Neuropharmacology Vol. 31, No. 4, pp. 337-341, 1992 Printedin Great Britain.All rightsreserved
0028-3908/92 $5.00 + 0.00 Copyright0 1992 PergamonPressplc
INHIBITION OF TRYPTOPHAN HYDROXYLASE BY (R)- AND (S)-l-METHYL-6,7-DIHYDROXY-1,2,3,4TETRAHYDROISOQUINOLINES (SALSOLINOLS) M.
OTA,~ P. DOSTERT,* T. HAMANAKA,’
T. NAGATSU~
and M. NAO?
‘Department of Psychiatry, Nagoya City University School of Medicine, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan, 2Farmitalia Carlo Erba, E&D-Erbamont Group, Milan, Italy, ‘Division of Molecular Genetics (II) Neurochemistry, Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-l 1, Japan and ‘Department of Biosciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, Japan (Accepted 10 October 1991)
Summary-The (R)- and (S)-enantiomers of salsolinol, the dopamine-derived tetrahydroisoquinolines, were found to inhibit the activity of tryptophan hydroxylase (TPH), prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition of TPH by salsolinols was found to be non-competitive with the substrate L-tryptophan. Tryptophan hydroxylase is composed of two elements with different kinetic properties in terms of cofactor (6R)-L-erylhro-5,6,7,8-tetrahydrobiopterin and these two elements were inhibited by salsolinols in competitive and uncompetitive ways, respectively. Stereoselectivity of salsolinol was not observed, concerning the potency and the type of inhibition on TPH. These data indicate that
salsolinols might be naturally occurring inhibitors of indoleamine metabolism. Key words-salsolinol,
tryptophan
hydroxylase, mastocytoma,
1-Methyl-6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline (salsolinol) is one of the 1,2,3,4-tetrahydroisoquinolines (TIQs), formed by condensation of dopamine with pyruvate, followed by decarboxylation (Brossi, 1982) and an asymmetric center at the C-l position of the salsolinol molecule gives rise to (R)- and (S)-enantiomers (Strolin-Benedetti, Bellotti, Pianezzola, Moro, Carminanti and Dostert, 1989). Another pathway of synthesis of salsolinol is the condensation of dopamine with acetaldehyde by the Pictet-Spengler reaction. The presence of salsolinol in humans was reported for the first time in the urine of Parkinsonian patients on L-DOPA medication (Sandier, Bonham-Carter, Hunter and Stern, 1973) and later found in the urine and cerebrospinal fluid (C.S.F.) of healthy volunteers (Collins, Nijm, Borge, Teas and Golfarb, 1979; Sjiiquist, Borg and Kvande, 1981). In the urine of healthy subjects, only the (R)-enantiomer of salsolinol was detected (Strolin-Benedetti et al., 1989). On the other hand, a large amount of (S)-salsolinol was found in port wine (Dostert, Strolin-Benedetti and Dordain, 1988). Moreover, high levels of salsolinol were detected in several regions of the brain, derived from intoxicated alcoholics (Sjsquist, Eriksson and Winblad 1982). These observations indicate that salsolinol may have some role in the development of intoxication with alcohol.
*To whom correspondence should be addressed. NP 31/4-C
L-tryptophan, (6R)tetrahydrobiopterin.
A number of clinical findings, implying an etiological role for serotonergic dysfunction in alcoholic organic brain diseases, have accumulated in recent years. For example, in patients with alcoholic amnestic disorder, sleep abnormalities (Martin, Loewenstein, Kaye, Ebert, Weingartner and Gillin, 1986) and reduced daily production of 6-hydroxymelatonin (Martin, Higa, Bums, Tamarkin, Ebert and Markey, 1984) were observed. The serotonin-uptake blocker, fluvoxamine maleate, effectively improved episodic memory in patients with alcoholic amnestic disorder (Martin, Adinoff, Eckardt, Stapleton, Bone, Rubinow, Lane and Linnoila, 1989). Furthermore, a reduction in levels of S-hydroxyindoleacetic acid in C.S.F. was documented in some alcoholics, whose brain function was not overtly impaired (Ballenger, Goodwin, Major and Brown, 1979; Banki, 1981). In order to find the effect of salsolinol on the enzymes involved in indoleamine metabolism, tryptophan hydroxylase [L-tryptophan, tetrahydropteridine : oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4, TPH] was chosen, because it is the ratelimiting enzyme in the synthesis of serotonin. The enzyme used in this experiment was prepared from mastocytoma P-8 15 cells, which produce serotonin and have high TPH activity. In this paper, the inhibition of TPH activity by (R)- and (S)-salsolinol is described. The inhibition is discussed in relation to the possible role of this amine as an inhibitor of indoleamine metabolism. 337
M. OTA
338 METHODS
(R)- and (S)-enantiomers of salsolinol were synthesized as previously reported (Teitel, O’Brien and Brossi, 1972). The chemicals, purchased from Sigma Chemical Co. (St Louis, Missouri, U.S.A.) were: phenylmethylsulfonyl fluoride (PMSF), antipain, peptstatin A, chymostatin, leupeptin, L-tryptophan, 5hydroxytryptophan (5HTP), NSD-1015 (m-hydroxybenzylhydrazine) and bovine y-globulin. Dithiothreitol (DTT), fl-mercaptoethanol and sodium octanesulfonate were from Nacalai Tesque (Kyoto, Japan). Sephadex G-25 was from Pharmacia Fine Chem. (Uppsala, Sweden). (6R)-L-eryfhro-5,6,7,8Tetrahydrobiopterin hydrochloride [(GR)BH,] was kindly donated by Dr Matsuura (Fujita Health University, Toyoake, Japan). Catalase, prepared from bovine liver, was purchased from BoehringerMannheim (Mannheim, Fed. Rep. Germany). Organic solvents were of HPLC grade. The murine mastocytoma cell line p-815, established in DBA mice (Dunn and Potter, 1957) and carried in DBAj2 mice (Hosoda and Glick, 1965), was kindly donated by Dr Hosoda (Aichi Cancer Institute, Nagoya, Japan). The ascitic fluid was taken and the cells were gathered by centrifugation at 1OOOgfor 10 min. The cells were washed twice with Hank’s balanced salt solution (HBSS) and then suspended in HBSS and stored at -80°C until use. The cells (250 mg protein) were diluted with the same volume as the extraction medium; 100 mM Tris-acetate buffer, pH 7.6, containing 2 mM PMSF, 4 mM DTT, 10 pgg/ml each of antipain, pepstatin A, chymostatin and leupeptin. The cell suspension was sonicated and centrifuged at 100,000 g for 30 min. The supernatant was passed through a Sephadex G-25 column (2 cm i.d. x 15 cm) equilibrated with 10 mM Tri-acetate buffer, pH 7.6, containing 1 mM PMSF. The fractions were eluted out by the same buffer in the void volume, gathered and used as the enzyme sample. The activity of TPH was measured by quantitative analysis of 5-hydroxytryptophan (5-HTP), produced from L-tryptophan in the presence of NSD1015, an inhibitor of aromatic L-amino acid decarboxylase, according to a previously reported method (Yamaguchi, Sawada, Kato and Nagatsu, 1981) with
et al.
