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Cysteamine reduces serum gonadotropin concentrations in adult male rats
Life Sciences, Vol. 30, pp. 245-252 Printed in the U.S.A.
Pergamon Press
CYSTEAMINE REDUCES SERUM GONADOTROPIN CONCENTRATIONS IN ADULT MALE RATS T.M. Badger, S.M. Sagar, W.J. Millard, J.B. Martin and P. Rosenblum Vincent Memorial Research Laboratories Departments of Obstetrics and Gynecology and Neurology Harvard Medical School and Massachusetts General Hospital Boston, MA 02114. (Received in final form November 24, 1981) Summar X We have examined the effects of cysteamine on the hypothalamicpituitary-gonadal axis of the adult male rat. A single subcutaneous injection of cysteamine (300 mg/kg) reduces significantly (p < 0.05) serum concentrations of LH, FSH and T. Cysteamine blocked LH secretion induced by castration and administration of naloxone and LHRH. Neither acute nor chronic treatment (7 days) altered the hypothalamic LHRH content. These results suggestthat cysteamine acts to reduce pituitary responsiveness to LHRH, resulting in lower mean serum gonadotropin and testosterone concentrations. It is possible, however, that cysteamine acts also at the hypothalamus to reduce LHRH secretion and/ or at the testes to reduce testosterone release. Cysteamine (B-mercaptoethylamine) is a metabolite of coenzyme A and a precursor to hypotaurine. Although taurine biosynthesis is not well understood at present, it appears that in the heart, metabolism of cysteine to taurine occurs via the cysteamine intermediate rather than by the major pathway via cysteine sulfic acid (1). To what extent the cysteamine pathway is utilized in other tissues is unknown. Cysteamine is one of the most potent radioprotective compounds known (2) and has been employed successfully to prevent severe liver damage after paracetamol (tylenol) poisoning (3-6). The ability of cysteamine to reduce cysteine concentrations in cells high in free (non-protein) cysteine has been applied to the treatment of patients with nephropathic cystinosis, an inherited error in cysteine metabolism resulting in free cysteine accumulation in lysosomes and eventual end stage renal failure in the first decade of life
(7-10). In the rat, cysteamine causes a rapid and reproducible onset of duodenal ulcers and has been proposed as a means of producing ulcers for investigations on the mechanisms of duodenal ulcerogenesis and possible means for its prevention (11-16). More recently, Szabo and Reichlin (17) have reported that oral administration of cysteamine produces an acute, partial depletion of somatostatin-like immunoreactivity in the gastrointestinal tract and hypothalamus of rats. We have recently confirmed the somatostatin depleting properties of cysteamine and extended these studies to show that subcutaneous injection of cysteamine reduces somatostatin-like immunoreactivity in various areas of the brain (18). In view of the actions of cysteamine in depleting somatostatin in the central nervous system, other neuroendocrine systems deserve further study. In addition, since cysteamine is used in humans for the treatment of such diseases as cystinosis, the study of the neuroendocrine consequences of its 0024-3205/82/030245-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
246
Cysteamine Reduces Serum T, LH and FSH
Vol.
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administration in humans is warranted. The present study was conducted therefore, to determine the effects of cysteamine on the hypothalamic-pituitarygonadal axis with particular interest in the concentrations of hypothalamic decapeptide luteinizing hormone releasing hormone (LHRH). Methods Materials. Cysteamine HCI (B-mercaptoethylamine) was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in saline and neutralized with NaOH. Doses are expressed as mg of the HCI salt. Highly purified synthetic LHRH was kindly provided by Drs. J. Rivier and W. Vale (Salk Institute). Anti LHRH and anti-ovine luteinizing hormone (LH) were obtained from Dr. T. Nett (Colarado State University) and Dr. G. Niswender (Colarado State University), respectively The ovine LH for iodinization was obtained from Dr. Leo Reichert (Albany Medical College). All other materials in the radioimmunoassay of LH and follicle stimulating hormone (FSH) were provided by the National Pituitary Agency. The standards used were NIAMDD-RAT-LH-RP-I and NIAMDD RAT FSH-RP-I. Tissue Preparation. In each experiment, trunk blood was collected following o decapitation and the serum stored at -20 C until assayed. The brain was removed immediately after decapitation, placed on ice and the hypothalamus and the median eminence rapidly dissected and placed in cold 2M acetic acid. The samples were then placed in boiling water for two minutes followed by sonication for 1 minute. After centrifuging at 1500 x g for 25 minutes, the supernate was dried under nitrogen, dissolved in phosphate-buffered saline (PBS) containing 0.10% bovine serum albumin and stored at -20°C. The pituitary was sonicated or homogenized in PBS and the supernate stored at -20°C. Total soluble protein in pituitary homogenates was determined on the supernate by the method of Lowry et al (19). Radioimmunoassays and Statistical Analysis. LH, FSH, LHRH and testosterone were measured by previously reported radioimmunoassays (20, 21). Statistical analyses were performed using the Student's t-test or analysis of variance followed by