Investigation of phytochemical constituents of anti-leukemic herbal drugs used by the traditional healers of Purulia, Birbhum and Bankura districts of West Bengal.

In the present work, 16 different plant drugs used by traditional healers from West Bengal were screened through in vitro cell line model. Herbal drugs used by traditional tribal healers in Purulia, Birbhum and Bankura districts of West Bengal were collected and screening against acute myeloid leukemia (AML) cell line (HL-60). Among 16 plant extracts, bark of Flacourtia indica (66.67%), leaf of Madhuca longifolia (69.17%), and leaf of Prosopis cineraria (68.08%) showed better cytotoxicity results than other herbals. Further, time-dependent study showed maximum cytotoxicity of the selected herbal extracts between 36 and 48 hours of treatment in both acute and chronic myeloid leukemia (CML) cell lines (HL-60 and K562). The LC-MS/MS analysis of the selected drugs revealed the presence of picrotoxinin and 10-deacetylbaccatin from F. indica, isoorientin and hirsutrin from M. longifolia, vitexin and rhoifolin in P. cineraria.

Restoring Light Sensitivity in Blind Retinae Using a Photochromic AMPA Receptor Agonist.

Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

Phytochemistry and biology of Loranthus parasiticus Merr, a commonly used herbal medicine.

Loranthus parasiticus Merr (L. parasiticus) is a member of Loranthaceae family and is an important medicinal plant with a long history of Chinese traditional use. L. parasiticus, also known as Sang Ji Sheng (in Chinese), benalu teh (in Malay) and baso-kisei (in Japanese), is a semiparasitic plant, which is mostly distributed in the southern and southwestern regions of China. This review aims to provide a comprehensive overview of the ethnomedicinal use, phytochemistry and pharmacological activity of L. parasiticus and to highlight the needs for further investigation and greater global development of the plant's medicinal properties. To date, pharmacological studies have demonstrated significant biological activities, which support the traditional use of the plant as a neuroprotective, tranquilizing, anticancer, immunomodulatory, antiviral, diuretic and hypotensive agent. In addition, studies have identified antioxidative, antimutagenic, antiviral, antihepatotoxic and antinephrotoxic activity. The key bioactive constituents in L. parasiticus include coriaria lactone comprised of sesquiterpene lactones: coriamyrtin, tutin, corianin, and coriatin. In addition, two proanthocyanidins, namely, AC trimer and (+)-catechin, have been recently discovered as novel to L. parasiticus. L. parasiticus usefulness as a medicinal plant with current widespread traditional use warrants further research, clinical trials and product development to fully exploit its medicinal value.

Over-expression of Mash1 improves the GABAergic differentiation of bone marrow mesenchymal stem cells in vitro.

Bone marrow mesenchymal stem cells (BMSCs) have been shown to be a promising cell type for the study of neuronal differentiation; however, few attempts had been made to differentiate these cells into inhibitory gamma-aminobutyric acid (GABA)ergic neurons. In this study, we over-expressed mammalian achaete-scute homologue-1 (Mash1), a basic helix-loop-helix (bHLH) transcription factor, in Sprague-Dawley rat BMSCs via lentiviral vectors, and then induced neuronal differentiation of these cells using conditioned medium. Our Western blot results show that, under conditions of differentiation, Mash1-overexpressing BMSCs exhibit an increased expression of neuronal markers and a greater degree of neuronal morphology compared to control, non-Mash1-overexpressing cells. Using immunocytochemistry, we observed increased expression of glutamic acid decarboxylase 67 (GAD67), as well as neuron-specific nuclear protein (NeuN) and ß3-tubulin, in Mash1-overexpressing BMSCs compared to control cells. Moreover, we also found the differentiated cells showed representative traces of action potentials in electrophysiological characterization. In conclusion, our study demonstrated that over-expression of Mash1 can improve GABAergic differentiation of BMSCs in vitro.

Amyloid precursor proteins interact with the heterotrimeric G protein Go in the control of neuronal migration.

Amyloid precursor protein (APP) belongs to a family of evolutionarily conserved transmembrane glycoproteins that has been proposed to regulate multiple aspects of cell motility in the nervous system. Although APP is best known as the source of ß-amyloid fragments (Aß) that accumulate in Alzheimer's disease, perturbations affecting normal APP signaling events may also contribute to disease progression. Previous in vitro studies showed that interactions between APP and the heterotrimeric G protein Goα-regulated Goα activity and Go-dependent apoptotic responses, independent of Aß. However, evidence for authentic APP-Go interactions within the healthy nervous system has been lacking. To address this issue, we have used a combination of in vitro and in vivo strategies to show that endogenously expressed APP family proteins colocalize with Goα in both insect and mammalian nervous systems, including human brain. Using biochemical, pharmacological, and Bimolecular Fluorescence Complementation assays, we have shown that insect APP (APPL) directly interacts with Goα in cell culture and at synaptic terminals within the insect brain, and that this interaction is regulated by Goα activity. We have also adapted a well characterized assay of neuronal migration in the hawkmoth Manduca to show that perturbations affecting APPL and Goα signaling induce the same unique pattern of ectopic, inappropriate growth and migration, analogous to defective migration patterns seen in mice lacking all APP family proteins. These results support the model that APP and its orthologs regulate conserved aspects of neuronal migration and outgrowth in the nervous system by functioning as unconventional Goα-coupled receptors.