a slight modification (Naoi, Hosoda, Ota, Takahashi and Nagatsu, 1990).The validity of the assay conditions was confirmed as reported previously. The enzyme sample (about 60 pg protein) was incubated at 37°C for 20min with 100nM r.-tryptophan in lOOn of 50 mM HEPESNaOH buffer, pH 7.6, containing 100 nM (6R)BH,, 100 nM j-mercaptoethanol, catalase (0.4mg protein) and 0.5 mM NSD-1015. The reaction was terminated by addition of 10~1 of 60% perchloric acid and the sample was mixed, centrifuged at 15,000 g for 10 min and filtered through a Millipore HV filter (pore size 0.45 pm). For quantification of 5-HTP, the sample was applied onto a Shimadzu HPLC apparatus, LCSA, connected to a Shimadzu fluorescence detector, FD-500. The column used was a pre-packed reversed-phase column, ODS-H (4 mm id. x 150 mm, Shimadzu Techno-Research, Kyoto, Japan) and the mobile phase was 90mM sodium acetate-35 mM citric acid buffer, pH 4.35, containing 130pM disodium EDTA and 2 mM sodium octanesulfonate, to which methanol was added to 11%. The flow rate was 0.8 ml/min. The fluorescence intensity at 345 nm was measured with excitation at 295 nm. Quantification of 5-HTP was carried out by comparison of the peak area with that of a standard. Protein concentration was measured according to Bradford (1976), using bovine y-globulin as standard. The values of the Michaelis constant (K,,,) and the maximum velocity (I’,,,,,), in terms of the substrate or the cofactor, were obtained by Lineweaver-Burk’s plot. The effects of (R)- and (S)-salsolinols on the activity of TPH were examined at concentrations from 1 nM to 1 mM. The activity was measured with the enzyme (6Opg protein), in the presence of 10 nM L-tryptophan and 100 FM (6R)BH, or 100pM L-tryptophan and 10,nM (6R)BH,. The activity of the enzyme was measured in the absence or presence of 20 PM (R)- or (S)-salsolinol. The type and the value of the inhibition constant (K,) were also estimated by Lineweaver-Burk’s plot. RESULTS
Kinetic properties of TPH used in these experiments are summarized in Table 1. The values of K,,,
Table I. Kinetic properties of a sample of TPH
In terms of substrate. t-tryptophan
(,“G)
v mar (pmol/min/mg protein)
14.8 k 1.7
262 k 21 Vm.ii2
VIns?,>
(pmol/min/mg protein)
(%)
(pmol/min/mg protein)
In terms of cofactor, 437 * 137 127 f 27 93.4 f 14.2 5.04 * 0.52 (6R)BH, Kinetic properties of TPH, prepared from murine mastocytoma cell line P-815. Each value represents the mean k SD of duplicate measurements of three experiments. The kinetic data of the enzyme activity, in terms of t-tryptophan, were determined from the data obtained using 100 PM (6R)BH,. The kinetic data of the enzyme activity, in terms of (6R)BH,, were determined from the data, obtained using 100 PM r-tryptophan.
Inhibition of TPH by (R)- and (S)-salsohnols (A) L -Tryptophan
(S)
339
(6R)BH4
Salsolinol (,uM)
Fig. 1. Inhibition of the activity of TPH by (R)- and (S)-salsohnols. In (A) the activity of TPH was obtained with 10&M L-tryptophan and 100pM (6R)BHI. In (B) activity of TPH was obtained with 100 PM L-tryptophan and 10 PM (6R)BH,. The activity of TRH for (A) was 99.1 + 4.9 pmol/min/mg protein and that for (B) was 67.9 + 2.1 pmol/min/mg protein. The relative activity of TPH was expressed as a percentage of the activity of control. Each point represents the means + SD of triphcate measurements. (O), OR)-salsolinol; (A), (~)-salsolinol.
and V,,, of the enzyme
in terms
of the substrate,
were 14.8 PM and 262 pmol/min/mg protein, respectively. On the contrary, in the case of the substrate, Lineweaver-Burk’s plot in terms of cofactor, (6~)BH~, showed that TPH was composed of two components with different kinetic characteristics (see Fig. 3, control). One component (termed as Component 1) had a low Km and V,,,,, value, 5 PM and 93 pmol/min/mg protein and the other (termed as Component 2) had a high Km and if,,, value, 127 FM and 437 pmol/min/mg protein, respectively. The effects of (R)- and (S)-salsolinols on the activity of TPH were examined, using a small concentration of L-tryptophan (10 PM) and a large concentration of the cofactor (IOOpM) (Fig. IA) and vice versa (Fig. IB), using IOOpM L-tryptophan and 10 FM (BR)BH,. Both enantiomers of salsolinol were L-tryptophan,
0.06
0.06
1
0.0
found to inhibit the activity of TPH in a dose-dependent manner, as shown in Fig. 1. One mM of(R)- and (S)-salsolinol inhibited the activity of TPH almost totally. The concentrations of salsolinol which gave the 50% inhibition of the activity of TPH (IC,) were almost identical between (R)- and (Qforrn, both in the presence of smaller concentration of the substrate and of the cofactor. The IC,, of salsolinol was found to be around 20 /LM, in regard to the substrate, which was almost the same as that in regard to the cofactor. The type of inhibition of the activity of TPH by salsolinol was characterized by plotting the data, according to Lineweaver-Burk, with various concentrations of L-tryptophan and (6R)BH,. The concentration of salsolinol was fixed at 20 /IM throughout these experiments. Figures 2 and 3 show typical results of the Lineweaver-Burk’s plot for three inde~ndent
0.1
l/[L-tryptophan]
0.2
0.3
(PM “)
Fig. 2. Effects of the concentration of L-tryptophan on activity of TPH in the absence and presence of 20 FM (R)or (S)-salsolinol. The activity of TPH was measured with nine different concentrations (from 3.9 to 1OOgM) of L-tryptophan and 100 PM (6R)BH,. The reciprocal of the reaction velocity was plotted against that of the concentration, according to Lineweaver and Burk. This figure represents one of three experiments, done in duplicate. (a), Control; (O), (R)-salsohnol; ( x ), (S)-salsolinol.
