Hyperforin, a Major Antidepressant Constituent of St. John’s Wort, Inhibits Serotonin Uptake by Elevating Free Intracellular

نویسنده

  • W. E. MÜLLER
چکیده

Extracts of Hypericum perforatum (St. John’s Wort) are widely used for the treatment of depressive disorders and are unspecific inhibitors of the neuronal uptake of several neurotransmitters. Previous studies have shown that hyperforin represents the reuptake inhibiting constituent. To characterize the mechanism of serotonin reuptake inhibition, kinetic analyses in synaptosomes of mouse brain were performed. Michaelis-Menten kinetics revealed that hyperforin (2 mM) induces a decrease in Vmax by more than 50% while only slightly decreasing Km, indicating mainly noncompetitive inhibition. By contrast, citalopram (1 nM) leads to an elevation of Km without changing Vmax. Monensin, a Na/H exchanger, showed similar properties as hyperforin (decrease of Vmax without changing Km). Compared with classical antidepressants, such as selective serotonin reuptake inhibitors and tricyclic antidepressants, hyperforin is only a weak inhibitor of [H]paroxetine binding relative to its effects on serotonin uptake. As monensin decreases serotonin uptake by increasing Na/H exchange, we compared the effects of hyperforin and monensin on the free intracellular sodium concentration ([Na]i) in platelets by measuring 1,3benzenedicarboxylic acid, 4,49-[1,4,10-trioxa-7,13-diazacyclopentadecan-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetraammonium salt (SBFI/AM) fluorescence. Both drugs elevated [Na]i over basal levels, with a maximal [Na ]i of 69 6 16.1 mM (50 mM hyperforin) and 140 6 9.1 mM (10 mM monensin). Citalopram at concentrations relevant for [H]serotonin uptake inhibition had no effect on [Na]i. Although the mode of action of hyperforin seems to be associated with elevated [Na]i similar to those levels found with monensin, the molecular mechanism might be different, as even at high concentrations, hyperforin does not elevate free intracellular sodium concentration ([Na]i) up to the extracellular level, as monensin does. Hyperforin represents the first substance with a known preclinical antidepressant profile that inhibits serotonin uptake by elevating [Na]i. Extracts of the medicinal plant Hypericum perforatum (St. John’s Wort) are broadly used in many countries to treat depressive disorders. Several recent reviews of controlled clinical studies with hypericum extract have come to the conclusion that it represents an effective antidepressant principle superior to placebo (Linde et al., 1996; Volz, 1997; Wheatley, 1998; Wong et al., 1998). In most clinical studies, hypericum extract was used at a dosage of 300 mg three times daily. A favorable side effect profile is considered its most relevant advantage. In agreement with the possible therapeutic potential, many recent pharmacological studies with hypericum extract also support its antidepressant activity. The extract inhibits the synaptosomal uptake of norepinephrine, serotonin, and dopamine, induces b-receptor down-regulation when given subchronically to rats, and is active in a large variety of behavioral models indicative of antidepressant activity (Bhattacharya et al., 1998; Butterweck et al., 1997; Chatterjee et al., 1998; Müller et al., 1998). However, monoamine oxidase (MAO)-Aand MAO-B-inhibiting properties of the extract are probably too weak to significantly contribute to its antidepressant activity (Cott, 1997; Müller et al., 1997). The extract contains a large number of constituents. It is not known which of these constituents is responsible for the antidepressant properties of the extract. The original assumption (Suzuki et al., 1984), that the naphthodianthrone derivative hypericin is specifically relevant by inhibiting MAO-A, has not been confirmed by several subsequent studies (Cott, 1997; Müller et al., 1997). Recently, the phloroglucinol derivative hyperforin became of increasing interest, as Received for publication March 5, 1999. 1 This study was supported by a research grant of Dr. Schwabe Arzneimittel (Karlsruhe, Germany), Lichtwer AG (Berlin, Germany), and by the Fond der Chemischen Industrie. ABBREVIATIONS: MAO, monoamine oxidase; SBFI/AM, 1,3-benzenedicarboxylic acid, 4,49-[1,4,10-trioxa-7,13-diazacyclopentadecan-7,13diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetraammonium salt; RT, room temperature; SSRI, selective serotonin reuptake inhibitor; [Na]i, free intracellular sodium concentration. 