The LRRC26 protein selectively alters the efficacy of BK channel activators.

Large conductance, Ca(2+)-activated K channel proteins are involved in a wide range of physiological activities, so there is considerable interest in the pharmacology of large conductance calcium-activated K (BK) channels. One potent activator of BK channels is mallotoxin (MTX), which produces a very large hyperpolarizing shift of the voltage gating of heterologously expressed BK channels and causes a dramatic increase in the activity of BK channels in human smooth muscle cells. However, we found that MTX shifted the steady-state activation of BK channels in native parotid acinar cells by only 6 mV. This was not because the parotid BK isoform (parSlo) is inherently insensitive to MTX as MTX shifted the activation of heterologously expressed parSlo channels by 70 mV. Even though MTX had a minimal effect on steady-state activation of parotid BK channels, it produced an approximate 2-fold speeding of the channel-gating kinetics. The BK channels in parotid acinar cells have a much more hyperpolarized voltage activation range than BK channels in most other cell types. We found that this is probably attributable to an accessory protein, LRRC26, which is expressed in parotid glands: expressed parSlo + LRRC26 channels were resistant to the actions of MTX. Another class of BK activators is the benzimidazalones that includes 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). Although the LRRC26 accessory protein strongly inhibited the ability of MTX to activate BK channels, we found that it had only a small effect on the action of NS-1619 on BK channels. Thus, the LRRC26 BK channel accessory protein selectively alters the pharmacology of BK channels.

Cys-loop receptor channel blockers also block GLIC.

The Gloeobacter ligand-gated ion channel (GLIC) is a bacterial homolog of vertebrate Cys-loop ligand-gated ion channels. Its pore-lining region in particular has a high sequence homology to these related proteins. Here we use electrophysiology to examine a range of compounds that block the channels of Cys-loop receptors to probe their pharmacological similarity with GLIC. The data reveal that a number of these compounds also block GLIC, although the pharmacological profile is distinct from these other proteins. The most potent compound was lindane, a GABA(A) receptor antagonist, with an IC50 of 0.2 µM. Docking studies indicated two potential binding sites for this ligand in the pore, at the 9' or between the 0' and 2' residues. Similar experiments with picrotoxinin (IC50 = 2.6 µM) and rimantadine (IC50 = 2.6 µM) reveal interactions with 2'Thr residues in the GLIC pore. These locations are strongly supported by mutagenesis data for picrotoxinin and lindane, which are less potent in a T2'S version of GLIC. Overall, our data show that the inhibitory profile of the GLIC pore has considerable overlap with those of Cys-loop receptors, but the GLIC pore has a unique pharmacology.

Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.

The rho 1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the rho 1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (pregnanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, allopregnanolone sulfate, and beta-estradiol) and probed how these steroids, as well as the nonsteroidal inhibitor picrotoxinin, modify GABA-elicited fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The fluorophore responds with changes in quantum yield to changes in the environment, allowing it to probe for structural changes taking place during channel activation or modulation. The results indicate that the modulators have specific effects on fluorescence changes suggesting that distinct conformational changes accompany inhibition. The findings are consistent with the steroids acting as allosteric inhibitors of the rho 1 GABA receptor and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the rho 1 GABA receptor.

GLC-3: a novel fipronil and BIDN-sensitive, but picrotoxinin-insensitive, L-glutamate-gated chloride channel subunit from Caenorhabditis elegans.