Al 0.0
I
0.1
0.2
0.3
l/[(GR)BHr] (CIM“)
Fig. 3. Effects of the concentration of (6R)BH, on the activity of TPH in the absence and presence of 20 RM (R)or (S)-salsolinol. The activity of TPH was measured with nine different concentrations of (6R)BH, (from 3.9 to 100 uM) and 100 uM L-trvutonhan. The reciurocal of the _. _
reaction velocity was plotted against that of the concentration of (6R)BH, according to Lineweaver and Burk. This
figure represents one of three experiments, done in duplicate. (O), Control; (O), (R)-salsolinol; ( x ), (S)-salsolinol.
M. OTAet 01.
340
experiments. Salsolinol inhibited the activity of TPH in a non-competitive way to L-tryptophan (Fig. 2). The inhibition constant, Ki, values of (R)- and (S)salsolinol obtained were 15.4 f 5.3 FM and 19.9 + 6.8 PM (mean f SD of duplicate measurements of three experiments), respectively. As shown in Fig. 3, (R)- and (S)-salsolinol inhibited both components and the inhibition of Component 2 was competitive to (6R)BH.,, while the inhibition of Component 1 was uncompetitive. The K, values for competitive inhibition of Component 2 by (R)- and (S)-salsolinol were 12.0 + 4.6 PM and 19.0 + 4.9 PM, respectively; K, values for uncompetitive inhibition of Component 1 by (R)- and (S)-salsolinol were 12.5 + 2.4 PM and 17.4 f 4.9 PM, respectively. These K, values were not statistically different between (R)- and (S)-salsolinol. Inhibition of TPH by salsolinol was not stereoselective for both substrate and cofactor. DISCUSSION
The effects of salsolinol on the activity of tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC), derived from the brain of the rat were described by Weiner and Collins (1978). In their report, racemic salsolinol inhibited the activity of TH in competition with a synthetic cofactor, (6RS)-methyl5,6,7,%tetrahydrobiopterin, with a K, value at the level of 10pM. In addition, they also reported that 100pM salsolinol did not appreciably affect the activity of DDC. On the other hand, the effects of tetrahydroisoquinolines, such as salsolinol, on indoleamine metabolism have not been reported. This paper reports in vitro inhibition of the activity of TPH by salsolinol, in respect to both the substrate and cofactor. The concentrations of (R)- and (S)salsolinols in the human brain have been reported (Ung-Chhun, Cheng, Pronger, Serrano, Chavez, Perez, Morales and Collins, 1985; Sjoquist et al., 1982). In the brain of patients with intoxicated alcoholism, the concentration of salsohnol in the caudate was up to 0.3 PM (Sjoquist et al., 1982). Considering the intracellular compartment of salsolinol with TPH, the concentration of salsolinol in the neurons may be much greater and compatible to the K, value of salsolinols, 10 PM. The inhibition of the activity of TPH by salsolinol was not stereoselective. This observation was different from the case of type A monoamine oxidase (MAO). The R form inhibited MAO much more effectively than S form (Bembenek, Abell, Chrisey, Rozwadowska, Gessner and Brossi, 1990). These results suggest that the binding site of salsolinol to TPH and type A MAO is not the same. Type A MAO may recognize the N-atom at C-2 position and a methyl group at C-l position but TPH cannot distinguish the stereoisomers at C-l position. The binding site of salsolinol to TPH may be quite different; salsolinol may be bound near at its hydroxy group of C-6 and/or C-7 position.
The influence of alcohol on indoleamine metabolism has mainly focused on aldehyde dehydrogenase, which shares the function on serotonin degrading process as reviewed by Truitt (1973). It has also been proposed that tetrahydroisoquinolines play a role in alcoholism (Davis and Walsh, 1970; Collins et al., 1979; Cohen and Collins, 1970), although their mechanism has not yet been fully clarified. This report supports the idea that the enhanced amount of salsolinol suppresses the activity of TPH resulting in the reduction of the amount of serotonin. It might be relevant to the observation that in the alcoholics the content of serotonin was decreased in the tissues or organs, such as platelets which store serotonin (Bailly, Vignau, Lauth, Racadot, Beuscart, Servant and Parquet, 1990). (R)-Salsolinol is known to be synthesized endogenously, whereas the (S)-form is derived from food or beverages, such as port wine (Strolin-Benedetti et al., 1989; Dostert, Strolin-Benedetti, Bellotti, Allievi and Dordain, 1990). (R)- and (S)-salsolinol inhibited the activity of TPH almost to the same degree in this experiment. This observation indicates that both enantiomers of salsolinol may inhibit the activity of TPH in vivo. The origin of salsolinol, detected in the brain, still remains to be clarified, since the transport of salsolinol through the blood-brain barrier has not been shown (Origitano, Hannigan and Collins, 1981). However, it is still noteworthy that the inhibition of salsolinol on TPH, a rate-controlling enzyme in the production of serotonin has been shown. Thus, dopamine-derived human alkaloids may regulate the level of serotonin in the human brain under physiological and pathological conditions, such as alcoholism, ageing and treatment
with L-DOPA.
