The effects of the administration of β-phenylethylamine on tyramine metabolism

European Journal of Pharmacology. 83 (1982) 277-282

277

Elsevier Biomedical Press

T H E E F F E C T S O F T H E A D M I N I S T R A T I O N OF J 3 - P H E N Y L E T H Y L A M I N E O N T Y R A M I N E METABOLISM P.S.

MCQUADE and A.V. JUORIO

*

Psychiatric Research Division, University Hospital, Saskatoon, Saskatchewan, S7N OXO Canada Received 14 April 1982, revised MS received 19 July 1982, accepted 27 July 1982

P.S. McQUADE and A.V. JUORIO, The effects of the aministration of fl-phenylethylamine on tyramine metabolism, European J. Pharmacol. 83 (1982) 277-282. The concentrations of p-tyramine (p-TA), m-tyramine (m-TA), dopamine (DA) and their principal metabolites, p-hydroxyphenylacetic acid (p-HPAA), m-hydroxyphenylacetic acid (m-HPAA) and homovanillic acid (HVA) were determined in the corpus striatum of Swiss mice at various times after the subcutaneous administration of fl-phenylethylamine (PE) (50 mg/kg). Initially p-TA concentrations were reduced but rapid synthesis was apparent up to 2 h after PE administration. PE treatment increased m-TA and these increases reached significance at I and 8 h. PE caused a bimodal increase in p-HPAA and m-HPAA concentrations with the first peak observed at 0.5-1 h due to initial release of p-TA and m-TA. Rapid synthesis of p-TA and m-TA resulted in increased acid concentrations at 4 h. HVA concentrations were increased up to 1 h after PE administration. The synthesis of p-TA and m-TA is related to that of DA probably as a result of the activation of tyrosine hydroxylase. PE may serve as a precursor for p-TA synthesis when the endogenous PE concentration is greatly elevated. fl-Phenylethylamine

p-Tyramine

p-Hydroxyphenylacetic acid

1. Introduction The central effects of fl-phenylethylamine (PE) have been known for fifty years. In 1933, Alles first described a central effect of this compound; animals awoke prematurely from anaesthesia after the administration of PE. The first observation of the behavioral syndrome caused by PE was by Schulte et al. (1941). This syndrome, characterized by an increase in spontaneous motor activity in rats, was very similar to that produced by amphetamine (Holtz et al., 1947). The administration of PE produces either a moderate increase or reduction in brain dopamine depending on dose, time or species used (Jonsson et al., 1966; Fuxe et al., 1967; Jackson and Smythe, 1973). PE also causes an elevation in the rat brain homovanillic

* To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Pres~';

m-Hydrophenylacetic acid

m-Tyramine

acid concentrations twenty minutes after injection (Antelman et al., 1977). Haag et al. (1961) observed that PE stimulates the release of catechols from bovine adrenals; this observation was confirmed in rat brain (Fuxe et al., 1967). The uptake of dopamine by caudate synaptosomes is also reduced in the presence of PE (Horn and Snyder, 1972). Direct actions of PE may be demonstrated after the iontophoretic application of this amine to spontaneously firing cells. Zarzecki et al. (1976) suggested that PE depressed the firing rate of spontaneously active caudate cells. PE also potentiated the DA response when both were simultaneously applied onto spontaneously firing caudate neurons (Jones and Boulton, 1980). The interaction between PE and the two naturally occurring tyramines (p-TA and m-TA) have now been investigated. This paper describes the effects of the administration of PE on the striatal concentrations of p-TA, m-TA and dopamine and of their respective major metabolits.

278 2. Materials and methods

2.1. Dissections and treatment Swiss male albino mice (20-25 g body weight) were treated with one acute injection (50 mg/kg) of fl-phenylethylamine hydrochloride (Sigma, St. Louis, Mo.) dissolved in saline. The mice were decapitated, their brains were removed and the caudate nuclei were dissected out and frozen on dry ice. Both nuclei from one mouse weighed approximately 33-36 rag.

