Presynaptic gating of excitation in the dorsal raphe nucleus by GABA.

The dorsal raphe nucleus (DR) controls forebrain serotonin neurotransmission to influence emotional states. GABA neurotransmission in the DR has been implicated in regulating sleep/wake states and influencing anxiety and aggression. To gain insight into how GABA regulates DR activity, we analyzed the organization of both GABA and glutamate axons in the rat DR using a high-resolution immunofluorescence technique, array tomography, as well as EM. This analysis revealed that a third or more of GABA-containing axons are organized in synaptic triads with a glutamatergic axon and a common postsynaptic target. Electrophysiological recordings showed that GABA has the capacity to presynaptically gate glutamate release in the DR through a combination of GABA-A and GABA-B receptor-mediated effects. Thus, GABA-glutamate synaptic triads are a common feature of the network architecture of the DR with the potential to regulate excitation of the nucleus.

The contribution of endocytosis to sensitization of nociceptors and synaptic transmission in nociceptive circuits.

ABSTRACT: Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.

Sensitive and Robust LC-MS/MS Analysis of Salivary Cortisol in Negative Mode.

Cortisol is one of the most important glucocorticoids involved in the regulation of human metabolism and physiological stress. Monitoring of levels of cortisol is of immense clinical benefit. In particular, salivary cortisol levels have been shown to correlate well with diurnal changes in cortisol levels in serum and have been used widely for monitoring of cortisol levels for diagnosis and prognosis purposes. We present a sensitive, fast, and robust quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) assay for salivary cortisol in negative mode. This assay employs protein precipitation followed by reversed-phase liquid chromatographic separation, negative-mode electrospray ionization (ESI), and MS/MS detection. This assay has a total run time of 5.8 minutes and a limit of quantification of 0.5 ng/mL with a linear range up to 100 ng/mL. No carryover was observed at 10 µg/mL. This assay also incorporates the routine monitoring of prednisolone, a potential interferent to salivary cortisol.

Discovery of 3,9-diazabicyclo[4.2.1]nonanes as potent dual orexin receptor antagonists with sleep-promoting activity in the rat.

Orexins are excitatory neuropeptides that regulate arousal and sleep. Orexin receptor antagonists promote sleep and offer potential as a new therapy for the treatment of insomnia. In this Letter, we describe the synthesis of constrained diazepanes having a 3,9 diazabicyclo[4.2.1]nonane bicyclic core with good oral bioavailability and sleep-promoting activity in a rat EEG model.

Different forms of glycine- and GABA(A)-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons.

BACKGROUND: Neurons in superficial (SDH) and deep (DDH) laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR) and GABAA-receptors (GABAARs) contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits) in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood. METHODS AND RESULTS: Here we compare fast inhibitory synaptic transmission in mouse (P17-37) SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23 degrees C). GlyR-mediated mIPSCs were detected in 74% (25/34) and 94% (25/27) of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18). Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 +/- 3.9 vs. 64.7 +/- 5.0 pA; n = 25 each), decay time was slower (8.5 +/- 0.8 vs. 5.5 +/- 0.3 ms), and frequency was lower (0.15 +/- 0.03 vs. 0.72 +/- 0.13 Hz) in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 +/- 2.4, n = 14 vs. 25. +/- 2.0 pA, n = 18) and frequencies (0.21 +/- 0.08 vs. 0.18 +/- 0.04 Hz) in both regions; however, decay times were slower (23.0 +/- 3.2 vs. 18.9 +/- 1.8 ms) in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 +/- 1.6 pS, n = 11 vs. 55.7 +/- 1.8, n = 8) and GABAARs (22.7 +/- 1.7 pS, n = 10 vs. 22.4 +/- 2.0 pS, n = 11) in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA), had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 muM) reduced GlyR-mediated mIPSC frequency in SDH and DDH, but did not affect other properties. Similar results were observed for GABAAR mediated mIPSCs, however, rise time was slowed by methAEA in SDH neurons. CONCLUSION: Together these data show that Gly- and GABAARs with clearly differing physiological properties and cannabinoid-sensitivity contribute to fast synaptic inhibition in mouse SDH and DDH.

The effects of haloperidol treatment on the distribution of NK1 receptor immunoreactive neurons in guinea-pig brain.

Previous studies have observed increased tachykinin NK(1) receptor immunoreactivity (NK(1)-IR) in the prefrontal cortex in subjects with schizophrenia. Since the subjects were medicated the possibility of a treatment effect could not be excluded. Thus, the present study was undertaken to determine the effect of chronic treatment with the antipsychotic drug, haloperidol, on the distribution of NK(1)-IR neurons in the guinea-pig brain. Guinea pigs were treated each day for 21 days with either haloperidol (1mg/kg) or vehicle and the brains were then processed for immunohistochemistry using an NK(1) receptor-specific polyclonal antibody. NK(1)-IR neurons and fibres were abundant in the forebrain cortex and caudate putamen and more sparsely distributed in a number of other brain regions. The relative density of NK(1)-IR neurons was significantly increased in the forebrain cortex, but not in the caudate putamen in guinea pigs treated with haloperidol. This study has shown that haloperidol causes region-specific changes to the density of NK(1)-IR neurons. Whether these changes are related to the therapeutic effects or to the side effects of haloperidol in individuals with schizophrenia, remains to be determined.

Actions of the dual FAAH/MAGL inhibitor JZL195 in a murine inflammatory pain model.

The analgesic efficacy of cannabinoids in chronic pain models is limited by side-effects. It has been proposed that this might be overcome by using agents which indirectly activate the endocannabinoid system. We examined the analgesic and side-effect profile of the dual FAAH/MAGL inhibitor JZL195 in an inflammatory pain model. The effect of systemic injections of a range of doses of JZL195 and the pan-cannabinoid receptor agonist WIN55212 were performed 1 day following intraplantar injection of CFA in C57BL/6 mice. JZL195 and WIN55212 both reduced mechanical allodynia and thermal hyperalgesia, and produced catalepsy and sedation in a dose dependent manner. Unlike WIN55212, JZL195 reduced allodynia at doses below those at which side-effects were observed. The effects of JZL195 and WIN55212 were abolished by co-application with the CB1 antagonist AM251. The CB2 antagonist also reduced the JZL195 anti-allodynia, and reversed the WIN55212 anti-allodynia. The reduction in allodynia produced by JZL195 was greater than that produced individually by the FAAH and MAGL inhibitors, URB597 and JZL184. These findings suggest that JZL195 reduces inflammation induced allodynia at doses below those which produce side-effects, and displays greater efficacy that FAAH or MAGL inhibitors. Thus, dual FAAH/MAGL inhibition has the potential to alleviate inflammatory pain with reduced cannabinoid-like side-effects.

Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia.

Despite increased understanding of the biological basis for sleep control in the brain, few novel mechanisms for the treatment of insomnia have been identified in recent years. One notable exception is inhibition of the excitatory neuropeptides orexins A and B by design of orexin receptor antagonists. Herein, we describe how efforts to understand the origin of poor oral pharmacokinetics in a leading HTS-derived diazepane orexin receptor antagonist led to the identification of compound 10 with a 7-methyl substitution on the diazepane core. Though 10 displayed good potency, improved pharmacokinetics, and excellent in vivo efficacy, it formed reactive metabolites in microsomal incubations. A mechanistic hypothesis coupled with an in vitro assay to assess bioactivation led to replacement of the fluoroquinazoline ring of 10 with a chlorobenzoxazole to provide 3 (MK-4305), a potent dual orexin receptor antagonist that is currently being tested in phase III clinical trials for the treatment of primary insomnia.

Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation.

Amphotropic murine leukemia virus (A-MuLV) utilizes the PiT2 sodium-dependent phosphate transporter as its cell surface receptor to infect mammalian cells. The process of A-MuLV infection requires cleavage of the R peptide from the envelope protein. This occurs within virions thereby rendering them competent to fuse with target cells. Envelope proteins lacking the inhibitory R peptide (e.g. envelope (R-) proteins) induce viral envelope-mediated cell-cell fusion (syncytium). Here we have performed studies to determine if cell signaling through protein kinases is involved in the regulation of PiT2-mediated A-MuLV envelope (R-)-induced syncytium formation. Truncated A-MuLV retroviral envelope protein lacking the inhibitory R peptide (R-) was used to induce viral envelope-mediated cell-cell fusion. Signaling through cyclic AMP to activate PKA was found to inhibit envelope-induced cell-cell fusion, whereas treatment of cells with PKA inhibitors H89, KT5720, and PKA Catalpha siRNA all enhanced this cell fusion process. It was noted that activation of PKC, as well as overexpression of PKCepsilon, up-regulated A-MuLV envelope protein-induced cell-cell fusion, whereas exposure to PKC inhibitors and expression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) inhibited syncytium formation. v-ras transformed NIH3T3 cells were highly susceptible to A-MuLV envelope-induced cell-cell fusion, whereas expression of a dominant-negative mutant of Ras (N17Ras) inhibited this cell fusion process. Importantly, activation of Raf-1 protein kinase also is required for A-MuLV envelope-induced syncytium formation. Expression of constitutively active BXB Raf supported, whereas expression of a dominant-negative mutant of Raf-1 (Raf301) blocked, A-MuLV-induced cell-cell fusion. These results indicate that specific cell signaling components are involved in regulating PiT2-mediated A-MuLV-induced cell-cell fusion. Selective pharmacological modulation of these signaling components may be an effective means of altering cell susceptibility to viral-mediated cytopathic effects.

Differential regulation of Cu, Zn- and Mn-superoxide dismutases by retinoic acid in normal and psoriatic human fibroblasts.

Superoxide dismutases' (SODs) expression is altered in several diseases including Alzheimer, atherosclerosis, cancer and psoriasis. Previously, we reported a marked increase in Mn-SOD and Cu,Zn-SOD functional activity in human dermal psoriatic fibroblasts. As retinoic acid (RA) has been used in the treatment of psoriasis and a mechanism for its beneficial effects is not understood, we investigated the effects of RA on SOD mRNA and protein expression levels in human normal and psoriatic fibroblasts. Prior to RA exposure, Cu,Zn-SOD protein and mRNA levels were similar in normal compared to psoriatic fibroblasts while Mn-SOD protein and mRNA levels were increased in psoriatic cells. However, in contrast to normal fibroblasts, exposure of psoriatic fibroblasts to 1 microM RA down-regulated Mn-SOD mRNA, and also decreased Mn-SOD activity by approximately 80% with no change in Mn-SOD protein levels. In contrast, Cu,Zn-SOD protein and enzymatic activity were modestly reduced by RA treatment in both normal and psoriatic fibroblasts. Furthermore, RA treatment of psoriatic fibroblasts also caused a decrease in Cu,Zn-SOD steady-state mRNA levels. These results indicate that RA can serve as a regulatory agent to down-regulate the steady-state levels of both Mn-SOD and Cu,Zn-SOD in psoriatic cells. These findings offer a new model for the antiinflammatory activity of RA when used in the treatment of psoriasis.

The synergistic activation of Raf-1 kinase by phorbol myristate acetate and hydrogen peroxide in NIH3T3 cells.

We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-epsilon activation as determined by the transient expression of dominant negative mutants of PKC-epsilon-K436R. Interestingly, H(2)O(2) produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H(2)O(2). This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H(2)O(2) occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-epsilon, along with a transient phosphorylation of both Raf-1 kinase and PKC.

Glutathione transferase zeta-catalyzed bioactivation of dichloroacetic acid: reaction of glyoxylate with amino acid nucleophiles.

Dichloroacetic acid (DCA) is a drinking water contaminant, a therapeutic agent, and a rodent carcinogen. Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloalkanoates and the cis-trans isomerization of maleylacetoacetate. GSTZ1-1 catalyzes the bioactivation of fluorine-lacking dihaloacetates to S-(alpha-halocarboxymethyl)glutathione, a reactive intermediate that covalently modifies and inactivates the enzyme or is hydrolyzed to glyoxylate. The purpose of this study was to examine the GSTZ1-1-catalyzed bioactivation of DCA, including the reaction of DCA-derived glyoxylate with amino acid nucleophiles and the characterization of the structures and kinetics of adduct formation by LC/MS. The binding of [1-(14)C]DCA-derived label to bovine serum albumin required both GSTZ1-1 and GSH, whereas the binding to dialyzed rat liver cytosolic protein was increased in the presence of GSH. Studies with model peptides (antiflammin-2 and IL-8 inhibitor) indicated that glyoxylate, rather than S-(alpha-chlorocarboxymethyl)glutathione, was the reactive species that modified amino acid nucleophiles. Both addition (+74 Da) and addition-elimination (+56 Da) adducts of glyoxylic acid were observed. Addition adducts (+74 Da) could not be characterized completely by mass spectrometry, whereas addition-elimination adducts (+56 Da) were characterized as 2-carboxy-4-imidazolidinones. 2-Carboxy-4-imidazolidinones were formed by the rapid equilibrium reaction of glyoxylate with the N-terminal amino group of antiflammin-2 to give an intermediate carbinolamine (K(eq) = 0.63 mM(-1)), which slowly eliminated water to give an intermediate imine (k(2) = 0.067 hour(-1)), which rapidly cyclized to give the 2-carboxy-4-imidazolidinone. Glucose 6-phosphate dehydrogenase was inactivated partially by glyoxylate when reactants were reduced with sodium borodeuteride, which may indicate that glyoxylate reacts with selective lysine epsilon-amino groups. The results of the present study demonstrate that GSTZ1-1 catalyzes the bioactivation of DCA to the reactive metabolite glyoxylate. The reaction of glyoxylate with cellular macromolecules may be associated with the multiorgan toxicity of DCA.