REFERENCES Bailly D., Vignau J., Lauth B., Racadot N., Beuscart R., Servant D. and Parquet P. J. (1990) Platelet serotonin decrease in alcoholic patients. Acta psychiat. stand. 81: 68-12. Ballenger J. C., Goodwin F. K., Major L. F. and Brown G. L. (1979) Alcohol and central serotonin metabolism in man. Archs gem. Psychiat. 36: 224-221. Banki C. M. (1981) Factors influencing monoamine metabolites and tryptophan in patients with alcohol dependence. J. Neural Transm. 50: 89-101. Bembenek M. E., Abel1 C. W., Chrisey L. A., Rozwadowska M. D., Gessner W. and Brossi A. (1990) Inhibition of monoamine oxidases A and B by simple isoquinoline alkaloids: racemic and optically active 1,2,3,4_tetrahydro, 3,4-dihydro-, and fully aromatic isoquinolines. J. med. Chem. 33: 147-152. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Analyt. Biochem. 72: 248-254.
Brossi A. (1982) Mammalian TIQs: products of condensation with aldehydes or pyruvic acids? Prog. chit. Biol. Res. 90: 123-133. Cohen G. and Collins M. (1970) Alkaloids from catecholamines in adrenal tissue: possible role in alcoholism. Science 167: 1749-1751.
Inhibition of TPH by (R)- and (S)-salsolinols Collins M. A., Nijm W. P., Borge G. F., Teas G. and Golfarb C. (1979) Dopamine-related tetrahydroisoquinolines: significant urinary excretion by alcoholics after alcohol consumption. Science 206: 1184-l 186. Davis V. E. and Walsh M. J. (1970) Alcohol, amines and alkaloids: a possible biochemical basis for alcohol addiction. Science 167: 1005-1007. Dostert P., Strolin-Benedetti M., Bellotti V., Allievi C. and Dordain G. (1990) Biosynthesis of salsolinol, a tetrahydroisoquinoline alkaloid, in healthy subjects. J. Neural Transm. 81: 215-223. Dostert P., Strolin-Benedetti M. and Dordain G. (1988) Dopamine-derived alkaloids in alcoholism and in Parkinson’s and Huntington’s diseases. J. Neural Transm. 74: 61-74. Dunn T. B. and Potter M. (1957) A transplantable mastcell neoplasm in the mouse. J. natn Cancer Inst. 18: 587601. Hosoda S. and Glick D. (1965) Biosynthesis of 5-hydroxytryptophan by neoplastic mouse mast cells. Biochim. biophys. Acta 111: 67-78.
Martin P. R., Adinoff B., Eckardt M. J., Stapleton J. M., Bone G. A. H., Rubinow D. R., Lane E. A. and Linnoila M. (1989) Effective pharmacotherapy of alcoholic amnestic disorder with fluvoxamine. Archs gen. Psychiat. 46: 617621.
Martin P. R., Higa S., Burns R. S., Tamarkin L., Ebert M. H. and Markey S. P. (1984) Decreased 6-hydroxymelatonin excretion in KorsakotI’s psychosis. Neurology 34: 966-968. Martin P. R., Loewenstein R. J., Kaye W. H., Ebert M. H., Weingartner H. and Gillin J. C. (1986) Sleep EEG in Korsakoff’s psychosis and Alzheimer’s disease. Neurology 36: 411414. Naoi M., Hosoda S., Ota M., Takahashi T. and Nagatsu T. (1990) Inhibition of tryptophan hydroxylase by food-derived carcinogenic heterocyclic amines, 3-amino-I-methyl-SH-pyrido[4,3-blindole and 3-amino1,4-dimethyl-SH-pyrido[4,3-blindole. Biochem. Pharmac. 41: 199-203.
341
Origitano T., Hannigan J. and Collins M. A. (1981) Rat brain salsolinol and blood-brain barrier. Brain Res. 224: 446-451.
Sandler M., Bonham-Carter S., Hunter K. R. and Stern G. M. (1973) Tetrahydroisoquinoline alkaloids: in uiuo metabolites of L-DOPA in man. Nature 241: 43943.
Sjijquist B., Borg S. and Kvande H. (1981) Salsolinol and methylated salsolinol in urine and cerebrospinal fluid from healthy volunteers. Subst. Alcohol Actions Misuse 2: 73-77.
Sjiiquist B., Eriksson A. and Winblad B. (1982) Salsolinol and catecholamines in human brain and their relation to alcoholism. Prog. clin. Biol. Res. 90: 5767. Strolin-Benedetti M., Bellotti V., Pianexxola E., Moro E., Carminati P. and Dostert P. (1989) . , Ratio of the R and S enantiomers of salsolinol in food and human urine. J. Neural Transm. 17: 47-53.
Teitel S., O’Brien J. and Brossi A. (1972) Alkaloids in mammalian tissues. 2. Synthesis of ( + ) and ( - ) substituted-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines. J. med. Chem. 15: 845-846.
Truitt E. B. Jr (1973) A biogenic amine hypothesis for alcohol tolerance. Ann. N. Y. Acad. Sci. 215: 177-182.
Ung-Chhun N., Cheng B. Y., Pronger D. A., Serrano P., Chavez B., Perez R. F., Morales J. and Collins A. (1985) Alkaloid adducts in human brain: coexistence of I-carboxylated and noncarboxylated isoquinolines and /?-carbolines in alcoholics and nonalcoholics. Prog. clin. Biol. Res. 183: 125-136. Weiner C. D. and Collins M. A. (1978) Tetrahydroisoquinolines derived from catecholamines or DOPA: effects on brain tyrosine hydroxylase activity. Biochem. Pharmac. 27: 2699-2703.
Yamaguchi T., Sawada M., Kato T. and Nagatsu T. (1981) Demonstration of tryptophan 5-monooxygenase activity in human brain by highly sensitive high-performance liquid chromatography with fluorometric detection. Biochem. Int. 2: 295-303.
