Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators of P2X7 receptors.

BACKGROUND AND PURPOSE: The P2X7 receptor is an ATP-gated ion channel predominantly expressed in immune cells and plays a key role in inflammatory processes. Ginseng is a well-known Chinese herb with both pro- and anti-inflammatory properties and many of its actions have been ascribed to constituent ginsenosides. We screened a number of ginsenoside compounds for pharmacological activity at P2X7 receptors, that might contribute to the reported immunomodulatory actions of ginseng. EXPERIMENTAL APPROACH: We used several assays to measure responses of P2X7 receptors, ATP-mediated dye uptake, intracellular calcium measurement and whole-cell patch-clamp recordings. HEK-293 cells stably expressing human P2X7 receptors were used in addition to mouse macrophages endogenously expressing P2X7 receptors. KEY RESULTS: Four ginsenosides of the protopanaxdiol series, Rb1, Rh2, Rd and the metabolite compound K (CK) potentiated the dye uptake responses of P2X7 receptors, whereas other ginsenosides tested were ineffective (1-10 µM). The potentiation was rapid in onset, required a threshold concentration of ATP (>50 µM) and had an EC50 of 1.08 µM. CK markedly enhanced ATP-activated P2X7 currents, probably via an extracellular site of action. One of the consequences of this potentiation effect is a sustained rise in intracellular Ca(2+) that could account for the decrease in cell viability in mouse macrophages after a combination of 500 µM ATP and 10 µM CK that are non-toxic when applied alone. CONCLUSIONS AND IMPLICATIONS: This study identifies selected ginsenosides as novel potent allosteric modulators of P2X7 channels that may account for some of the reported immune modulatory actions of protopanaxdiol ginsenosides in vivo.

Facilitatory effect of Ca2+ on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells.

1. The facilitatory effect of external calcium ions (Ca2+o) on the alpha1-adrenoceptor-activated non-selective cation current (Icat) was investigated in rabbit portal vein cells using noise and voltage-jump relaxation analysis of the whole-cell macroscopic current. 2. Micromolar concentrations of Ca2+o potentiated the peak amplitude of Icat at a holding potential (Vh) of -50 mV. The effective [Ca2+]o which produced a 50% potentiation (EC50) was 3 microM. 3. From noise analysis the estimated single channel conductance (gamma) was approximately 23 pS with [Ca2+]o between 3 and 100 microM, whereas in < 10 nM or 1 microM Ca2+o gamma was approximately 10 pS. 4. The spectral density function of Icat at negative potentials could be described by the sum of two Lorentzians in every [Ca2+]o examined. The time constant of the lower frequency Lorentzian component (tau1) was about 11 ms in < 10 nM Ca2+o and was about 45 ms in micromolar concentrations of Ca2+o (1-100 microM). In contrast, the time constant of the higher frequency component (tau2) was similar in < 10 nM Ca2+o and 100 microM Ca2+o (between 1 and 2 ms). 5. The lower frequency Lorentzian component was responsible for about half the total current variance in < 10 nM Ca2+o whereas in micromolar concentrations of Ca2+o it was responsible for most of the measured current variance. 6. In voltage-jump experiments, on stepping the voltage from -50 to +50 mV the instantaneous current was followed by an exponential decline of Icat. Stepping back to -30 mV produced an exponential inward relaxation (Irelax,-30 mV) leading to an increase in the steady-state amplitude of Icat in micromolar concentrations of Ca2+o, but this relaxation was not observed in < 10 nM Ca2+o. The relative amplitude of Irelax,-30 mV increased in an [Ca2+]o-dependent manner (EC50 was 2 microM) although the time constant of this relaxation (taurelax,-30 mV) remained unchanged (about 60 ms between 2 and 100 microM Ca2+o). 7. The data suggest that Ca2+o produces marked changes in the kinetics and single channel conductance of cation channels, which may account for the facilitatory effect of micromolar concentrations of Ca2+o on the peak amplitude of Icat.

Modulation of Ca(2+)-activated Cl- currents in rabbit portal vein smooth muscle by an inhibitor of mitochondrial Ca2+ uptake.

1. The effects of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an inhibitor of mitochondrial Ca2+ uptake, was investigated on the properties of Ca(2+)-activated chloride currents (ICl(Ca)) in rabbit portal vein smooth muscle cells using the perforated patch whole-cell voltage-clamp technique to ascertain whether this Ca2+ uptake process influences the time course of the subsarcolemmal Ca2+ signal that activates ICl(Ca). 2. In cells bathed in either physiological calcium (2 mM Cao2+) or high calcium (10 mM Cao2+) external solutions, application of CCCP (1-2 microM) evoked an inward current and prolonged the exponential decay time constant (tau) of Ca(2+)-activated Cl- 'tail' currents (Itail) evoked by Ca2+ influx through voltage-dependent calcium channels (VDCCs). The effect of CCCP on tau was greater in cells where the amplitude of Itail was relatively large and, in different cells, the effect of CCCP on tau was positively correlated with the amplitude of Itail. 3. CCCP abolished spontaneously occurring transient Ca(2+)-activated Cl- currents (STICs), but did not alter their time course before complete block. 4. Thapsigargin and cyclopiazonic acid (inhibitors of the sarcoplasmic Ca(2+)-ATPase) inhibited STICs, but did not affect the decay of Itail or STICs. 5. In conclusion, when Ca2+ enters the cell through VDCCs, the time course of the consequent Ca2+ signal in the subsarcolemmal domain containing Ca(2+)-activated chloride channels appears to be regulated by Ca2+ uptake into mitochondria. In contrast, inhibition of Ca2+ uptake by the sarcoplasmic reticulum ATPase does not seem to influence the time course of ICl(Ca).