1. We report the cloning and expression of a novel Caenorhabditis elegans polypeptide, GLC-3, with high sequence identity to previously cloned L-glutamate-gated chloride channel subunits from nematodes and insects. 2. Expression of glc-3 cRNA in XENOPUS oocytes resulted in the formation of homo-oligomeric L-glutamate-gated chloride channels with robust and rapidly desensitizing currents, an EC(50) of 1.9+/-0.03 mM and a Hill coefficient of 1.5+/-0.1. GABA, glycine, histamine and NMDA all failed to activate the GLC-3 homo-oligomer at concentrations of 1 mM. The anthelminthic, ivermectin, directly and irreversibly activated the L-glutamate-gated channel with an EC(50) of 0.4+/-0.02 microM. 3. The GLC-3 channels were selective for chloride ions, as shown by the shift in the reversal potential for L-glutamate-gated currents after the reduction of external Cl(-) from 107.6 to 62.5 mM. 4. Picrotoxinin failed to inhibit L-glutamate agonist responses at concentrations up to 1 mM. The polycyclic dinitrile, 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile (BIDN), completely blocked L-glutamate-induced chloride currents recorded from oocytes expressing GLC-3 with an IC(50) of 0.2+/-0.07 microM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L-glutamate-gated currents recorded from the GLC-3 receptor with an IC(50) of 11.5+/-0.11 microM. 5. In this study, we detail the unusual antagonist pharmacology of a new GluCl subunit from C. elegans. Unlike all other native and recombinant nematode GluCl reported to date, the GLC-3 receptor is insensitive to picrotoxinin, but is sensitive to two other channel blockers, BIDN and fipronil. Further study of this receptor may provide insights into the molecular basis of non-competitive antagonism by these compounds.

Interaction of the neurotoxic pesticides ivermectin and lindane with the enteric GABAA receptor-ionophore complex in the guinea-pig.

In isolated segments of guinea-pig small intestine, gamma-aminobutyric acid (GABA) (3-300 microM), the GABAA receptor agonist 3-aminopropane sulphonic acid (3-APS) (3-300 microM) and ivermectin (1-300 microM) caused concentration-dependent nerve-mediated cholinergic contractions sensitive to tetrodotoxin (1 microM) and hyoscine (1 microM). The EC50 values were 30.2 +/- 4.3, 24.6 +/- 3.1 and 4.8 +/- 0.6 microM, respectively. Picrotoxinin (10 microM), an allosteric blocker of the Cl- channel associated with GABAA receptors, non-competitively antagonized the contractile response caused by each agonist. Like picrotoxinin, lindane (10, 30 microM) caused a dose-related shift to the right of the concentration-response curve to GABA, 3-APS and ivermectin with depression of the maximum response. SR 95531 (3 microM), a competitive antagonist of GABAA receptors, caused a parallel dextral shift of the concentration-response curve to ivermectin with an apparent single point pA2 value of 6.5. Our results suggest that ivermectin and lindane, two neurotoxic pesticides interfering with central GABAErgic transmission, exert agonist and non-competitive antagonist properties at the enteric GABAA receptor-ionophore complex. This peripheral complex can thus be considered as an additional target for the action of both these compounds.

Picrotoxinin and phorbol-12-myristate-13-acetate stimulate the secretion of multiple forms of somatostatin from cultured rat brain cells.

The molecular forms of somatostatin (SRIF) secreted by cultured fetal rat brain cells were resolved using reverse phase high performance liquid chromatography followed by radioimmunoassay. Multiple forms of SRIF-like immunoactivity were detected in media from cells treated with either picrotoxinin, phorbol-12-myristate-13-acetate, or high potassium. For stimulated cells, elevated levels of an SRIF-28-like molecule, an SRIF-14-like molecule, and a hydrophobic SRIF-like molecule were observed compared to basal conditions. All three forms of SRIF-like molecules were also detected in acid extracts of whole cells. The data are consistent with the possibility that secretion of multiple SRIFs , including SRIF-28, may be regulated by multiple effectors and mechanisms.

Evidence for association of a high affinity avermectin binding site with the benzodiazepine receptor.

Avermectin B1a concentrations corresponding to the KD value of a specific high affinity binding site for [3H]avermectin B1a are sufficient to stimulate the specific high affinity binding of [3H]flunitrazepam to its receptors. This stimulation of [3H]flunitrazepam binding by low concentrations of avermectin B1a was enhanced by chloride ions and picrotoxinin and reduced by the GABA agonists THIP and PSA. The similar modulation by chloride ions, GABA agonists and picrotoxinin and the resistance against washing of membranes of avermectin B1a bound to its specific high affinity binding site or to the site modulating [3H]flunitrazepam binding indicate a close association of the specific high affinity binding site for [3H]avermectin B1a with the GABA-benzodiazepine receptor complex.

Interaction of barbiturates with adenosine receptors in rat brain.

