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Effect of α-methyl-p-tyrosine on the circadian variation of plasma corticosterone in rats
EUROPEAN JOURNAL OF PHARMACOLOGY 11 (1970) 266-268. NORTH-HOLLANDPUBLISHINGCOMPANY Short communication
EFFECT OF ot-METHYL-p-TYROSINE ON THE CIRCADIAN VARIATION OF PLASMA CORTICOSTERONE
IN RATS a
U. SCAPAGNINI b, G.R. VAN LOON c, G.P. MOBERG d and W.F. GANONG Departments of Physiology and Anatomy, School of Medicine, University of California, San Francisco, California 94122, USA
Received 25 March 1970
Accepted 16 June 1970
U. SCAPAGNINI, G.R. VAN LOON, G.P. MOBERG and W.F. GANONG, Effect of Ot-methyl-p-tyrosine on the circadian variation of plasma corticosterone in rats European J. Pharmacol. 11 (1970) 266-268. Alpha-methyl-p-tyrosine (a-MT) increased plasma corticosterone in most rats tested, and at all tunes of day. Hypothalamic catecholamine content fell in rats in which O~MTincreased plasma corticosterone, but not in rats m which it did not. The data are consistent with the hypothesis that an adrenergic neural system inhibits ACTH secretion. Corticosterone Orcadmn
Hypothalamic Ot-Methyl-p-tyrosine
1. INTRODUCTION Some authors have reported that they can find no correlation between brain catecholamines and ACTH secretion (Smelik, 1967; Carr and Moore, 1968), while others have suggested that catecholamines stimulate ACTH secretion (Endr~czi et al., 1963;Krieger and Krieger, 1965; Naumenko, 1968). More recently, evidence has been presented for a central adrenergic neura/ system that inhibits ACTH secretion (Van Loon et al., 1969; Van Loon and Ganong, 1969; Van Loon et al., 1970; Bhattacharya and Marks, 1969). Circadian variation in plasma adrenocortical steroids is well established (Critchlow, 1963) and a a Supported by USPHS Grant AM06704 and Brooks Fund. b Lecturer in Anatomy; formerly NATO Postdoctoral Research Fellow from Department of Pharmacology (II Chair), Umverslty of Naples, Italy. c Bay Area Heart Research Committee Fellow. d USPHS Postdoctoral Research Fellow of the National Institute of Neurological Diseases and Stroke; supported by Fellowship 9 FO2 NS40741.
Norepmephrlne Dopamme
24 hr rhythm in brain catecholamine content has also been reported (Reis and Wurtman, 1968). In this study, we have investigated the effect of a-MT, a drug which inhibits catecholamine synthesis, on the diurnal fluctuation in plasma corticosterone in the rat.
2. MATERIALS AND METHODS Male Sprague-Dawley rats (Berkeley Pacific Co., Berkeley, California) weighing 200-250 g were caged in pairs in a room at 22 -+ I°C with automatic light control that provided 14 hr light (0600-2000 Pacific Standard Time) and 10 hr dark each day. Food and water were given ad libitum. The methyl ester of a-MT (Corrodi et al., 1966), 250mg/kg of body weight was administered intraperitoneally in water, 0.5 ml[100 g of body weight, 9 hours prior to killing. Control animals were injected i.p. with water. The animals were killed by decapitation at 0400, 0800, 1200, 1600, 2000, and 2400 hours. Trunk blood was
U.Scapagnmi et aL, ~rain catecholamines and plasma cortwosterone collected and centrifuged and the plasma promptly frozen. Brains were removed and placed on dry ice. The hypothalamus was quickly dissected out of each, weighed, homogenized m 5% trlchloroacetic acid and frozen at - 4 ° C for subsequent analysis of catecholamine content. Plasma corticosterone was estimated by the method of Givner and Rochefort (1965). Norepinephrine and dopamine were estimated fluorometrically by a slight modification (Weiner, 1969) of the method of Udenfriend and Zaltsman-Nirenberg (1963). Catecholamine analyses were carried out on pools of 2 or 3 hypothalami. For purposes of analysis, the plasma corticosterone levels were first de ~mined and then the hypothalami of selected rats x th plasma corticosterone values higher than the highest control values were analyzed separately from those of rats with plasma corticosterone values in the normal range. The data were analyzed statistically b y Student's t test.
267
3. RESULTS Alpha-MT produced a mean overall increase in plasma corticosterone 9 hr after injection at the six times of day examined (table 1). The greatest increment in plasma corticosterone after a-MT was seen at 0800 hours and the smallest at 2000 hours. Most of the individual animals injected with a-MT showed an increase in plasma corticosterone, b u t some did not. This was a consistent finding in each of three separate experiments. Hypothalamic norepinephrine and dopamine contents were low in the rats which responded to a-MT with an increase in plasma corticosterone, b u t were normal in those which failed to show an increase (table 2). All the animals appeared to be in good health, and there were no obvious changes in behavior.
Table 1 Effect of 0t-MT on the circadian variation of plasma cortlcosterone. Values are/zg/100 ml, mean + standard error of all animals tested. Figures m parentheses are numbers of determinations. Time of day (hr) Treatment 0800 Cont, ol &-MT
1200
1600
2000
2400
2.6 + 0.2 (35)
4.7 +1.7 (9)
10.7 + 1.4 * (8)
25.5 _+2.7 * (12)
13 5 + 1.8 * (8)
40.7 + 4.2 (46)
33.4 + 5.7 (8)
28.9 + 3.6 * (10)
35.1 _+2.3 (15)
27.8 + 3.1 * (16)
0400 6.1 + 1.3 * (8) 42.6 + 5.3 (8)
* p<0.01 as compared to 0800 Each t~-MTvalue is statistically different from the corresponding control value at p < 0.01.
Table 2 Correlation between plasma corticosterone and hypothalamlc contents of norepinephrine and dopamine at 0800 hr after t~-methyl-p-tyrosine methyl ester, 250 mg/kg i.p. 9 hr earher. Values are means + standard errors. Plasma
Hypothalamlc contents (t~g/g)
Cortlcosterone (ug/100 ml)
Norepmephrine
Dopamlne
51.6 + 1.2 *** 2.9 _+0.4 3.9 + 0.2
0.56 __.0.15 *** 2.01 + 0.28 1.95 + 0.10
0.08 + 0.02 *** 0.39 + 0.02 0.37 + 0.02
Treatment
o~-methyl-p-tyrosme * (n = 18) ot-methyl-p-tyrosine ** (n = 10) Control (n = 25)
* Ammals in which plasma corticosterone was above all control values. Animals in which plasma corticosterone was lower than the htghest control. *** p < 0.01 compared to control. **
268
U.Scapagntm et al , Brain catecholamines and plasma corttcosterone
4. DISCUSSION Alpha-MT produced an increase in plasma corticosterone at all times of day examined. It is apparent that most but not all the animals responded to a-MT with an increase in plasma corticosterone, and that in those animals in which corticosterone was not increased, t~-MT did not deplete hypothalamic catecholamines. The cause of this failure to produce depletion in some animals is unknown, but several possibilities should be considered. These include injection of the drug into intestine rather than intraperltoneally, more rapid excretion of the drug by some animals, poor absorption from the injection site, decreased uptake across the blood-brain barrier, decreased uptake at neuronal cell membrane, etc. However, the cause of this two-population effect of c(-MT on plasma corticosterone is not relevant for the present study. The drug merely provided a tool which allowed us to demonstrate an inverse relationship between hypothalamic content of catecholamines and plasma cortlcosterone m the rat after administration of a-MT. The increase in corticosterone could be explained by removal of a.MT of a tonic inhibitory influence of brain catecholamines on the hypothalamo-pltuitaryadrenal axis, and is in keeping with our previous hypothesis of an adrenergic neural system that inhibits ACTH secretion (Van Loon et al., 1969). Other investigators have also reported an increase an plasma cortlcosterolds after t~-MT (Carr and Moore, 1968, Krieger and Rizzo, 1969), and some have related this effect to possible stress of peritoneal irritation by the insoluble drug (Carr and Moore, 1968). In the present study, the soluble form of a-MT was used to circumvent th~s problem. Furthermore, repeated injections of th~s soluble form of a-MT in dogs caused an increase m both morning and afternoon levels of plasma 17-hydroxycorhcosteroids (Van Loon and Genong, unpublished). On the other hand, chronic treatment with a-MT failed to increase adrenal weight m rats (Wemer, Kragt and Genong, unpublished).
