title: Specific binding and characteristics of geissoschizine methyl ether, an indole alkaloid of Uncaria Hook, in the rat brain.
abstract: BACKGROUND
  Geissoschizine methyl ether (GM) is an indole alkaloid that is a component of Uncaria Hook, and has been identified as the active component responsible for the anti-aggressive effects of the Uncaria Hook-containing traditional Japanese medicine, yokukansan. Recently, GM was shown to reach the brain by crossing the blood-brain barrier in rats following the oral administration of yokukansan. This finding suggested that there may be specific binding sites for GM in the brain. Here we show evidence that tritium-labeled GM ([(3)H]GM) binds specifically to several brain areas of rats.
  
  
  METHODS
  Male rats were used. [(3)H]GM was synthesized from a demethylated derivative of GM. Specific binding sites of [(3)H]GM on brain sections were determined by quantitative autoradiography, and maximum binding densities (Bmax) and dissociation constants (Kd) were calculated. Several chemical compounds were used to clarify the molecules that recognize [(3)H]GM in the completion-binding assay. Emulsion microautoradiography was also performed to identify the cells that bind [(3)H]GM.
  
  
  RESULTS
  Specific binding of [(3)H]GM was observed in the frontal cortex, including the prefrontal cortical region (e.g., prelimbic cortex (PrL)), hippocampus, caudate putamen, amygdala, central medial thalamic nucleus, dorsal raphe nucleus (DR), and cerebellum. Bmax ranged between 0.65 and 8.79pmol/mg tissue, and Kd was between 35.0 and 232.6nM. Specific binding with relatively high affinity (Kd less than 62nM) was dense in the frontal cortical region, moderate in the DR, and sparse in the cerebellum. The specific binding of [(3)H]GM in the PrL was significantly replaced by the serotonin 1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), 5-HT2A receptor antagonist ketanserin, 5-HT2B receptor agonist BW 723C86, 5-HT2C receptor agonist RO60-0175, adrenergic α2A receptor antagonist yohimbine, L-type Ca(2+) channel blocker verapamil, and μ-opioid receptor antagonist naloxone. Similar results were obtained in the frontal cortex and DR, but not in the cerebellum. Microautoradiography revealed that [(3)H]GM signals were distributed throughout the frontal cortex, which included neuron-like large cells.
  
  
  CONCLUSIONS
  These results demonstrate that specific binding sites for GM exist in rat brain tissue, and suggest that the pharmacological actions of GM are mainly associated with 5-HT receptors in the frontal cortex and DR. These results provide an insight into the neuropharmacology of GM and GM-containing herbal medicines.
authors.affiliation: Tsumura Research Laboratories, Kampo Scientific Strategies Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan. Electronic address: mizoguchi_kazushige@mail.tsumura.co.jp.
authors.forename: Kazushige
authors.initials: K
authors.lastname: Mizoguchi
authors.affiliation: Tsumura Research Laboratories, Kampo Scientific Strategies Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Kushida
authors.affiliation: Tsumura Research Laboratories, Kampo Scientific Strategies Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Kanno
authors.affiliation: Kampo Formulation Development Center, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Igarashi
authors.affiliation: Kampo Formulation Development Center, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nishimura
authors.affiliation: Tsumura Research Laboratories, Kampo Scientific Strategies Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ikarashi
authors.affiliation: Tsumura Research Laboratories, Kampo Scientific Strategies Division, Tsumura & Co., 3586 Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300-1192, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kase
mesh_terms: D000818:Animals; D001345:Autoradiography; D001812:Blood-Brain Barrier; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D026121:Indole Alkaloids / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D032070:Uncaria / Q000737:chemistry*
chemical_list: D026121:Indole Alkaloids; C110832:geissoschizine methylether
keywords: Autoradiography; Brain; Frontal cortex; Geissoschizine methyl ether; Rat; Serotonin receptor