0028-3908/92 $5.00 + 0.00 Copyright0 1992 PergamonPressplc
INHIBITION OF TRYPTOPHAN HYDROXYLASE BY (R)- AND (S)-l-METHYL-6,7-DIHYDROXY-1,2,3,4TETRAHYDROISOQUINOLINES (SALSOLINOLS) M.
OTA,~ P. DOSTERT,* T. HAMANAKA,’
T. NAGATSU~
and M. NAO?
‘Department of Psychiatry, Nagoya City University School of Medicine, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan, 2Farmitalia Carlo Erba, E&D-Erbamont Group, Milan, Italy, ‘Division of Molecular Genetics (II) Neurochemistry, Institute for Comprehensive Medical Science, School of Medicine, Fujita Health University, Toyoake, Aichi 470-l 1, Japan and ‘Department of Biosciences, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466, Japan (Accepted 10 October 1991)
Summary-The (R)- and (S)-enantiomers of salsolinol, the dopamine-derived tetrahydroisoquinolines, were found to inhibit the activity of tryptophan hydroxylase (TPH), prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition of TPH by salsolinols was found to be non-competitive with the substrate L-tryptophan. Tryptophan hydroxylase is composed of two elements with different kinetic properties in terms of cofactor (6R)-L-erylhro-5,6,7,8-tetrahydrobiopterin and these two elements were inhibited by salsolinols in competitive and uncompetitive ways, respectively. Stereoselectivity of salsolinol was not observed, concerning the potency and the type of inhibition on TPH. These data indicate that
salsolinols might be naturally occurring inhibitors of indoleamine metabolism. Key words-salsolinol,
tryptophan
hydroxylase, mastocytoma,
1-Methyl-6,7-dihydroxy1,2,3,4-tetrahydroisoquinoline (salsolinol) is one of the 1,2,3,4-tetrahydroisoquinolines (TIQs), formed by condensation of dopamine with pyruvate, followed by decarboxylation (Brossi, 1982) and an asymmetric center at the C-l position of the salsolinol molecule gives rise to (R)- and (S)-enantiomers (Strolin-Benedetti, Bellotti, Pianezzola, Moro, Carminanti and Dostert, 1989). Another pathway of synthesis of salsolinol is the condensation of dopamine with acetaldehyde by the Pictet-Spengler reaction. The presence of salsolinol in humans was reported for the first time in the urine of Parkinsonian patients on L-DOPA medication (Sandier, Bonham-Carter, Hunter and Stern, 1973) and later found in the urine and cerebrospinal fluid (C.S.F.) of healthy volunteers (Collins, Nijm, Borge, Teas and Golfarb, 1979; Sjiiquist, Borg and Kvande, 1981). In the urine of healthy subjects, only the (R)-enantiomer of salsolinol was detected (Strolin-Benedetti et al., 1989). On the other hand, a large amount of (S)-salsolinol was found in port wine (Dostert, Strolin-Benedetti and Dordain, 1988). Moreover, high levels of salsolinol were detected in several regions of the brain, derived from intoxicated alcoholics (Sjsquist, Eriksson and Winblad 1982). These observations indicate that salsolinol may have some role in the development of intoxication with alcohol.
*To whom correspondence should be addressed. NP 31/4-C
L-tryptophan, (6R)tetrahydrobiopterin.
A number of clinical findings, implying an etiological role for serotonergic dysfunction in alcoholic organic brain diseases, have accumulated in recent years. For example, in patients with alcoholic amnestic disorder, sleep abnormalities (Martin, Loewenstein, Kaye, Ebert, Weingartner and Gillin, 1986) and reduced daily production of 6-hydroxymelatonin (Martin, Higa, Bums, Tamarkin, Ebert and Markey, 1984) were observed. The serotonin-uptake blocker, fluvoxamine maleate, effectively improved episodic memory in patients with alcoholic amnestic disorder (Martin, Adinoff, Eckardt, Stapleton, Bone, Rubinow, Lane and Linnoila, 1989). Furthermore, a reduction in levels of S-hydroxyindoleacetic acid in C.S.F. was documented in some alcoholics, whose brain function was not overtly impaired (Ballenger, Goodwin, Major and Brown, 1979; Banki, 1981). In order to find the effect of salsolinol on the enzymes involved in indoleamine metabolism, tryptophan hydroxylase [L-tryptophan, tetrahydropteridine : oxygen oxidoreductase (5-hydroxylating), EC 1.14.16.4, TPH] was chosen, because it is the ratelimiting enzyme in the synthesis of serotonin. The enzyme used in this experiment was prepared from mastocytoma P-8 15 cells, which produce serotonin and have high TPH activity. In this paper, the inhibition of TPH activity by (R)- and (S)-salsolinol is described. The inhibition is discussed in relation to the possible role of this amine as an inhibitor of indoleamine metabolism. 337
M. OTA
338 METHODS
(R)- and (S)-enantiomers of salsolinol were synthesized as previously reported (Teitel, O’Brien and Brossi, 1972). The chemicals, purchased from Sigma Chemical Co. (St Louis, Missouri, U.S.A.) were: phenylmethylsulfonyl fluoride (PMSF), antipain, peptstatin A, chymostatin, leupeptin, L-tryptophan, 5hydroxytryptophan (5HTP), NSD-1015 (m-hydroxybenzylhydrazine) and bovine y-globulin. Dithiothreitol (DTT), fl-mercaptoethanol and sodium octanesulfonate were from Nacalai Tesque (Kyoto, Japan). Sephadex G-25 was from Pharmacia Fine Chem. (Uppsala, Sweden). (6R)-L-eryfhro-5,6,7,8Tetrahydrobiopterin hydrochloride [(GR)BH,] was kindly donated by Dr Matsuura (Fujita Health University, Toyoake, Japan). Catalase, prepared from bovine liver, was purchased from BoehringerMannheim (Mannheim, Fed. Rep. Germany). Organic solvents were of HPLC grade. The murine mastocytoma cell line p-815, established in DBA mice (Dunn and Potter, 1957) and carried in DBAj2 mice (Hosoda and Glick, 1965), was kindly donated by Dr Hosoda (Aichi Cancer Institute, Nagoya, Japan). The ascitic fluid was taken and the cells were gathered by centrifugation at 1OOOgfor 10 min. The cells were washed twice with Hank’s balanced salt solution (HBSS) and then suspended in HBSS and stored at -80°C until use. The cells (250 mg protein) were diluted with the same volume as the extraction medium; 100 mM Tris-acetate buffer, pH 7.6, containing 2 mM PMSF, 4 mM DTT, 10 pgg/ml each of antipain, pepstatin A, chymostatin and leupeptin. The cell suspension was sonicated and centrifuged at 100,000 g for 30 min. The supernatant was passed through a Sephadex G-25 column (2 cm i.d. x 15 cm) equilibrated with 10 mM Tri-acetate buffer, pH 7.6, containing 1 mM PMSF. The fractions were eluted out by the same buffer in the void volume, gathered and used as the enzyme sample. The activity of TPH was measured by quantitative analysis of 5-hydroxytryptophan (5-HTP), produced from L-tryptophan in the presence of NSD1015, an inhibitor of aromatic L-amino acid decarboxylase, according to a previously reported method (Yamaguchi, Sawada, Kato and Nagatsu, 1981) with
et al.
