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.     

Shuffling the Deck Anew: How NR3 Tweaks NMDA Receptor Function

The N-methyl-D: -aspartate (NMDA) receptors are the most complex members in the family of ionotropic glutamate receptors. They are involved in long-term potentiation and underlie higher cognitive functions like memory formation and learning. On the other hand, overstimulation of NMDA receptors (NMDARs), leading to a massive influx of Ca(2+) ions into the cell, is linked to neurodegenerative disorders such as for example Huntington's disease and epilepsy. NMDARs are generally considered to be heteromeric tetramers and are conventionally thought to assemble from NR1 splice variants and NR2 subunits, which determine crucial channel properties. With the recent discovery of the functionally different NR3 subunits, many of the known features of NMDARs are being reassessed: The presence of NR3 in NMDARs decreases Mg(2+) sensitivity and Ca(2+) permeability and reduces agonist-induced current responses. Between altering those essential key characteristics of conventional NMDARs and forming a new class of excitatory glycine receptors when coassembling with NR1, the NR3 subunits give rise to a functionally entirely new array of "NMDA" receptors. Understanding the multifaceted influence of NR3 is imperative to further the understanding of the complex role of NMDARs in neurotransmission and higher brain functions.     

Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons

In the present study, we have examined any possible involvement of L-type Ca²⁺ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca²⁺ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca²⁺ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca²⁺]_i changes. These GTS-mediated [Ca²⁺]_i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg₃ are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca²⁺ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.     

Prenatal stress on the kinetic properties of Ca2+ and K+ channels in offspring hippocampal CA3 pyramidal neurons

Prenatal stress is known to cause neuronal loss and oxidative damage in the hippocampus of offspring rats. To further understand the mechanisms, the present study was undertaken to investigate the effects of prenatal stress on the kinetic properties of high-voltage-activated (HVA) Ca(2+) and K(+) channels in freshly isolated hippocampal CA3 pyramidal neurons of offspring rats. Pregnant rats in the prenatal stress group were exposed to restraint stress on days 14-20 of pregnancy three times daily for 45 min. The patch clamp technique was employed to record HVA Ca(2+) and K(+) channel currents. Prenatal stress significantly increased HVA Ca(2+) channel disturbance including the maximal average HVA calcium peak current amplitude (-576.52+/-7.03 pA in control group and -702.05+/-6.82 pA in prenatal stress group, p<0.01), the maximal average HVA Ca(2+) current density (-40.89+/-0.31 pA/pF in control group and -49.44+/-0.37 pA/pF in prenatal stress group, p<0.01), and the maximal average integral current of the HVA Ca(2+) channel (106.81+/-4.20 nA ms in control group and 133.49+/-4.59 nA ms in prenatal stress group, p<0.01). The current-voltage relationship and conductance--voltage relationship of HVA Ca(2+) channels and potassium channels in offspring CA3 neurons were not affected by prenatal stress. These data suggest that exposure of animals to stressful experience during pregnancy can exert effects on calcium ion channels of offspring hippocampal neurons and that the calcium channel disturbance may play a role in prenatal stress-induced neuronal loss and oxidative damage in offspring brain.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Copper inhibits P2Y2-dependent Ca signaling through the effects on thapsigargin-sensitive Ca stores in HTC hepatoma cells

Purinergic P2Y₂ G-protein coupled receptors play a key role in the regulation of hepatic Ca²⁺ signaling by extracellular ATP. The concentration of copper in serum is about 20 μM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y₂ receptors in a model liver cell line. Exposure to a P2Y₂ agonist UTP increased [Ca²⁺]_i by stimulating Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca²⁺]_i evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y₂ receptors in a concentration-dependent manner with the IC₅₀ of about 15 μM. These results suggest that copper inhibits P2Y₂ receptors through the effects on thapsigargin-sensitive Ca²⁺ stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.     

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.     

