European Journal of Pharmacology, 41 (1977) 291--299 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
291
EFFECT OF DEXAMETHASONE ON MONOAMINE OXIDASE INHIBITION BY IPRONIAZID IN RAT BRAIN JOHN W. VEALS, CHRYZANTA A. KORDUBA and SAMSON SYMCHOWICZ
Schering Corporation, Bloomfield, New Jersey, U.S.A. Received 4 December 1975, revised MS received 23 April 1976, accepted 7 October 1976
J.W. VEALS, C.A. KORDUBA and S. SYMCHOWICZ, Effect of dexamethasone on monoamine oxidase inhibition by iproniazid in rat brain, European J. Pharmacol. 41 (1977) 291--299. Chronic (6 days) dexamethasone administration caused a slight decrease of rat brain MAO enzyme activity which was reflected by lower levels of 14C-homovanillic acid (HVA) and increased levels of 14C-3-methoxytyramine (3MT) following intracisternal injection of 14C-dopamine (DA). Opposite results with dexamethasone were obtained in iproniazid (MAO-inhibited)-treated rats. In these animals, brain MAO enzyme activity was significantly increased by dexamethasone. This effect increased with the duration of dexamethasone treatment and appeared to be dose dependent. In the brain areas tested (hypothalamus, midbrain, cerebellum, pons and medulla, olfactory, rest of brain) increases of MAO enzyme activity were also indicated by lower levels of 14C-3MT and increased levels of 14C-HVA formed from intracisternally injected radiolabeled DA. Treatment with other glucocorticoids (16(~-methyldichlorisone, 16~-methylprednisone and prednisolone) had a similar effect on 14C-DA metabolism. On the other hand, desoxycorticosterone, progesterone, estradiol and testosterone, did not exhibit this property. The data indicate that chronic glucocortieoid treatment may have a slight inhibitory effect on brain MAO and also has the ability to partially reverse or antagonize the inhibition of MAO caused by iproniazid. Brain MAO
COMT activity
14C-DA metabolism
1. Introduction Irreversible inhibition of monoamine oxidase (MAO) by hydrazines such as iproniazid may be antagonized by pretreatment with short-acting reversible MAO inhibitors such as harmaline (Pletscher and Besendorf, 1959) and harmine (Horita and McGrath, 1960). Pretreatment with these short-acting MAO inhibitors may occupy the active sites of MAO, protecting the enzyme from the irreversible effects of hydrazine compounds and therefore the MAO recovery rate is much more rapid, similar to that seen following the reversible inhibitor alone (Horita and McGrath, 1960; Dubnick et al., 1963; Horita and Chinn, 1964). This antagonism prevented the usual catecholamine and 5-hydroxytryp-
Iproniazid
Steroids
tamine increasing effect of iproniazid in rat brains (Pletscher and Besendorf, 1959; Ehringer et al., 1961; Horita and Chinn, 1964). We have reported previously that several glucocorticoids alter the metabolism of catecholamines in rats treated with the MAO inhibitor iproniazid (Korduba et al., 1970). Steroid treatment is known to counteract the activity of several drugs (Szabo et al., 1974) and glucocorticoids have been shown to lower MAO activity (Schweppe, 1951; Ho-Van-Hap, 1967). Thus, a possible interaction between glucocorticoids and iproniazid was indicated and it was of interest to further examine the effects of dexamethasone and other steroids on MAO inhibition by iproniazid.
