Molecular mechanisms of large-conductance ca (2+) -activated potassium channel activation by ginseng gintonin.

Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca(2+)]i through G protein-coupled LPA receptors. Large-conductance Ca(2+)-activated K(+) (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca(2+)]i and are regulated by various Ca(2+)-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca(2+) bowl and the regulator of K(+) conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca(2+)-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca(2+) bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.

Inhibitory effects of berberine against morphine-induced locomotor sensitization and analgesic tolerance in mice.

We previously reported that a methanolic extract of Coptis japonica, which is a well-known traditional oriental medicine, inhibits morphine-induced conditioned place preference (CPP) in mice. Berberine is a major component of Coptis japonica extract, and it has been established that the adverse effects of morphine on the brain involve dopamine (DA) receptors. However, to our knowledge, no study has investigated the inhibitory effects of berberine on morphine-induced locomotor sensitization and analgesic tolerance in mice. Here, we investigated the effects of berberine on morphine-induced locomotor sensitization and on the development of analgesic tolerance. Furthermore, we examined the effects of berberine treatment on N-methyl-D-aspartate (NMDA) receptor channel activity expressed in Xenopus laevis oocytes. Berberine was found to completely block both morphine-induced locomotor sensitization and analgesic tolerance, and reduce D(1) and NMDA receptor bindings in the cortex. Moreover, berberine markedly inhibited NMDA current in Xenopus laevis oocytes expressing NMDA receptor subunits. Our results suggest that the inhibitory effects of berberine on morphine-induced locomotor sensitization and analgesic tolerance are closely related to the modulation of D1 and NMDA receptors, and that berberine should be viewed as a potential novel means of attenuating morphine-induced sensitization and analgesic tolerance.

Modification of radiation response in mice by Panax ginseng and diethyldithiocarbamate.

The aim of this study was to determine the effect of Panax ginseng and its fractions on jejunal crypt survival, endogenous spleen colony formation and apoptosis in jejunal crypt cells of mice irradiated with high- and low-dose of gamma-irradiation. The radioprotective effect of ginseng was compared with the effect of diethyldithiocarbamate (DDC). Ginseng administration before irradiation protected the jejunal crypts (p < 0.005), increased the formation of endogenous spleen colony (p < 0.005) and reduced the frequency of radiation-induced apoptosis (p < 0.05). The radioprotective effect on jejunal crypts and apoptosis in the DDC-treated group appeared similar to that in the ginseng--treated groups. Treatment with DDC showed no significant modifying effects on the formation of endogenous spleen colony. In the experiment on the effect of fractions of ginseng, the result indicated that the lipophilic non-polar compounds (Fraction 1), lipophilic-acidic compounds (Fraction 2), free sugars (Fraction 7) and saponin compounds (Fraction 8) might have a major radioprotective effect. Although the mechanisms of this inhibitory effect remain to be elucidated, these results indicated that ginseng might be a useful radioprotector, especially since it is a relatively nontoxic natural product. Further studies are needed to fully characterize the protective nature of ginseng extract and its components.

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam binding in rat brain by the prolonged ventricular infusion of ginsenoside Rc and Rg1.

In the present study, we have investigated the effects of centrally administered ginsenoside Rc and Rg1 on the modulation of the NMDA receptor and GABA(A)receptor binding in rat brain. The NMDA receptor binding was analysed by quantitative autoradiography using [(3)H]MK-801 binding, and the GABA(A)receptor bindings were analysed by using [(3)H]muscimol and [(3)H]flunitrazepam binding in rat brain slices. Rats were infused with ginsenoside Rc or Rg1 (10 microg/10 microl h(-1), i.c.v.) for 7 days, through pre-implanted cannula using osmotic minipumps (Alzet, model 2ML). The levels of [(3)H]MK-801 binding were highly decreased in part of the parietal layers of the cortex and cingulated by ginsenoside Rc and Rg1. The levels of [(3)H]muscimol binding were strongly elevated in almost all regions of the frontal cortex by the treatment of ginsenoside Rc but decreased by ginsenoside Rg1. However, the [(3)H]flunitrazepam binding was not modulated by ginsenoside Rc or Rg1 infusion. These results suggest that prolonged infusion of ginsenosides could differentially modulate [(3)H]MK-801 and [(3)H]muscimol binding in a region-specific manner.

Effect of ginsenosides, active components of ginseng, on capsaicin-induced pain-related behavior.

Our recent study demonstrated that ginsenosides had antinociceptive effects by reducing some types of pain-related behavior in mice (Yoon et al., 1998. Ginsenosides induce differential antinociception and inhibit substance P-induced nociceptive response in mice. Life Science 62, PL319-PL325). In the present study we further investigated whether ginsenosides produce antinociceptive effects through an action at central or peripheral site(s) and whether these effects are mediated by the opioid system. Intraperitoneally injected ginsenosides suppressed in a dose-dependent manner the pain-related behavior produced by capsaicin injection into the plantar surface of the hind paw; the ED(50) was 49 mg/kg [26-92 mg/kg, 95% confidence interval (C.I.)]. Intrathecally or intracerebroventricularly administered ginsenosides also suppressed the capsaicin-induced pain-related behavior in a dose-dependent manner; the ED(50)s were 1.72 mg/kg (0.8-3.72 mg/kg, 95% C.I.) and 1. 48 mg/kg (0.8-2.6 mg/kg, 95% C.I.), respectively. On the other hand, subcutaneously injected ginsenosides to the plantar surface prior to the capsaicin injection did not alter the pain-related behavior. Naloxone pretreatment was without effect in blocking the antinociceptive effect of intrathecally administered ginsenosides. Intraperitoneally injected ginsenosides also did not significantly affect the motor response of animals. These results suggest that ginsenosides produce antinociceptive effects through their action at the spinal and/or supraspinal site(s), not at nociceptors in the periphery. In addition, the results suggest that the antinociceptive effects are not mediated by opioid receptors.

Preparation of monoclonal antibody against ginsenoside Rf and its enzyme immunoassay.

A rapid and sensitive indirect competitive enzyme immunoassay method has been developed for quantitating ginsenoside Rf (Rf) in crude total Panax ginseng saponins and in rat plasma using high titer mouse monoclonal antibody (mAb) raised against a conjugate of Rf and bovine serum albumin (BSA). The isotype of mAb against Rf was IgG3 with a K chain. The presence of Rf inhibited the binding of the mouse anti-Rf mAb to a Rf-BSA solid phase coating antigen. The working range was 0.01-10 ng/assay and detection limits were 20 pg in various ginseng extract fractions or 34 pg in rat plasma per assay. The anti-Rf mAb cross-reacted with ginsenoside Rg2 by 57.5%, but not with other ginsenosides. However, this anti-Rf mAb did not cross-react with BSA or cellubiose, which is a carbohydrate component of Rf. Using this standard curve, we could measure the amount of Rf in ginseng total extract, ginseng total saponins, protopanaxadiol saponins, and propanaxatriol saponins. We could also measure the amount of Rf in rat plasma after the oral administration of Rf and found that Rf reached a maximum level in rat plasma after 16 h. These results indicate that the anti-Rf mAb could be useful for the quantitation of Rf in crude ginseng fractions and in body fluids.

Protopanaxatriol ginsenosides inhibit glucose uptake in primary cultured rabbit renal proximal tubular cells by arachidonic acid release.

Ginsenosides are involved in protective action against renal dysfunction and the regulation of renal functions. However, the effects of ginsenosides on glucose reabsorption are not yet known in renal proximal tubular cells. The aim of this study was to examine the effects of ginsenosides, protopanaxadiol (PD) saponin and protopanaxatriol (PT) saponin, on alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its mechanism of action in primary cultured rabbit renal proximal tubular cells (PTCs). The alpha-MG uptake was inhibited by 90% by 0.5mM phloridizin and by removal of Na+ in the PTCs. These are typical characteristics described for the proximal tubule. To determine the time- and dose-dependent effects of PD and PT saponins on alpha-MG uptake, PTCs were incubated with different concentrations of PD and PT saponins (10-100 microg/ml) and for different time periods (from 10 min to 24 h). PT saponin (>/=50 microg/ml) from 30 min inhibited alpha-MG uptake; however, PD saponin did not alter the alpha-MG uptake at any doses and time periods. In the kinetic analysis of alpha-MG uptake, PT saponin produced a significant decrease in Vmax. The PT saponin induced inhibition of alpha-MG uptake was blocked by mepacrine, a phospholipase A2 inhibitor. In addition, PT saponin increased [3H] arachidonic acid release by 218% of that of control, and this effect was also completely blocked by mepacrine. In conclusion, PT saponin inhibited, in part, alpha-MG uptake through the phospholipase A2 signal pathway in primary cultured rabbit renal PTCs.

Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay.

We have developed an enzyme immunoassay (EIA) to quantify trace amounts of ginsenoside Rf (Rf), one of the glycosides of protopanaxatriol from Panax ginseng. A carrier protein of bovine serum albumin (BSA) was coupled to the carbohydrate component of Rf using the periodate oxidation method. Antibodies were raised in rabbits using Rf-BSA conjugate as the immunogen and competitive indirect EIA was used for the determination of Rf. The working range was 0.01-10 ng per assay. The anti-Rf antiserum cross-reacted with ginsenoside Rg2 (105%), which is also a component of Panax ginseng and has a very similar chemical structure to Rf. These results suggest that the anti-Rf antiserum could also be used for the quantitation of ginsenoside Rg2 as well as ginsenoside Rf. In a comparison of EIA and HPLC the linear regression equation and correlation coefficient for the two methods were y(EIA) = 1.31x (HPLC)-11.48 and 0.98, respectively.

Effects of ginsenosides on Ca2+ channels and membrane capacitance in rat adrenal chromaffin cells.

We investigated the effects of ginseng total saponins (GTS) and five ginsenosides on voltage-dependent Ca2+ channels and membrane capacitance using rat adrenal chromaffin cells. In this study, cells were voltage-clamped in a whole-cell recording mode and a perforated patch-clamp technique was used. The inward Ca2+ currents (I(Ca)) was elicited by depolarization and the change in cell membrane capacitance (deltaCm) was monitored. The application of GTS (100 microg/ml) induced rapid and reversible inhibition of the Ca2+ current by 38.8 +/- 3.6% (n = 16). To identify the particular single component that seems to be responsible for Ca2+ current inhibition, the effects of five ginsenosides (ginsenoside Rb1, Rc, Re, Rf, and Rg1) on the Ca2+ current were examined. The inhibitions to the Ca2+ current by Rb1, Rc, Re, Rf, and Rg1 were 15.3 +/- 2.2% (n = 5); 36.9 +/- 2.4% (n = 7); 28.1 +/- 1.9% (n = 12); 19.0 +/- 2.5% (n = 10); and 16.3 +/- 1.6% (n = 15), respectively. The order of inhibitory potency (100 microM) was Rc > Re > Rf > Rg1 > Rb1. A software based phase detector technique was used to monitor membrane capacitance change (deltaCm). The application of GTS (100 microg/ml) induced inhibitory effects on deltaCm by 60.8 +/- 9.7% (n = 10). The inhibitions of membrane capacitance by Rb1, Rc, Re, Rf, and Rg1 were 35.3 +/- 5.5% (n = 7); 41.8 +/- 7.0% (n = 8); 40.5 +/- 5.9% (n = 9); 51.2 +/- 7.6% (n = 9); and 35.9 +/- 5.1% (n = 10), respectively. The inhibitory potencies of the ginsenosides on deltaCm were Rf > Rc > Re > Rg1 > Rb1. Therefore, we found that GTS and ginsenosides exerted inhibitory effects on both Ca2+ currents and deltaCm in rat adrenal chromaffin cells. These results suggest that ginseng saponins regulate catecholamine secretion from adrenal chromaffin cells and this regulation could be the cellular basis of antistress effects induced by ginseng.

