0013-7227/92/1303-1445$03.00/0 Endocrinology Copyright Q 1992 by The Endocrine Society
Vol. 130, No. 3
Printed
Pyridostigmine-Mediated Evidence for Somatostatin WILLIAM B. WEHRENBERG, AND ANDREA GIUSTINA
STEPHEN
Growth Hormone Involvement* D. WIVIOTT,
DONALD
in U.S.A.
Release:
M. VOLTZ,
Department of Health Science, University of Wisconsin, Milwaukee, Wisconsin 53211; and the Cattedra di Clinka Medica, University of Bresciu (A.G.), Brescia, Italy 25125
ABSTRACT. Pyridostigmine (PD), a choline&erase inhibitor, has been shown to elicit GH release when given alone and to potentiate the GH response to GH-releasing hormone (GHRH) in man. Numerous experiments have indirectly indicated that somatostatin (SS) inhibition is its likely mechanism of action. This study sought to establish the ability of PD to induce GH release in the rat, determine the dose-response relationship, and test the hypothesis that SS inhibition is the method of action. Three experiments were performed to monitor the GH response to PD. I1 Five erou~s of male rata were food deurived for ‘72 h. The groups we; then treated iv with saline, SS antibody (SSab), and 10,100, and 1000 rg/kg PD, respectively. Blood samples were drawn before and after treatment. II) Two groups of male rata were pretreated iv with GHRH antibody (GHRH-ab) and either SS-ab or normal sheep serum (NSS). Blood samples were drawn every 30 min for 8.5 h, during which time each animal was injected with PD (10 rg/kg) in the third hour and again in the sixth hour. III) Male rats received a PD injection (10 rg/kg, iv) during a spontaneous GH trough period and a second PD injection during a spontaneous GH peak period. Blood samples
were drawn at regular intervals preceding and following treatments. In Exp I, PD induced a clear 4- to 5-fold increase in GH concentrations in food-deprived rats. The maximal GH responses occurred after the 10 and 100 &kg doses, although the pattern and duration were different with-these two doses. In Exp II, PD induced an approximately a-fold increase in GH values in animals pretreated with GHRH-ab and NSS, but failed to induce a change in GH in the animals treated with GHRHab and SS-ab. In Exp III, PD failed to induce any change in GH concentration when administered during spontaneous GH peaks or troughs. The first two experiments suggest that PD increases GH secretion in the rat via inhibition of SS. The failure of PD to alter GH during a spontaneous peak is consistent with the current hypothesis that the level of SS is low at this time. Its failure to alter GH during trough periods may be related to very high SS tone. In conclusion, our results support the hypothesis that PD acts via inhibition of SS secretion. (Endocrinology 130: 1445-1450,1992)
T
HERE is considerable evidence that the cholinergic system is involved in the mediation of GH secretion (1, 2). Specifically, there is evidence that cholinergic agonists and antagonists play opposite roles in controlling GH secretion. Cholinergic agonists, including pilocarpine and paraoxon, have been shown to elicit GH secretion and potentiate the GH response to GH-releasing hormone (GHRH) in rats (3-5). Other agonists, such as esserene and edrophonium, have been shown to have similar effects in dogs (6,7) and humans (8), respectively. Consistent with this function, cholinergic antagonists have been shown to block the GH response to various stimuli. In humans, sleep-induced GH release has been suppressed by the cholinergic antagonist methscopolamine bromide (9). Pirenzepine blocks the stimulatory
effect of L-dopa, clonidine, and amorphine in humans (10) and is effective in blocking the GH response to GHRH in animals (3) and humans (11-13). Similarly, atropine blocks the GH response to clonidine and exercise in humans (14) and inhibits GHRH-induced GH release in animals (4, 15) and humans (13). Pyridostigmine (PD), a cholinergic agonist with both muscarinic and nicotinic actions, has been shown to elicit a GH response in humans when given alone (13,16). PD also has been reported to produce a synergistic GH response when given in combination with GHRH in man. This effect has been reported in short children (17) and in normal (13, 18-20) and obese subjects (11, 21). In certain studies the results of PD administration have been intriguing. PD has reportedly failed to induce GH secretion nocturnally (22-24) and has an impaired effect in type 1 diabetic individuals with an exaggerated GH response to GHRH (25). It is hypothesized that both of these models are characterized by low somatostatin (SS) tone, suggesting that the method of action of PD involves SS. The aim of our study was to establish the ability of
Received August 5, 1992. Address requests for reprints to: Dr. William B. Wehrenberg, Denartment of Health Sciences. Universitv of Wisconsin. P.O. Box 413. -Milwaukee, Wisconsin 53201: *This work was supported by NIH Grants ROl-DK-33324 and Research Career Development Award K04-DK-01374 (to W.B.W.). 1445
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 09 November 2015. at 20:58 For personal use only. No other uses without permission. . All rights reserved.
1446
PD MECHANISM
PD to elicit GH secretion in rats, determine a doseresponse relationship, and determine whether the method of action of PD-mediated GH release is SS inhibition.
Materials
and Methods
AnimaLs All animals used in these studies were acquired, maintained, and used in accordance with the guidelines established by the NIH. Adult male Sprague-Dawiey rats were maintained in a temperature- and humidity-controlled environment and exposed to a 14-h light, 10-h dark lighting schedule (lights on at 0600 h). Experimentation This study was conducted as a series of three experiments. Exp I. Five groups of male rats were fitted with chronic indwelling venous catheters under ether anesthesia 3 days before experimentation (26). The animals were food deprived immediately after surgery for 72 h. On the day of the experiment, a control blood sample (0.3 ml) was drawn at 0 min. The groups were then treated in the following manner: saline (0.5 ml, iv; n = 4), SS antibody (SS-ab; 0.5 ml, iv; n = 5), PD (10 rg/kg, iv; n = 5), PD (100 pg/kg, iv; n = 5), and PD (1000 rg/kg, iv; n = 5). Blood samples were taken 5,10,15,30,60,90, and 120 min after treatment. Blood samples were centrifuged, and plasma was separated and then frozen for determination of GH by RIA. Exp ZZ.Male rats, anesthetized with sodium pentobarbital (40 mg/kg, ip), were fitted with chronic indwelling venous catheters 5 days before experimentation, as described previously (26). On the day of experimentation, all animals were treated with GHRH antiserum (GHRH-ab; 0.25 ml, iv) at -30 min. Animals were then divided into two groups; one received SS-ab (0.5 ml, iv; n = 6), and the other received normal sheep serum (NSS; 0.5 ml, iv; n = 6) at -29 min. Control blood samples were taken at 30-min intervals from O-2.5 h. Each animal was then injected with PD (10 rg/kg, iv) at 2.75 h and again at 5.5 h. After each of the PD injections, blood samples were drawn at 15, 30, 60, 90,120, and 150 min, for a total experiment time of 8.5 h. Blood samples were handled as described above; however, the red blood cells were resuspended in normal saline and returned to the animals at approximately 3-h intervals. Exp ZZZ.Six days before experimentation, male rats (n = 8) were fitted with chronic indwelling venous catheters using sodium pentobarbital anesthesia (40 mg/kg, ip). On the day of experimentation, control blood samples were drawn at 20-min intervals for 1 h. Each animal then received a PD injection (10 rg/kg, iv), followed by blood sampling at 5, 10, 15, 20, 40, 60, 80, and 100 min. Each animal then received a second PD injection (10 pg/kg, iv), with subsequent blood samples drawn at the same intervals. Injections of PD were specifically designed to be administered at either a trough or a peak period of spontaneous GH secretion (26, 27).
OF ACTION
Endo. Vol130.NoR
Antibody preparation Rat GHRH-Ab was prepared by immunizing rabbits with a mixture of synthetic rat GHRH and methylated BSA emulsified in Freund’s adjuvant, as described previously (28). This antiserum is specific for rat GHRH. It does not cross-react with GHRH from other species or with peptides with considerable sequence homology with GHRH, including secretin, vasoactive intestinal peptide, and glucagon. The antibody dilution required to bind approximately 50% of a rat [‘251]GHRH-labeled trace was 1:80,000. The dose administered was based on our observation that 0.25 ml GHRH-ab is effective in neutralizing endogenous GHRH, as evidenced by an absence of pulsatile GH secretion (29). SS-ab was prepared by immunizing sheep with synthetic SS. Complete characterization of the SS-Ab has been previously described (30). Briefly, the antibody was partially purified by ammonium sulfate purification. It is directed toward the midportion of the somatostatin molecule. The antibody dilution required to bind approximately 50% of a [1251]Tyr-SS-labeled trace was 1:25,000. The dose administered was based on our observation that 0.5 ml SS-ab effectively neutralizes endogenous SS in the rat (31). RZA procedures Plasma GH concentrations were measured in duplicate by a double antibody procedure, using materials supplied by the National Pituitary Agency of the NIH. Blood samples were assayed in aliquots of either 10 or 50 ~1.GH concentrations are expressed in terms of the rat GH RP-2 standard. Assay sensitivity was approximately 0.08 rig/tube. Within-assay variation averaged less than lo%, and between-assay variation averaged less than 16%. Statistical analyses Data are expressed as the mean f SEM. Significant treatment effects of PD in food-deprived rats (Exp I) were detected by analysis of variance, with consideration given for repeated measures in the same animals (32). The Mann-Whitney U test was used to determine significant treatment effects of PD on GH secretion in normal rats treated with GHRH-ab and SSab (Exp II). Results of PD administration to normal rats during peak and trough GH secretion (Exp III) are presented as individual observations. To determine whether PD augmented GH secretion during a peak or trough, we determined the change in GH concentrations 5, 10, and 15 min postinjection us. the level in the preinjection sample. These values should be significantly greater than zero if augmentation occurred. This hypothesis was evaluated by t test. A significance level of P < 0.05 was used. In less than 1% of the cases,blood samples were missed due to technical problems during the experiments. In these instances, GH values were estimated by calculating the average GH concentration from the immediately preceding and following blood samples.
Results Exp I This experiment
administration
was designed to determine
whether
of PD could induce GH secretion in ani-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 09 November 2015. at 20:58 For personal use only. No other uses without permission. . All rights reserved.
PD MECHANISM
mals with high SS concentrations induced by food deprivation. Baseline GH concentrations were below 10 ng/ ml in all food-deprived rats. After saline administration, GH concentrations remained low, between 5-15 rig/ml (Fig. 1). In contrast, SS-ab-treated animals (Fig. 1) showed a significant increase in GH (P < 0.05) after antibody administration. GH values rose and remained elevated for approximately 60 min. Values began to decrease after this time, but GH was still significantly elevated 90 min after injection. The effects of PD administration are depicted in Fig. 2. Animals receiving 10 pg/kg PD had the greatest GH response, as measured by the area under the curve (Fig. 2, inset). This group also exhibited the most sustained response, with GH levels rising from below 10 rig/ml to
80
5
60
B 40 6
O’b
.-.
0
T
I
I
I
30
60
90
120
Minutes
FIG. 1. Mean plasma GH concentrations in 72-h food-deprived rats. Saline (n = 4) or SS-ab (n = 5) was administered after the 0 min blood sample. Animals receiving SS-ab showed a significant increase in plasma GH after antibody administration. Inset, Area under the GH curve (AUC) for the same two groups of food-deprived animals. *, P
Vol. 130, No. 3
Printed
Pyridostigmine-Mediated Evidence for Somatostatin WILLIAM B. WEHRENBERG, AND ANDREA GIUSTINA
STEPHEN
Growth Hormone Involvement* D. WIVIOTT,
DONALD
in U.S.A.
Release:
M. VOLTZ,
Department of Health Science, University of Wisconsin, Milwaukee, Wisconsin 53211; and the Cattedra di Clinka Medica, University of Bresciu (A.G.), Brescia, Italy 25125
ABSTRACT. Pyridostigmine (PD), a choline&erase inhibitor, has been shown to elicit GH release when given alone and to potentiate the GH response to GH-releasing hormone (GHRH) in man. Numerous experiments have indirectly indicated that somatostatin (SS) inhibition is its likely mechanism of action. This study sought to establish the ability of PD to induce GH release in the rat, determine the dose-response relationship, and test the hypothesis that SS inhibition is the method of action. Three experiments were performed to monitor the GH response to PD. I1 Five erou~s of male rata were food deurived for ‘72 h. The groups we; then treated iv with saline, SS antibody (SSab), and 10,100, and 1000 rg/kg PD, respectively. Blood samples were drawn before and after treatment. II) Two groups of male rata were pretreated iv with GHRH antibody (GHRH-ab) and either SS-ab or normal sheep serum (NSS). Blood samples were drawn every 30 min for 8.5 h, during which time each animal was injected with PD (10 rg/kg) in the third hour and again in the sixth hour. III) Male rats received a PD injection (10 rg/kg, iv) during a spontaneous GH trough period and a second PD injection during a spontaneous GH peak period. Blood samples
were drawn at regular intervals preceding and following treatments. In Exp I, PD induced a clear 4- to 5-fold increase in GH concentrations in food-deprived rats. The maximal GH responses occurred after the 10 and 100 &kg doses, although the pattern and duration were different with-these two doses. In Exp II, PD induced an approximately a-fold increase in GH values in animals pretreated with GHRH-ab and NSS, but failed to induce a change in GH in the animals treated with GHRHab and SS-ab. In Exp III, PD failed to induce any change in GH concentration when administered during spontaneous GH peaks or troughs. The first two experiments suggest that PD increases GH secretion in the rat via inhibition of SS. The failure of PD to alter GH during a spontaneous peak is consistent with the current hypothesis that the level of SS is low at this time. Its failure to alter GH during trough periods may be related to very high SS tone. In conclusion, our results support the hypothesis that PD acts via inhibition of SS secretion. (Endocrinology 130: 1445-1450,1992)
T
HERE is considerable evidence that the cholinergic system is involved in the mediation of GH secretion (1, 2). Specifically, there is evidence that cholinergic agonists and antagonists play opposite roles in controlling GH secretion. Cholinergic agonists, including pilocarpine and paraoxon, have been shown to elicit GH secretion and potentiate the GH response to GH-releasing hormone (GHRH) in rats (3-5). Other agonists, such as esserene and edrophonium, have been shown to have similar effects in dogs (6,7) and humans (8), respectively. Consistent with this function, cholinergic antagonists have been shown to block the GH response to various stimuli. In humans, sleep-induced GH release has been suppressed by the cholinergic antagonist methscopolamine bromide (9). Pirenzepine blocks the stimulatory