the method of least significant difference (22). The Acute Effects of Cysteamine on the Hypothalamic-Pituitary-Gonadal-Axis (HPG). Adult male rats (n = 10/group) were injected subcutaneously (SC) with 300 mg/kg cysteamine or saline. Twenty-four hours later each rat was decapitated and the trunk blood, brain and pituitar~ collected. These samples were prepared as described above and frozen at -20 C until the radioimmunoassays were performed for LH, FSH, T and LHRH. The Effects of Chronic Cysteamine on the HPG. Ten groups of adult male rats (n = i0) were injected SC once daily with saline or 300 mg/kg cysteamine for i, 2, 3, 4 or 7 days. Twenty-four hours following the last injection, these rats were decapitated and the trunk blood brain and pituitary of each rat were ' o collected, prepared as described above and stored at -20 C until radioimmunoassays could be performed. Time Course of Cysteamine Effects on Serum Gonadotropins. To determine the time course with which cysteamine reduces serum LH and FSH concentrations, rats (n = 6-10/group) were injected SC with either saline or 300 mg/kg cysteamine and decapitated at 0.5, i, 2, 4, 8, 24, 72 or 160 hours later. The trunk blood, brains and pituitaries were collected, prepared as described above and frozen at -20°C until LH radioimmunoassays could be performed. Dose Response of Cysteamine on Serum LH. Adult male rats (n = 10/group) were injected SC with saline or 3, 9, 30, 90, 200 or 300 mg/kg cysteamJne and decapitated 2 hours later. Two hours was selected because the results from the time course experiments indicated this to be the earliest point at which a
Vol. 30, No. 3, 1982
Cysteamine Reduces Serum T, LH and FSH
247
maximal reduction occurred in serum LH. The trunk blood was collected and the serum stored -20°C until LH radioimmunoassay could be performed. The Effects of Cysteamine on the Castration Induced Gonadotropin Rise. Male rats age 60 days (n = 10/group) were castrated under ether anesthesia. At the time of the surgery, each animal received either 300 mg/kg cysteamine or saline as a single subcutaneous injection. Twenty-four hours thereafter, each rat was decapitated and the blood, brain and pituitaries collected. The Effects of Cysteamine on LHRH Stimulated LH Secretion. To determine the effects of cysteamine on pituitary function, adult male rats (n = 10/group)were injected SC with 300 mg/kg of cysteamine. Two hours later, 150 ng of LHRH or 0.5 mg/kg naloxone were injected SC and each rat decapitated 15 minutes thereafter. The blood was collected and the sera stored at -20°C until LH radioimmunoassays could be performed.
EXPERIMENT ~ SERUM FSH
EXPERIMENT 2
PITUITARY FSH
PITUITARY FSH
SERUM FSH
~'~ 2°° f
0
t'
~00
I
0~
SERUM LH
SERUM LH
~,oo
10
~ 2o
200
0
I
I
~
0.4
o ~ I
~
PITUITARY LH
"~ ~200 ~f 400600 f~
0
HYPOTHALAMIC LHRH
08
,
t0
O~
SERUM T
ol
0
PITUITARY LH
2o
0
~
HYPOTHALAM~C LHRR
SERUM T
5
0.8
4
ME
HYPOTH
~ 0.4 I
0
[ ] Saline
[ ] Cysteamine FIG. 1
A single subcutaneous injection of cysteamine (300 mg/kg) or saline was administered at i000 hours and the animals were decapitated 24 hours later. The results from two identical experiments are shown. The values are means (Jr SE) and n = 10/group. * = p ~ 0.05; • * = p ~ 0.01. Results Acute Effects. Figure 1 shows the effects of a single subcutaneous injection of cysteamine (300 mg/kg) on hypothalamic LHRH, pituitary gonadotropins and serum LH, FSH and testosterone concentrations of adult male rats measured
248
Cysteamine Reduces Serum T, LH and FSH
Vol.
30, No.
3, 1982
24 hours postinjection. Serum LH, FSH and testosterone concentrations are reduced significantly (p < 0.05) in cysteamine treated animals. Furthermore, pituitary concentrations of LH, but not FSH were reduced significantly (p ~ 0.05). LHRH concentrations in the median eminence and the hypothalamus were not affected significantly. Chronic Effects. Since acute cysteamine treatment did not affect hypothalamic LHRH, a second experiment was conducted to determine if chronic exposure to cysteamine would result in an alteration in hypothalamic LHRH concentrations. As shown in Figure 2, even chronic cysteamine treatment had no significant affect on hypothalamic LHRH concentrations. Serum LH, FSH and testosterone concentrations were decreased 24 hours following a single injection and remained low for the next six days of cysteamine treatment. Although pituitary LH concentrations were reduced significantly (p<_ 0.05) after a single cysteamine injection, pituitary FSH concentrations did not decrease until after three days of treatment.
5°°f SERUM FSH
-~ 300 I PITUITARY FSH
400 I 500
200 "
SERUM LH
30
500
>
zo
~. 400
~
4o
~.~oo t
~,
o
SERUM T
I
o~
0.8
~
6
,~ ~ 0.4
~ ~ \
4
0
~ 0
1 2 5 4
PITUITARY LH I
HYPOTHALAM IC LHRH
2
0
7
0
i
2 3 4 7
DAYS O N CYSTEAMINE FIG. 2 Cysteamine (300 mg/kg) or saline was injected as a single daily subcutaneous administration for I, 2, 3, 4, or 7 days. The values are means (+ SE) and n = 10/group. Numbers at the base of each bar are p v a l u e s as determined by analysis of variance followed by the method of least significant difference (21). Time Course. Serum LH concentrations are reduced significantly (p ~ 0.025) 2 hours following a single injection of cysteamine (Figure 3). The levels remained low for at least 24 hours and had begun to return toward normal by day three. By day 7, serum LH concentrations did not differ significantly from controls but were still slightly reduced.