0022-3565/99/2903-1363$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 290, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 290:1363–1368, 1999 1363 at A PE T Jornals on A uust 5, 2017 jpet.asjournals.org D ow nladed from it represents the major reuptake-inhibiting component of hypericum extract (Chatterjee et al., 1998; Müller et al., 1998). Moreover, it leads to elevated extracellular brain concentrations of serotonin, norepinephrine, and dopamine after administration to rats (Kaehler et al., 1999), down-regulates b-receptors (Müller et al., 1997), is active in several behavioral models indicative of antidepressant activity (Chatterjee et al., 1998; Bhattacharya et al., 1998), leads to specific changes of the rat and human EEG typical for selective serotonin reuptake inhibitors (SSRIs; Dimpfel et al., 1998; Schellenberg et al., 1998), and reaches plasma levels in the rat needed to inhibit synaptosomal uptake of several neurotransmitters (about 700 nM; Biber et al., 1998) at a single dose of hypericum extract of 300 mg/kg, which is effective in most behavioral studies (Bhattacharya et al., 1998; Butterweck et al., 1998). In humans, hyperforin plasma levels of about 600 nM were seen after a single dose of 600 mg of hypericum extract (Biber et al., 1998). It also seems to be relevant for the antidepressant activity of hypericum extract in humans (Laakmann et al., 1998). Thus, hyperforin has to be considered an important antidepressant constituent of hypericum extract. Contrary to all other antidepressant drugs known, hyperforin not only potently inhibits the synaptosomal uptake of norepinephrine, dopamine, and serotonin, but also the uptake of the two amino acid transmitters, g-aminobutyric acid and L-glutamate (Chatterjee et al., 1998). This might point to a different mechanism of action, probably not associated with specific binding sites at the transporter molecules. Accordingly, we have performed several additional experiments comparing the molecular basis of the serotonin uptake inhibition by hyperforin with that of SSRIs like citalopram and with the uptake inhibition by the sodium ionophore monensin (Reith and O’Reilly, 1990; Izenwasser et al., 1992). Monensin has been shown to elevate [Na]i in a variety of different cell types in vitro (Izenwasser et al., 1992; Adovelande and Schrével, 1996; Rochdi et al., 1996). It has been used for many years in veterinary medicine to treat gastrointestinal infections like coccidiosis in cattle (Bergen and Bates, 1984). As it unspecifically enhances intracellular sodium concentration, it is rather toxic. Monensin is a very hydrophilic molecule and is probably not reaching the brain. In the present study, it was exclusively used as a tool for in vitro experiments to elevate free intracellular sodium by an unspecific physiochemical mechanism independent of the physiological systems regulating sodium conductance. Experimental Procedures Synaptosomal Uptake of Serotonin Synaptosomal preparations from the frontal cortex of female NMRI mice were used for serotonin uptake. The tissue was homogenized in ice-cold sucrose solution (0.32 M) and diluted with 10 ml of the homogenizing medium. The nuclear fraction was eliminated by centrifugation at 750g for 10 min (Beckman Centrifuge, Model J2– 21; Beckman Coulter, Fullerton, CA). The supernatant was centrifugated at 17400g for 20 min to obtain the crude synaptosomal pellets. The pellets were suspended in 11 ml of ice-cold Krebs-HEPES-buffer (150 mM NaCl, 10 mM HEPES, 6.2 mM KCl, 1.2 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Glucose, 10 mM Pargylin, and 0.1% ascorbic acid; pH 7.4 at 37°C), aliquoted in microtiter plates, incubated in the presence of varying concentrations of the drugs tested at 37°C for 15 min in a shaking water bath, and cooled on ice. The H-labeled tracer (2.9 nM serotonin) was added and the uptake started by incubation at 37°C for 4 min, during which time uptake is linearly dependent on time. The probes were cooled, immediately filtered through Whatman GF/B glass fiber filters, and washed three times with ice-cold buffer solution with a Brandel cell harvester. Filters were placed in plastic scintillation vials containing 4 ml of Lumasafe scintillation cocktail (Packard, Meriden, CT). Radioactivity was measured after 12 h. Nonspecific uptake was determined in parallel probes maintained throughout on ice or containing unlabeled serotonin (1 mM serotonin).

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تاریخ انتشار 1999