effect of L-dopa, clonidine, and amorphine in humans (10) and is effective in blocking the GH response to GHRH in animals (3) and humans (11-13). Similarly, atropine blocks the GH response to clonidine and exercise in humans (14) and inhibits GHRH-induced GH release in animals (4, 15) and humans (13). Pyridostigmine (PD), a cholinergic agonist with both muscarinic and nicotinic actions, has been shown to elicit a GH response in humans when given alone (13,16). PD also has been reported to produce a synergistic GH response when given in combination with GHRH in man. This effect has been reported in short children (17) and in normal (13, 18-20) and obese subjects (11, 21). In certain studies the results of PD administration have been intriguing. PD has reportedly failed to induce GH secretion nocturnally (22-24) and has an impaired effect in type 1 diabetic individuals with an exaggerated GH response to GHRH (25). It is hypothesized that both of these models are characterized by low somatostatin (SS) tone, suggesting that the method of action of PD involves SS. The aim of our study was to establish the ability of
Received August 5, 1992. Address requests for reprints to: Dr. William B. Wehrenberg, Denartment of Health Sciences. Universitv of Wisconsin. P.O. Box 413. -Milwaukee, Wisconsin 53201: *This work was supported by NIH Grants ROl-DK-33324 and Research Career Development Award K04-DK-01374 (to W.B.W.). 1445
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 09 November 2015. at 20:58 For personal use only. No other uses without permission. . All rights reserved.
1446
PD MECHANISM
PD to elicit GH secretion in rats, determine a doseresponse relationship, and determine whether the method of action of PD-mediated GH release is SS inhibition.
Materials
and Methods
AnimaLs All animals used in these studies were acquired, maintained, and used in accordance with the guidelines established by the NIH. Adult male Sprague-Dawiey rats were maintained in a temperature- and humidity-controlled environment and exposed to a 14-h light, 10-h dark lighting schedule (lights on at 0600 h). Experimentation This study was conducted as a series of three experiments. Exp I. Five groups of male rats were fitted with chronic indwelling venous catheters under ether anesthesia 3 days before experimentation (26). The animals were food deprived immediately after surgery for 72 h. On the day of the experiment, a control blood sample (0.3 ml) was drawn at 0 min. The groups were then treated in the following manner: saline (0.5 ml, iv; n = 4), SS antibody (SS-ab; 0.5 ml, iv; n = 5), PD (10 rg/kg, iv; n = 5), PD (100 pg/kg, iv; n = 5), and PD (1000 rg/kg, iv; n = 5). Blood samples were taken 5,10,15,30,60,90, and 120 min after treatment. Blood samples were centrifuged, and plasma was separated and then frozen for determination of GH by RIA. Exp ZZ.Male rats, anesthetized with sodium pentobarbital (40 mg/kg, ip), were fitted with chronic indwelling venous catheters 5 days before experimentation, as described previously (26). On the day of experimentation, all animals were treated with GHRH antiserum (GHRH-ab; 0.25 ml, iv) at -30 min. Animals were then divided into two groups; one received SS-ab (0.5 ml, iv; n = 6), and the other received normal sheep serum (NSS; 0.5 ml, iv; n = 6) at -29 min. Control blood samples were taken at 30-min intervals from O-2.5 h. Each animal was then injected with PD (10 rg/kg, iv) at 2.75 h and again at 5.5 h. After each of the PD injections, blood samples were drawn at 15, 30, 60, 90,120, and 150 min, for a total experiment time of 8.5 h. Blood samples were handled as described above; however, the red blood cells were resuspended in normal saline and returned to the animals at approximately 3-h intervals. Exp ZZZ.Six days before experimentation, male rats (n = 8) were fitted with chronic indwelling venous catheters using sodium pentobarbital anesthesia (40 mg/kg, ip). On the day of experimentation, control blood samples were drawn at 20-min intervals for 1 h. Each animal then received a PD injection (10 rg/kg, iv), followed by blood sampling at 5, 10, 15, 20, 40, 60, 80, and 100 min. Each animal then received a second PD injection (10 pg/kg, iv), with subsequent blood samples drawn at the same intervals. Injections of PD were specifically designed to be administered at either a trough or a peak period of spontaneous GH secretion (26, 27).