292 2. Materials and methods
2.1. General Male albino rats obtained from Carworth Farms (Wilmington, Mass., CFE strain) weighing 220--250 g were used in all experiments. Prior to use, the animals were housed under normal laboratory conditions for at least 1 week. F o o d and water were given ad libiturn. ~4C-dopamine (6.3 mCi/mmol), 2-14C tryptamine bisuccinate (47.3 mCi/mmol), and S-adenosyl-L-methionine-methyl-~4C (48.5 mCi/mmol) were obtained from New England Nuclear Corp. (Boston, Mass.). Purity of radiolabeled materials was confirmed by paper or thin layer chromatography. 3,4-Dihydroxybenzoic acid was purchased from Calbiochem, Calif.; S-adenosyl-L-methionine chloride and iproniazid phosphate were from Sigma Chemical Co., St. Louis, Mo. Dexamethasone (9a-fluoro-16a-methylprednisolone), 16~-methylprednisone, 16a-methyldichlorisone, desoxycorticosterone, estradiol, progesterone and testosterone were prepared at Schering Corp. For injection, drugs were suspended in 0.4% aqueous methylcellulose and given by the i.p. route. Unless otherwise specified, dexamethasone was given daily (9.00 a.m.) at a dose of 4 mg/kg i.p. for 6 days, the last dose given 4 h before sacrifice, and iproniazid phosphate (100 mg/kg i.p. of base equivalent) given 20 h before sacrifice.
J.W. VEALS ET AL in 0.01% hydrochloric acid in ethanol containing ascorbic acid (0.05%) and centrifuged at 70,000 × g for 15 min at 0°C. Aliquots of the clear supernatant were counted by liquid scintillation spectrometry to determine total (tissue) radioactivity content. The rest of the supernatant was subjected to paper chromatography to identify and quantify the tissue levels of ~4CCatecholamines and their metabolites. A detailed description of this procedure has been reported previously (Symchowicz and Korduba, 1967).
2.3. Monoamine oxidase (MAO) activity MAO activity was measured in tissue homogenates, as described by Wurtman and Axelrod (1963); the tissues were homogenized in 100 volumes of 1.15% KC1 and 50 pl aliquots were used for the assay, except in iproniazid-treated rats where the issues were homogenized in 2 volumes of the 1.15% KC1 and 100 pl aliquots were taken for MAO assay. 1 pg of 2J4C-tryptamine bisuccinate {47.3 mCi/mmol, final concentration 7.81 X 10 -6 M) was used as substrate for MAO and incubated for 20 rain at 37°C. For in vitro drug effect studies the tissues were homogenized in 100 volumes of 1.15% KC1 and 50 pl aliquots were used for assay. Dexamethasone or iproniazid was preincubated with tissue homogenate for 15 or 10 min, respectively, before the addition of ~4Ctryptamine.
2.2. Studies with ~4C-dopamine 2.4. COMT assay Animals were treated with dexamethasone and 14C-DA (1 pCi in 20 pl of Elliot's 'B' irrigating solution, Travenol Laboratories, Morton, Grove, Ill.) was administered intracisternally (Schanberg et al., 1967) 2 h before sacrifice. In some experiments, the animals were pretreated with iproniazid phosphate as indicated. The animals were sacrificed by cervical dislocation, the brains were removed, dissected at 4°C as described by Glowinski and Iversen (1966) and placed on dry ice. The frozen tissues were weighed and homogenized
COMT was determined by the method of McCaman (1965) utilizing S-adenosyl-L-methionine-methyl-14C and 3,4-dihydroxybenzoic acid as substrates. Brain tissue was homogenized in 5 volumes of 1.15% KC1 and centrifuged at 14,000 X g for 20 rain. An aliquot (0.075 ml) of the supernatant was added to 0.5 ml of 0.08 M (pH 7.8) phosphate buffersubstrate solution containing MgC12 (5 × 10 -3 M), 3,4-dihydroxybenzoic acid (1 × 10 -3 M) and S-adenosyl-L-methionine-meth-
DEXAMETHASONE
AND MAO INHIBITION
BY IPRONIAZID
yl-~4C (6 X 10 -s M). The reaction was kept at 37°C for 30 min and stopped with the addition of 0.075 ml 3 N HC1. The radioactive product (~4C-3-methoxy-4-hydroxybenzoic acid) was extracted into 2.5 ml ethyl acetate, Radioactivity in the ethyl acetate was determined in a liquid scintillation counter. The quantity of product formed was determined from the known specific activity of 14C-S-adenosyl-L-methionine and the enzyme activity calculated as nmoles of product formed per g of tissue per hour.
