Biomedical subjects

Hyewhon Rhim

Publications and source records attributed to Hyewhon Rhim.

At least 37 records · Page 2Linked to original sources

PubMed2005

Protective effects of ginseng saponins on 3-nitropropionic acid-induced striatal degeneration in rats.

The precise cause of neuronal cell death in Huntington's disease (HD) is not known. Systemic administration of 3-nitropropionic acid (3-NP), an irreversible succinate dehydrogenase inhibitor, not only induces a cellular ATP depletions but also causes a selective striatal degeneration similar to that seen in HD. Recent accumulating reports have shown that ginseng saponins (GTS), the major active ingredients of Panax ginseng, have protective effects against neurotoxin insults. In the present study, we examined in vitro and in vivo effects of GTS on striatal neurotoxicity induced by repeated treatment of 3-NP in rats. Here, we report that systemic administration of GTS produced significant protections against systemic 3-NP- and intrastriatal malonate-induced lesions in rat striatum with dose-dependent manner. GTS also improved significantly 3-NP-caused behavioral impairment and extended survival. However, GTS itself had no effect on 3-NP-induced inhibition of succinate dehydrogenase activity. To explain the mechanisms underlying in vivo protective effects of GTS against 3-NP-induced striatal degeneration, we examined in vitro effect of GTS against 3-NP-caused cytotoxicity using cultured rat striatal neurons. We found that GTS inhibited 3-NP-induced intracellular Ca(2+) elevations. GTS restored 3-NP-caused mitochondrial transmembrane potential reduction in cultured rat striatal neurons. GTS also prevented 3-NP-induced striatal neuronal cell deaths with dose-dependent manner. The EC(50) was 12.6 +/- 0. 7microg/ml. These results suggest that in vivo protective effects of GTS against 3-NP-induced rat striatal degeneration might be achieved via in vitro inhibition of 3-NP-induced intracellular Ca(2+) elevations and cytotoxicity of striatal neurons.

Analysis of Variance↗

PubMed2004

Stereospecificity of ginsenoside Rg3 action on ion channels.

Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-ginsenoside and 20(S)-ginsenoside are epimers. We have shown previously that the mixture of 20(R)- and 20(S)-ginsenosides regulates ion channel activity. However, it was not clear which epimer was responsible. We investigated the structure-activity relationship of the ginsenoside Rg3 stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(R)-Rg3) and 20-S-proto-panaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyr-anoside], (20(S)-Rg3) in regulating voltage-dependent Ca2+, K+ or Na+ channel currents and 5-HT3A and a3b4 nicotinic acetylcholine (nACh) receptor channel currents expressed in Xenopus oocytes. 20(S)-Rg3 but not 20(R)-Rg3 inhibited the Ca2+, K+ and Na+ channel currents in a dose- and voltage-dependent manner. The fact that only 20(S)-Rg3 is active indicates that its hydroxyl group may be geometrically better aligned with the hydroxyl acceptor group in the ion channels than that of 20(R)-Rg3. However, both Rg3 stereoisomers inhibited 5-HT3A and a3beta4 nACh receptor channel currents. These results indicate that the selectivity of action of the Rg3 stereoisomers differs between voltage-dependent and ligand-gated ion channels.

Animals↗

PubMed2004

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits.

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.

Barium↗

PubMed2004

Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons.

We previously reported that ginseng, a well-known herbal medicine, inhibited NMDA receptors in cultured hippocampal neurons. Here, we further examined the detailed mechanism of ginseng-mediated inhibition using its main active ingredient, ginsenoside Rg3. Co-application of ginsenoside Rg3 with increasing concentrations of NMDA did not change the EC50 of NMDA to the receptor, suggesting that ginsenoside Rg3 inhibits NMDA receptors without competing with the NMDA-binding site. Ginsenoside Rg3-mediated inhibition also occurred in a distinctive manner from the well-characterized NMDA receptor open channel blocker, MK-801. However, ginsenoside Rg3 produced its effect in a glycine concentration-dependent manner and shifted the glycine concentration-response curve to the right without changing the maximal response, suggesting the role of ginsenoside Rg3 as a competitive NMDA receptor antagonist. We also demonstrated that ginsenoside Rg3 significantly protected neurons against NMDA insults. Therefore, these results suggest that ginsenoside Rg3 protects NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons.