Ginsenoside Rf, a trace component of ginseng root, produces antinociception in mice.

Ginseng root, a traditional oriental medicine, contains more than a dozen biologically active saponins called ginsenosides, including one present in only trace amounts called ginsenoside-Rf (Rf). Previously, we showed that Rf inhibits Ca2+ channels in mammalian sensory neurons through a mechanism requiring G-proteins, whereas a variety of other ginsenosides were relatively ineffective. Since inhibition of Ca2+ channels in sensory neurons contributes to antinociception by opioids, we tested for analgesic actions of Rf. We find dose-dependent antinociception by systemic administration of Rf in mice using two separate assays of tonic pain: in the acetic acid abdominal constriction test, the ED50 was 56+/-9 mg/kg, a concentration similar to those reported for aspirin and acetaminophen in the same assay; in the tonic phase of the biphasic formalin test, the ED50 was 129+/-32 mg/kg. Rf failed to affect nociception measured in three assays of acute pain: the acute phase of the formalin test, and the thermal (49 degrees C) tail-flick and increasing-temperature (3 degrees C/min) hot-plate tests. The simplest explanation is that Rf inhibits tonic pain without affecting acute pain, but other possibilities exist. Seeking a cellular explanation for the effect, we tested whether Rf suppresses Ca2+ channels on identified nociceptors. Inhibition was seen on large, but not small, nociceptors. This is inconsistent with a selective effect on tonic pain, so it seems unlikely that Ca2+ channel inhibition on primary sensory neurons can fully explain the behavioral antinociception we have demonstrated for Rf.

Ginsenosides induce differential antinociception and inhibit substance P induced-nociceptive response in mice.

Ginsenosides are main pharmacoactive molecules of ginseng. The antinociceptive activity of ginsenosides after intrathecal (i.t.) injection was examined in formalin test. We also investigated the effects of ginsenosides on substance P (SP) induced-pain behaviors by i.t. treatment using mice. Pretreatment of ginsenosides by i.t. induced the inhibition of biting and licking of hind paw injected with 1% formalin with dose-dependent manner. The ED50 was 23 (19-28, 95% C.I.) microg/mouse for acute phase and 15 (9-23, 95% C.I.) microg/mouse for tonic phase. Interestingly, cotreatment of ginsenosides with SP also inhibited SP-induced pain behaviors (scratching, licking or biting of hind portion of body) with dose-dependent manner. The ED50 for the inhibition of SP-induced pain behavior by ginsenosides was 30 (11-85, 95% C.I.) microg/mouse. These results suggest that ginsenosides have antinociceptive activity in formalin test and this effect is due to blocking of SP-induced nociceptive information to postsynaptic site(s) at the spinal level.

A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.

Ginseng root extract inhibits calcium channels in rat sensory neurons through a similar path, but different receptor, as mu-type opioids.

The effect of Panax ginseng root extract on Ca2+ current of adult rat trigeminal ganglion neurons was investigated using whole-cell patch-clamp methods. The application of P. ginseng root extract (100 micrograms/ml) produced rapid, reversible reduction of the Ca2+ current by 22 +/- 4%. Treatment with pertussis toxin (250 ng/ml) for 16 h reduced the inhibition to 4 +/- 1%. The continual presence of 1 microM DAGO, a selective mu-opioid agonist that inhibits Ca2+ channels, occluded further inhibition of Ca2+ current by P. ginseng root extract. Yohimbine, phaclofen, atropine, and naloxone--antagonists of alpha 2-adrenergic, GABAB, muscarinic, and opiate receptors, respectively--did not block the inhibitory effect on Ca2+ current of P. ginseng root extract. Thus, P. ginseng root extract acts on sensory neurons through a similar pathway as mu-type opioids: both inhibit Ca2+ channels through pertussis toxin-sensitive GTP-binding proteins. However, the receptor for P. ginseng root extract is not an alpha 2-adrenergic, GABAB, muscarinic, or opioid receptor.