293
14C-3MT LEVELS
12o, ic~
15::.:.':| CONTROL i " , / / 1 DEXAMETHASONE
1
e p'
291
EFFECT OF DEXAMETHASONE ON MONOAMINE OXIDASE INHIBITION BY IPRONIAZID IN RAT BRAIN JOHN W. VEALS, CHRYZANTA A. KORDUBA and SAMSON SYMCHOWICZ
Schering Corporation, Bloomfield, New Jersey, U.S.A. Received 4 December 1975, revised MS received 23 April 1976, accepted 7 October 1976
J.W. VEALS, C.A. KORDUBA and S. SYMCHOWICZ, Effect of dexamethasone on monoamine oxidase inhibition by iproniazid in rat brain, European J. Pharmacol. 41 (1977) 291--299. Chronic (6 days) dexamethasone administration caused a slight decrease of rat brain MAO enzyme activity which was reflected by lower levels of 14C-homovanillic acid (HVA) and increased levels of 14C-3-methoxytyramine (3MT) following intracisternal injection of 14C-dopamine (DA). Opposite results with dexamethasone were obtained in iproniazid (MAO-inhibited)-treated rats. In these animals, brain MAO enzyme activity was significantly increased by dexamethasone. This effect increased with the duration of dexamethasone treatment and appeared to be dose dependent. In the brain areas tested (hypothalamus, midbrain, cerebellum, pons and medulla, olfactory, rest of brain) increases of MAO enzyme activity were also indicated by lower levels of 14C-3MT and increased levels of 14C-HVA formed from intracisternally injected radiolabeled DA. Treatment with other glucocorticoids (16(~-methyldichlorisone, 16~-methylprednisone and prednisolone) had a similar effect on 14C-DA metabolism. On the other hand, desoxycorticosterone, progesterone, estradiol and testosterone, did not exhibit this property. The data indicate that chronic glucocortieoid treatment may have a slight inhibitory effect on brain MAO and also has the ability to partially reverse or antagonize the inhibition of MAO caused by iproniazid. Brain MAO
COMT activity
14C-DA metabolism
1. Introduction Irreversible inhibition of monoamine oxidase (MAO) by hydrazines such as iproniazid may be antagonized by pretreatment with short-acting reversible MAO inhibitors such as harmaline (Pletscher and Besendorf, 1959) and harmine (Horita and McGrath, 1960). Pretreatment with these short-acting MAO inhibitors may occupy the active sites of MAO, protecting the enzyme from the irreversible effects of hydrazine compounds and therefore the MAO recovery rate is much more rapid, similar to that seen following the reversible inhibitor alone (Horita and McGrath, 1960; Dubnick et al., 1963; Horita and Chinn, 1964). This antagonism prevented the usual catecholamine and 5-hydroxytryp-
Iproniazid
Steroids
tamine increasing effect of iproniazid in rat brains (Pletscher and Besendorf, 1959; Ehringer et al., 1961; Horita and Chinn, 1964). We have reported previously that several glucocorticoids alter the metabolism of catecholamines in rats treated with the MAO inhibitor iproniazid (Korduba et al., 1970). Steroid treatment is known to counteract the activity of several drugs (Szabo et al., 1974) and glucocorticoids have been shown to lower MAO activity (Schweppe, 1951; Ho-Van-Hap, 1967). Thus, a possible interaction between glucocorticoids and iproniazid was indicated and it was of interest to further examine the effects of dexamethasone and other steroids on MAO inhibition by iproniazid.