Animals↗

PubMed2004

Synthesis and biological evaluation of novel T-type Ca2+ channel blockers.

A small molecule library of piperazinylalkylisoxazole derivatives containing about 600 compounds was designed, synthesized and evaluated for blocking effects on T-type Ca(2+) channel. Several ligands were identified to possess high inhibitory activity against the T-type Ca(2+) channel. The compound 21 with trifluoromethyl substituents at C(3)-position of phenyl group (R(1)) and C(2)-position of phenyl group (R(2)) showed the highest inhibitory activity with IC(50) value of 1.02 microM, which is comparable to that of mibefradil.

Calcium↗

PubMed2004

3,4-Dihydroquinazoline derivatives as novel selective T-type Ca2+ channel blockers.

For LVA T-type Ca2+ channel blockers, 3,4-dihydroquinazoline derivatives as new scaffolds were prepared and evaluated for the inhibitory activity against two members of the recombinant T-type Ca2+ channel family. Among them, 8a (KYS05001, IC50=0.9 microM) was nearly equipotent with mibefradil (IC50=0.84 microM) and inhibited LVA T-type Ca2+ channel with greater efficacy than HVA Ca2+ channel.

Animals↗

PubMed2004

Ginseng saponins induce store-operated calcium entry in Xenopus oocytes.

1 We investigated the effect of the active ingredients of Panax ginseng, ginsenosides, on store-operated Ca2+ entry (SOCE) using a two-electrode voltage clamp technique in Xenopus oocytes in which SOCE is monitored through Ca(2+)-activated Cl- currents. 2 Under hyperpolarizing voltage clamp conditions, treatment with ginsenosides produced a biphasic Ca(2+)-activated Cl- current consisting of a rapid transient inward current and a slowly developing secondary sustained inward current. The transient inward current was inactivated rapidly, whereas the sustained inward current persisted for nearly 10 min. The effect of ginsenosides on the biphasic current was dose-dependent and reversible. The EC50 was 42.8+/-11.6 and 46.6+/-7.1 microg ml(-1) for the transient and sustained inward current, respectively. 3 In the absence of extracellular Ca2+ ginsenosides induced only a transient inward current but in the presence of extracellular Ca2+ ginsenosides induced the biphasic current. Magnitudes of the sustained currents were dependent on extracellular Ca2+ concentration. Sustained inward current induced by ginsenosides, but not transient inward current, and ginsenoside-induced store-operated Ca2+ (SOC) currents (ISOC) were blocked by La3+, a Ca2+ channel blocker, suggesting that the sustained inward current and ISOC was derived from an influx of extracellular Ca2+. 4 Treatment with 2-APB and heparin, which are IP3 receptor antagonists, inhibited the ginsenoside-induced biphasic current. Treatment with the PLC inhibitor, U73122, also inhibited the ginsenoside-induced biphasic current. Intraoocyte injection of ATP-gammaS, but not adenylyl AMP-PCP, induced a persistent activation of ginsenoside-induced sustained current but did not affect the transient current. 5 In rat hippocampal neurons, ginsenosides inhibited both carbachol-stimulated intracellular Ca2+ release and intracellular Ca2+ depletion-activated SOCE. 6 These results indicate that ginsenoside might act as a differential regulator of intracellular Ca2+ levels in neurons and Xenopus oocytes.

Animals↗

PubMed2004

Ginsenosides inhibit NMDA receptor-mediated epileptic discharges in cultured hippocampal neurons.