2.2. Estimation of p- and m-hydroxyphenylacetic acid The tissues were homogenized in 0.1 M zinc sulfate and proteins were precipitated using 0.1 M barium hydroxide. The supernatant was then percolated through a DEAE-Sephadex column and the acids extracted with ethyl acetate. The acids, after derivatization using pentafluoropropionic anhydride and hexafluoroisopropanol, were quantified using a gas chromatograph equipped with an electron capture detector (McQuade et al., 1981). Alternatively the acids were quantified as their methyl heptafluorobutyric diesters using gas chromatography-mass spectrometry (Durden and Boulton, 1981).

2.3. Estimation of p-tyramine and m-tyramine Striatal tissue was weighed, homogenized in 0.1 N HCI containing disodium edetate (EDTA, 1 m g / m l ) and directly derivatized with 5-dimethylaminonaphthalene sulfonyl (dansyl) chloride. The resultant derivatives after extraction into benzene were transferred to silica gel TLC plates, separated, eluted and estimated by the high resolution mass spectrometric selected ion monitoring (integrated ion current) technique. Deuterated p- or m-TA were added as internal standards at the beginning of the procedure (Philips et al., 1974; 1975).

2.4. Estimation of dopamine DA was estimated fluorometrically using the pooled striata from two mice (Laverty and Shar-

man, 1965). After separation on a Dowex 50W-X4 ion exchange column and acetylation, a fluorophore was developed by condensing the DA with 1,2-diaminoethane. The fluorophore was extracted into isobutanol and estimated fluorometrically. A check on the recovery of 100 ng of DA was carried out in each experiment. The recovery was 83 - 3% (12) ( m e a n - S . E . M . , number of experiments in parentheses). The results were corrected accordingly.

2.5. Estimation of homovanillic acid The pooled striata of five mice were homogenized in 0.1 M hydrochloric acid, deproteinized with 0.4 M perchloric acid and extracted with nbutyl acetate. The HVA was estimated fluorometrically (And~n et al., 1963). Checks on the recovery of 200 ng of added HVA were carried out in every experiment. The percentage recovery was 77--+2 (I0) ( m e a n - S . E . M . , number of experiments in parentheses) with the tissue results corrected accordingly.

3. Results

3.1. Amine changes The endogenous concentrations of the tyramines and DA after the administration of PE at the various time intervals are presented in table 1. The p-TA concentration was initially decreased at 0.5 h (to 43% of controls), but rapid synthesis was apparent up to 2 h after PE injection. By 4 h, p-TA concentrations had returned to the control values. Concentrations of m-TA, in contrast, were significantly elevated at 1 h (to 124% of control and 8 h (to 146% of control) after PE injection. DA concentrations tended to be slightly, but not significantly, increased at 0.5 and 4 h.

3.2. Acid changes The corresponding concentrations of p-HPAA, m-HPAA and HVA are given in table 2. p-HPAA concentrations were significantly increased at 0.5, 1, 2 and 4 h after PE treatment. The increases were

279 TABLE I Effect of the subcutaneous administration of fl-phenylethylamine (50 mg/kg) on the concentration of p-tyramine (p-TA), m-tyramine (m-TA) and dopamine (DA) in the mouse striatum. Values are means (-+ S.E.M., number of experiments in parentheses) in n g / g of fresh tissue.

Controls Treated

Time h

p-TA ng/g

m-TA ng/g

DA ng/g

0.5 1 2 4 8

23 -+1.3 (9) 9.8-+0.8 (11) b 29 -+-2.6(11) a 38 --+5.3 (9) ~ 26 -+-4.0 (7) 20 -+1.1 (7)

6.8-+0.3 (9) 8.1 ±0.8 (I I) 8.4-+0.3 (9) b 8.1--+0.8 (8) 7.2+0.8 (6) 9.9-+0.8 (5) h

9500-+- 650 (13) 10300-+ 750 (8) 10000-+ 450 (6) 9200-+ 1 100 (7) 12000-+1000 (7) 10000-+ I 000 (7)

Student's t-test: a P<0.05; b P<0.01, with respect to controls.

