Screening and isolation of a natural dopamine D1 receptor antagonist using cell-based assays.
To develop a cell-based assay to screen for human dopamine D(1) receptor agonists or antagonists from medicinal plant extracts, a stable Chinese hamster ovary (CHO) cell line (CHO-D1R) expressing the human dopamine D(1) receptor was established using an expression vector containing a scaffold attachment region (SAR) element. CHO-D1R cells showed specific binding to [(3)H]-SCH23390 with high affinity (K(d)=1.47+/-0.17 nM) and dose-dependent responses for the dopamine-mediated stimulation of cAMP concentrations (EC(50)=20.6+/-1.44 nM). The screening of medicinal plant extracts using cell-based cAMP assays revealed that an extract of Gleditsia sinensis Lam., which is known to be rich in saponin, had strong antagonist activity for the D(1) receptor. From the activity-guided fractionation and chemical structural analysis of the G. sinensis extract, a compound called gleditsioside F was isolated and was identified to have antagonist activity for the D(1) receptor. Gleditsioside F showed very effective D(1) antagonist activity by inhibiting ligand binding to the D(1) receptor as well as by inhibiting dopamine-mediated increases in cAMP concentration.
Yu
Sungryul
S
Graduate School of Biotechnology, Kyung Hee University, Seochundong 1, Yongin-si, Gyeonggi-do 446-701, Republic of Korea.
Park
Jeong Soo
JS
Paredes
Verenice
V
Song
Myoung-Chong
MC
Baek
Nam-In
NI
Lee
Sang-Ik
SI
Lim
Jong-Soon
JS
Cho
Nam-Young
NY
Yoon
Jaeseung
J
Baek
Kwanghee
K
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001688:Biological Products / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018492:Dopamine Antagonists / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D031501:Gleditsia / Q000737:chemistry; D006801:Humans; D008024:Ligands; D017447:Receptors, Dopamine D1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012503:Saponins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
D001688:Biological Products; D018492:Dopamine Antagonists; D008024:Ligands; D017447:Receptors, Dopamine D1; D012503:Saponins; D054852:Small Molecule Libraries