Alpha 1-adrenoceptor activation of a non-selective cation current in rabbit portal vein by 1,2-diacyl-sn-glycerol.

1. The transduction mechanisms involved in the activation and modulation of the noradrenaline-activated cation current (Icat) were investigated with whole-cell patch clamp techniques in rabbit portal vein smooth muscle cells. 2. Intracellular application of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S, 500 microM) evoked a 'noisy' inward current at -50 mV with a similar current-voltage relationship and reversal potential to the current evoked by bath application of noradrenaline (100 microM). Guanosine 5-O-(2-thiodiphosphate) (GDP beta S, 1 mM) markedly inhibited noradrenaline-activated Icat. 3. The phospholipase C (PLC) inhibitor U73122 inhibited the amplitude of the noradrenaline-activated Icat in a concentration- and time-dependent manner and the IC50 was about 180 nM. U73122 had similar effects on the cation current evoked by GTP gamma S. 4. Intracellular application of myo-inositol 1,4,5-trisphosphate (IP3, 100 microM) from the patch pipette did not activate any membrane current in cells where intracellular calcium concentration ([Ca2+]i) was buffered to 14 nM, but subsequent addition of noradrenaline evoked Icat. 5. Bath application of the 1,2-diacyl-sn-glycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 microM) activated Icat, whereas the phorbol ester phorbol 12,13-dibutyrate (PDBu, 0.1-5 microM) failed to activate Icat, in every cell examined. Icat activated by OAG after bath application of PDBu was not significantly different from OAG-activated Icat in the absence of PDBu. The DAG lipase inhibitor RHC80267 (10 microM) activated Icat in some cells, whereas the DAG kinase inhibitor R59949 (10 microM) never activated Icat. 6. Bath application of the protein kinase C inhibitor chelerythrine (1-10 microM) had no effect on either OAG-or noradrenaline-activated Icat. 7. It is concluded that noradrenaline activates Icat via a G-protein coupled to PLC and that the resulting DAG product plays a central role in the activation of cation channels via a protein kinase C-independent mechanism.

Activation of potassium currents by inhibitors of calcium-activated chloride conductance in rabbit portal vein smooth muscle cells.

1. The conventional whole-cell recording technique was used to study the effects of the chloride channel inhibitors ethacrynic acid, anthracene-9-carboxylic acid (A-9-C) and indanyloxyacetic acid (IAA) on membrane currents in rabbit portal vein smooth muscle cells at a holding potential of 0 mV. 2. Using a pipette solution that contained 1 x 10(-4) M 1,2-bis (2-aminophenoxy)-ethane-N,N,N,N,-tetraacetic acid (BAPTA) and a normal bathing solution the addition of ethacrynic acid (2 x 10(-4) M to 1 x 10(-3) M) inhibited spontaneous transient outward currents (STOCs) and evoked a concentration-dependent current at a holding potential of 0 mV. A similar current was activated by IAA (5 x 10(-4) M to 1 x 10(-3) M) but not by A-9-C (1-5 x 10(-3) M) at a holding potential of 0 mV. 3. The amplitude of the current evoked by ethacrynic acid and IAA was linearly related to potential between -30 and 0 mV and displayed outward rectification at positive potentials. The current induced by A-9-C was evident only at potentials positive to +20 mV. 4. Glibenclamide (1 x 10(-5) M) abolished the current evoked by ethacrynic acid and IAA at potentials negative to +10 mV and partially inhibited the current positive to +10 mV. The glibenclamide-insensitive current at positive potentials was completely inhibited by 1 x 10(-3) M TEA. The A-9-C-evoked current was insensitive to glibenclamide and abolished by 1 x 10(-3) M TEA. 5. The glibenclamide-sensitive current activated by ethacrynic acid was not sustained and declined to control levels in the continued presence of ethacrynic acid. However, the outwardly rectifying current recorded at +50 mV was well maintained over the same period. 6. Outwardly rectifying currents evoked by ethacrynic acid and A-9-C were observed with a pipette solution containing 1 x 10(-2) M BAPTA in cells bathed in Ca-free extracellular solution containing 5 x 10(-4) M BAPTA and 1 x 10(-5) M cyclopiazonic acid. 7. It is concluded that all three chloride-channel blockers activated an outwardly rectifying, TEA-sensitive current. Moreover, ethacrynic acid and IAA evoked an additional glibenclamide-sensitive current which was present at all potentials between -30 and +50 mV.