TABLE 2 Effect of the subcutaneous administration of fl-phenylethylamine (50 mg/kg) on the concentration of p-hydroxyphenylacetic acid (p-HPAA), m-hydroxyphenyl-acetic acid (m-HPAA) and HVA in the mouse striatum. Values are means (+S.E.M., number of experiments in parentheses) in n g / g of fresh tissue. Time h Saline controls Treated

0.5 1 2 4 8

p-HPAA ng/g

3 1 +. - 2.3(9) 97-+ 9 (6) c 86--~ 7.8 (6) c 50± 7.6(7) b'd 120-+ 15 (6)c'd 38-+ 3.8(5)

m-HPAA ng/g

HVA ng/g

14--' 1.6(9) 117-+17 (6) c 93-'- 7.8 (6) c 41-+ 8.4(7) b'd 85-+21 (6) c'a 7-+ 1.7(5) a

1000 ÷ 60(13) 2200--+-200 (5) c 1800-'-130 (6) c 1200-+ 70 (6) I 100-+ 70 (8) 1200± 50 (8)

Student's t-test: a P<0.05; b P<0.02; c P<0.001, with respect to controls. The treatment of the results by Dunnett's test indicates that p-HPAA and m-HPAA values at 2 h are significantly different from those at I h (d P<0.05); similarly the values obtained at 4 h are significantly different from those at 2 h (d P<0.05).

b i p h a s i c w i t h t h e g r e a t e s t i n c r e a s e s o b s e r v e d a t 0.5 and 4h after PE administration (with values of 313 a n d 387% o f c o n t r o l s r e s p e c t i v e l y ) . Less marked increases were observed at 1 or 2 h (277 a n d 161% o f c o n t r o l s r e s p e c t i v e l y ) . T h e p a t t e r n o f the m-HPAA concentration increases was very similar to that of the p-isomer. The greatest conc e n t r a t i o n s w e r e o b s e r v e d a t 0.5, 1 a n d 4 h a f t e r P E t r e a t m e n t ( t o 836, 6 6 4 a n d 607% o f c o n t r o l s ) . At 8 h the m-HPAA concentrations were reduced t o 50% o f t h e c o n t r o l c o n c e n t r a t i o n s . T h e H V A c o n c e n t r a t i o n s w e r e i n c r e a s e d o n l y a t t h e t w o initial t i m e s (0.5 a n d 1 h). The concentrations of m-HPAA and p-HPAA,

w h i l e s i g n i f i c a n t l y i n c r e a s e d a b o v e t h e c o n t r o l valu e s a t 2 h, w e r e s i g n i f i c a n t l y d e c r e a s e d r e l a t i v e to their corresponding concentrations at 1 and 4h ( t a b l e 2).

4. Discussion T h e s t r i k i n g r e d u c t i o n in p - T A c o n c e n t r a t i o n s e e n a t 0.5 h f o l l o w i n g P E ( t a b l e 1) is v e r y s i m i l a r to the reduction in p-TA concentrations observed after the administration of the antipsychotic drugs ( J u o r i o , 1977). T h i s e f f e c t m a y b e d u e t o t h e stimulation of dopamine synthesis observed after

280 antipsychotic drug administration (Burkard et al., 1967). Since dopamine levels did not change concomitant with large increases in HVA concentrations, it was obvious that DA synthesis and breakdown were stimulated under our conditions. PE has previously been shown to stimulate DA synthesis in rat striatum, striatal slices or synaptosomes (Antelman et al., 1977; Snodgrass and Uretsky, 1978; Roberts and Patrick, 1979); since tyrosine hydroxylase (TH) is the key enzyme in regulating DA synthesis, it is possible that PE may cause the enzyme to be activated as is apparent after antipsychotic treatment. Tyrosine hydroxylase exists in both a particulate and soluble form in the rat striatum (Kuczenski and Mandeli, 1972). PE is highly lipophilic and thus readily crosses the blood brain barrier (Oldendorf, 1971). It is possible that PE penetrates the membranes and thus may facilitate or activate a membrane associated enzyme such as TH. Alternatively, the apparent stimulating actions of PE may be caused by PE, in a manner already proposed for amphetamine, affecting the endogenous distribution of an inhibitory pool of dopamine, thus reducing the effective dopamine concentration and releasing T H from feedback regulation (Patrick et al., 1981). TH, after either being released from feedback inhibition or possibly activated, could also account for the increased concentrations of m-TA. TH after activation might use phenylalanine as a substrate to produce m-tyrosine (Ishimitsu et al., 1980) which may then be rapidly decarboxylated to form m-TA (Mitoma et al., 1957). An activated T H might also utilize most of the available tyrosine to form dopamine and so p-TA concentrations would decline as was seen 0.5 h after PE administration. This assumes that the normal synthesis route for p-TA is the decarboxylation of p-tyrosine (Lovenberg et al., 1962). In the presence of an increased concentration of a better decarboxylase substrate such as L-DOPA, the decarboxylase enzyme molecules might be nearly saturated, thus excluding p-tyrosine, a very poor substrate for the decarboxylase. The very rapid increase in p-TA concentrations observed by 2 h (table 1) suggested that the exogenous PE may be serving as a precursor for p-TA synthesis. Nakajima et al. (1964) found that PE

administration to rabits pretreated with an inhibitor of MAO, caused a large increase in brain PE concentrations with the highest concentration occurring in the caudate. Jonsson et al. (1975) described a mixed function oxidase present in liver which was capable of hydroxylating PE. Boulton et al. (1974) observed that both p-TA and m-TA were synthesized after the intraperitoneal injection of deuterated PE. Finally, Silkaitis and Mosnaim (1976) have proposed that PE could be hydroxylated by rabbit brain to form p-TA. Should PE be acting as a substrate, this might account for the very large p-HPAA and m-HPAA concentrations encountered after PE administration. If the concentrations of p-HPAA and m-HPAA at the initial times are regarded as an index of turnover of the respective amines, it is apparent that the amines are being metabolized at a very high rate indeed. Alternatively, the large accumulations of the acids may be due to the inability of the cells to dispose of or transport them out of the striatum. Thus one observes a large accumulation of the acids which gradually declines with time (i.e. until 2h) at which time p-TA and m-TA synthesis is complete (table 2). In order to return to normal amine concentrations, another large release of amines occurs followed by a large increase in the acid concentrations (the high values at 4 h after PE injection), thus explaining the decline at 2 h and subsequent increase at 4 h in the acid concentrations (table 2). This long time course for both the amine and acid production suggested to us that the tyramines and p-HPAA and m-HPAA were produced within the striatum. Nakajima et al. (1964) had previously demonstrated that in mice pretreated with an inhibitor of monoamine oxidase the PE concentrations in the brain were maximally increased ten minutes after an intraperitoneal injection of PE and were approximately back to normal levels by thirty minutes after the injection. In further support of this position, Oldendorf (1971) found that low concentrations of p-TA failed to penetrate into the brain and thus the concentrations observed at 1 and 2 h would also be too small to have originated peripherally. The initial decrease in p-TA concentrations is also difficult to reconcile with a peripheral origin of these compounds as most of the PE would be

281 cleared from the bloodstream by thirty min. PE stimulates DA synthesis, possibly by activating TH; m-TA concentrations may thus increase after this activation of TH. This enzyme also appears to play a key role in p-TA synthesis, sequestering p-tyrosine for DA synthesis and thus interferring with p-TA synthesis. Should DA synthesis and p-TA synthesis be increased simultaneously, p - T A m a y p l a y a r o l e in t h e f e e d b a c k i n h i b i t i o n o f DA synthesis. This may be a possible neuromodulatory role for p-TA and/or m-TA.

Acknowledgements We thank Dr. A.A. Boulton for his encouragement and critical comments; Drs. D.A. Durden and C. Kazakoff for supervising the mass spectrometric and GC-MS analyses; Dr. B.A. Davis for synthesizing the deuterated compounds; D. Chou, M. Mizuno, S. Smith, G. Wheatley and E.P. Zarycki for expert technical assistance; the University of Saskatchewan for a scholarship (P.S. McQuade) and Saskatchewan Health for continuing financial support.

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