Vol.
30, No.
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Cysteamine Reduces Serum T, LH and FSH
40
249
Saline a--a Cysleamine
30
-.a 20
t0
_
I
0
I
I
I
I
I
I
I
I
//~
2 4 6 8 HOURS POSTINdECTION
FIG.
I
I
i
I
3
I
I
I
5
I
7
DAYS POSTINdECTION 3
Cysteamlne (300 mg/kg) or saline was injected subcutaneously at 0800 hours and each rat was decapitated at i, 2, 4 or 8 hours and i, 3 or 7 days thereafter. The values are means (+ SE) and n = 6/group. 80% Bo = 80% of total specific binding. * = p < 0.05 and • * = p ~ 0.025. P values were determined by analysis of variance followed by the method of least significant difference (21). Dose Response. Table 1 shows the effects of cysteamine (3-300 mg/kg) on serum LH. Although serum LH concentrations appeared to be reduced at 200 mg/kg cysteamine, the first dose to elicit a significant (p < 0.025) reduction in serum LH was 300 mg/kg (as compared to saline controlsT. TABLE I Serum LH Concentrations:
Cysteamine Dose Response Serum LH ng/ml
Treatment Saline Cysteamine
3 mg/kg 9 30 90 200 300
36.76 30.16 50.12 36.64 33.73 25.36 12.13
+ + + + + + +
7.94 7 48 9 08 7 01 8 24 7 67 1 03*
Adult male rats were injected with saline or cysteamine at 0900 hours and decapitated 2 hours later. Analysis of variance followed by the method of least significant difference was used to determine statistical differences ** = p < 0.025. Blockade of Castration. Castration induces a significant rise in serum LH concentrations by twenty-four hours (20). This postcastratzon rise was observed in control rats (21 ~ 2.8 vs. 100 ~ 3.1), as shown in Figure 4. In rats injected with the higher dose of cysteamine (350 mg/kg) at the time of orchiectomy, this rise in serum LH concentrations was partially blocked
250
Cysteamine
Reduces
Serum T, LH and FSH
Vol.
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3, 1982
(p ! 0.01). At the lower cysteamine dose (35 mg/kg), however, the rise in serum LH was enhanced significantly (p < 0.05). Serum FSH levels were reduced significantly (p ! 0.05) by the 350 mg/kg dose,:: but unaffected by the lower dose. 5
EXPERIMENT t
EXPERIMENT 2
4 3 2
0
2000"i 150
"~:~ 100 I
~/]
..
0
35 350
0
35 350
CYSTEAMIIVE ('rng/kg] FIG.
4
Three groups of male rats (n = 10/group) were castrated under ether anesthesia and simultaneously injected SC with 35 or 350 mg/kg cysteamine or saline. Twenty-four hours later, all rats were decapitated. The values are means (+ SE). The results from two identical experiments are presented. * = p < 0.05; ** = p < 0.01. Serum LH concentrations of intact rats were 21 + 2.8 ng/ml.-Effects of C ~ s t e a m i n e on Naloxone and LHRH Stimulation of LH Secretion. As shown in Figure 5, rats injected with cysteamine (300 mg/kg) had significantly lower (p ! 0.01) serum LH concentrations when measured 2 hours later and those injected with naloxone or LHRH had significantly elevated serum LH concentrations when compared to saline-saline controls. Cysteamine treatment reduced significantly (p ! 0.01) the naloxone and LHRH stimulated rise in serum LH concentrations as compared to the appropriate saline control rats. Discussion The effects of cysteamine on somatostatin concentrations in brain and gut are important because they indicate that cysteamine can reduce significantly the brain content of a neuropeptide that has regulatory actions on the pituitary gland. We were interested in determining whether cysteamine had similar effects on another neuroendocrine system,the h y p o t h a l a m i c - p i t u i t a r y - g o n a d a l axis. The present study was initiated to determine the effects of cysteamine on hypothalamic LHRH concentrations. It is clear from our data that no significant reductions in radioimmunoassayable hypothalamic LHRH occurs with cysteamine
Vol.
30, No.
3, 1982
Cysteamine
Reduces
Serum T. LH and FSH
251
administration, regardless of whether cysteamine is administered acutely or chronically. There are, however, substantial effects on the remainder of the
240
D
200 ~\
..j
Saline Cysteamine
t60 ~120-
4O 0
SALINE
LHRH FIG.
Cysteamine
(300 mg/kg)
NALOXONE
5
or saline was injected
SC into adult male rats
(n = lO/group). Two hours later, LHRH (150 ng), naloxone (0.5 mg/kg) or saline was injected SC and the animals were decapitated 15 minutes thereafter. The values are means (+ SE). * = p ! 0.05; • * = p ! 0.025; *** = p ~ 0.01. hypothalamic-pituitary-gonadal axis. Cysteamine injections reduce concentrations of pituitary and serum gonadotropins and serum testosterone in a time and dose dependent fashion. Significant reductions of serum LH and FSH were observed between one and two hours after a subcutaneous injection of 300 mg/kg cysteamine and remained low for a remarkably long period (3-4 days). Cysteamine reduces serum LH concentrations to a greater extent than FSH. Serum testosterone concentrations are also reduced and appear to be correlated with the reductions in LH concentrations. We studied the effects of cysteamine on stimulated LH and FSH release under three separate conditions; castration, stimulation of the hypothalamus with naloxone and stimulation of the pituitary with LHRH. The castration induced rise in serum LH and FSH was blocked by 300 mg/kg cysteamine, indicating that the stimulated LH release as well as basal LH secretion is inhibited by cysteamine. At a lower dose of cysteamine (35 mg/kg) the castration response was enhanced, indicating that there may be a biphasic response related to the dose. When naloxone, a specific narcotic antagonist, is administered to rats and humans, LH secretion is stimulated, presumably by stimulation of hypothalamic LHRH release. To determine if LHRH could be released in cysteamine treated rats, we injected naloxone and measured the subsequent rise in serum LH concentrations as a bioassay for LHRH secretion. Interestingly, naloxone administration does not stimulate LH secretion in cysteamine treated rats. This effect could be explained by the action of cysteamine on at least two sites, the hypothalamus or the pituitary. Therefore, LHRH was employed to determine the responsiveness of the pituitary of cysteamine treated rats. The results of these experiments indicate that the pituitaries of such rats are less responsive to LHRH. These data suggest that some or all of the inhibition of naloxone stimulated LH secretion could be explained by an action at the level of the pituitary. It is possible, however, that cysteamine is acting at both the pituitary and the hypothalamus to reduce serum gonadotropin concentrations.
252
Cysteamine Reduces Serum T, LH and FSH
Vol. 30, No. 3, 1982
Acknowledgements This work was supported by the Vincent Memorial Research Fund and NIH Grants HD145686 (TMB) and AM26252 (JBM). Post-doctoral fellows were supported by the National Eye Institute (SMS) and the National Institute of Arthritis (WJM). Send reprint requests to Dr. T.M. Badger, Vincent I, Massachusetts General Hospital, Boston, MA 02114. References i. 2. 3. 4. 5. 6. 7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
R. HUXTABLE and R. BRESSLER, Taurine. pg. 45, Raven Press, New York (1976) Z.M. BACQ, Chemical Protection Asainst Ionizing Radiation, p. 16 Charles C. Thomas, Springfield (1965). L.F. PRESCOTT, R.W. NEWTON, C.P. SWANSON, N. WRIGHT, A.P.W. FORREST, H. MATTHEW, Lancet 1 588-592 (1974). L.F. PRESCOTT, J. PARK, G.R. SUTHERLAND, l.J. SMITH AND A.T PROUDFOOT, Lancet 2 109-113 (1976). J.R. MITCHELL, D.J. JOLLOW, W.Z. POTTER, J.R. GILLETTE, B.B. BRODLE, J. Pharm. Exp. Therp. 187 211-217 (1973). A.E.M. MCLEAN and P.A. DAY, Biochem. Pharm. 24 37-42 (1975). J.G. THOENE, R.G. OSHIMA, A.C. CRAWHALL and D.L. OLSON and J.D. SCHENIDER, J. Clin. Invest. 58 180-189 (1976). M. YUDKOFF, J.W. FOREMAN and S. STANTON, N. Eng. J. Med. 304 141-145 (1981). E.P. GIRARDIN, M.S. DEWOLFE, J . E . S . CROCKER, J. P e d i a t r . 94 838-840 (1979). I.P. ROY, A.C. POLLAND, Lancet 2 729-730 (1978). H. SELYE and S.SZABO, Nature 244 458-459 (1973). S. SZABO and E.S. REYNOLDS, Environmental Health Perspectives ii 135140 (1975). S. SZABO, Am. J. Path. 93 273-276 (1978). S. SZABO, L.R. HAITH and S.E. REYNOLDS, Am. J. Dig. Dis. 24 471-477 (1979). S. SZABO, Lancet 1 880-882 (1979). P. KIRKEGAARD, S.S. POULSON, F.B. hOUD, C. HALSE and J. CHRISTIANSEN, Scand. J. Gastroent. 15 621-624 (1980). S. SZABO and S. RECHLIN, Regulatory Peptides 1 (Suppl. i) SIII-I18 (1981). S.M. SAGAR, D. LANDRY, W.J. MILLARD, T.M. BADGER, M.A. ARNOLD and J.B. MARTIN, Am. Acad. Neurol. 33 26 (1981). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-269 (1951). T.M. BADGER, C.E. WILCOX, E.R. MEYER, R.D. BELL AND T.J. CICERO, Endocrinol. 102 136-142 (1978). T.M. BADGER, P.M. ROSENBLUM, R.E. CLEMENT and J.S. LOUGHLIN, Proc. Soc. Exp. Biol. Med. 165 253-259 (1980). G.W. SNEDECOR and W.G. COCHRAN, Statistical Methods. p. 272 Iowa State University Press, Ames (1967).