Src tyrosine kinase inhibitor PP2 markedly enhances Ras-independent activation of Raf-1 protein kinase by phorbol myristate acetate and H2O2.

Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H2O2) resulted in synergistic activation of Raf-1 kinase (Lee, M., Petrovics, G., and Anderson, W. B. (2003) Biochem. Biophys. Res. Commun. 311, 1026-1033). In this study we have demonstrated that PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H2O2 to activate Raf-1 kinase, whereas it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H2O2 treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H2O2.

Protein kinase Cepsilon mediates PMA-induced growth inhibition of low population density NIH 3T3 fibroblasts.

Phorbol 12-myristate-13-acetate (PMA), a potent tumor promoter and activator of most protein kinase C (PKC) isotypes, was found to significantly inhibit the growth of low population density (1-5% confluency) NIH 3T3 cells. Higher cell population density (above 10% confluency) provided protection from this growth inhibitory effect of PMA. PMA-induced growth arrest is accompanied by an elevation in the level of p21(Cip1) protein, along with cell cycle arrest at the G1/S transition. Activation of PKC is required for this growth inhibitory response since the pan PKC inhibitor GF109203 blocked this effect of PMA. However, the classical PKC inhibitor Gö6976 had no effect, strongly suggesting the involvement of novel PKC isotypes (delta and/or epsilon). Overexpression of PKCepsilon, but not PKCdelta, was found to potentiate PMA-induced growth inhibition. Overexpression of a kinase-inactive dominant-negative mutant of PKCepsilon (K437R) decreased the growth inhibitory effect of PMA and also blocked the PMA-induced increase in the level of p21(Cip1) protein. Taken together, these results indicate that PMA has a cell population density-dependent effect on the growth of NIH 3T3 cells and that the PMA growth inhibitory effect at low cell population density is mediated through activation of PKCepsilon.

Mass spectral characterization of dichloroacetic acid-modified human glutathione transferase zeta.

Glutathione transferase zeta (GSTZ1-1) is widely expressed in eukaryotic species, and four human allelic variants of hGSTZ1-1 have been described. GSTZ1-1 catalyzes the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate and the biotransformation of a range of alpha-haloalkanoic acids. GSTZ1-1-catalyzed biotransformation of fluorine-lacking alpha,alpha-dihaloalkanoic acids, including dichloroacetic acid (DCA), results in the mechanism-based inactivation and covalent modification of the enzyme. The objective of this study was to investigate further the DCA-induced inactivation of hGSTZ1c-1c and to explore the mechanism of inactivation by characterization of the sites and types of DCA-induced covalent modifications. The partition ratio for the DCA-induced, mechanism-based inactivation of hGSTZ1c-1c was (5.7 +/- 0.5) x 10(2), and the k(cat) for the biotransformation of DCA was 39 min(-)(1). Inactivation of hGSTZ1c-1c in vitro was limited at high enzyme concentrations and was inhibited by glyoxylate. The stoichiometry of DCA binding to hGSTZ1c-1c was approximately 0.5 mol of DCA/mol of enzyme monomer. A single DCA-derived adduct was observed and was assigned to cysteine-16 by a combination of matrix-assisted laser-desorption-ionization time-of-flight and electrospray-ionization quadrupole ion-trap mass spectrometry and by analysis of [1-(14)C]DCA binding to C16A hGSTZ1c-1c. The DCA-derived adduct contained both glutathione and the carbon skeleton of DCA, presumably in a dithioacetal linkage. Also, cysteine-16 formed a mixed disulfide bond with glutathione. These data support a mechanism of inactivation whereby glutathione displaces a chlorine atom from DCA, and cysteine-16 in the enzyme active site displaces the second chlorine atom to result in a covalently modified and inactivated enzyme. These findings explain the DCA-induced inactivation of GSTZ1-1 observed in humans and rats.