Epilepsy or the occurrence of spontaneous recurrent epileptiform discharges (SREDs, seizures) is one of the most common neurological disorders. Shift in the balance of brain between excitatory and inhibitory functions due to different types of structural or functional alterations may cause epileptiform discharges. N-Methyl-D-aspartate (NMDA) receptor dysfunctions have been implicated in modulating seizure activities. Seizures and epilepsy are clearly dependent on elevated intracellular calcium concentration ([Ca2+]i) by NMDA receptor activation and can be prevented by NMDA antagonists. This perturbed [Ca2+]i levels is forerunner of neuronal death. However, therapeutic tools of elevated [Ca2+]i level during status epilepticus (SE) and SREDs have not been discovered yet. Our previous study showed fast inhibition of ginseng total saponins and ginsenoside Rg3 on NMDA receptor-mediated [Ca2+]i in cultured hippocampal neurons. We, therefore, examined the direct modulation of ginseng on hippocampal neuronal culture model of epilepsy using fura-2-based digital Ca2+ imaging and neuronal viability assays. We found that ginseng total saponins and ginsenoside Rg3 inhibited Mg2+ free-induced increase of [Ca2+]i and spontaneous [Ca2+]i oscillations in cultured rat hippocampal neurons. These results suggest that ginseng may play a neuroprotective role in perturbed homeostasis of [Ca2+]i and neuronal cell death via the inhibition of NMDA receptor-induced SE or SREDs.

Animals↗

PubMed2003

Prevention of ginsenoside-induced desensitization of Ca2+-activated Cl- current by microinjection of inositol hexakisphosphate in Xenopus laevis oocytes: involvement of GRK2 and beta-arrestin I.

We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca(2+)-activated Cl(-) currents via Galpha(q/11)-phospholipase C-beta3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl(-) channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl(-) channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl(-) channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP(6)), which is known to bind beta-arrestins and interfere with beta-arrestin-induced receptor down-regulation, and cRNAs coding beta-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 +/- 4.1, 9.2 +/- 2.3, and 2.6 +/- 2.2% of the first responses, respectively. Preintraoocyte injection of InsP(6) before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration-, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC(50) was 13.9 +/- 8.7 microM. Injection of cRNA coding beta-arrestin I but not beta-arrestin II blocked InsP(6) effect on prevention of ginsenoside-induced Cl(-) channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl(-) current. However, the GRK2-caused loss of ginsenoside effect on Cl(-) current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl(-) channel desensitization is mediated via activation of GRK2 and beta-arrestin I.

Animals↗

PubMed2003

Enhanced learning and memory in mice lacking Na+/Ca2+ exchanger 2.

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) plays a role in regulation of intracellular Ca(2+) concentration via the forward mode (Ca(2+) efflux) or the reverse mode (Ca(2+) influx). To define the physiological function of the exchanger in vivo, we generated mice deficient for NCX2, the major isoform in the brain. Mutant hippocampal neurons exhibited a significantly delayed clearance of elevated Ca(2+) following depolarization. The frequency threshold for LTP and LTD in the hippocampal CA1 region was shifted to a lowered frequency in the mutant mice, thereby favoring LTP. Behaviorally, the mutant mice exhibited enhanced performance in several hippocampus-dependent learning and memory tasks. These results demonstrate that NCX2 can be a temporal regulator of Ca(2+) homeostasis and as such is essential for the control of synaptic plasticity and cognition.

Animals↗

PubMed2003

Effects of ginsenoside Rg2 on the 5-HT3A receptor-mediated ion current in Xenopus oocytes.