292 2. Materials and methods
2.1. General Male albino rats obtained from Carworth Farms (Wilmington, Mass., CFE strain) weighing 220--250 g were used in all experiments. Prior to use, the animals were housed under normal laboratory conditions for at least 1 week. F o o d and water were given ad libiturn. ~4C-dopamine (6.3 mCi/mmol), 2-14C tryptamine bisuccinate (47.3 mCi/mmol), and S-adenosyl-L-methionine-methyl-~4C (48.5 mCi/mmol) were obtained from New England Nuclear Corp. (Boston, Mass.). Purity of radiolabeled materials was confirmed by paper or thin layer chromatography. 3,4-Dihydroxybenzoic acid was purchased from Calbiochem, Calif.; S-adenosyl-L-methionine chloride and iproniazid phosphate were from Sigma Chemical Co., St. Louis, Mo. Dexamethasone (9a-fluoro-16a-methylprednisolone), 16~-methylprednisone, 16a-methyldichlorisone, desoxycorticosterone, estradiol, progesterone and testosterone were prepared at Schering Corp. For injection, drugs were suspended in 0.4% aqueous methylcellulose and given by the i.p. route. Unless otherwise specified, dexamethasone was given daily (9.00 a.m.) at a dose of 4 mg/kg i.p. for 6 days, the last dose given 4 h before sacrifice, and iproniazid phosphate (100 mg/kg i.p. of base equivalent) given 20 h before sacrifice.
J.W. VEALS ET AL in 0.01% hydrochloric acid in ethanol containing ascorbic acid (0.05%) and centrifuged at 70,000 × g for 15 min at 0°C. Aliquots of the clear supernatant were counted by liquid scintillation spectrometry to determine total (tissue) radioactivity content. The rest of the supernatant was subjected to paper chromatography to identify and quantify the tissue levels of ~4CCatecholamines and their metabolites. A detailed description of this procedure has been reported previously (Symchowicz and Korduba, 1967).
2.3. Monoamine oxidase (MAO) activity MAO activity was measured in tissue homogenates, as described by Wurtman and Axelrod (1963); the tissues were homogenized in 100 volumes of 1.15% KC1 and 50 pl aliquots were used for the assay, except in iproniazid-treated rats where the issues were homogenized in 2 volumes of the 1.15% KC1 and 100 pl aliquots were taken for MAO assay. 1 pg of 2J4C-tryptamine bisuccinate {47.3 mCi/mmol, final concentration 7.81 X 10 -6 M) was used as substrate for MAO and incubated for 20 rain at 37°C. For in vitro drug effect studies the tissues were homogenized in 100 volumes of 1.15% KC1 and 50 pl aliquots were used for assay. Dexamethasone or iproniazid was preincubated with tissue homogenate for 15 or 10 min, respectively, before the addition of ~4Ctryptamine.
2.2. Studies with ~4C-dopamine 2.4. COMT assay Animals were treated with dexamethasone and 14C-DA (1 pCi in 20 pl of Elliot's 'B' irrigating solution, Travenol Laboratories, Morton, Grove, Ill.) was administered intracisternally (Schanberg et al., 1967) 2 h before sacrifice. In some experiments, the animals were pretreated with iproniazid phosphate as indicated. The animals were sacrificed by cervical dislocation, the brains were removed, dissected at 4°C as described by Glowinski and Iversen (1966) and placed on dry ice. The frozen tissues were weighed and homogenized
COMT was determined by the method of McCaman (1965) utilizing S-adenosyl-L-methionine-methyl-14C and 3,4-dihydroxybenzoic acid as substrates. Brain tissue was homogenized in 5 volumes of 1.15% KC1 and centrifuged at 14,000 X g for 20 rain. An aliquot (0.075 ml) of the supernatant was added to 0.5 ml of 0.08 M (pH 7.8) phosphate buffersubstrate solution containing MgC12 (5 × 10 -3 M), 3,4-dihydroxybenzoic acid (1 × 10 -3 M) and S-adenosyl-L-methionine-meth-
DEXAMETHASONE
AND MAO INHIBITION
BY IPRONIAZID
yl-~4C (6 X 10 -s M). The reaction was kept at 37°C for 30 min and stopped with the addition of 0.075 ml 3 N HC1. The radioactive product (~4C-3-methoxy-4-hydroxybenzoic acid) was extracted into 2.5 ml ethyl acetate, Radioactivity in the ethyl acetate was determined in a liquid scintillation counter. The quantity of product formed was determined from the known specific activity of 14C-S-adenosyl-L-methionine and the enzyme activity calculated as nmoles of product formed per g of tissue per hour.
293
14C-3MT LEVELS
12o, ic~
15::.:.':| CONTROL i " , / / 1 DEXAMETHASONE
1
e p'