OF ACTION
Endo. Vol130.NoR
Antibody preparation Rat GHRH-Ab was prepared by immunizing rabbits with a mixture of synthetic rat GHRH and methylated BSA emulsified in Freund’s adjuvant, as described previously (28). This antiserum is specific for rat GHRH. It does not cross-react with GHRH from other species or with peptides with considerable sequence homology with GHRH, including secretin, vasoactive intestinal peptide, and glucagon. The antibody dilution required to bind approximately 50% of a rat [‘251]GHRH-labeled trace was 1:80,000. The dose administered was based on our observation that 0.25 ml GHRH-ab is effective in neutralizing endogenous GHRH, as evidenced by an absence of pulsatile GH secretion (29). SS-ab was prepared by immunizing sheep with synthetic SS. Complete characterization of the SS-Ab has been previously described (30). Briefly, the antibody was partially purified by ammonium sulfate purification. It is directed toward the midportion of the somatostatin molecule. The antibody dilution required to bind approximately 50% of a [1251]Tyr-SS-labeled trace was 1:25,000. The dose administered was based on our observation that 0.5 ml SS-ab effectively neutralizes endogenous SS in the rat (31). RZA procedures Plasma GH concentrations were measured in duplicate by a double antibody procedure, using materials supplied by the National Pituitary Agency of the NIH. Blood samples were assayed in aliquots of either 10 or 50 ~1.GH concentrations are expressed in terms of the rat GH RP-2 standard. Assay sensitivity was approximately 0.08 rig/tube. Within-assay variation averaged less than lo%, and between-assay variation averaged less than 16%. Statistical analyses Data are expressed as the mean f SEM. Significant treatment effects of PD in food-deprived rats (Exp I) were detected by analysis of variance, with consideration given for repeated measures in the same animals (32). The Mann-Whitney U test was used to determine significant treatment effects of PD on GH secretion in normal rats treated with GHRH-ab and SSab (Exp II). Results of PD administration to normal rats during peak and trough GH secretion (Exp III) are presented as individual observations. To determine whether PD augmented GH secretion during a peak or trough, we determined the change in GH concentrations 5, 10, and 15 min postinjection us. the level in the preinjection sample. These values should be significantly greater than zero if augmentation occurred. This hypothesis was evaluated by t test. A significance level of P < 0.05 was used. In less than 1% of the cases,blood samples were missed due to technical problems during the experiments. In these instances, GH values were estimated by calculating the average GH concentration from the immediately preceding and following blood samples.
Results Exp I This experiment
administration
was designed to determine
whether
of PD could induce GH secretion in ani-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 09 November 2015. at 20:58 For personal use only. No other uses without permission. . All rights reserved.
PD MECHANISM
mals with high SS concentrations induced by food deprivation. Baseline GH concentrations were below 10 ng/ ml in all food-deprived rats. After saline administration, GH concentrations remained low, between 5-15 rig/ml (Fig. 1). In contrast, SS-ab-treated animals (Fig. 1) showed a significant increase in GH (P < 0.05) after antibody administration. GH values rose and remained elevated for approximately 60 min. Values began to decrease after this time, but GH was still significantly elevated 90 min after injection. The effects of PD administration are depicted in Fig. 2. Animals receiving 10 pg/kg PD had the greatest GH response, as measured by the area under the curve (Fig. 2, inset). This group also exhibited the most sustained response, with GH levels rising from below 10 rig/ml to
80
5
60
B 40 6
O’b
.-.
0
T
I
I
I
30
60
90
120
Minutes
FIG. 1. Mean plasma GH concentrations in 72-h food-deprived rats. Saline (n = 4) or SS-ab (n = 5) was administered after the 0 min blood sample. Animals receiving SS-ab showed a significant increase in plasma GH after antibody administration. Inset, Area under the GH curve (AUC) for the same two groups of food-deprived animals. *, P