Treatment with ginsenosides, major active ingredients of Panax ginseng, produces a variety of pharmacological or physiological responses with effects on the central and peripheral nervous systems. Recent reports showed that ginsenoside Rg2 inhibits nicotinic acetylcholine receptor-mediated Na+ influx and channel activity. In the present study, we investigated the effect of ginsenoside Rg2 on human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity, which is also one of the ligand-gated ion channel families. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. The ginsenoside Rg2 itself had no effect on the oocytes that were injected with H2O as well as on the oocytes that were injected with the 5-HT3A receptor cRNA. In the oocytes that were injected with the 5-HT3A receptor cRNA, the pretreatment of ginsenoside Rg2 inhibited the 5-HT-induced inward peak current (I5-HT) The inhibitory effect of ginsenoside Rg2 on I5-HT was dose dependent and reversible. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 was 22.3 +/- 4.6 microM. The inhibition of I5-HT by ginsenoside Rg2 was non-competitive and voltage-independent. These results indicate that ginsenoside Rg2 might regulate the 5-HT3A receptors that are expressed in Xenopus oocytes. Further, this regulation on the ligand-gated ion channel activity by ginsenosides might be one of the pharmacological actions of Panax ginseng.

Animals↗

PubMed2003

Increased production of tumor necrosis factor-alpha induces apoptosis after traumatic spinal cord injury in rats.

We showed previously that, after spinal cord injury (SCI), tumor necrosis factor-alpha (TNF-alpha) may serve as an external signal, initiating apoptosis in neurons and oligodendrocytes. To further characterize the apoptotic cascade initiated by TNF-alpha after SCI, we examined the expression of TNF-alpha, inducible nitric oxide (NO) synthase (iNOS), and the level of NO after SCI. Western blots and reverse transcription polymerase chain reactions showed an early upregulation of TNF-alpha after injury. A peak TNF-alpha expression was observed within 1 h of injury. By 4 h after injury, the expression of iNOS and the level of NO were markedly increased in the injured spinal cord. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive cells were also first observed in the lesioned area 4 h after SCI. The largest number of TUNEL-positive cells was observed between 24-48 h after SCI. Injecting a neutralizing antibody against TNF-alpha into the lesion site after injury significantly reduced the expression of iNOS, the level of NO and the number of TUNEL-positive cells in the injured spinal cord. Injecting the NOS inhibitors, N(G)-monomethyl-L-arginine monoacetate and S-methylisothiourea sulfate, or an NO scavenger, carboxy-PTIO, into the lesion site also significantly reduced the level of NO and the degree of DNA laddering in the injured spinal cord. These data suggest that after SCI, apoptosis induced by TNF-alpha may be mediated in part by NO via upregulation of iNOS, induced in response to TNF-alpha.

Animals↗

PubMed2003

Role of K(+) channels in frequency regulation of spontaneous action potentials in rat pituitary GH(3) cells.

The frequency of spontaneous action potentials (SAP) is important in the regulation of hormone secretion. The decrease in K(+) conductance is known as a primary mechanism for increasing SAP frequency. To investigate the nature of K(+) channels that contribute to the frequency regulation of the SAP in rat clonal pituitary GH(3) cells, the effect of various K(+) channel blockers on the SAP and membrane currents were recorded using the patch-clamp technique. A classical inward rectifying K(+) channel blocker, Cs(+) (5 mM), caused an increase in firing frequency and depolarization in after-hyperpolarization (AHP) voltage. An ETHER-A-GO-GO(ERG) type K(+) channel blocker, E-4031 (5 microM), caused no significant effect on the SAP. Tetraethylammonium (TEA, 10 mM) decreased firing frequency and increased the duration of SAP. These effects were not changed by the presence of high concentration of Ca(2+) buffer (10 mM EGTA or BAPTA) in pipette solutions. In voltage-clamp experiments, Cs(+) and E-4031 did not affect outwardly rectifying K(+) currents, but significantly inhibited inwardly rectifying K(+) currents recorded in isotonic K(+) solution. However, the kinetics of Cs(+)-sensitive current and E-4031-sensitive current were distinctive: the time to peak was more immediate and the decay rate was slower in Cs(+)-sensitive current than in E-4031-sensitive current. These results imply that Cs(+) and E-4031 inhibit the distinct components of inwardly rectifying K(+) currents, and that the contribution of the Cs(+)-sensitive current can be immediate on repolarization and can last more effectively over pacemaking potential range than E-4031-sensitive current.