Dual effect of external Ca2+ on noradrenaline-activated cation current in rabbit portal vein smooth muscle cells.

1. The influence of divalent cations was investigated on the noradrenaline-activated non-selective cation current (Icat) in freshly dispersed smooth muscle cells of rabbit portal vein. 2. Reduction of external calcium concentration ([Ca2+]o) from 1.5 mM to 'test' levels of 100-500 microM during stimulation of Icat produced a sustained potentiation of Icat, which indicates an inhibitory action of Cao2+. With 'test' concentrations lower than 100 microM Cao2+ the amplitude of Icat was initially potentiated and then the current subsequently declined. Under these conditions re-addition of 1.5 mM Cao2+ transiently activated Icat which demonstrates an additional facilitatory action of [Ca2+]o. The half-maximal [Ca2+]o for the facilitatory and inhibitory action on Icat was 6 and 400 microM, respectively. 3. When [Ca2+]o was decreased to very low levels (< 10 nM) by inclusion of 1 mM BAPTA in nominally Ca(2+)-free external solution, Icat could still be evoked by noradrenaline. This suggested that Ca2+ is not obligatory for activation of Icat but merely modulatory. 4. In 1.5 mM Cao2+ the current-voltage (I-V) relationship of Icat was S-shaped with a reversal potential (Er) of about +9 mV. The I-V characteristics and Er of the potentiated current in 200 microM Cao2+ were not markedly different from those in 1.5 mM Cao2+. 5. During the noradrenaline-induced Icat, removal of external Mg2+ caused a small increase in the amplitude of Icat which was not as great as that seen on reduction of [Ca2+]o and there was no facilitatory effect of Mgo2+. 6. These results indicate that external Ca2+ has a dual action on Icat, both facilitating and inhibiting the current. This dual action is not shared with external Mg2+.

Effect of temperature on spontaneous Ca2+-activated Cl- currents in rabbit portal vein cells.

The effect of temperature on the time course of spontaneous transient inward (Ca2+-activated Cl-) currents (STICs) was investigated with the perforated patch technique in rabbit portal vein cells. STICs decayed exponentially at temperatures between 17 degrees C and 35 degrees C. The time constant of the decay (tau ) was decreased with increasing temperature and had a temperature coefficient, Q10, of 2.6. The temperature sensitivity of tau could be described by the Arrhenius equation which gave an activation energy of 16kcal/mol. This result indicates that STIC decay is governed by a single first-order reaction and provides further support for the hypothesis that the decay is due to the gating properties of Ca2+-activated Cl- channels.

Synergistic action of histamine and adenosine triphosphate on the response to noradrenaline in rabbit pulmonary artery smooth muscle cells.

The interaction between histamine, adenosine triphosphate (ATP) and noradrenaline was studied with the perforated-patch technique in single cells isolated from the rabbit pulmonary artery. In these cells all of the agents activated caffeine-sensitive currents. In potassium-free conditions at a holding potential of -50 mV bath applied histamine, in concentrations that did not produce a response, greatly enhanced the magnitude of inward currents evoked by ionophoretic application of noradrenaline. These inward currents were calcium-activated chloride currents, ICl(Ca). In potassium-containing solutions at a holding potential of 0 mV, bath applied histamine potentiated the outward currents (calcium-activated potassium currents (IK(Ca)) induced by noradrenaline. This synergistic action was rapid in onset, sustained during the continued presence of histamine and reversible. Bath application of noradrenaline inhibited the response to ionophoretically applied noradrenaline but not the caffeine-induced currents. ATP also stimulated ICl(Ca) and IK(Ca) through a mechanism dependent on the caffeine-sensitive intracellular calcium store and also potentiated the currents activated by noradrenaline. It is concluded that one explanation for the phenomenon of potentiation in smooth muscle is convergence of several distinct pharmacological receptors to a common cellular mechanism.

Properties of spontaneous inward currents in rabbit pulmonary artery smooth muscle cells.

Spontaneous inward and outward currents were studied with perforated patch recording in freshly dispersed rabbit pulmonary artery smooth muscle cells. With physiological potassium concentrations, spontaneous outward and inward currents were recorded at negative membrane potentials. Ion substitution experiments revealed that the outward and inward currents were respectively potassium and chloride conductance increases. Both conductances were abolished by bath application of caffeine (2-10 mM), which releases calcium from internal stores. The rise time and half-decay time of spontaneous potassium currents were both about 25 ms. The spontaneous chloride current has a rise time of 30 ms and decayed exponentially with a time constant (tau) of 70 ms at -50 mV. The tau value was increased by depolarization and increased e-fold for a change of 99 mV in membrane potential. In every cell examined when the spontaneous currents occurred as biphasic events, typically between -20 mV and -40 mV, outward currents preceded inward currents in over 90% of these events whereas the inward current always preceded the outward current in caffeine- and noradrenaline-evoked responses. An explanation for these data is that there may be localization of some chloride channels with respect to the caffeine-sensitive calcium store.