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

There is increasing evidence that a functional interaction exists between interleukin-1β (IL-1β) and N-methyl-d-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1β on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1β (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca²⁺-activated K⁺ (K_Ca) channels. IL-1β (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1β significantly inhibited ChTX-sensitive K_Ca currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1β. These results strongly suggest that IL-1β increases the neuronal excitability by inhibition of ChTX-sensitive K_Ca channels activated by Ca²⁺ influx through both NMDA receptors and voltage-gated Ca²⁺ channels.     

Influence of purinergic modulators on eEPSCs in rat CA3 hippocampal neurons: Contribution of ionotropic ATP receptors

ATP is considered to impact on fast synaptic transmission in several regions of the CNS, including the CA1 and CA3 areas of the hippocampus. The existing paradigm suggests that ATP induces synaptic responses in CA3 pyramidal cells, and a fast ATP-mediated component is observed in cultured hippocampal slices mainly under conditions of a synchronous discharge from multiple presynaptic inputs. We confirmed the existence of a fast ATP-mediated component within electrically evoked EPSCs (eEPSCs) in CA3 neurons of acute slices of the rat hippocampus using a whole-cell patch-clamp recording mode. In approximately 50% of the examined cells, eEPSCs were not completely inhibited by co-applied glutamate receptor antagonists, NBQX (50 μM) and D-APV (25 μM). The residual current was sensitive to ionotropic P2X receptor antagonists, such as suramin (25 μM) and NF023 (2 μM). Known purinergic receptor modulators, ivermectin (10 μM) and PPADS (10 μM), practically did not affect EPSCs, whereas a nonhydrolyzable ATP analog, ATPγS (100 μM), slightly decreased the EPSC amplitude. Moreover, ATPγS (100 μM) at a holding potential of −70 mV generated a slow inward current in most recorded neurons, which was insensitive to glutamate receptor antagonists. This fact is indicative of the ionotropic P2X receptor activation. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 21–29, January–February, 2008.     

1,25-Dihydroxyvitamin D3 induces Ca-mediated apoptosis in adipocytes via activation of calpain and caspase-12

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Functional GABA(B) receptors expressed in cultured calvarial osteoblasts

In immature and mature primary cultured rat calvarial osteoblasts, both mRNA and corresponding proteins were constitutively expressed for 2 splice variants of GABA(B) receptor (GABA(B)R) subunits but not for any known GABA(A) and GABA(C) receptor subunits. The agonist for GABA(B)R baclofen significantly inhibited cAMP formation induced by forskolin in a manner sensitive to the antagonist 2-hydroxysaclofen. Similar expression was seen with mRNA for GABA(B)R-1a and -1b splice variants in the murine calvarial osteoblast cell line MC3TC-E1 cells cultured for 7-21 days in vitro (DIV). In these MC3T3-E1 cells, baclofen not only inhibited the activity of alkaline phosphatase, but also exacerbated Ca2+ accumulation, throughout the culture period up to 28 DIV. These results suggest that GABA may play an unidentified role in mechanisms associated with cellular proliferation, differentiation, and/or development through functional GABA(B)R constitutively expressed in cultured osteoblasts.     

Involvement of K+ channels and calcium-dependent pathways in the action of T3 on amino acid accumulation and membrane potential in Sertoli cells of immature rat testis

The purpose of this study was to investigate the involvement of calcium in K+ currents and its effects on amino acid accumulation and on the membrane potential regulated by tri-iodo-L-thyronine (T3) in Sertoli cells. Immature rat testes were pre-incubated for 30 min in Krebs-Ringer bicarbonate buffer and incubated for 60 min in the presence of [14C]methylaminoisobutyric acid with and without T3 or T4 (dose-response curve). Specific channel blockers or chelating agents were added at different concentrations during pre-incubation and incubation periods to study the basal amino acid accumulation and a selected concentration of each drug was chosen to analyze the influence on the stimulatory hormone action. All amino acid accumulation experiments were carried out in a Dubnoff metabolic incubator at 32 degrees C, pH 7.4 and gassed with O2:CO2 (95:5; v/v). Seminiferous tubules from immature Sertoli cell-enriched testes were used for the electrophysiology experiments. Intracellular recording of the Sertoli cells was carried out in a chamber perfused with KRb with/without T3, T4 or blockers and the membrane potential was monitored. We found that T3 and T4 stimulated alpha-[1-14C] methylaminoisobutyric acid accumulation in immature rat testes and induced a membrane hyperpolarization in Sertoli cells. The action of T3 on amino acid accumulation and on the hyperpolarizing effect was inhibited by the K(+)-ATP channel blocker tolbutamide as well as the voltage-dependent Ca2+ channel blocker verapamil. These results clearly demonstrate for the first time the existence of an ionic mechanism related to Ca2+ and K+ fluxes in the rapid, nongenomic action of T3.     