Pergamon Press
CYSTEAMINE REDUCES SERUM GONADOTROPIN CONCENTRATIONS IN ADULT MALE RATS T.M. Badger, S.M. Sagar, W.J. Millard, J.B. Martin and P. Rosenblum Vincent Memorial Research Laboratories Departments of Obstetrics and Gynecology and Neurology Harvard Medical School and Massachusetts General Hospital Boston, MA 02114. (Received in final form November 24, 1981) Summar X We have examined the effects of cysteamine on the hypothalamicpituitary-gonadal axis of the adult male rat. A single subcutaneous injection of cysteamine (300 mg/kg) reduces significantly (p < 0.05) serum concentrations of LH, FSH and T. Cysteamine blocked LH secretion induced by castration and administration of naloxone and LHRH. Neither acute nor chronic treatment (7 days) altered the hypothalamic LHRH content. These results suggestthat cysteamine acts to reduce pituitary responsiveness to LHRH, resulting in lower mean serum gonadotropin and testosterone concentrations. It is possible, however, that cysteamine acts also at the hypothalamus to reduce LHRH secretion and/ or at the testes to reduce testosterone release. Cysteamine (B-mercaptoethylamine) is a metabolite of coenzyme A and a precursor to hypotaurine. Although taurine biosynthesis is not well understood at present, it appears that in the heart, metabolism of cysteine to taurine occurs via the cysteamine intermediate rather than by the major pathway via cysteine sulfic acid (1). To what extent the cysteamine pathway is utilized in other tissues is unknown. Cysteamine is one of the most potent radioprotective compounds known (2) and has been employed successfully to prevent severe liver damage after paracetamol (tylenol) poisoning (3-6). The ability of cysteamine to reduce cysteine concentrations in cells high in free (non-protein) cysteine has been applied to the treatment of patients with nephropathic cystinosis, an inherited error in cysteine metabolism resulting in free cysteine accumulation in lysosomes and eventual end stage renal failure in the first decade of life
(7-10). In the rat, cysteamine causes a rapid and reproducible onset of duodenal ulcers and has been proposed as a means of producing ulcers for investigations on the mechanisms of duodenal ulcerogenesis and possible means for its prevention (11-16). More recently, Szabo and Reichlin (17) have reported that oral administration of cysteamine produces an acute, partial depletion of somatostatin-like immunoreactivity in the gastrointestinal tract and hypothalamus of rats. We have recently confirmed the somatostatin depleting properties of cysteamine and extended these studies to show that subcutaneous injection of cysteamine reduces somatostatin-like immunoreactivity in various areas of the brain (18). In view of the actions of cysteamine in depleting somatostatin in the central nervous system, other neuroendocrine systems deserve further study. In addition, since cysteamine is used in humans for the treatment of such diseases as cystinosis, the study of the neuroendocrine consequences of its 0024-3205/82/030245-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
246
Cysteamine Reduces Serum T, LH and FSH
Vol.
30, No. 3, 1982
administration in humans is warranted. The present study was conducted therefore, to determine the effects of cysteamine on the hypothalamic-pituitarygonadal axis with particular interest in the concentrations of hypothalamic decapeptide luteinizing hormone releasing hormone (LHRH). Methods Materials. Cysteamine HCI (B-mercaptoethylamine) was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in saline and neutralized with NaOH. Doses are expressed as mg of the HCI salt. Highly purified synthetic LHRH was kindly provided by Drs. J. Rivier and W. Vale (Salk Institute). Anti LHRH and anti-ovine luteinizing hormone (LH) were obtained from Dr. T. Nett (Colarado State University) and Dr. G. Niswender (Colarado State University), respectively The ovine LH for iodinization was obtained from Dr. Leo Reichert (Albany Medical College). All other materials in the radioimmunoassay of LH and follicle stimulating hormone (FSH) were provided by the National Pituitary Agency. The standards used were NIAMDD-RAT-LH-RP-I and NIAMDD RAT FSH-RP-I. Tissue Preparation. In each experiment, trunk blood was collected following o decapitation and the serum stored at -20 C until assayed. The brain was removed immediately after decapitation, placed on ice and the hypothalamus and the median eminence rapidly dissected and placed in cold 2M acetic acid. The samples were then placed in boiling water for two minutes followed by sonication for 1 minute. After centrifuging at 1500 x g for 25 minutes, the supernate was dried under nitrogen, dissolved in phosphate-buffered saline (PBS) containing 0.10% bovine serum albumin and stored at -20°C. The pituitary was sonicated or homogenized in PBS and the supernate stored at -20°C. Total soluble protein in pituitary homogenates was determined on the supernate by the method of Lowry et al (19). Radioimmunoassays and Statistical Analysis. LH, FSH, LHRH and testosterone were measured by previously reported radioimmunoassays (20, 21). Statistical analyses were performed using the Student's t-test or analysis of variance followed by the method of least significant difference (22). The Acute Effects of Cysteamine on the Hypothalamic-Pituitary-Gonadal-Axis (HPG). Adult male rats (n = 10/group) were injected subcutaneously (SC) with 300 mg/kg cysteamine or saline. Twenty-four hours later each rat was decapitated and the trunk blood, brain and pituitar~ collected. These samples were prepared as described above and frozen at -20 C until the radioimmunoassays were performed for LH, FSH, T and LHRH. The Effects of Chronic Cysteamine on the HPG. Ten groups of adult male rats (n = i0) were injected SC once daily with saline or 300 mg/kg cysteamine for i, 2, 3, 4 or 7 days. Twenty-four hours following the last injection, these rats were decapitated and the trunk blood brain and pituitary of each rat were ' o collected, prepared as described above and stored at -20 C until radioimmunoassays could be performed. Time Course of Cysteamine Effects on Serum Gonadotropins. To determine the time course with which cysteamine reduces serum LH and FSH concentrations, rats (n = 6-10/group) were injected SC with either saline or 300 mg/kg cysteamine and decapitated at 0.5, i, 2, 4, 8, 24, 72 or 160 hours later. The trunk blood, brains and pituitaries were collected, prepared as described above and frozen at -20°C until LH radioimmunoassays could be performed. Dose Response of Cysteamine on Serum LH. Adult male rats (n = 10/group) were injected SC with saline or 3, 9, 30, 90, 200 or 300 mg/kg cysteamJne and decapitated 2 hours later. Two hours was selected because the results from the time course experiments indicated this to be the earliest point at which a
Vol. 30, No. 3, 1982
Cysteamine Reduces Serum T, LH and FSH
247
maximal reduction occurred in serum LH. The trunk blood was collected and the serum stored -20°C until LH radioimmunoassay could be performed. The Effects of Cysteamine on the Castration Induced Gonadotropin Rise. Male rats age 60 days (n = 10/group) were castrated under ether anesthesia. At the time of the surgery, each animal received either 300 mg/kg cysteamine or saline as a single subcutaneous injection. Twenty-four hours thereafter, each rat was decapitated and the blood, brain and pituitaries collected. The Effects of Cysteamine on LHRH Stimulated LH Secretion. To determine the effects of cysteamine on pituitary function, adult male rats (n = 10/group)were injected SC with 300 mg/kg of cysteamine. Two hours later, 150 ng of LHRH or 0.5 mg/kg naloxone were injected SC and each rat decapitated 15 minutes thereafter. The blood was collected and the sera stored at -20°C until LH radioimmunoassays could be performed.
EXPERIMENT ~ SERUM FSH
EXPERIMENT 2
PITUITARY FSH
PITUITARY FSH
SERUM FSH
~'~ 2°° f
0
t'
~00
I
0~
SERUM LH
SERUM LH
~,oo
10
~ 2o
200
0
I
I
~
0.4
o ~ I
~
PITUITARY LH
"~ ~200 ~f 400600 f~
0
HYPOTHALAMIC LHRH
08
,
t0
O~
SERUM T
ol
0
PITUITARY LH
2o
0
~
HYPOTHALAM~C LHRR
SERUM T
5
0.8
4
ME
HYPOTH
~ 0.4 I
0
[ ] Saline
[ ] Cysteamine FIG. 1
A single subcutaneous injection of cysteamine (300 mg/kg) or saline was administered at i000 hours and the animals were decapitated 24 hours later. The results from two identical experiments are shown. The values are means (Jr SE) and n = 10/group. * = p ~ 0.05; • * = p ~ 0.01. Results Acute Effects. Figure 1 shows the effects of a single subcutaneous injection of cysteamine (300 mg/kg) on hypothalamic LHRH, pituitary gonadotropins and serum LH, FSH and testosterone concentrations of adult male rats measured
248
Cysteamine Reduces Serum T, LH and FSH
Vol.
30, No.
3, 1982
24 hours postinjection. Serum LH, FSH and testosterone concentrations are reduced significantly (p < 0.05) in cysteamine treated animals. Furthermore, pituitary concentrations of LH, but not FSH were reduced significantly (p ~ 0.05). LHRH concentrations in the median eminence and the hypothalamus were not affected significantly. Chronic Effects. Since acute cysteamine treatment did not affect hypothalamic LHRH, a second experiment was conducted to determine if chronic exposure to cysteamine would result in an alteration in hypothalamic LHRH concentrations. As shown in Figure 2, even chronic cysteamine treatment had no significant affect on hypothalamic LHRH concentrations. Serum LH, FSH and testosterone concentrations were decreased 24 hours following a single injection and remained low for the next six days of cysteamine treatment. Although pituitary LH concentrations were reduced significantly (p<_ 0.05) after a single cysteamine injection, pituitary FSH concentrations did not decrease until after three days of treatment.
5°°f SERUM FSH
-~ 300 I PITUITARY FSH
400 I 500
200 "
SERUM LH
30
500
>
zo
~. 400
~
4o
~.~oo t
~,
o
SERUM T
I
o~
0.8
~
6
,~ ~ 0.4
~ ~ \
4
0
~ 0
1 2 5 4
PITUITARY LH I
HYPOTHALAM IC LHRH
2
0
7
0
i
2 3 4 7
DAYS O N CYSTEAMINE FIG. 2 Cysteamine (300 mg/kg) or saline was injected as a single daily subcutaneous administration for I, 2, 3, 4, or 7 days. The values are means (+ SE) and n = 10/group. Numbers at the base of each bar are p v a l u e s as determined by analysis of variance followed by the method of least significant difference (21). Time Course. Serum LH concentrations are reduced significantly (p ~ 0.025) 2 hours following a single injection of cysteamine (Figure 3). The levels remained low for at least 24 hours and had begun to return toward normal by day three. By day 7, serum LH concentrations did not differ significantly from controls but were still slightly reduced.
Vol.
30, No.
3, 1982
Cysteamine Reduces Serum T, LH and FSH
40
249
Saline a--a Cysleamine
30
-.a 20
t0
_
I
0
I
I
I
I
I
I
I
I
//~
2 4 6 8 HOURS POSTINdECTION
FIG.
I
I
i
I
3
I
I
I
5
I
7
DAYS POSTINdECTION 3
Cysteamlne (300 mg/kg) or saline was injected subcutaneously at 0800 hours and each rat was decapitated at i, 2, 4 or 8 hours and i, 3 or 7 days thereafter. The values are means (+ SE) and n = 6/group. 80% Bo = 80% of total specific binding. * = p < 0.05 and • * = p ~ 0.025. P values were determined by analysis of variance followed by the method of least significant difference (21). Dose Response. Table 1 shows the effects of cysteamine (3-300 mg/kg) on serum LH. Although serum LH concentrations appeared to be reduced at 200 mg/kg cysteamine, the first dose to elicit a significant (p < 0.025) reduction in serum LH was 300 mg/kg (as compared to saline controlsT. TABLE I Serum LH Concentrations:
Cysteamine Dose Response Serum LH ng/ml
Treatment Saline Cysteamine
3 mg/kg 9 30 90 200 300
36.76 30.16 50.12 36.64 33.73 25.36 12.13
+ + + + + + +
7.94 7 48 9 08 7 01 8 24 7 67 1 03*
Adult male rats were injected with saline or cysteamine at 0900 hours and decapitated 2 hours later. Analysis of variance followed by the method of least significant difference was used to determine statistical differences ** = p < 0.025. Blockade of Castration. Castration induces a significant rise in serum LH concentrations by twenty-four hours (20). This postcastratzon rise was observed in control rats (21 ~ 2.8 vs. 100 ~ 3.1), as shown in Figure 4. In rats injected with the higher dose of cysteamine (350 mg/kg) at the time of orchiectomy, this rise in serum LH concentrations was partially blocked
250
Cysteamine
Reduces
Serum T, LH and FSH
Vol.
30, No.
3, 1982
(p ! 0.01). At the lower cysteamine dose (35 mg/kg), however, the rise in serum LH was enhanced significantly (p < 0.05). Serum FSH levels were reduced significantly (p ! 0.05) by the 350 mg/kg dose,:: but unaffected by the lower dose. 5
EXPERIMENT t
EXPERIMENT 2
4 3 2
0
2000"i 150
"~:~ 100 I
~/]
..
0
35 350
0
35 350
CYSTEAMIIVE ('rng/kg] FIG.
4
Three groups of male rats (n = 10/group) were castrated under ether anesthesia and simultaneously injected SC with 35 or 350 mg/kg cysteamine or saline. Twenty-four hours later, all rats were decapitated. The values are means (+ SE). The results from two identical experiments are presented. * = p < 0.05; ** = p < 0.01. Serum LH concentrations of intact rats were 21 + 2.8 ng/ml.-Effects of C ~ s t e a m i n e on Naloxone and LHRH Stimulation of LH Secretion. As shown in Figure 5, rats injected with cysteamine (300 mg/kg) had significantly lower (p ! 0.01) serum LH concentrations when measured 2 hours later and those injected with naloxone or LHRH had significantly elevated serum LH concentrations when compared to saline-saline controls. Cysteamine treatment reduced significantly (p ! 0.01) the naloxone and LHRH stimulated rise in serum LH concentrations as compared to the appropriate saline control rats. Discussion The effects of cysteamine on somatostatin concentrations in brain and gut are important because they indicate that cysteamine can reduce significantly the brain content of a neuropeptide that has regulatory actions on the pituitary gland. We were interested in determining whether cysteamine had similar effects on another neuroendocrine system,the h y p o t h a l a m i c - p i t u i t a r y - g o n a d a l axis. The present study was initiated to determine the effects of cysteamine on hypothalamic LHRH concentrations. It is clear from our data that no significant reductions in radioimmunoassayable hypothalamic LHRH occurs with cysteamine
Vol.
30, No.
3, 1982
Cysteamine
Reduces
Serum T. LH and FSH
251
administration, regardless of whether cysteamine is administered acutely or chronically. There are, however, substantial effects on the remainder of the
240
D
200 ~\
..j
Saline Cysteamine
t60 ~120-
4O 0
SALINE
LHRH FIG.