The effects of barbiturates on radioligand binding to inhibitory Ri adenosine receptors of rat brain membranes were investigated. Binding of the adenosine receptor agonist (-)N6-phenylisopropyl[3H]adenosine and the antagonist 1,3-diethyl-8-[3H]phenylxanthine was inhibited by several barbiturates. This inhibition was concentration-dependent and occurred in the range of pharmacologically effective concentrations. Pentobarbital was the most potent of the barbiturates tested with a Ki of 92 mumol/l. The (+)isomers of hexobarbital and mephobarbital were more potent than the respective (-)isomers. Barbituric acid itself did not displace either radioligand in concentrations up to 1 mmol/l. The inhibitory effect of pentobarbital was reversed by a single wash of membranes preincubated with the barbiturate. The presence of pentobarbital caused a decrease of the affinity of the receptor for the antagonist radioligand but did not alter the number of binding sites, suggesting a competitive antagonism. The effects of pentobarbital on radioligand binding to the receptor were not changed by the presence of picrotoxinin nor by the absence of chloride ions. This indicates that they are not mediated via the picrotoxinin binding site. The barbiturates could not be classified as either agonists or antagonists at the Ri adenosine receptor. The presence of GTP did not influence the inhibition of radioligand binding by pentobarbital; this is also observed for antagonists, whereas the affinity of agonists is markedly reduced by GTP. Binding of antagonists to the receptor is enthalpy-driven; the interaction of pentobarbital with the receptor was entropy-driven and the same was true for agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of gamma-aminobutyric acid (GABA) and ethylenediamine (EDA) on Limulus and Helix central neurones and rat cerebellar and sympathetic ganglion neurones.

Intracellular recordings were made from central Limulus and Helix neurones and extracellular recordings from rat cerebellar Purkinje cells and sympathetic ganglia. The actions of gamma-aminobutyric acid (GABA) and ethylenediamine (EDA) and related analogues on these preparations were investigated. On Limulus neurones inhibited by GABA, EDA and piperazine were 81 and 186 times respectively less potent than GABA. Both the GABA and EDA events were chloride mediated, having similar reversal potentials and were reversibly antagonised by picrotoxinin. The EDA response persisted in high magnesium Ringer. On Helix neurones inhibited by GABA, EDA was 92 times less potent while on neurones excited by GABA, EDA was 9.25 times less potent. The other analogues tested had little or no GABA-like effect on either preparation. On rat cerebellar Purkinje cells, EDA was equipotent with GABA and both compounds were antagonised by bicuculline. Flurazepam only potentiated the action of EDA on 3 out of 23 cells tested while the GABA response of all 23 cells was potentiated by the benzodiazepine. Diaminopropionic acid was a weak inhibitor of cerebellar Purkinje cell firing but flurazepam potentiated this response in 6 out of 10 cells tested. On rat cervical ganglion neurones, EDA was half the potency of GABA and likewise the other analogues were less potent than GABA as depolarising agents. Incubation with glutamic acid decarboxylase inhibitors had no effect on the EDA response. Cross desensitisation between GABA and EDA was demonstrated using the ganglion preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for similarities between cyclodiene type insecticides and picrotoxinin in their action mechanisms.

Evidence has been obtained to indicate that cyclodiene-type insecticides, e.g., heptachlor epoxide and gamma-BHC, mimic the action of picrotoxinin. These insecticides inhibit the GABA (gamma-aminobutyric acid)-stimulated chloride uptake in the coxal muscle of the American cockroach, and directly compete against [3H]a-dihydropicrotoxinin for binding in the rat brain synaptosomes. Moreover, several cyclodiene-resistant insect strains are also resistant to picrotoxinin. This cross-resistance is specific to picrotoxinin and does not extend to other neuroexcitants. These insecticides, like picrotoxinin, cause central nerve excitation by stimulating transmitter release. Similarity in molecular structures also has been pointed out. These results indicate that some of the nerve excitation symptoms that insecticides cause are likely due to their interaction with picrotoxinin receptor.

On the mechanism by which dopamine inhibits prolactin release in the anterior pituitary.

An in vitro perfusion system was used to assess the effects of chloride channel blockers, dopamine (DA) receptor agonists and antagonists, and GABA receptor agonists and antagonists on prolactin release from the mouse anterior pituitary. Dopamine and muscimol inhibited prolactin release (IC50 = 6 X 10(-8)M and 10(-5)M respectively). The GABA receptor antagonist bicuculline blocked the inhibition of prolactin release by muscimol but not dopamine. The dopamine receptor antagonist chlorpromazine blocked the dopamine- but not muscimol-induced inhibition of prolactin release. Haloperidol, however, reversed both the muscimol and dopamine induced inhibition of prolactin release. Furthermore, the chloride channel blocker picrotoxinin blocked the inhibition of prolactin release elicited by both dopamine and muscimol. These later results suggest that the anterior pituitary dopamine receptor which mediates the inhibition of prolactin release may be coupled to a picrotoxinin sensitive chloride ionophore and that haloperidol may affect the function of both DA and GABA receptors in the anterior pituitary.