a slight modification (Naoi, Hosoda, Ota, Takahashi and Nagatsu, 1990).The validity of the assay conditions was confirmed as reported previously. The enzyme sample (about 60 pg protein) was incubated at 37°C for 20min with 100nM r.-tryptophan in lOOn of 50 mM HEPESNaOH buffer, pH 7.6, containing 100 nM (6R)BH,, 100 nM j-mercaptoethanol, catalase (0.4mg protein) and 0.5 mM NSD-1015. The reaction was terminated by addition of 10~1 of 60% perchloric acid and the sample was mixed, centrifuged at 15,000 g for 10 min and filtered through a Millipore HV filter (pore size 0.45 pm). For quantification of 5-HTP, the sample was applied onto a Shimadzu HPLC apparatus, LCSA, connected to a Shimadzu fluorescence detector, FD-500. The column used was a pre-packed reversed-phase column, ODS-H (4 mm id. x 150 mm, Shimadzu Techno-Research, Kyoto, Japan) and the mobile phase was 90mM sodium acetate-35 mM citric acid buffer, pH 4.35, containing 130pM disodium EDTA and 2 mM sodium octanesulfonate, to which methanol was added to 11%. The flow rate was 0.8 ml/min. The fluorescence intensity at 345 nm was measured with excitation at 295 nm. Quantification of 5-HTP was carried out by comparison of the peak area with that of a standard. Protein concentration was measured according to Bradford (1976), using bovine y-globulin as standard. The values of the Michaelis constant (K,,,) and the maximum velocity (I’,,,,,), in terms of the substrate or the cofactor, were obtained by Lineweaver-Burk’s plot. The effects of (R)- and (S)-salsolinols on the activity of TPH were examined at concentrations from 1 nM to 1 mM. The activity was measured with the enzyme (6Opg protein), in the presence of 10 nM L-tryptophan and 100 FM (6R)BH, or 100pM L-tryptophan and 10,nM (6R)BH,. The activity of the enzyme was measured in the absence or presence of 20 PM (R)- or (S)-salsolinol. The type and the value of the inhibition constant (K,) were also estimated by Lineweaver-Burk’s plot. RESULTS
Kinetic properties of TPH used in these experiments are summarized in Table 1. The values of K,,,
Table I. Kinetic properties of a sample of TPH
In terms of substrate. t-tryptophan
(,“G)
v mar (pmol/min/mg protein)
14.8 k 1.7
262 k 21 Vm.ii2
VIns?,>
(pmol/min/mg protein)
(%)
(pmol/min/mg protein)
In terms of cofactor, 437 * 137 127 f 27 93.4 f 14.2 5.04 * 0.52 (6R)BH, Kinetic properties of TPH, prepared from murine mastocytoma cell line P-815. Each value represents the mean k SD of duplicate measurements of three experiments. The kinetic data of the enzyme activity, in terms of t-tryptophan, were determined from the data obtained using 100 PM (6R)BH,. The kinetic data of the enzyme activity, in terms of (6R)BH,, were determined from the data, obtained using 100 PM r-tryptophan.
Inhibition of TPH by (R)- and (S)-salsohnols (A) L -Tryptophan
(S)
339
(6R)BH4
Salsolinol (,uM)
Fig. 1. Inhibition of the activity of TPH by (R)- and (S)-salsohnols. In (A) the activity of TPH was obtained with 10&M L-tryptophan and 100pM (6R)BHI. In (B) activity of TPH was obtained with 100 PM L-tryptophan and 10 PM (6R)BH,. The activity of TRH for (A) was 99.1 + 4.9 pmol/min/mg protein and that for (B) was 67.9 + 2.1 pmol/min/mg protein. The relative activity of TPH was expressed as a percentage of the activity of control. Each point represents the means + SD of triphcate measurements. (O), OR)-salsolinol; (A), (~)-salsolinol.
and V,,, of the enzyme
in terms
of the substrate,
were 14.8 PM and 262 pmol/min/mg protein, respectively. On the contrary, in the case of the substrate, Lineweaver-Burk’s plot in terms of cofactor, (6~)BH~, showed that TPH was composed of two components with different kinetic characteristics (see Fig. 3, control). One component (termed as Component 1) had a low Km and V,,,,, value, 5 PM and 93 pmol/min/mg protein and the other (termed as Component 2) had a high Km and if,,, value, 127 FM and 437 pmol/min/mg protein, respectively. The effects of (R)- and (S)-salsolinols on the activity of TPH were examined, using a small concentration of L-tryptophan (10 PM) and a large concentration of the cofactor (IOOpM) (Fig. IA) and vice versa (Fig. IB), using IOOpM L-tryptophan and 10 FM (BR)BH,. Both enantiomers of salsolinol were L-tryptophan,
0.06
0.06
1
0.0
found to inhibit the activity of TPH in a dose-dependent manner, as shown in Fig. 1. One mM of(R)- and (S)-salsolinol inhibited the activity of TPH almost totally. The concentrations of salsolinol which gave the 50% inhibition of the activity of TPH (IC,) were almost identical between (R)- and (Qforrn, both in the presence of smaller concentration of the substrate and of the cofactor. The IC,, of salsolinol was found to be around 20 /LM, in regard to the substrate, which was almost the same as that in regard to the cofactor. The type of inhibition of the activity of TPH by salsolinol was characterized by plotting the data, according to Lineweaver-Burk, with various concentrations of L-tryptophan and (6R)BH,. The concentration of salsolinol was fixed at 20 /IM throughout these experiments. Figures 2 and 3 show typical results of the Lineweaver-Burk’s plot for three inde~ndent
0.1
l/[L-tryptophan]
0.2
0.3
(PM “)
Fig. 2. Effects of the concentration of L-tryptophan on activity of TPH in the absence and presence of 20 FM (R)or (S)-salsolinol. The activity of TPH was measured with nine different concentrations (from 3.9 to 1OOgM) of L-tryptophan and 100 PM (6R)BH,. The reciprocal of the reaction velocity was plotted against that of the concentration, according to Lineweaver and Burk. This figure represents one of three experiments, done in duplicate. (a), Control; (O), (R)-salsohnol; ( x ), (S)-salsolinol.