Action Potentials↗

PubMed2002

Inhibitory effect of ginsenosides on NMDA receptor-mediated signals in rat hippocampal neurons.

Alternative medicines such as herbal products are increasingly being used for preventive and therapeutic purposes. Ginseng is the best known and most popular herbal medicine used worldwide. In spite of some beneficial effects of ginseng on the CNS, little scientific evidence shows at the cellular level. In the present study, we have examined the direct modulation of ginseng on the activation of glutamate, especially NMDA, receptors in cultured hippocampal neurons. Using fura-2-based digital imaging techniques, we found ginseng total saponins inhibited NMDA-induced but less effectively glutamate-induced increase in [Ca2+]i. Ginseng total saponins also modulated Ca2+ transients evoked by depolarization with 50mM KCl along with its own effects on [Ca2+]i. Furthermore, we demonstrated that ginsenoside Rg3 is an active component for ginseng actions on NMDA receptors. The data obtained suggest that the inhibition of NMDA receptors by ginseng, in particular by ginsenoside Rg3, could be one of the mechanisms for ginseng-mediated neuroprotective actions.

Animals↗

PubMed2002

17Beta-estradiol inhibits high-voltage-activated calcium channel currents in rat sensory neurons via a non-genomic mechanism.

There is increasing evidence that estrogen influences electrical activity of neurons via stimulation of membrane receptors. Although the presence of intracellular estrogen receptors and their responsiveness in dorsal root ganglion (DRG) primary sensory neurons were reported, rapid electrical responses of estrogen in DRG neurons have not been reported yet. Therefore the current study was initiated to examine the rapid effects of estrogen on Ca2+ channels and to determine its detailed mechanism in female rat DRG neurons using whole-cell patch-clamp recordings. Application of 17beta-estradiol (1 microM) caused a rapid inhibition on high-voltage-activated (HVA)-, but not on low-voltage-activated (LVA)-Ca2+ currents. This rapid estrogen-mediated inhibition was reproducible and dose-dependent. This effect was also sex- and stereo-specific; it was greater in cells isolated from intact female rats and was more effective than that of 17alpha-estradiol, the stereoisomer of the endogenous 17alpha-estradiol. In addition, ovariectomy reduced the inhibition significantly but this effect was restored by administration of estrogen in ovariectomized subjects. Occlusion experiments using selective blockers revealed 17beta-estradiol mainly targeted on both L- and N-type Ca2+ currents. Overnight treatment of cells with pertussis toxin profoundly reduced 17beta-estradiol-mediated inhibition of the currents. On the other hand, estradiol conjugated to bovine serum albumin (EST-BSA) produced a similar extent of inhibition as 17beta-estradiol did. These results suggest that 17beta-estradiol can modulate L- and N-type HVA Ca2+ channels in rat DRG neurons via activation of pertussis toxin-sensitive G-protein(s) and non-genomic pathways. It is likely that such effects are important in estrogen-mediated modulation of sensory functions at peripheral level.

Animals↗

PubMed2002

Ginseng and ginsenoside Rg3, a newly identified active ingredient of ginseng, modulate Ca2+ channel currents in rat sensory neurons.

There is increasing evidence that ginseng influences pain modulation. In spite of extensive behavior studies, the detailed mechanism of ginseng actions at the cellular level and the identity of the active substance have not been elucidated yet. Whole-cell patch-clamp recordings were used to examine the modulation of high-voltage-activated Ca2+ channel currents by ginseng total saponins and its various individual ginsenosides in rat dorsal root ganglion neurons. Application of ginseng total saponins suppressed Ca2+ channel currents in a dose-dependent manner. Occlusion experiments using selective blockers revealed that ginseng total saponins could modulate L-, N-, and P-type currents. The co-application of ginseng total saponins and the gamma-opioid receptor agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), produced non-additive effects in most cells tested and each effect was significantly relieved by a depolarizing prepulse. Overnight treatment of cells with pertussis toxin profoundly reduced the inhibition. Furthermore, we now report that ginsenoside Rg3, among the major fractions of ginseng saponins, is a newly identified active component for the inhibition. These results suggest that the modulation of Ca2+ channels by ginseng total saponins, in particular by ginsenoside Rg3, could be part of the pharmacological basis of ginseng-mediated antinociception.

