Stimulation of Glutamate, Aspartate and Gamma-Aminobutyric Acid Release from Synaptosomes by Hyperforin

Hyperforin is one pharmacologically active constituent of the medicinal herb Hypericum perforatum. The mechanism of its antidepressant-like activity is currently considered to be the inhibition of synaptic reuptake of neurotransmitters. Here, we will demonstrate that it also stimulates the release of glutamate from rat cortical synaptosomes, and that this effect is preceded by an increase in their free calcium [Ca²⁺]_i levels. These hyperforin-related effects were also observed in the absence of Ca²⁺ in the medium. Although we noted enhanced glutamate, aspartate and GABA release under the influence of hyperforin, the release of various other amino acids was not enhanced. In contrast, reserpine did not influence the release of any of the amino acids studied. Adding hyperforin to synaptosomal suspension decreased their pHi, which returned to basal levels under certain incubation conditions. It also prevented the generation of ATP-induced pH gradients of isolated synaptic vesicles, and preformed pH-gradients were reversed by it. We will discuss the implications of our studies in understanding the mechanisms of hyperforin activity in relation to current findings on its pharmacological activity profile. Our observations suggest that neurotransmitter release stimulation from synaptosomes and the previously reported reuptake inhibitory properties of hyperforin are consequences of its effects on synaptosomal ionic homeostasis, and that it is not a reserpine-like agent.

