The anxiogenic β-carboline FG-7142 increases in vivo and in vitro tyrosine hydroxylation in the prefrontal cortex

Systemic administration of the anxiogenic β-carboline FG-7142, a benzodiazepine inverse agonist, results in a regionally selective increase in dopamine (DA) utilization in the anteromedial prefrontal cortex (PFC). We have examined both in vivo and in vitro tyrosine hydroxylation in the PFC and other mesotelencephalic DA system terminal fields in order to determine if FG-7142 effects changes in DA synthesis, and to determine if the β-carboline biochemically activates certain DA neurons through an action occurring at the cell body level (impulse-dependent regulation) or at the terminal field level (presynaptic regulation). FG-7142 increased in vivo tyrosine hydroxylation in the PFC and in the ventral tegmental area, midbrain source of the DA innervation of the PFC; no changes were observed in mesolimbic or nigrostriatal regions. The β-carboline also increased in vitro tyrosine hydroxylation in the PFC, but decreased tyrosine hydroxylation in striatal slices. The effects of FG-7142 were blocked by the benzodiazepine antagonist RO 15-1788. Another β-carboline inverse agonist, methyl-β-carboline-carboxylate, also increased in vitro tyrosine hydroxylation in the PFC. GABA exerted opposite effects to those of the β-carbolines, decreasing in vitro tyrosine hydroxylation increasing DA synthesis in the CP. These data indicate that the benzodiazepine inverse agonists increase both in vivo and in vitro tyrosine hydroxylation in the PFC, and that the β-carboline may act to increase DA synthesis at both the terminal field and the cell body level.     

Stress and β-carbolines decrease the density of low affinity gaba binding sites: An effect reversed by diazepam

Cerebral cortex membranes from rats habituated to manipulations preceding decapitation (habituated rats) had 40% higher GABA binding than membranes from naive animals. Diazepam (5 X 10(-6) M), added to membranes from naive rats, increased GABA binding to the level of habituated rats, but failed to induce any further increase in membranes from the latter animals. Vice versa, beta-carbolines (FG 7142, beta-CCE, DMCM) added to membranes from habituated rats lowered GABA binding to the level of naive animals, but caused no further decrease in the membranes from this last group. Diazepam removed the effect of beta-carbolines in membranes from habituated rats. It is suggested that handling represents a stressful stimulus for naive animals and that stress lowers GABA binding by releasing an endogenous ligand for benzodiazepine receptors possessing similar properties to beta-carbolines. Finally, the results indicate that the emotional status of animals from which brain tissue is obtained should be considered when connections between GABA and benzodiazepine receptors are studied.     

Modulation of GABA-stimulated chloride influx by β-carbolines in rat brain membrane vesicles

We have shown that GABA-stimulated chloride influx into membrane vesicles from the rat cerebral cortex can be enhanced or inhibited by β-carbolines. The β-carbolines can produce a full spectrum of responses from maximal enhancement to maximal inhibition.     

β-Adrenergic agonists suppress chronic/relapsing experimental allergic encephalomyelitis (CREAE) in Lewis rats

Chronic/relapsing experimental allergic encephalomyelitis (CREAE) serves as an animal model for relapsing/remitting multiple sclerosis. Treatment with the β-adrenergic agonist isoproterenol or the β₂-adrenergic agonist terbutaline significantly suppressed both the first acute attack and the number of relapses in CREAE Lewis rats. The number of relapses was decreased even when treatment with β-adrenergic agonist was started after the onset of the first acute attack of CREAE. β-adrenergic receptor number was increased significantly on splenocytes from CREAE rats as compared to healthy controls or CFA-injected rats. Terbutaline treatment of CREAE rats lowered the splenocyte receptor number to normal values.     

Enhanced aggregation and β structure of amyloid β peptide after coincubation with C1Q

Several lines of evidence now suggest that aggregation of soluble amyloid β peptide (Aβ) into a cross β sheet configuration may be an important factor in mediating potential neurotoxicity of Aβ. Synthetic Aβ has been shown to self aggregate in vitro. Here, we demonstrate that coincubation of freshly solubilized Aβ with C1q, a complement component known to bind Aβ in vitro and to colocalize with Aβ in vivo, results in as much as a 7-fold enhancement of Aβ aggregation, as well as a 2–4-fold enhancement of β structure within aggregates. The addition of C1q to preformed Aβ aggregates also results in significantly increased resistance to aggregate resolubilization. © 1994 Wiley-Liss, Inc.     

Differential effects of morphine withdrawal on cerebral β1- and β2-adrenergic receptors

Effect of morphine dependence and its withdrawal on the ³H-dihydroalprenolol (³H-DHA) binding for βadrenergic receptors,β₁ andβ₂, was examined by a computerized analysis of biphasic Hofstee plots. The relative density ofβ₁ andβ₂ receptors in the rat cerebral cortex was found to be approximately 70% and 30%, respectively. In rats rendered dependent on morphine by a subcutaneous implantation of a morphine pellet, the ³H-DHA binding toβ₁ andβ₂ receptors was not altered. During the stage of withdrawal induced by administration of naloxone, however, the ³H-DHA binding to the cerebral particulate fractions was increased, and this increase was due to the increased binding sites inβ₁ but not inβ₂ receptor. On the other hand, the apparent affinities ofβ₁ andβ₂ receptors for atenolol and salbutamol, selective antagonists forβ₁- andβ₂-adrenergic receptors, respectively, were not altered under these experimental conditions. These results suggest that an abrupt increase in cerebralβ₁-receptor binding sites occurs at morphine withdrawal, and the occurrence of such a supersensitivity in cerebralβ₁ receptor may be involved in the exhibition and/or maintenance of the abstinence syndrome in morphine-dependent subjects.     

β-Carboline inhibition of benzodiazepine receptor binding in vivo

The in vivo potencies of 4 β-carboline derivatives as inhibitors of benzodiazepine receptor binding were investigated. For all 4 derivatives maximal inhibition was seen within 2 min after i.v. application but decreased continuously for the next 20 min and after this time accounted for less than one-third of maximal inhibition. The in vivo potencies of the β-carbolines as inhibitors of benzodiazepine receptor binding are much smaller than one would expect from their affinities measured in vitro. Thus, it is assumed than only a small portion of the i.v. dose reaches the brain.     

The antihyperalgesic activity of a selective P2X7 receptor antagonist, A-839977, is lost in IL-1αβ knockout mice

The pro-inflammatory cytokine interleukin-1β (IL-1β) has been implicated in both inflammatory processes and nociceptive neurotransmission. Activation of P2X7 receptors is the mechanism by which ATP stimulates the rapid maturation and release of IL-1β from macrophages and microglial cells. Recently, selective P2X7 receptor antagonists have been shown to reduce inflammatory and neuropathic pain in animal models. However, the mechanisms underlying these analgesic effects are unknown. The present studies characterize the pharmacology and antinociceptive effects of a structurally novel P2X7 antagonist. A-839977 potently (IC₅₀ = 20–150 nM) blocked BzATP-evoked calcium influx at recombinant human, rat and mouse P2X7 receptors. A-839977 also potently blocked agonist-evoked YO-PRO uptake and IL-1β release from differentiated human THP-1 cells. Systemic administration of A-839977 dose-dependently reduced thermal hyperalgesia produced by intraplantar administration of complete Freund's adjuvant (CFA) (ED₅₀ = 100 μmol/kg, i.p.) in rats. A-839977 also produced robust antihyperalgesia in the CFA model of inflammatory pain in wild-type mice (ED₅₀ = 40 μmol/kg, i.p.), but the antihyperalgesic effects of A-839977 were completely absent in IL-1αβ knockout mice. These data demonstrate that selective blockade of P2X7 receptors in vivo produces significant antinociception in animal models of inflammatory pain and suggest that the antihyperalgesic effects of P2X7 receptor blockade in an inflammatory pain model in mice are mediated by blocking the release of IL-1β.     