REFERENCES Bhattacharya, A.N. and B.H. Marks, 1969, Reserpine- and chloropromazme-mduced changes In hypothalamo-
hypophyseal-adrenax system m rats in the presence and absence of hypothermia, J. Pharmacol. Exptl. Therap. 165, 108. Carr, L.A. and K E. Moore, 1968, Effects of reserpine and 0emethyltyrosme on brain catecholamlnes and the pituitary-adrenal response to stress, Neuroendocrinology 3,285. Corrodl, H., K. Fuxe and T. H~Skfelt, 1966, Refillment of the catecholamlne stores with 3,4-dihydroxyphenylalanine after depletion reduced by inhibition of tyrostnehydroxylase, Life ScL 5,605. Crltchlow, V , 1963, The role of light m the neuroendocrIne system, m: Advances in Neuroendocrlnology, ed A V. Nalbandov (University of Illinois Press, Urbana) p. 377. Endr6czl, E , G. Schrelberg and K Llssak, 1963, The role of central nervous actwating and inhibitory structures in the control of pitultary-adrenocortlcal function Effect of intracerebral chohnergic and adrenergic stimulation, Acta Physiol. Acad. Scl. Hung. 24, 211 Gwner, M. and J. Rochefort, 1965, An improved assay of cortlcosterone in rat serum and adrenal tissue, Steroids 6, 486. Krieger, D.T and H.P Krleger, 1965, The effects of intrahypothalamic rejection of drugs on ACTH release In cat, Proc. Intern. Congres. Endocrmol, 2nd ed. (London, 1964) part I, p. 640. Krleger, D.T. and F. Rlzzo, 1969, Serotonm mediation of circadian periodicity of plasma 17-hydroxycortlcosterolds, Am. J. Physiol. 217, 1703. Naumenko, E.F., 1968, Hypothalamic chemoreactwe structures and the regulation of the pituitary-adrenal function. Effect of local injection of norepmephrlne, carbachol and serotonm into the brain of guinea pigs w~th intact brain and after mesencephalic transection, Brmn Res. 11, 1 Rels, D.J. and R.J. Wurtman, 1968, Diurnal changes m brain noradrenahne, Life Sci. 7, 91. Smelik, P.G., 1967, ACTH secretion after depletion of hypothalamlc monoamles by reserpine implants, Neuroendocrinology 2,267. Van Loon, G.R., L. Hflger, R. Cohen and W.F Ganong, Evidence for a hypotbalamlc adrenerglc system that inhibits ACTH secretion m the dog, Federation Proc. 28, 438. Van Loon, G.R. and W.F Ganong, 1969, Effect of drugs which alter catecholamine metabolism on the inhibition of stress-reduced ACTH secretion produced by L-dopa, Physiologist 12,381 Van Loon, G R., U. Scapagnini, G.P. Moberg and W.F. Ganong, 1970, Evidence for adrenergic inhibition of ACTH secretion in the rat, Federation Proc., in press Udenfnend, S. and P. Zaltsman-Ntrenberg, 1963, Norepinephrme and 3,4-hydroxyphenethylamIne turnover m guinea pig brain m wvo, Science 142, 394. Welner, R.I., 1969, Relationship of brain catecholammes to gonadotropin secretion and the onset of puberty, Ph D. Thesis, Umverslty of Cahfornm, San Francisco
EFFECT OF ot-METHYL-p-TYROSINE ON THE CIRCADIAN VARIATION OF PLASMA CORTICOSTERONE
IN RATS a
U. SCAPAGNINI b, G.R. VAN LOON c, G.P. MOBERG d and W.F. GANONG Departments of Physiology and Anatomy, School of Medicine, University of California, San Francisco, California 94122, USA
Received 25 March 1970
Accepted 16 June 1970
U. SCAPAGNINI, G.R. VAN LOON, G.P. MOBERG and W.F. GANONG, Effect of Ot-methyl-p-tyrosine on the circadian variation of plasma corticosterone in rats European J. Pharmacol. 11 (1970) 266-268. Alpha-methyl-p-tyrosine (a-MT) increased plasma corticosterone in most rats tested, and at all tunes of day. Hypothalamic catecholamine content fell in rats in which O~MTincreased plasma corticosterone, but not in rats m which it did not. The data are consistent with the hypothesis that an adrenergic neural system inhibits ACTH secretion. Corticosterone Orcadmn
Hypothalamic Ot-Methyl-p-tyrosine
1. INTRODUCTION Some authors have reported that they can find no correlation between brain catecholamines and ACTH secretion (Smelik, 1967; Carr and Moore, 1968), while others have suggested that catecholamines stimulate ACTH secretion (Endr~czi et al., 1963;Krieger and Krieger, 1965; Naumenko, 1968). More recently, evidence has been presented for a central adrenergic neura/ system that inhibits ACTH secretion (Van Loon et al., 1969; Van Loon and Ganong, 1969; Van Loon et al., 1970; Bhattacharya and Marks, 1969). Circadian variation in plasma adrenocortical steroids is well established (Critchlow, 1963) and a a Supported by USPHS Grant AM06704 and Brooks Fund. b Lecturer in Anatomy; formerly NATO Postdoctoral Research Fellow from Department of Pharmacology (II Chair), Umverslty of Naples, Italy. c Bay Area Heart Research Committee Fellow. d USPHS Postdoctoral Research Fellow of the National Institute of Neurological Diseases and Stroke; supported by Fellowship 9 FO2 NS40741.
Norepmephrlne Dopamme
24 hr rhythm in brain catecholamine content has also been reported (Reis and Wurtman, 1968). In this study, we have investigated the effect of a-MT, a drug which inhibits catecholamine synthesis, on the diurnal fluctuation in plasma corticosterone in the rat.
2. MATERIALS AND METHODS Male Sprague-Dawley rats (Berkeley Pacific Co., Berkeley, California) weighing 200-250 g were caged in pairs in a room at 22 -+ I°C with automatic light control that provided 14 hr light (0600-2000 Pacific Standard Time) and 10 hr dark each day. Food and water were given ad libitum. The methyl ester of a-MT (Corrodi et al., 1966), 250mg/kg of body weight was administered intraperitoneally in water, 0.5 ml[100 g of body weight, 9 hours prior to killing. Control animals were injected i.p. with water. The animals were killed by decapitation at 0400, 0800, 1200, 1600, 2000, and 2400 hours. Trunk blood was
U.Scapagnmi et aL, ~rain catecholamines and plasma cortwosterone collected and centrifuged and the plasma promptly frozen. Brains were removed and placed on dry ice. The hypothalamus was quickly dissected out of each, weighed, homogenized m 5% trlchloroacetic acid and frozen at - 4 ° C for subsequent analysis of catecholamine content. Plasma corticosterone was estimated by the method of Givner and Rochefort (1965). Norepinephrine and dopamine were estimated fluorometrically by a slight modification (Weiner, 1969) of the method of Udenfriend and Zaltsman-Nirenberg (1963). Catecholamine analyses were carried out on pools of 2 or 3 hypothalami. For purposes of analysis, the plasma corticosterone levels were first de ~mined and then the hypothalami of selected rats x th plasma corticosterone values higher than the highest control values were analyzed separately from those of rats with plasma corticosterone values in the normal range. The data were analyzed statistically b y Student's t test.
