Mechanistic evaluation of a novel cyclohexenone derivative's functionality against nociception and inflammation: An in-vitro, in-vivo and in-silico approach.

The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharmacological evaluation as a putative anti-nociceptive and anti-inflammatory agent in mice. Initial in vitro studies revealed that CHD inhibited both cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes and it also reduced mRNA expression of COX-2 and the pro-inflammatory cytokines TNF-α and IL-1ß. It was then shown that CHD dose dependently inhibited chemically induced tonic nociception in the abdominal constriction assay and also phasic thermal nociception (i.e. anti-nociception) in the hot plate and tail immersion tests in comparison with aspirin and tramadol respectively. The thermal test outcomes indicated a possible moderate centrally mediated anti-nociception which, in the case of the hot plate test, was pentylenetetrazole (PTZ) and naloxone reversible, implicating GABAergic and opioidergic mechanisms. CHD was also effective against both the neurogenic and inflammatory mediator phases induced in the formalin test and it also disclosed anti-inflammatory activity against the phlogistic agents, carrageenan, serotonin, histamine and xylene compared with standard drugs in edema volume tests. In silico studies indicated that CHD possessed preferential affinity for GABAA, opioid and COX-2 target sites and this was supported by molecular dynamic simulations where computation of free energy of binding also favored the formation of stable complexes with these sites. These findings suggest that CHD has prospective anti-nociceptive and anti-inflammatory properties, probably mediated through GABAergic and opioidergic interactions supplemented by COX-2 and 5-LOX enzyme inhibition in addition to reducing pro-inflammatory cytokine expression. CHD may therefore possess potentially beneficial therapeutic effectiveness in the management of inflammation and pain.

Avances sobre el conocimiento y monitorización de la anestesia adecuada / Advances in knowledge and monitoring of adequate anaesthesia

Con la aparición de la Teoría de la cascada anestésica en el año 2005 y el conocimiento de los circuitos de reverberación sostenida, el concepto de la anestesia y de cómo se producen los distintos eventos relacionados en su proceso ha despertado a un sinfín de consideraciones directamente relacionadas con estructuras anatómicas encadenadas del sistema nervioso central y, aún más, de estructuras moleculares –situs y locus- que intervienen definitivamente en la producción de un fenómeno hasta ahora tan ambiguo como este. El, cada día, más creciente conocimiento de los receptores GABAA, NMDA, glutamato, glicina, de sus subunidades o de los canales iónicos transmembrana; de su ubicación y actuación por acción de los fármacos anestésicos; pone de relieve que las teorías hasta ahora conocidas mantenían una importante laguna que no explicaba el efecto anestésico ni sus parcelas. La hipótesis de que los anestésicos generales producen hipnosis, amnesia e inmovilidad por actuación en diversas partes del sistema nervioso central y por distintos mecanismos moleculares, arrancó hace tan solo diez años. Desde entonces, hemos aprendido la teoría unitaria de la narcosis se ha modificado desarrollando numerosos componentes, cada uno de los cuales interviene en microcircuitos neuronales discretos. Estas redes se caracterizan por células específicas, interconexiones y sistemas de neurotransmisión determinados, lo que expresa objetivos moleculares –locus- diferentes para los anestésicos generales y ello abre nuevas expectativas al desarrollo de fármacos específicos, desprovistos de efectos secundarios (AU)

UIT the onset of the Anesthetic Cascade Theory in 2005 and the knowledge of sustained reverberation circuits, the events related with the anesthetic process and the anesthesia concept have woke up a lot of considerations about the connections of anatomic structures of central nervous system and, even more, about molecular structures –situs and locus- definitively implicated in the production of such ambiguous phenomenon like this. The growing knowledge about GABAA, NMDA, glutamate and glycine receptors, their subunits and their ionic channels; the knowledge about the location and their activation mediated by anesthetic drugs remarks that actual theories had an important defect because they didn´t explain the anesthetic effect or its components. The hypothesis that explains the effect of general anesthetics as producers of amnesia, hypnosis and immobility operating on different structures of central nervous system and by different molecular mechanisms started just ten years ago. Since then, we have learnt that narcosis unitary theory ha been modified developing new components, each one of them works are characterized by specific cell types, interconnections and specific neurotransmitter system that reveal different molecular targets –locus- of general anesthetic agents, so the knowledge of this new locus opens a new era in the development of new specific anesthetic drugs without unwanted side-effects (AU)

Microfluidic gradient-generating device for pharmacological profiling.

We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cell-based sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K+ channels (hERG) and ligand-gated GABA(A) receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC50 and EC50 values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor.

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

A single amino acid change within the ion-channel domain of the gamma-aminobutyric acid rho1 receptor accelerates desensitization and increases taurine agonism.

BACKGROUND: GABAC receptors are part of the ligand-gated ion channel family of receptors that share some functional and structural features: e.g., they have four putative transmembrane domains (TM1-TM4) and the TM2-segment is presumed to form the ion-channel. GABAC receptors open chloride channels and do not desensitize even after long exposures to GABA. These receptors are highly expressed in vertebrate retina, where they may play a unique role due to their unusual biophysical and pharmacologic characteristics. METHODS: To determine whether the TM2 domain plays a role in the process of desensitization of GABAC receptors, we used site-directed mutagenesis to produce several permutations within the leucine (L9') residue of the TM2 domain of the human GABArho1 subunit. Recombinant receptors were expressed in Xenopus laevis oocytes and their functional and pharmacologic properties were studied by using a two-microelectrode, voltage-clamp. RESULTS: Several amino acid changes led to receptors that did not generate GABA-currents, whereas an Asp for Leu mutation in the well-conserved L9' position of the rho1 subunit (L301D-rho1) generated a fast-desensitizing, bicuculline-resistant receptor that was antagonized by TPMPA, a specific GABAC receptor antagonist. Moreover, in contrast with wild-type rho1 receptors, which are practically not gated by taurine, L301D-rho1 mutant receptors generated substantial taurine-currents. CONCLUSIONS: Substitution of L9' residue in the TM2 region of GABArho1 receptor for an amino acid residue with an acidic lateral chain greatly accelerates its desensitization rate and increases taurine-agonism. This mutant will be useful to study mechanisms involved in gating and desensitization of GABAC receptors in particular, and of neurotransmitter receptors in general.

Complementary regulation of anaesthetic activation of human (alpha6beta3gamma2L) and Drosophila (RDL) GABA receptors by a single amino acid residue.

1. The influence of a transmembrane (TM2) amino acid located at a homologous position in human beta1 (S290) and beta3 (N289) GABAA receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2. GABA-evoked currents mediated by human wild-type (WT) alpha6beta3gamma2L or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated alpha6beta3gamma2L, but not RDL, receptors. 3. GABA-evoked currents mediated by human mutant GABAA receptors expressing the RDL methionine residue (i.e. alpha6beta3N289Mgamma2L) were potentiated by propofol or pentobarbitone with approximately 2-fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4. Incorporation of the homologous GABAA beta1-subunit residue in the RDL receptor (i.e. RDLM314S) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous beta3-subunit residue (i.e. RDLM314N) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5. The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (alpha6beta3gamma2L) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed.

Separate domains for desensitization of GABA rho 1 and beta 2 subunits expressed in Xenopus oocytes.

Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA beta2 subunits from the brain and rho1 subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic chimeric receptor/channel was created from the beta2 and rho1 subunits for this investigation. The chimera was constructed from the extracellular N-domain of the beta2 subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain of the rho1 subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA beta2, rho1, and beta2/rho1 chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the beta2 subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated current decay.

Channel gating in the absence of agonist by a homo-oligomeric molluscan GABA receptor expressed in Xenopus oocytes from a cloned cDNA.

We have previously described the isolation of a complementary DNA (cDNA) from the freshwater mollusc Lymnaea stagnalis encoding a polypeptide that exhibits approximately 50% identity to the beta-subunits of vertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor. When expressed in Xenopus laevis oocytes from in vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at -60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of approximately 20 pS channels, with an open probability greater than 90% (at a pipette potential of -100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (-100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABAA receptor alpha 1-subunit, but the channels gated by these receptors did not open spontaneously.

Small N-terminal deletion by splicing in cerebellar alpha 6 subunit abolishes GABAA receptor function.

Sequence variation was found in cDNA coding for the extracellular domain of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit. About 20% of polymerase chain reaction (PCR)-amplified alpha 6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226-255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (alpha 6S) and long (wild-type) forms of the alpha 6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the alpha 6S subunit with the GABAA receptor beta 2 and gamma 2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by beta 2 gamma 2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the alpha 6 subunit-containing receptors.

The agonist binding site of the gamma-aminobutyric acid type A channel is not formed by the extracellular cysteine loop.

The amino-terminal extracellular domain of the subunits comprising the gamma-aminobutyric acid (GABA) receptor contains two cysteine residues (designated at relative positions 1 and 15) separated by 13 amino acids. These two cysteines (presumably disulfide bonded) are located approximately 150 amino acids from the amino terminus. There is significant homology in the amino acid sequence of this cysteine loop both between the different subunits of the GABA receptor and with subunits of other members of this ligand-gated ion channel superfamily (nicotinic acetylcholine- and glycine-activated ion channels). A number of highly conserved amino acids within the cysteine loop have been postulated to play a role in agonist binding. Here, using site-directed mutagenesis and oocyte expression, we have examined the effects of mutating amino acids comprising the cysteine loop on the activation of recombinant GABA channels composed of rat alpha 1, beta 2 and gamma 2 subunits. Preventing the formation of the putative cysteine-cysteine disulfide bond in any of the subunits, by mutating the cysteine at position 15 to serine, prevented the functional expression of that subunit. For example, coexpression of gamma C15S with wild-type alpha and beta subunits resulted in GABA-activated currents with properties identical to those of GABA-activated currents from coexpression of alpha and beta subunits alone. These properties included sensitivity to activation by GABA (similar EC50 values), blockade by Zn2+, and lack of modulation by the benzodiazepine diazepam. We also mutated conserved amino acids in the beta subunit that had been specifically proposed to form the GABA binding site (beta R6, beta Y8, and beta D11). These mutations (as well as several others within or adjacent to the cysteine loop) produced either a very moderate effect or no effect on GABA sensitivity, suggesting that these particular amino acids do not play a key role in activation of the GABA channel. The data presented in this study support a role for the cysteine loop in subunit assembly, rather than channel activation.