Al 0.0
I
0.1
0.2
0.3
l/[(GR)BHr] (CIM“)
Fig. 3. Effects of the concentration of (6R)BH, on the activity of TPH in the absence and presence of 20 RM (R)or (S)-salsolinol. The activity of TPH was measured with nine different concentrations of (6R)BH, (from 3.9 to 100 uM) and 100 uM L-trvutonhan. The reciurocal of the _. _
reaction velocity was plotted against that of the concentration of (6R)BH, according to Lineweaver and Burk. This
figure represents one of three experiments, done in duplicate. (O), Control; (O), (R)-salsolinol; ( x ), (S)-salsolinol.
M. OTAet 01.
340
experiments. Salsolinol inhibited the activity of TPH in a non-competitive way to L-tryptophan (Fig. 2). The inhibition constant, Ki, values of (R)- and (S)salsolinol obtained were 15.4 f 5.3 FM and 19.9 + 6.8 PM (mean f SD of duplicate measurements of three experiments), respectively. As shown in Fig. 3, (R)- and (S)-salsolinol inhibited both components and the inhibition of Component 2 was competitive to (6R)BH.,, while the inhibition of Component 1 was uncompetitive. The K, values for competitive inhibition of Component 2 by (R)- and (S)-salsolinol were 12.0 + 4.6 PM and 19.0 + 4.9 PM, respectively; K, values for uncompetitive inhibition of Component 1 by (R)- and (S)-salsolinol were 12.5 + 2.4 PM and 17.4 f 4.9 PM, respectively. These K, values were not statistically different between (R)- and (S)-salsolinol. Inhibition of TPH by salsolinol was not stereoselective for both substrate and cofactor. DISCUSSION
The effects of salsolinol on the activity of tyrosine hydroxylase (TH) and DOPA decarboxylase (DDC), derived from the brain of the rat were described by Weiner and Collins (1978). In their report, racemic salsolinol inhibited the activity of TH in competition with a synthetic cofactor, (6RS)-methyl5,6,7,%tetrahydrobiopterin, with a K, value at the level of 10pM. In addition, they also reported that 100pM salsolinol did not appreciably affect the activity of DDC. On the other hand, the effects of tetrahydroisoquinolines, such as salsolinol, on indoleamine metabolism have not been reported. This paper reports in vitro inhibition of the activity of TPH by salsolinol, in respect to both the substrate and cofactor. The concentrations of (R)- and (S)salsolinols in the human brain have been reported (Ung-Chhun, Cheng, Pronger, Serrano, Chavez, Perez, Morales and Collins, 1985; Sjoquist et al., 1982). In the brain of patients with intoxicated alcoholism, the concentration of salsohnol in the caudate was up to 0.3 PM (Sjoquist et al., 1982). Considering the intracellular compartment of salsolinol with TPH, the concentration of salsolinol in the neurons may be much greater and compatible to the K, value of salsolinols, 10 PM. The inhibition of the activity of TPH by salsolinol was not stereoselective. This observation was different from the case of type A monoamine oxidase (MAO). The R form inhibited MAO much more effectively than S form (Bembenek, Abell, Chrisey, Rozwadowska, Gessner and Brossi, 1990). These results suggest that the binding site of salsolinol to TPH and type A MAO is not the same. Type A MAO may recognize the N-atom at C-2 position and a methyl group at C-l position but TPH cannot distinguish the stereoisomers at C-l position. The binding site of salsolinol to TPH may be quite different; salsolinol may be bound near at its hydroxy group of C-6 and/or C-7 position.
The influence of alcohol on indoleamine metabolism has mainly focused on aldehyde dehydrogenase, which shares the function on serotonin degrading process as reviewed by Truitt (1973). It has also been proposed that tetrahydroisoquinolines play a role in alcoholism (Davis and Walsh, 1970; Collins et al., 1979; Cohen and Collins, 1970), although their mechanism has not yet been fully clarified. This report supports the idea that the enhanced amount of salsolinol suppresses the activity of TPH resulting in the reduction of the amount of serotonin. It might be relevant to the observation that in the alcoholics the content of serotonin was decreased in the tissues or organs, such as platelets which store serotonin (Bailly, Vignau, Lauth, Racadot, Beuscart, Servant and Parquet, 1990). (R)-Salsolinol is known to be synthesized endogenously, whereas the (S)-form is derived from food or beverages, such as port wine (Strolin-Benedetti et al., 1989; Dostert, Strolin-Benedetti, Bellotti, Allievi and Dordain, 1990). (R)- and (S)-salsolinol inhibited the activity of TPH almost to the same degree in this experiment. This observation indicates that both enantiomers of salsolinol may inhibit the activity of TPH in vivo. The origin of salsolinol, detected in the brain, still remains to be clarified, since the transport of salsolinol through the blood-brain barrier has not been shown (Origitano, Hannigan and Collins, 1981). However, it is still noteworthy that the inhibition of salsolinol on TPH, a rate-controlling enzyme in the production of serotonin has been shown. Thus, dopamine-derived human alkaloids may regulate the level of serotonin in the human brain under physiological and pathological conditions, such as alcoholism, ageing and treatment
with L-DOPA.