Analgesics, Opioid↗

PubMed2002

Effects of ginsenoside Rg2 on human neuronal nicotinic acetylcholine receptors.

Ginseng saponins, major active components of ginseng root used by folk medicine in the treatment of various diseases, produce multiple pharmacological responses having many effects on the central and peripheral nervous system. Specifically, ginsenoside Rg(2) has been shown to block the nicotinic acetylcholine receptors in bovine chromaffin cells. We have studied the effect of Rg(2) on different types of human neuronal nicotinic acetylcholine receptors (nAChRs), both homomeric and heteromeric, expressed in Xenopus oocytes. Rg(2) did not affect the acetylcholine (ACh)-induced currents in alpha(7) human receptors, however Rg(2) affected the peak currents, and mainly the desensitization of heteromeric receptors alpha(3)beta(4), alpha(3)beta(2), alpha(4)beta(4), and alpha(4)beta(2). Both effects, a diminution of peak current and an increase of desensitization, are dose-dependent and are very similar for all the receptors. The mechanism of action has been studied in more detail in alpha(3)beta(4) and alpha(4)beta(2) receptors where we found a negligible shift in the ACh dose-response curves and a persistence of the Rg(2) effects at high ACh concentrations, indicative of a noncompetitive antagonism. A lack of voltage dependence on the reduction of the peak currents induced by ACh also suggests that Rg(2) does not act as an open channel blocker of human nAChR. The results indicate that Rg(2) acts specifically on heteromeric human nAChRs modulating their desensitization and suggest a possible mechanism by which this saponin contributes to the multiple therapeutic effects of ginseng.

Animals↗

PubMed2002

Functional expression of a novel ginsenoside Rf binding protein from rat brain mRNA in Xenopus laevis oocytes.

We have shown that ginsenoside Rf (Rf) regulates voltage-dependent Ca(2+) channels through pertussis toxin (PTX)-sensitive G proteins in rat sensory neurons. These results suggest that Rf can act through a novel G protein-linked receptor in the nervous system. In the present study, we further examined the effect of Rf on G protein-coupled inwardly rectifying K(+) (GIRK) channels after coexpression with size-fractionated rat brain mRNA and GIRK1 and GIRK4 (GIRK1/4) channel cRNAs in Xenopus laevis oocytes using two-electrode voltage-clamp techniques. We found that Rf activated GIRK channel in a dose-dependent and reversible manner after coexpression with subfractions of rat brain mRNA and GIRK1/4 channel cRNAs. This Rf-evoked current was blocked by Ba(2+), a potassium channel blocker. The size of rat brain mRNA responding to Rf was about 6 to 7 kilobases. However, Rf did not evoke GIRK current after injection with this subfraction of rat brain mRNA or GIRK1/4 channel cRNAs alone. Other ginsenosides, such as Rb(1) and Rg(1), evoked only slight induction of GIRK currents after coexpression with the subfraction of rat brain mRNA and GIRK1/4 channel cRNAs. Acetylcholine and serotonin almost did not induce GIRK currents after coexpression with the subfraction of rat brain mRNA and GIRK1/4 channel cRNAs. Rf-evoked GIRK currents were not altered by PTX pretreatment but were suppressed by intracellularly injected guanosine-5'-(2-O-thio) diphosphate, a nonhydrolyzable GDP analog. These results indicate that Rf activates GIRK channel through an unidentified G protein-coupled receptor in rat brain and that this receptor can be cloned by the expression method demonstrated here.

Animals↗