Brain β-endorphin-like immunoreactivity in adult rats given β-endorphin neonatally

β-endorphin-like immunoreactivity was measured by radioimmunoassay in the brains of adult rats treated neonatally with β-endorphin, naloxone, or vehicle. After treatment with β-endorphin, the decreases observed in β-endorphin-like immunoreactivity in the hypothalamus, pineal, midbrain, pons-medulla, hippocampus, striatum, frontal cortex, occipital cortex, and posterior cortex were highly significant but the 23% decrease in the thalamus was not significantly different from that of control rats. Neonatal administration of naloxone only resulted in a significant decrease in β-endorphin-like immunoreactivity in the hypothalamus. In contrast, no differences were discernible in content of either β-endorphin-like immunoreactivity or ACTH-like immunoreactivity in the pituitary of rats treated with β-endorphin, naloxone, or vehicle in the neonatal period. These same rats had shown an increased threshold to painful thermal stimulation by the tail-flick test after administration of either β-endorphin or naloxone at birth. The results suggest that neonatally injected β-endorphin may alter the levels of β-endorphin-like immunoreactivity in rat brain as well as the response to pain.     

Dopamine uptake inhibitory capacities of β-carboline and 3,4-dihydro-β-carboline analogs of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) oxidation products

Potentially endogenous beta-carboline and 3,4-dihydro-beta-carboline alkaloidal compounds were compared, generally as 2-methylated (quaternary) and normethylated pairs, to the neurotoxin, 1-methyl-4-phenyl-dihydropyridinium ion (MPP+), with respect to inhibition of [3H]dopamine uptake into rat striatal synaptosomal preparations. Although less potent than MPP+, several compounds displayed IC50 values for inhibition in the moderate range (12-24 microM). Notably, quaternization generally did not improve inhibitory potency, and the 3,4-dihydro-compounds often were more effective inhibitors than their heteroaromatic analogs. The partially competitive nature of inhibition by one of the more effective pairs, 2-methyl-harmine and harmine, was consistent with uptake of the beta-carbolines by the synaptosomal dopamine uptake system, as was the fact that the accumulation of 2-[14C]methyl-harmine was significantly reduced by low Na+ media and by nomifensine, a potent inhibitor of the dopamine transporter. When viewed with reports that certain 2-methyl-beta-carbolines show MPP+-like toxicity in vitro and in vivo, these studies support the proposal that a mammalian beta-carbolinium compound may be taken up by nigrostriatal neurons and provoke the neuronal degeneration underlying Parkinson's disease.     

Metabolites of the β-amyloid precursor protein generated by β-secretase localise to the trans-golgi network and late endosome in 293 cells

Deposition of β-amyloid occurs in the brains of all sufferers of Alzheimer's disease. β-amyloid is proteolytically derived from the β-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and β-secretase cleavage site of the Alzheimer's amyloid precursor protein. The β-secretase cleavage event occurs at the extreme N-terminus of the β-amyloid peptide. Our antiserum to the N-terminus of the β-amyloid peptide (NTβ4) specifically recognises β-secretase cleaved species as opposed to intact βAPP. NTβ4 specifically immunoprecipitates a 13 kDa fragment of βAPP (p13) which is potentially amyloidogenic. We have used these anti-sera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic βAPP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of βAPP fragments possessing the Asp-1 N-terminus of β-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network. © 1996 Wiley-Liss, Inc.     

β-Adrenoceptors regulate myoelectric activity in the small intestine of rats: stimulation by β2 and inhibition by β3 subtypes

Abstract Using β-adrenergic agonists and antagonists this study investigated the importance of three different adrenoceptor subtypes for the regulation of migrating myoelectric complexes (MMCs) of the upper small intestine in conscious, naive rats. After a control period of 60 min with four activity fronts, agonists were given as an intravenous infusion for another 60 min. The non-selective β-adrenoceptor agonist isoprenaline (1 μg kg^?1 min^?1) inhibited MMCs and induced irregular spiking during the infusion period. This effect was blocked by intravenous administration of a bolus dose of either the non-selective β-adrenoceptor antagonist propranolol (1 mg kg^?1), or the β₂-antagonist ICI 118 551 (1 mg kg^?1), both given prior to isoprenaline. However, acebutolol (1 mg kg^?1), a selective β₁-antagonist, failed to antagonize the effect of isoprenaline. Furthermore, prenalterol, a selective β₁-agonist (12.5–800.0 μg kg^?1 min^?1), had no effect on the MMC pattern, whereas the β₂-selective agonist ritodrine (25–100 μg kg^?1 min^?1) induced a myoelectric pattern similar to one induced by isoprenaline. The partial β₃-adrenoceptor agonist D7114 (50–100 μg kg^?1 min^?1), disrupted the MMCs and induced quiescence. Neither of the antagonists, i.e. propranolol (1 mg kg^?1), acebutolol (1 mg kg^?1) nor ICI 118 551 (1 mg kg^?1), given alone induced changes in the MMC pattern. In conclusion, β₂-adrenoceptors in particular but also β₃-adrenoceptors seem to be of importance in the regulation of small intestinal motility by disrupting the regular MMC pattern in rats.     

Pineal response to isoproterenol in rats chronically treated with electroconvulsive shock

Chronic electroconvulsive shock (ECS) has been shown to induce a downregulation of β₁-adrenergic receptors in the rat cerebral cortex. Because the secretion of melatonin in the pineal gland is regulated primarily by β₁-adrenoceptors, in the present study we investigated the effect of chronic administration of ECS on pineal β-adrenergic responsiveness to isoproterenol. To this purpose, young adult male rats received once daily for 8 days ECS (80 mA 0.5 s) or sham ECS. On the day after the last ECS or sham treatment, they were injected with isoproterenol hydrochloride (1 mg/kg SC) or volume-matched saline at 1600 h. Two hours later they were killed by decapitation. Results showed that the isoproterenol-induced increase in the pineal melatonin content was blunted in rats treated with ECS as compared to sham-treated animals (shock × druginteraction = p < 0.01). These data indicate that chronic ECS treatment affects β₁ receptor-mediated melatonin production in the pineal gland. Further studies need to elucidate whether the blunted melatonin response to isoproterenol in ECS-treated rats is due to a downregulation of pinealocyte β-adrenergic receptors.     

Increase of cyclic GMP in cerebellum by methyl-6, 7-dimethoxy-4-ethyl-β-carboline-3-car☐ylate (DMCM)

The intraperitoneal administration of DMCM (0.5–3 mg/kg) produced a dose-related increase in the content of cyclic GMP in the rat cerebellar cortex. The effect of DMCM on cyclic GMP was abolished by pretreatment with benzodiazepine receptor ligands, diazepam and Ro15-1788 and by the GABA agonist muscimol. The results suggest that DMCM increases cerebellar cyclic GMP content through a direct action on benzodiazepine receptors located in the cerebellar cortex. The interaction between DMCM and the GABAergic system associated with benzodiazepine receptors is discussed. Cerebellar cyclic GMP content can be used as a biochemical index to differentiate agonists and antagonists for benzodiazepine receptors.     

Effects of β-endorphin and its fragments on inhibitory avoidance behavior in rats

The effects on retrieval of a one-trial learning inhibitory avoidance response of β-endorphin, α-endorphin, and γ-endorphin, given prior to test have been studied in rats. β-Endorphin (β-LPH_61–91) in a relatively low dose (1.5 μg sc. or 50 ng icv.) facilitated inhibitory avoidance behavior, while a higher dose (10 μg sc. or 100 ng icv.) caused bimodal changes (facilitation in 50% of the animals and attenuation in another 40%. Peripheral injection of γ-endorphin attenuated inhibitory avoidance behaviour in a dose-dependent manner. The C-terminus of β-endorphin (β-LPH_78–91) was ineffective. α-Endorphin facilitated inhibitory avoidance behavior in a dose-dependent manner. Naltrexone pretreatment antagonized the bimodal effect of β-endorphin: following pretreatment with the opiate antagonist the low latency component disappeared, but the facilitatory effect of the neuropeptide remained the same.It is suggested that β-endorphin carries more than one bit of behavioral information. Inherent activities either related or unrelated to naltrexone-sensitive opiate receptors as well as biotransformation into α- and γ-endorphin may contribute to the multiple behavioral effects of this neuropeptide.     

Pro-opiocortin fragments in human and rat brain: β-endorphin and α-MSH are the predominant peptides

The ‘pro-opiocortin’ fragments, β-lipotropin, β-endorphin, ACTH and α-MSH, were estimated in discrete areas of rat and human brain and pituitaries by means of radioimmunoassay in combination with gelfiltration. These peptides exihibited parallel patterns of distribution, but with β-endorphin and α-MSH predominant in the brain of rat and man, and, in contrast, their respective precursors, β-LPH and ACTH predominant in the adenohypophysis of rat and man. These data may be indicative of important differences in post-translational processing of ‘pro-opiocortin’ between these contrasting tissues.     

δ‐Catenin/NPRAP: A new member of the glycogen synthase kinase‐3β signaling complex that promotes β‐catenin turnover in neurons

Through a multiprotein complex, glycogen synthase kinase‐3β (GSK‐3β) phosphorylates and destabilizes β‐catenin, an important signaling event for neuronal growth and proper synaptic function. δ‐Catenin, or NPRAP (CTNND2), is a neural enriched member of the β‐catenin superfamily and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ‐catenin expression is also affected by GSK‐3β signaling and participates in the molecular complex regulating β‐catenin turnover in neurons. Immunofluorescent light microscopy revealed colocalization of δ‐catenin with members of the molecular destruction complex: GSK‐3β, β‐catenin, and adenomatous polyposis coli proteins in rat primary neurons. GSK‐3β formed a complex with δ‐catenin, and its inhibition resulted in increased δ‐catenin and β‐catenin expression levels. LY294002 and amyloid peptide, known activators of GSK‐3β signaling, reduced δ‐catenin expression levels. Furthermore, δ‐catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ‐catenin, like that of β‐catenin, is regulated by proteasome‐mediated degradation. Coimmunoprecipitation experiments showed that δ‐catenin overexpression promoted GSK‐3β and β‐catenin interactions. Primary cortical neurons and PC12 cells expressing δ‐catenin treated with proteasome inhibitors showed increased ubiquitinated β‐catenin forms. Consistent with the hypothesis that δ‐catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ‐catenin showed enhanced β‐catenin turnover. These studies identify δ‐catenin as a new member of the GSK‐3β signaling pathway and further suggest that δ‐catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β‐catenin in neuronal cells. © 2010 Wiley‐Liss, Inc.     

Electrophysiological investigation of β, β′-iminodipropionitrile neuropathy: Intracellular recordings in spinal cord

β, β′-Iminodipropionitrile (IDPN) was given to cats (50 mg/kg/week for 5 weeks) to induce giant axonal swellings in the proximal internodes of motor axons. Conventional intracellular recording techniques were used to investigate the influence of the axon swellings on axonal impulse conduction and generation of action potentials in unidentified lumbosacral motoneurons (MN).Action potentials recorded from axon swellings, verified by lack of orthodromically or antidromically elicited EPSPs or IPSPs, afterhyperpolarization potentials or initial segment-somaldendritic (IS-SD) inflections, were variable in shape. Some were indistinguishable from recordings in normal axons. Component or extra potentials occurred in 45% of recordings from axon swellings; their position on the action potential depended on the direction of impulse invasion into the swelling. Many action potentials were broad, with amplitudes less than 30 mV. Impulse conduction was estimated to be blocked in 19% of motor axons tested.Action potentials recorded in MN of IDPN treated cats resembled in many aspects those recorded in chromatolytic MN, with increased latencies upon antidromic stimulation and decreased IS conduction times and SD thresholds; other parameters did not differ significantly. The minimal interval between two stimuli which each evoked action potentials increased from3.3 ± 0.1to5.8 ± 0.5ms. IS-SD portions of the action potentials could not be fractionated in 49% of cells regardless of interpulse interval. Many MN failed to follow frequencies as low as 10 Hz. Delayed depolarizations were observed in 14% of MN recordings. Repetitive action potentials were elicited by single stimuli in 14% of MN and more frequently by orthodromic than antidromic stimulation. Action potentials could often be elicited in the same MN by stimulation of more than one ventral root filament. The incidence of this ephaptic transmission or crosstalk was estimated to be 12%. The findings are discussed in terms of the influence of proximal axon swellings on action potential generation in MN, propagation along non-homogeneous regions of axons and functional chromatolysis.