REFERENCES Bailly D., Vignau J., Lauth B., Racadot N., Beuscart R., Servant D. and Parquet P. J. (1990) Platelet serotonin decrease in alcoholic patients. Acta psychiat. stand. 81: 68-12. Ballenger J. C., Goodwin F. K., Major L. F. and Brown G. L. (1979) Alcohol and central serotonin metabolism in man. Archs gem. Psychiat. 36: 224-221. Banki C. M. (1981) Factors influencing monoamine metabolites and tryptophan in patients with alcohol dependence. J. Neural Transm. 50: 89-101. Bembenek M. E., Abel1 C. W., Chrisey L. A., Rozwadowska M. D., Gessner W. and Brossi A. (1990) Inhibition of monoamine oxidases A and B by simple isoquinoline alkaloids: racemic and optically active 1,2,3,4_tetrahydro, 3,4-dihydro-, and fully aromatic isoquinolines. J. med. Chem. 33: 147-152. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Analyt. Biochem. 72: 248-254.
Brossi A. (1982) Mammalian TIQs: products of condensation with aldehydes or pyruvic acids? Prog. chit. Biol. Res. 90: 123-133. Cohen G. and Collins M. (1970) Alkaloids from catecholamines in adrenal tissue: possible role in alcoholism. Science 167: 1749-1751.
Inhibition of TPH by (R)- and (S)-salsolinols Collins M. A., Nijm W. P., Borge G. F., Teas G. and Golfarb C. (1979) Dopamine-related tetrahydroisoquinolines: significant urinary excretion by alcoholics after alcohol consumption. Science 206: 1184-l 186. Davis V. E. and Walsh M. J. (1970) Alcohol, amines and alkaloids: a possible biochemical basis for alcohol addiction. Science 167: 1005-1007. Dostert P., Strolin-Benedetti M., Bellotti V., Allievi C. and Dordain G. (1990) Biosynthesis of salsolinol, a tetrahydroisoquinoline alkaloid, in healthy subjects. J. Neural Transm. 81: 215-223. Dostert P., Strolin-Benedetti M. and Dordain G. (1988) Dopamine-derived alkaloids in alcoholism and in Parkinson’s and Huntington’s diseases. J. Neural Transm. 74: 61-74. Dunn T. B. and Potter M. (1957) A transplantable mastcell neoplasm in the mouse. J. natn Cancer Inst. 18: 587601. Hosoda S. and Glick D. (1965) Biosynthesis of 5-hydroxytryptophan by neoplastic mouse mast cells. Biochim. biophys. Acta 111: 67-78.
Martin P. R., Adinoff B., Eckardt M. J., Stapleton J. M., Bone G. A. H., Rubinow D. R., Lane E. A. and Linnoila M. (1989) Effective pharmacotherapy of alcoholic amnestic disorder with fluvoxamine. Archs gen. Psychiat. 46: 617621.
Martin P. R., Higa S., Burns R. S., Tamarkin L., Ebert M. H. and Markey S. P. (1984) Decreased 6-hydroxymelatonin excretion in KorsakotI’s psychosis. Neurology 34: 966-968. Martin P. R., Loewenstein R. J., Kaye W. H., Ebert M. H., Weingartner H. and Gillin J. C. (1986) Sleep EEG in Korsakoff’s psychosis and Alzheimer’s disease. Neurology 36: 411414. Naoi M., Hosoda S., Ota M., Takahashi T. and Nagatsu T. (1990) Inhibition of tryptophan hydroxylase by food-derived carcinogenic heterocyclic amines, 3-amino-I-methyl-SH-pyrido[4,3-blindole and 3-amino1,4-dimethyl-SH-pyrido[4,3-blindole. Biochem. Pharmac. 41: 199-203.
341
Origitano T., Hannigan J. and Collins M. A. (1981) Rat brain salsolinol and blood-brain barrier. Brain Res. 224: 446-451.
Sandler M., Bonham-Carter S., Hunter K. R. and Stern G. M. (1973) Tetrahydroisoquinoline alkaloids: in uiuo metabolites of L-DOPA in man. Nature 241: 43943.
Sjijquist B., Borg S. and Kvande H. (1981) Salsolinol and methylated salsolinol in urine and cerebrospinal fluid from healthy volunteers. Subst. Alcohol Actions Misuse 2: 73-77.
Sjiiquist B., Eriksson A. and Winblad B. (1982) Salsolinol and catecholamines in human brain and their relation to alcoholism. Prog. clin. Biol. Res. 90: 5767. Strolin-Benedetti M., Bellotti V., Pianexxola E., Moro E., Carminati P. and Dostert P. (1989) . , Ratio of the R and S enantiomers of salsolinol in food and human urine. J. Neural Transm. 17: 47-53.
Teitel S., O’Brien J. and Brossi A. (1972) Alkaloids in mammalian tissues. 2. Synthesis of ( + ) and ( - ) substituted-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines. J. med. Chem. 15: 845-846.
Truitt E. B. Jr (1973) A biogenic amine hypothesis for alcohol tolerance. Ann. N. Y. Acad. Sci. 215: 177-182.
Ung-Chhun N., Cheng B. Y., Pronger D. A., Serrano P., Chavez B., Perez R. F., Morales J. and Collins A. (1985) Alkaloid adducts in human brain: coexistence of I-carboxylated and noncarboxylated isoquinolines and /?-carbolines in alcoholics and nonalcoholics. Prog. clin. Biol. Res. 183: 125-136. Weiner C. D. and Collins M. A. (1978) Tetrahydroisoquinolines derived from catecholamines or DOPA: effects on brain tyrosine hydroxylase activity. Biochem. Pharmac. 27: 2699-2703.
Yamaguchi T., Sawada M., Kato T. and Nagatsu T. (1981) Demonstration of tryptophan 5-monooxygenase activity in human brain by highly sensitive high-performance liquid chromatography with fluorometric detection. Biochem. Int. 2: 295-303.