Cysteamine
(300 mg/kg)
NALOXONE
5
or saline was injected
SC into adult male rats
(n = lO/group). Two hours later, LHRH (150 ng), naloxone (0.5 mg/kg) or saline was injected SC and the animals were decapitated 15 minutes thereafter. The values are means (+ SE). * = p ! 0.05; • * = p ! 0.025; *** = p ~ 0.01. hypothalamic-pituitary-gonadal axis. Cysteamine injections reduce concentrations of pituitary and serum gonadotropins and serum testosterone in a time and dose dependent fashion. Significant reductions of serum LH and FSH were observed between one and two hours after a subcutaneous injection of 300 mg/kg cysteamine and remained low for a remarkably long period (3-4 days). Cysteamine reduces serum LH concentrations to a greater extent than FSH. Serum testosterone concentrations are also reduced and appear to be correlated with the reductions in LH concentrations. We studied the effects of cysteamine on stimulated LH and FSH release under three separate conditions; castration, stimulation of the hypothalamus with naloxone and stimulation of the pituitary with LHRH. The castration induced rise in serum LH and FSH was blocked by 300 mg/kg cysteamine, indicating that the stimulated LH release as well as basal LH secretion is inhibited by cysteamine. At a lower dose of cysteamine (35 mg/kg) the castration response was enhanced, indicating that there may be a biphasic response related to the dose. When naloxone, a specific narcotic antagonist, is administered to rats and humans, LH secretion is stimulated, presumably by stimulation of hypothalamic LHRH release. To determine if LHRH could be released in cysteamine treated rats, we injected naloxone and measured the subsequent rise in serum LH concentrations as a bioassay for LHRH secretion. Interestingly, naloxone administration does not stimulate LH secretion in cysteamine treated rats. This effect could be explained by the action of cysteamine on at least two sites, the hypothalamus or the pituitary. Therefore, LHRH was employed to determine the responsiveness of the pituitary of cysteamine treated rats. The results of these experiments indicate that the pituitaries of such rats are less responsive to LHRH. These data suggest that some or all of the inhibition of naloxone stimulated LH secretion could be explained by an action at the level of the pituitary. It is possible, however, that cysteamine is acting at both the pituitary and the hypothalamus to reduce serum gonadotropin concentrations.
252
Cysteamine Reduces Serum T, LH and FSH
Vol. 30, No. 3, 1982
Acknowledgements This work was supported by the Vincent Memorial Research Fund and NIH Grants HD145686 (TMB) and AM26252 (JBM). Post-doctoral fellows were supported by the National Eye Institute (SMS) and the National Institute of Arthritis (WJM). Send reprint requests to Dr. T.M. Badger, Vincent I, Massachusetts General Hospital, Boston, MA 02114. References i. 2. 3. 4. 5. 6. 7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
R. HUXTABLE and R. BRESSLER, Taurine. pg. 45, Raven Press, New York (1976) Z.M. BACQ, Chemical Protection Asainst Ionizing Radiation, p. 16 Charles C. Thomas, Springfield (1965). L.F. PRESCOTT, R.W. NEWTON, C.P. SWANSON, N. WRIGHT, A.P.W. FORREST, H. MATTHEW, Lancet 1 588-592 (1974). L.F. PRESCOTT, J. PARK, G.R. SUTHERLAND, l.J. SMITH AND A.T PROUDFOOT, Lancet 2 109-113 (1976). J.R. MITCHELL, D.J. JOLLOW, W.Z. POTTER, J.R. GILLETTE, B.B. BRODLE, J. Pharm. Exp. Therp. 187 211-217 (1973). A.E.M. MCLEAN and P.A. DAY, Biochem. Pharm. 24 37-42 (1975). J.G. THOENE, R.G. OSHIMA, A.C. CRAWHALL and D.L. OLSON and J.D. SCHENIDER, J. Clin. Invest. 58 180-189 (1976). M. YUDKOFF, J.W. FOREMAN and S. STANTON, N. Eng. J. Med. 304 141-145 (1981). E.P. GIRARDIN, M.S. DEWOLFE, J . E . S . CROCKER, J. P e d i a t r . 94 838-840 (1979). I.P. ROY, A.C. POLLAND, Lancet 2 729-730 (1978). H. SELYE and S.SZABO, Nature 244 458-459 (1973). S. SZABO and E.S. REYNOLDS, Environmental Health Perspectives ii 135140 (1975). S. SZABO, Am. J. Path. 93 273-276 (1978). S. SZABO, L.R. HAITH and S.E. REYNOLDS, Am. J. Dig. Dis. 24 471-477 (1979). S. SZABO, Lancet 1 880-882 (1979). P. KIRKEGAARD, S.S. POULSON, F.B. hOUD, C. HALSE and J. CHRISTIANSEN, Scand. J. Gastroent. 15 621-624 (1980). S. SZABO and S. RECHLIN, Regulatory Peptides 1 (Suppl. i) SIII-I18 (1981). S.M. SAGAR, D. LANDRY, W.J. MILLARD, T.M. BADGER, M.A. ARNOLD and J.B. MARTIN, Am. Acad. Neurol. 33 26 (1981). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-269 (1951). T.M. BADGER, C.E. WILCOX, E.R. MEYER, R.D. BELL AND T.J. CICERO, Endocrinol. 102 136-142 (1978). T.M. BADGER, P.M. ROSENBLUM, R.E. CLEMENT and J.S. LOUGHLIN, Proc. Soc. Exp. Biol. Med. 165 253-259 (1980). G.W. SNEDECOR and W.G. COCHRAN, Statistical Methods. p. 272 Iowa State University Press, Ames (1967).