References

• 1 Barrie A, Chieregatti E, Miloso M, Benfenati F, Valtorta F. Epidemial growth factor-mediated inhibition of neurotransmitter glutamate release from rat forebrain synaptosomes.  Mol Pharmacol. 1996;  49 399-403
• 2 Bhattacharya S K, Chakrabarti A, Chatterjee S S. Activity profiles of two hyperforin-containing Hypericum extracts in behavioral models.  Pharmacopsychiat. 1998;  31 ((Suppl)) 22-29
• 3 Bombardelli E, Morazzoni P. Hypericum perforatum.  Fitoterapia. 1995;  66 43-68
• 4 Butterweck V, Petereit F, Winterhoff H, Nahrstedt A. Solubilized hypericin and pseudohypericin from Hypericum perforatum exert antidepressant activity in forced swimming test.  Planta Med. 1998;  64 291-294
• 5 Butterweck V, Jürgenliemk G, Nahrstedt A, Winterhoff H. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test.  Planta Med. 2000;  66 3-6
• 6 Chatterjee S S, Koch E, Nöldner M, Biber A, Erdelmeier C. Hyperforin and hypericum extract: Interaction with some neurotransmitter systems.  Phytomedicine. 1996;  3 ((Suppl. 1)) 106
• 7 Chatterjee S S, Nöldner M, Koch E, Erdelmeier C. Antidepressant activity of Hypericum perforatum and hyperforin: The neglected possibility.  Pharmacopsychiat. 1998;  31 ((Suppl)) 7-15
• 8 Chatterjee S S, Bhattacharya S K, Wonnemann M, Singer A, Müller W E. Hyperforin as a possible antidepressant component of Hypericum extracts.  Life Sci. 1998;  63 499-510
• 9 Chatterjee S S, Erdelmeier C, Klessing K. Stable hyperforin salts: Method for producing them and their use in the treatment of Alzheimer's disease. Int.Pat. PCT Wo 99/41220 1999
• 10 Chatterjee S S, Filippov V, Lishko P, Maximyuk O, Nöldner M, Kristhal O. Hyperforin attenuates various ionic conductance mechanisms in the isolated hippocampal neurones of rat.  Life Sci. 1999;  65 2395-2405
• 11 Connop B P, Thies R L, Beyreuther K, Ida N, Reiner P B. Novel effects of FCCP [carbonyl cyanide p-(Trifluoro-methoxy)phenylhydrazone] on amyloid precursor protein processing.  J Neurochem. 1999;  72 1457-1465
• 12 Dunkley P R, Rostas J AP, Heath J W, Powis D A. The preparation and use of synaptosomes for studying secretion of catecholamines. Chapter 19 in “In vitro methods for studying secretion”. Eds.: Poisner and Torifaró Elsevier Science 1987: 315-334
• 13 Fontana G, Blaustein M P. Calcium buffering and free Ca²⁺ in rat brain synaptosomes.  J Neurochem. 1993;  60 843-850
• 14 Gobbi M, DallaValle F, Ciapparelli C, Diomede L, Morazzoni P, Verotta L, Caccia S, Cervo L, Mennini T. Hypericum perforatum L. extract does not inhibit 5-HT transporter in rat brain cortex.  Naunyn-Schmiedeberg's Arch Pharmacol. 1999;  360 262-269
• 15 Gurevich A I, Dobrynin V N, Kolosov M N, Popravko S A, Ryabova I D, Chernov B K, Derbentseva N A, Aizenman B E, Garagulya A D. Hyperforin an antibiotic from Hypericum perforatum.  Antibiotiki. 1971;  16 510-513
• 16 Hell J W, Maycox P R, Jahn R. Energy dependence and functional reconstitution of γ-aminobutyric acid carrier from synaptic vesicles.  J Biol Chem. 1990;  265 2111-2117
• 17 Kaehler S T, Sinner C, Chatterjee S S, Philippu A. Hyperforin enhances the extracellular concentrations of catecholamines, serotonine and glutamate in rat locus coeruleus.  Neurosci Lett. 1999;  262 199-202
• 18 Kasai H, Takahashi N. Multiple kinetic components and the Ca²⁺ requirements of exocytosis.  Phil Trans R Soc Lond B. 1999;  354 331-335
• 19 Klusa V, Germane S, Nöldner M, Chatterjee S S. Hypericum extract and hyperforin: Cognitive function modulating properties in rodents.  Pharmacopsychiat. 2001;  34 ( Suppl1) 61-69
• 20 Koch E, Chatterjee S S. Hyperforin stimulates intracellular calcium mobilization and enhances extracellular acidification in DDT₁-MF² smooth muscle cells.  Pharmacopsychiat. 2001;  ( Suppl1) 34 70-73
• 21 Lindroth P, Mopper K. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde.  Anal Chem. 1979;  51 1667-1674
• 22 Lowry O H, Rosebrough N J, Farr A L, Randall R J. Protein determination with the Folin phenol reagent.  J Biol Chem. 1951;  193 265-275
• 23 Müller W E, Singer A, Wonnemann M, Hafner U, Rolli M, Schäfer C. Hyperforin represents the neurotransmitter reuptake inhibiting constituent of Hypericum extract.  Pharmacopsychiat. 1998;  31 ((Suppl)) 16-21
• 24 Müller W E, Chatterjee S S. Hyperforin and the antidepressant activity of St. John's Wort.  Pharmacopsychiat. 1998;  31 ((Suppl)) 1-60
• 25 Nicholls D G, Sihra T S, Sanchez-Prieto J. Calcium-dependent and- independent release of glutamate from synaptosomes monitored by continuous fluorometry.  J Neurochem. 1987;  49 50-57
• 26 Nöldner M, Chatterjee S S, Erdelmeier C. Pharmacological studies leading to the identification of hyperforin as a CNS-active component of Hypericum perforatum.  Naunyn-Schmiedeberg's Arch Pharmacol. 1998;  358 ((Suppl. 2)) R 495
• 27 Roseth S, Fykse E M, Fonnum F. The effect of arachidonic acid and free fatty acids on vesicular uptake of glutamate and γ-aminobutyric acid.  Eur J Pharmacol. 1998;  341 281-288
• 28 Schrader E, Meier B, Brattström A. Hypericum treatment of mild-moderate depression in a placebo-controlled study. A prospective, double-blind, randomised, placebo-controlled, multicentre study.  Hum Psychopharmacol. 1998;  13 163-169
• 29 Scotti A L, Chatton J Y, Reuter H. Roles of Na⁺-Ca²⁺ exchange and of mitochondria in the regulation of presynaptic Ca²⁺ and spontaneous glutamate release.  Phil Trans R Soc Lond B. 1999;  354 357-364
• 30 Singer A, Wonnemann M, Müller W E. Hyperforin, a major antidepressant constituent of St. John's Wort, inhibits uptake by elevating free intracellular Na⁺.  J Pharmacol Exp Ther. 1999;  290 1363-1368
• 31 Sokal A, Bartosz G. Uncouplers of mitochondrial oxidative phosphorlation are not substrates of erythrocyte glutathione-S-conjugate pump.  Arch Biochem Biophys. 1998;  349 113-121
• 32 Svoboda K, Mainen Z F. Synaptic [Ca²⁺]: Intracellular stores spill their guts.  Neuron. 1999;  22 427-430
• 33 Tretter L, Chinopoulus C, Adam-Vizi V. Plasma membrane depolarization and disturbed Na⁺ homeostasis induced by protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon in isolated nerve terminals.  Mol Pharmacol. 1998;  53 734-741
• 34 Wonnemann M, Singer A, Müller W E. Inhibition of synaptosomal uptake of ³H-L-glutamate and ³H-GABA by hyperforin, a major constituent of St. John's Wort: The role of amiloride sensitive sodium conductive pathways.  Neuropsychopharmacol. 2000;  23 188-197
• 35 Zoccarato F, Cavallini L, Alexandre A. The pH-sensitive dye acridine orange as a tool to monitor exocytosis / endocytosis in synaptosomes.  J Neurochem. 1999;  72 625-633