267
3. RESULTS Alpha-MT produced a mean overall increase in plasma corticosterone 9 hr after injection at the six times of day examined (table 1). The greatest increment in plasma corticosterone after a-MT was seen at 0800 hours and the smallest at 2000 hours. Most of the individual animals injected with a-MT showed an increase in plasma corticosterone, b u t some did not. This was a consistent finding in each of three separate experiments. Hypothalamic norepinephrine and dopamine contents were low in the rats which responded to a-MT with an increase in plasma corticosterone, b u t were normal in those which failed to show an increase (table 2). All the animals appeared to be in good health, and there were no obvious changes in behavior.
Table 1 Effect of 0t-MT on the circadian variation of plasma cortlcosterone. Values are/zg/100 ml, mean + standard error of all animals tested. Figures m parentheses are numbers of determinations. Time of day (hr) Treatment 0800 Cont, ol &-MT
1200
1600
2000
2400
2.6 + 0.2 (35)
4.7 +1.7 (9)
10.7 + 1.4 * (8)
25.5 _+2.7 * (12)
13 5 + 1.8 * (8)
40.7 + 4.2 (46)
33.4 + 5.7 (8)
28.9 + 3.6 * (10)
35.1 _+2.3 (15)
27.8 + 3.1 * (16)
0400 6.1 + 1.3 * (8) 42.6 + 5.3 (8)
* p<0.01 as compared to 0800 Each t~-MTvalue is statistically different from the corresponding control value at p < 0.01.
Table 2 Correlation between plasma corticosterone and hypothalamlc contents of norepinephrine and dopamine at 0800 hr after t~-methyl-p-tyrosine methyl ester, 250 mg/kg i.p. 9 hr earher. Values are means + standard errors. Plasma
Hypothalamlc contents (t~g/g)
Cortlcosterone (ug/100 ml)
Norepmephrine
Dopamlne
51.6 + 1.2 *** 2.9 _+0.4 3.9 + 0.2
0.56 __.0.15 *** 2.01 + 0.28 1.95 + 0.10
0.08 + 0.02 *** 0.39 + 0.02 0.37 + 0.02
Treatment
o~-methyl-p-tyrosme * (n = 18) ot-methyl-p-tyrosine ** (n = 10) Control (n = 25)
* Ammals in which plasma corticosterone was above all control values. Animals in which plasma corticosterone was lower than the htghest control. *** p < 0.01 compared to control. **
268
U.Scapagntm et al , Brain catecholamines and plasma corttcosterone
4. DISCUSSION Alpha-MT produced an increase in plasma corticosterone at all times of day examined. It is apparent that most but not all the animals responded to a-MT with an increase in plasma corticosterone, and that in those animals in which corticosterone was not increased, t~-MT did not deplete hypothalamic catecholamines. The cause of this failure to produce depletion in some animals is unknown, but several possibilities should be considered. These include injection of the drug into intestine rather than intraperltoneally, more rapid excretion of the drug by some animals, poor absorption from the injection site, decreased uptake across the blood-brain barrier, decreased uptake at neuronal cell membrane, etc. However, the cause of this two-population effect of c(-MT on plasma corticosterone is not relevant for the present study. The drug merely provided a tool which allowed us to demonstrate an inverse relationship between hypothalamic content of catecholamines and plasma cortlcosterone m the rat after administration of a-MT. The increase in corticosterone could be explained by removal of a.MT of a tonic inhibitory influence of brain catecholamines on the hypothalamo-pltuitaryadrenal axis, and is in keeping with our previous hypothesis of an adrenergic neural system that inhibits ACTH secretion (Van Loon et al., 1969). Other investigators have also reported an increase an plasma cortlcosterolds after t~-MT (Carr and Moore, 1968, Krieger and Rizzo, 1969), and some have related this effect to possible stress of peritoneal irritation by the insoluble drug (Carr and Moore, 1968). In the present study, the soluble form of a-MT was used to circumvent th~s problem. Furthermore, repeated injections of th~s soluble form of a-MT in dogs caused an increase m both morning and afternoon levels of plasma 17-hydroxycorhcosteroids (Van Loon and Genong, unpublished). On the other hand, chronic treatment with a-MT failed to increase adrenal weight m rats (Wemer, Kragt and Genong, unpublished).
REFERENCES Bhattacharya, A.N. and B.H. Marks, 1969, Reserpine- and chloropromazme-mduced changes In hypothalamo-
hypophyseal-adrenax system m rats in the presence and absence of hypothermia, J. Pharmacol. Exptl. Therap. 165, 108. Carr, L.A. and K E. Moore, 1968, Effects of reserpine and 0emethyltyrosme on brain catecholamlnes and the pituitary-adrenal response to stress, Neuroendocrinology 3,285. Corrodl, H., K. Fuxe and T. H~Skfelt, 1966, Refillment of the catecholamlne stores with 3,4-dihydroxyphenylalanine after depletion reduced by inhibition of tyrostnehydroxylase, Life ScL 5,605. Crltchlow, V , 1963, The role of light m the neuroendocrIne system, m: Advances in Neuroendocrlnology, ed A V. Nalbandov (University of Illinois Press, Urbana) p. 377. Endr6czl, E , G. Schrelberg and K Llssak, 1963, The role of central nervous actwating and inhibitory structures in the control of pitultary-adrenocortlcal function Effect of intracerebral chohnergic and adrenergic stimulation, Acta Physiol. Acad. Scl. Hung. 24, 211 Gwner, M. and J. Rochefort, 1965, An improved assay of cortlcosterone in rat serum and adrenal tissue, Steroids 6, 486. Krieger, D.T and H.P Krleger, 1965, The effects of intrahypothalamic rejection of drugs on ACTH release In cat, Proc. Intern. Congres. Endocrmol, 2nd ed. (London, 1964) part I, p. 640. Krleger, D.T. and F. Rlzzo, 1969, Serotonm mediation of circadian periodicity of plasma 17-hydroxycortlcosterolds, Am. J. Physiol. 217, 1703. Naumenko, E.F., 1968, Hypothalamic chemoreactwe structures and the regulation of the pituitary-adrenal function. Effect of local injection of norepmephrlne, carbachol and serotonm into the brain of guinea pigs w~th intact brain and after mesencephalic transection, Brmn Res. 11, 1 Rels, D.J. and R.J. Wurtman, 1968, Diurnal changes m brain noradrenahne, Life Sci. 7, 91. Smelik, P.G., 1967, ACTH secretion after depletion of hypothalamlc monoamles by reserpine implants, Neuroendocrinology 2,267. Van Loon, G.R., L. Hflger, R. Cohen and W.F Ganong, Evidence for a hypotbalamlc adrenerglc system that inhibits ACTH secretion m the dog, Federation Proc. 28, 438. Van Loon, G.R. and W.F Ganong, 1969, Effect of drugs which alter catecholamine metabolism on the inhibition of stress-reduced ACTH secretion produced by L-dopa, Physiologist 12,381 Van Loon, G R., U. Scapagnini, G.P. Moberg and W.F. Ganong, 1970, Evidence for adrenergic inhibition of ACTH secretion in the rat, Federation Proc., in press Udenfnend, S. and P. Zaltsman-Ntrenberg, 1963, Norepinephrme and 3,4-hydroxyphenethylamIne turnover m guinea pig brain m wvo, Science 142, 394. Welner, R.I., 1969, Relationship of brain catecholammes to gonadotropin secretion and the onset of puberty, Ph D. Thesis, Umverslty of Cahfornm, San Francisco