Reinforcing effects of compounds lacking intrinsic efficacy at α1 subunit-containing GABAA receptor subtypes in midazolam- but not cocaine-experienced rhesus monkeys

Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors ('α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration

Structural studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa : Inhibitor complexes with Anions and Acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules

Modulation of lipopolysaccharide-induced neuronal response by activation of the enteric nervous system.

International audienceBackground:Evidence continues to mount concerning the importance of the enteric nervous system (ENS) incontrolling numerous intestinal functions in addition to motility and epithelial functions. Nevertheless, little isknown concerning the direct participation of the ENS in the inflammatory response of the gut during infectious orinflammatory insults. In the present study we analyzed the ENS response to bacterial lipopolysaccharide, inparticular the production of a major proinflammatory cytokine, tumor necrosis factor-alpha (TNF-α).Methods:TNF-αexpression (measured by qPCR, quantitative Polymerase Chain Reaction) and production(measured by ELISA) were measured in human longitudinal muscle-myenteric plexus (LMMP) and rat ENS primarycultures (rENSpc). They were either treated or not treated with lipopolysaccharide (LPS) in the presence or not ofelectrical field stimulation (EFS). Activation of extracellular signal-regulated kinase (ERK) and 5?-adenosinemonophosphate-activated protein kinase (AMPK) pathways was analyzed by immunocytochemistry and Westernblot analysis. Their implications were studied using specific inhibitors (U0126, mitogen-activated protein kinasekinase, MEK, inhibitor and C compound, AMPK inhibitor). We also analyzed toll-like receptor 2 (TLR2) expression andinterleukin-6 (IL-6) production after LPS treatment simultaneously with EFS or TNF-α-neutralizing antibody.Results:Treatment of human LMMP or rENSpc with LPS induced an increase in TNF-αproduction. Activation of theENS by EFS significantly inhibited TNF-αproduction. This regulation occurred at the transcriptional level. Signalinganalyses showed that LPS induced activation of ERK but not AMPK, which was constitutively activated in rENSpcneurons. Both U0126 and C compound almost completely prevented LPS-induced TNF-αproduction. In the presenceof LPS, EFS inhibited the ERK and AMPK pathways. In addition, we demonstrated using TNF-α-neutralizing antibody thatLPS-induced TNF-αproduction increased TLR2 expression and reduced IL-6 production.Conclusions:Our results show that LPS induced TNF-αproduction by enteric neurons through activation of thecanonical ERK pathway and also in an AMPK-dependent manner. ENS activation through the inhibition of thesepathways decreased TNF-αproduction, thereby modulating the inflammatory response induced by endotoxin

Neural bases for addictive properties of benzodiazepines

Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability