophyseal portal plasmas . The finding that neither the amplitude nor the periodicity of the GH pulses was altered following B-endorphin antiserum administration, together with the lack of effect on basal plasma IRI levels, suggests that circulating B-endorphin does not play a physiologically important role in maintaining basal GH and IRI secretion. Of interest is the fact that the animals exhibited a behavioral response following antiserum administration . Previous reports have documented that exogenous administration of endorphin results in a variety of behavioral changes including generalized muscular rigidity (18), marked hyperactivity (19), and wet-dog shaken (20) . The finding that immunized animals were rendered flaccid for a 15-30 min period suggests that circulating ß-endorphin may play a role in modulating behavioral excitability . The administration of the opiate antagonists naloxone and naltrexone also failed to significantly alter the GH rhythm or plasma IRI levels . These findings are in contrast to previous reports (4,5) that aaloxone significantly suppressed basal GH levels in the rat, although the dose of naloxone (0 .2 mg/ kg) employed in those studies was different from the present one, and only single samples of blood were obtained . The present findings are in agreement with those of Cocchi et al (6) and Martin and co-workers (21) . In the latter study, various doses of aaloxone ranging from 0 .4-20 mg/kg were employed and
Vol . 25, No . 23, 1979
Grawth Hormone and Insulin Secretion
1989
ao significant effect on pulsatile GH secretion was demonstrated . Moreover, studies is other species including goat (22) and man (23,24) have failed to demonstrate a significant effect of naloxone on basal GH secretion . In conclusion, the failure of B-endorphin antiserum, aaloaone, and naltrexone to influence basal GH and insulin secretion argues against a direct tonic participation of endogenous opioid peptides in GH and insulin regulation is the normal physiologic state . While these findings do not support the view that endogenous opiate-like peptides play a physiologically important role in maintaining basal GH and insulin secretion, they do not exclude the possibility that they are involved is mediating neuroendocrine responses to pain and stress . Açknowledgements This research was supported by grants from the McGill University-Montreal Children's Hospital Research Institute and the Medical Research Council of Canada . The stimulating contribution of Dr . Eleanor Colle is gratefully acknowledged . The technical assistance of Carol Fascia and James MacLean, and the secretarial assistance of Mary Youakim are appreciated . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
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