Brain active transmembrane water cycling measured by MR is associated with neuronal activity.

PURPOSE: fMRI is widely used to study brain activity. Unfortunately, conventional fMRI methods assess neuronal activity only indirectly, through hemodynamic coupling. Here, we show that active, steady-state transmembrane water cycling (AWC) could serve as a basis for a potential fMRI mechanism for direct neuronal activity detection. METHODS: AWC and neuronal actitivity in rat organotypic cortical cultures were simultaneously measured with a hybrid MR-fluorescence system. Perfusion with a paramagnetic MRI contrast agent, Gadoteridol, allows NMR determination of the kinetics of transcytolemmal water exchange. Changes in intracellular calcium concentration, [Cai2+ ] were used as a proxy of neuronal activity and were monitored by fluorescence imaging. RESULTS: When we alter neuronal activity by titrating with extracellular [K+ ] near the normal value, we see an AWC response resembling Na+ -K+ -ATPase (NKA) Michaelis-Menten behavior. When we treat with the voltage-gated sodium channel inhibitor, or with an excitatory postsynaptic inhibitor cocktail, we see AWC decrease by up to 71%. AWC was found also to be positively correlated with the basal level of spontaneous activity, which varies in different cultures. CONCLUSIONS: These results suggest that AWC is associated with neuronal activity and NKA activity is a major contributor in coupling AWC to neuronal activity. Although AWC comprises steady-state, homeostatic transmembrane water exchange, our analysis also yields a simultaneous measure of the average cell volume, which reports any slower net transmembrane water transport.

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.

Ginkgolide X is a potent antagonist of anionic Cys-loop receptors with a unique selectivity profile at glycine receptors.

The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and gamma-aminobutyric acid type A receptors (GlyRs and GABA(A)Rs, respectively) in the fluorescence-based FLIPR(TM) Membrane Potential assay. The compound inhibited the signaling of all GABA(A)R subtypes included in the study with high nanomolar/low micromolar IC(50) values, except the rho 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC(50) values at the homomeric alpha1 and alpha2 GlyRs, whereas it was inactive at the heteromeric alpha 1 beta and alpha 2 beta subtypes at concentrations up to 300 microm. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6' M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA(A)R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a alpha1 GlyR homology model revealed two distinct putative binding sites formed by the 6' M2 residues together with the 2' residues and the 10' and 13' residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor.

Conformational variability of the glycine receptor M2 domain in response to activation by different agonists.

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop.

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.

Espectro de acción antiepiléptica de la DL-4-hidroxi, 4-etil,4-fenil butiramida y sus homólogos inferiores / Profile of the antiepileptic action of DL-4-hydroxy, 4-ethyl, 4-phenyl butiramide and its inferior homologues

Se estudio la actividad farmacológica en animales de una serie nueva de compuestos anticonvulsionantes, la DL-4-hidroxil, 4-etil, 4-fenil butiramida (HEPB) y sus homólogos inferiores propionamida (HEPP) y acetamida (HEPA). La neurotoxidad fue determinada con un rotarod y se indujeron cuadros convulsivos con electrochoque supramáximo (MES), pentilentetrazol (TSC), estricnina (STR) y picrotoxina (PIC). HEPP es menos neurotóxica que HEPB y HEPA y altera el comportamiento de los ratones solamente a dosis altas. Los tres compuestos presentaron un amplio espectro de acción anticonvulsionante. Ellos son muy potentes para inhibir cuadros convulsivos inducidos con 4-AP, BIC, TSC y PTZ en dosis no tóxicas administradas ip, pero son inefectivas contra convulsiones inducidas con pic y STR. Los índices terapéuticos (IT = DT 50/D 50) fueron más elevados para HEPP. En consecuencia los resultados indican que los tres compuestos probados pueden servir para el tratamiento de convulsiones generalizadas tipo ausencias. Puesto que HEPP es el compuesto menos neurotóxico se ha seleccionado para los estudios toxicológicos y neuroquímicos