Modulation of Ca2+-currents by sequential and simultaneous activation of adenosine A1 and A2A receptors in striatal projection neurons

D₁- and D₂-types of dopamine receptors are located separately in direct and indirect pathway striatal projection neurons (dSPNs and iSPNs). In comparison, adenosine A₁-type receptors are located in both neuron classes, and adenosine A_2A-type receptors show a preferential expression in iSPNs. Due to their importance for neuronal excitability, Ca²⁺-currents have been used as final effectors to see the function of signaling cascades associated with different G protein-coupled receptors. For example, among many other actions, D₁-type receptors increase, while D₂-type receptors decrease neuronal excitability by either enhancing or reducing, respectively, Ca_V1 Ca²⁺-currents. These actions occur separately in dSPNs and iSPNs. In the case of purinergic signaling, the actions of A₁- and A_2A-receptors have not been compared observing their actions on Ca²⁺-channels of SPNs as final effectors. Our hypotheses are that modulation of Ca²⁺-currents by A₁-receptors occurs in both dSPNs and iSPNs. In contrast, iSPNs would exhibit modulation by both A₁- and A_2A-receptors. We demonstrate that A₁-type receptors reduced Ca²⁺-currents in all SPNs tested. However, A_2A-type receptors enhanced Ca²⁺-currents only in half tested neurons. Intriguingly, to observe the actions of A_2A-type receptors, occupation of A₁-type receptors had to occur first. However, A₁-receptors decreased Ca_V2 Ca²⁺-currents, while A_2A-type receptors enhanced current through Ca_V1 channels. Because these channels have opposing actions on cell discharge, these differences explain in part why iSPNs may be more excitable than dSPNs. It is demonstrated that intrinsic voltage-gated currents expressed in SPNs are effectors of purinergic signaling that therefore play a role in excitability.     

Protection of cultured rat cortical neurons from excitotoxicity by asarone,a major essential oil component in the rhizomes of Acorus gramineus

Previous reports have shown that the methanol extract and the essential oil from Acori graminei Rhizoma (AGR) inhibited excitotoxic neuronal cell death in primary cultured rat cortical cells. In the present study, an active principle was isolated from the methanol extract by biological activity-guided fractionations and identified as asarone. We evaluated neuroprotective actions and action mechanisms of the isolated asarone as well as the alpha- and the beta-asarone obtained commercially. The isolated asarone inhibited the excitotoxicity induced by the exposure of cortical cultures for 15 min to 300 microM NMDA in a concentration-dependent manner, with the IC50 of 56.1 microg/ml. The commercially obtained alpha- and beta-asarone exhibited more potent inhibitions of the NMDA-induced excitotoxicity than the isolated asarone. Their respective IC50 values were 18.2 and 26.5 microg/ml. The excitotoxicity induced by glutamate (Glu) was also inhibited, but with much less potency than the toxicity induced by NMDA. The IC50 values for the alpha-, beta-, and the isolated asarone were 89.7, 121.7, and 279.5 microg/ml, respectively. Based on the receptor-ligand binding studies using a use-dependent NMDA receptor-channel blocker [3H]MK-801, asarone inhibited the specific bindings in a concentration-dependent fashion. These results indicate that asarone, the major essential oil component in AGR, exhibits neuroprotective action against the NMDA- or Glu-induced excitotoxicity through the blockade of NMDA receptor function. The alpha-asarone was found to exhibit more potent inhibition of [3H]MK-801 bindings, which is consistent with its more potent neuroprotective action than the beta- or the isolated asarone.     

The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation.These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor.