Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Both 17beta-estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age-related memory changes. There is growing evidence that a number of estrogen receptor-mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory.Low levels of estradiol (10nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen-stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S-nitroso-N-acetylpenicillamine (SNAP: 119%; 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl-cGMP: 173% of control). Estradiol-stimulated dendritic branching was reversed by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase.     

Deleterious effect of β-estradiol in a rat model of transient forebrain ischemia

Estrogen has demonstrated great potential as a therapeutic agent in focal ischemic brain injury, as exogenous β-estradiol has proven beneficial in a variety of focal stroke models. In contrast, the relatively few studies of estrogen’s efficacy in transient forebrain ischemia have produced inconsistent results. The present study was therefore designed to clarify estrogen’s neuroprotective potential in selective hippocampal neuronal injury resulting from four-vessel occlusion in the rat. Female Wistar rats (normal, ovariectomized, or ovariectomized and estradiol-treated) received 5 or 10 min of ischemia. No differences in hippocampal cell loss were found amongst the groups with 10 min of ischemia. Amongst the groups with 5 min of ischemia, the mildest injury was found in the ovariectomized animals, which lost only 32% of their CA1 pyramidal cells. In comparison, mean cell losses were 54% and 49%, respectively, in intact females and in ovariectomized animals with estradiol replacement. Linear regression analysis demonstrated a highly significant relationship between cell loss and plasma estradiol levels. The mechanism by which exogenous and endogenous estrogen exacerbated the injury is unclear, as estrogen has many neuroprotective effects. On the other hand, many other reported effects of estrogen in hippocampal area CA1 might confer increased sensitivity to ischemia, either by modulating the excitatory effects of glutamate or by modifying the inhibitory effects of GABA. Determining how to modulate the various competing effects of estrogen is of both theoretical and practical importance, as it is now clear that one cannot assume that estrogen administration will always improve outcome in cerebral ischemia.     

17beta-estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius

The effects of 17beta-estradiol (17betaE2) on spontaneous and excitatory amino acid (EAA) induced nucleus tractus solitarius (NTS) neuronal activity were investigated by electrophysiological and immunohistochemical experiments in ovariectomized female Sprague-Dawley rats. Out of 62 NTS neurons tested, 42 were inhibited (68%) following iontophoretic application of 17betaE2 in a current-dependent manner. The averaged firing rate decreased from 3.06+/-0.40 to 0.78+/-0.17 Hz. The inhibitory responses were rapid in onset (within 1 min) and variable in duration (2-4 min). The inhibitory effects of 17betaE2 were blocked by simultaneously applied 17betaE2 antagonist ICI182,780, but not by GABA antagonist, bicuculline and phaclofen. L-Glutamate, AMPA or NMDA enhanced the activity of 71, 73 or 69% of NTS cells tested, respectively. The excitatory effects of EAA were significantly inhibited in the presence of 17betaE2. Fluorescent immunohistochemistry revealed that all subnuclei of the NTS contained high levels of estrogen receptors (ERs) immunoreactivity. These results suggest that 17betaE2 inhibits spontaneous and EAA-induced NTS neuronal activity through 17betaE2 activation of ERs.     

Inhibitory effect of 17beta-estradiol in the parabrachial nucleus is mediated by GABA

In the present investigation, electrophysiological recordings of thalamic relay neurons were used to investigate the role of estrogen as a modulator of visceral afferent information through the PBN to forebrain structures. Experiments were done in anaesthetized (sodium thiobutabarbitol; 100 mg/kg) male and ovariectomized female rats supplemented for 7 days prior with either 17beta-estradiol (OVX-E(2)) or saline (OVX-S). A portion of the right cervical vagus was isolated for the electrical activation (0.8 Hz, 2 ms duration) of visceral afferents. The evoked single and multi-unit activity was recorded via a recording electrode in the ventrobasal thalamus. Exogenous microinjection of 17beta-estradiol (0.1, 0.25 and 0.5 microM; 200 nl) into the parabrachial nucleus (PBN) produced a significant, dose-dependent attenuation in the magnitude of visceral afferent activation-evoked responses of neurons recorded in the thalamus in both male and OVX-E(2) groups. No effect on evoked thalamic activity was observed following injection of estrogen into the PBN of OVX-S animals. Co-injection of estrogen with the GABA(A) receptor antagonist, bicuculine (0.1 microM; 200 nl) but not phaclofen (GABA(B); 0.1, 0.5 or 1 microM; 200 nl) resulted in an increase in the evoked thalamic response in males (55+/-11%) and OVX-E(2) female (68+/-15%) rats. These studies suggest that estrogen inhibits neurotransmission in the PBN via an interaction with the GABA(A) receptor to modulate the flow of visceral information to the thalamus.     

Acute and long-term effects of 17β-estradiol on Gi/o coupled neurotransmitter receptor function in the female rat brain as assessed by agonist-stimulated [S]GTPγS binding

Estrogens exert effects on mood, mental state, memory and other central nervous system (CNS) functions by modulating neurotransmitter receptor systems in the brain. Studies were designed to investigate the effect of 17β-estradiol (E₂) on agonist-stimulated [³⁵S]GTPγS binding in membranes to assess the first step in the intracellular signal transduction cascade in a functional assay following: (1) an acute, one-time bolus subcutaneous injection, or (2) 14-day continuous exposure by a slow-release pellet implanted subcutaneously. In rats treated with E₂ acutely, the maximal response produced by activation of serotonin_1A (5-HT_1A) receptors was decreased 25% in the hippocampus, cortex, and amygdala. Similarly, acute E₂ administration desensitized 5-HT_1B and GABA_B receptors in hypothalamus and cerebellum, respectively, and cannabinoid receptors in hippocampus and cortex. Although the maximal responses were decreased, acute E₂ treatment did not alter the EC₅₀ of any of the aforementioned receptors. The incubation of membranes prepared from the cortex of ovariectomized (OVX) rats with E₂ (1 μM) in vitro did not alter 5-HT_1A or cannabinoid receptor-mediated [³⁵S]GTPγS binding. By contrast to acute treatment in vivo, 14-day E₂ administration to OVX rats did not alter the maximal responses produced by activation of 5-HT_1A, 5-HT_1B, GABA_B, or cannabinoid receptors in any of the brain regions examined. Thus, it is concluded that acute E₂ administration in vivo modulates multiple G_i/o coupled receptors in various regions of the female rat brain. Because these effects are observed only in vivo, it is concluded that cytosolic, nuclear and/or extraneuronal factors are required.     

Serotonergic agents modulate antidepressant-like effect of the neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice

The present study demonstrated the antidepressant-like effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α, 5α THP) in mouse forced swim test of depression and its modulation by different serotonergic agents. Pretreatment with the selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), the 5-HT releaser, fenfluramine (10 mg/kg, i.p.), the 5-HT_1A receptor agonist, 8-OH-DPAT (0.1 mg/kg, s.c.), the 5-HT_1B/1C receptor agonist, TFMPP (4 mg/kg, s.c.) and the 5-HT_2A/1C receptor agonist, DOI (2 mg/kg, s.c.) potentiated the antidepressant-like effect of 3α, 5α THP. At these doses the serotonergic agents per se did not modify the duration of immobility. However, fluoxetine (20 mg/kg, i.p.), fenfluramine (20 mg/kg, i.p.) or imipramine (5 or 20 mg/kg, i.p.) not only reduced immobility but also enhanced the antidepressant-like effect of 3α, 5α THP. Such a potentiating effect of the 5-HT_1A or the 5-HT_2A/1C receptor agonist was not antagonized by the sub-effective dose (0.1 mg/kg, s.c.) of their respective antagonists p-MPPI or ketanserin. Pretreatment with p-CPA (300×3 mg/kg, i.p.), a depleter of 5-HT neuronal store failed to block the influence of fluoxetine and fenfluramine on antidepressant-like effect of 3α, 5α THP. The accelerated effect of 3α, 5α THP in presence of serotonergic agents was antagonized by the GABA_A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). These findings for the first time demonstrate that serotonergic agents potentiate the antidepressant-like action of 3α, 5α THP, by enhancing the GABAergic tone as a likely consequence of increased brain content of this neurosteroid.     

Spatial working memory improvement by an α2-adrenoceptor agonist dexmedetomidine is not mediated through α2C-adrenoceptor

1. Aged α_2C-adrenoceptor knockout and wild type mice were used to investigate whether α_2C-adrenoceptors are involved in mediating the beneficial effects of α₂-adrenoceptor agonist, dexmedetomidine, on spatial working memory.

2. A win-stay task in the radial arm maze was used to dissociate the effects of dexmedetomidine on working vs. reference memory. In addition, the animals were tested in simple response habit learning in the T-maze.

3. Knockout mice made more working memory errors after the change of the baited arm in radial arm maze, but after training reached again as accurate level of performance as wild type controls. Dexmedetomidine 5 and 10 μg/kg alleviated the increase in spatial working memory errors after the change of the baited arm in knockout mice. Knockout and wild type mice performed equally well in T-maze, and dexmedetomidine had no effect on this simple response learning.

4. The present results indicate that α₂-adrenoceptor agonists have a selective effect on spatial working memory not only in monkeys but also in mice. Further, this study confirms our earlier finding that the presence of α_2C-adrenoceptors is not necessary for the spatial working memory enhancing effect of α₂-adrenoceptor agonists.

17β-Estradiol reduces cortical lesion size in the glutamate excitotoxicity model by enhancing extracellular lactate: a new neuroprotective pathway

Estrogens play an important role in neuronal function and in protecting neurones in the cerebral cortex against pathological conditions. An in vivo model of glutamate excitotoxicity in which glutamate is applied to the cortex of rats through a microdialysis probe has been used to investigate the neuroprotective processes initiated by 17β-estradiol. Rats were pre-treated with 17β-estradiol (i.v.) before local application of 100 mM glutamate into the cortex through a microdialysis probe. Pre-treatment with 17β-estradiol significantly reduced the size of the glutamate-induced cortical lesion. In the cortical microdialysates collected from the probe, a peak of lactate was observed immediately after glutamate application. After 17β-estradiol pre-treatment this peak of lactate was significantly higher with estradiol than without 120 min after glutamate application, reaching 700% basal level at the end of measurement. The level of extracellular glucose was markedly decreased with and without 17β-estradiol pre-treatment. Local blockage of neuronal lactate transporters with α-cyano-4-hydroxycinnamate (4-CIN) completely abolished the neuroprotective effect of 17β-estradiol and induced a larger cortical lesion. An accumulation of extracellular lactate was observed after inhibition of the lactate transporters suggesting that transport of lactate into neurones is necessary for the neuroprotective effect of 17β-estradiol. The anti-estrogen tamoxifen also abolished the neuroprotective effect of 17β-estradiol on the lesion size and inhibited the production of lactate. These results suggest a new neuroprotective mechanism of 17β-estradiol by activating glutamate-stimulated lactate production, which is estrogen receptor-dependent.     

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABAA receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ ]flunitrazepam (2 nM), [ ]muscimol (5 nM), and 4 nM [ ]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ ]flunitrazepam and [ ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ ]flunitrazepam as well as [ ]muscimol binding (E_max) in the ethanol-dependent rat cerebellum as compared to control group (p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ ]TBPS at lower concentrations (100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ ]TBPS binding (−E_max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid_A (GABA_A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.     

Cataleptic effect of neurosteroid 3α-hydroxy-5α-pregnan-20-one in mice: modulation by serotonergic agents

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) induced catalepsy in mice is modified by dopaminergic, adenosinergic and GABAergic agents. In light of serotonergic agents being implicated in antipsychotic-induced catalepsy and their ability to increase brain neurosteroid content, the present study was undertaken to investigate the effect of various 5-HT agents on catalepsy induced by 3α,5α-THP in mice. Pretreatment with selective serotonin reuptake inhibitor, fluoxetine (5 mg/kg, i.p.), 5-HT releaser, fenfluramine (10 mg/kg, i.p.), 5-HT_1A receptor agonist, 8-OH-DPAT (0.3 mg/kg, s.c.), 5-HT_1B/1C receptor agonist, TFMPP (3 mg/kg, i.p.), 5-HT_2A/1C receptor agonist, DOI (1.5 mg/kg, s.c.) and 5-HT₃ agonist, 2-methylserotonin (5 mg/kg, i.p.) potentiated the catalepsy induced by exogenous administration of 3α,5α-THP. Furthermore, FGIN 1–27, an MDR agonist that increases endogenous content of 3α,5α-THP although per se failed to exhibit any cataleptic effect but enhanced the cataleptic response in combination with these serotonergic agents. The potentiating action of 5-HT_1A, 5-HT_2A/1C or 5-HT₃ receptor agonist on 3α,5α-THP induced catalepsy was not blocked by prior administration of sub-effective dose (1 mg/kg, s.c.) of their respective receptor antagonists pindolol, ritanserin or ondansetron or by pretreatment with serotonergic neurotoxin 5,7-DHT (100 μg/mouse, i.c.v.). However this effect of different serotonergic agents was antagonized by the GABA_A receptor antagonist, bicuculline (1 mg/kg, i.p.) or the 3α-hydroxysteroid oxidoreductase enzyme inhibitor, indomethacin (5 mg/kg, i.p.). The 5-HT agents enhance neurosteroid-induced catalepsy by increasing GABAergic tone, likely as a consequence of increased brain content of 3α,5α-THP.     

Relationships between β- and α2-adrenoceptors and G coupling proteins in the human brain: effects of age and suicide

Interactions between brain α₂- and β-adrenoceptors are of interest in physiological (aging) and pathological (major depression) processes involving both receptors. In this study, total β-adrenoceptors and β_1/2-subtypes were quantitated in postmortem human brains to investigate their relationships with α_2A-adrenoceptors and specific G proteins during the process of aging and in brains of suicide victims. Analysis of [³H]CGP12177 binding, in the presence of CGP20712A (β₁-antagonist), indicated that the predominant β-adrenoceptor in the frontal cortex is the β₁-subtype (65–75%). The density of total β- (r=−0.60, n=44) or β₁-adrenoceptors (r=−0.78, n=22), but not the β₂-subtype, declined with aging (3–80 years). The density of total β- or β₁-adrenoceptors, but not the β₂-subtype, correlated with the number of α₂-adrenoceptors quantitated in the same brains with the agonist [³H]UK14304 (r=0.71–0.81) or the antagonist [³H]RX821002 (r=0.61–0.66). Interestingly, the ratios α₂/β- or α₂/β₁-adrenoceptors did not correlate with the age of the subject at death, indicating that the proportion of α₂/β-adrenoceptors in brain remains rather constant during the process of aging. The density of β-adrenoceptors correlated with the immunodensity of Gαs (r=0.55) and Gβ (r=0.61) proteins, and that of α₂-adrenoceptors with those of Gαi_1/2 (r=0.88) and Gβ (r=0.65). In brains of suicides, compared to controls, the ratio between α₂- and β- or β₁-adrenoceptors (α₂-full agonist sites/β-sites) was greater (1.3- to 2.0-fold; P<0.05). The results demonstrate a close interdependence between brain α₂- and β-adrenoceptors during aging, and in brains of suicides. The quantitation of the α_2A/β-adrenoceptor ratio could represent a relevant neurochemical index in the study of brain pathologies in which both receptors are involved.     

β-Endorphin-induced hyperglycemia is mediated by increased central sympathetic outflow to adrenal medulla

Synthetic human β-endorphin increased plasma glucose concentration when administered intracisternally in chronically cannulated, conscious, unrestrained, adult male rats. This hyperglycemic effect of β-endorphin was blocked by prior systemic administration of naloxone, supporting mediation of the effect at opioid receptors in brain. Adrenal denervation blocked the β-endorphin-induced increase in plasma glucose, supporting a thesis that this effect is mediated at least in part by increased epinephrine secretion. The hyperglycemic response to intracerebral β-endorphin was also blocked by either intracerebral hemicholinium-3 or somatostatin, supporting both a cholinergic link and a somatostatin neuron in the brain mechanism regulating endorphin-induced stimulation of sympathetic outflow.     

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.     

GSK‐3β inhibitors reverse cocaine‐induced synaptic transmission dysfunction in the nucleus accumbens

Nucleus accumbens receives glutamatergic projection from the prefrontal cortex (PFC) and dopaminergic input from the Ventral tegmental area (VTA). Recent studies have suggested a critical role for serine/threonine kinase glycogen synthase kinase 3β (GSK3β) in cocaine‐induced hyperactivity; however, the effect of GSK3β on the modulation of glutamatergic and dopaminergic afferents is unclear. In this study, we found that the GSK3 inhibitors, LiCl (100 mg/kg, i.p.) or SB216763 (2.5 mg/kg, i.p.), blocked the cocaine‐induced hyperlocomotor activity in rats. By employing single‐unit recordings in vivo, we found that pretreatment with either SB216763 or LiCl for 15 min reversed the cocaine‐inhibited firing frequency of medium spiny neuron (MSN) in the nucleus accumbens (NAc). Preperfusion of SB216763 (5 μM) ameliorated the inhibitory effect of cocaine on both the α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) (up to 99 ± 6.8% inhibition) and N‐methyl‐D‐aspartic acid receptor (NMDAR)‐mediate EPSC (up to 73 ± 9.7% inhibition) in the NAc in brain slices. The effect of cocaine on AMPA and NMDA receptor‐mediate excitatory postsynaptic current (EPSC) were mimicked by the D₁‐like receptor agonist SKF 38393 and blocked by the D₁‐like receptor antagonist SCH 23390, whereas D₂‐like receptor agonist or antagonist failed to mimic or to block the action of cocaine. Preperfusion of SB216763 for 5 min also ameliorated the inhibitory effect of SKF38393 on both AMPA and NMDA receptor‐mediated components of EPSC, indicate the effect of SB216763 on cocaine was via the D₁‐like receptor. Moreover, cocaine inhibited the presynaptic release of glutamate in the NAc, and SB216763 reversed this effect. In conclusion, D₁ receptor–GSK3β pathway, which mediates glutamatergic transmission in the NAc core through a presynaptic mechanism, plays an important role in acute cocaine‐induced hyperlocomotion.     

β-Adrenoceptor subtypes in the human brain: autoradiographic localization

The distribution and characteristics of beta-adrenoceptors in postmortem human brain was studied using quantitative autoradiographic techniques. 125I-Cyanopindolol was used as a ligand. High densities of beta-adrenoceptors were found in the caudate, putamen, different cortical areas and layers and the hippocampal formation. Low densities were present in other areas such as the thalamus, hypothalamus, midbrain and cerebellar cortex. Specific beta 1 and beta 2 antagonists were used to visualize and quantify separately the two subtypes of beta-adrenoceptors. Computer analysis of the competition curves obtained revealed that the putamen was enriched in beta 1 sites while the cerebellum contained predominantly beta 2 adrenoceptors. The regional distribution of beta-adrenoceptor subtypes was found to be similar to that seen in the rat brain.     

The influence of heparin and indol on the catalytic properties of α- and β/δ -thrombins

Heparin was shown to form an equimolar complex with α- and β/δ -forms of thrombin. The formation of the complex resulted in inhibition of the TAME esterase activity of thrombin ( by 40% form α- and 17% for β/δ-form ) and in stimulation of its BAME esterase activity ( by 50% for α- and 64% for β/δ-form ). Heparin caused the 70% inhibition of the activity of both forms of the enzyme towards the synthetic amid substrate Bz-Phe-Val-Arg-pNA; at the same time it had little if any effect on the enzyme activity towards Tos-Gly-Pro-Arg-pNA and stimulated the α- and β/δ-thrombins activities towards H-D-Phe-Pip-Arg-pNA by 16% and 57% respectively. In the case of both ester and amid substrates heparin exerted its effect on kcat, but had no effect on Km(app).Indol was shown to activate the TAME hydrolysis catalyzed by α- and β/δ-thrombins. The identity of the binding site for indol and for the additional TAME molecule in the effect of substrate activation was demonstrated. Heparin did not prevent the effects of indol and substrate activation of the thrombin-catalyzed hydrolysis of ester substrates. Moreover the kinetic parameters of indol activation are similar for the free enzyme and its complex with heparin indicating the different localization of the binding sites for indol and heparin in the molecule of thrombin.     

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.     

Attenuation of β‐amyloid‐induced tauopathy via activation of CK2α/SIRT1: Targeting for cilostazol

β‐Amyloid (Aβ) deposits and hyperphosphorylated tau aggregates are the chief hallmarks in the Alzheimer's disease (AD) brains, but the strategies for controlling these pathological events remain elusive. We hypothesized that CK2‐coupled SIRT1 activation stimulated by cilostazol suppresses tau acetylation (Ac‐tau) and tau phosphorylation (P‐tau) by inhibiting activation of P300 and GSK3β. Aβ was endogenously overproduced in N2a cells expressing human APP Swedish mutation (N2aSwe) by exposure to medium containing 1% fetal bovine serum for 24 hr. Increased Aβ accumulation was accompanied by increased Ac‐tau and P‐tau levels. Concomitantly, these cells showed increased P300 and GSK3β P‐Tyr216 expression; their expressions were significantly reduced by treatment with cilostazol (3–30 μM) and resveratrol (20 μM). Moreover, decreased expression of SIRT1 and its activity by Aβ were significantly reversed by cilostazol as by resveratrol. In addition, cilostazol strongly stimulated CK2α phosphorylation and its activity, and then stimulated SIRT1 phosphorylation. These effects were confirmed by using the pharmacological inhibitors KT5720 (1 μM, PKA inhibitor), TBCA (20 μM, inhibitor of CK2), and sirtinol (20 μM, SIRT1 inhibitor) as well as by SIRT1 gene silencing and overexpression techniques. In conclusion, increased cAMP‐dependent protein kinase‐linked CK2/SIRT1 expression by cilostazol can be a therapeutic strategy to suppress the tau‐related neurodegeneration in the AD brain. © 2013 Wiley Periodicals, Inc.     

Microinjection of carbachol in the lateral hypothalamus produces opposing actions on nociception mediated by α1- and α2-adrenoceptors

Electrical stimulation of the lateral hypothalamus (LH) produces antinociception partially blocked by intrathecal α-adrenergic antagonists, but the mechanism underlying this effect is not clear. Evidence from immunological studies demonstrates that substance P-immunoreactive neurons in the LH project near the A7 catecholamine cell group, a group of noradrenergic neurons in the pons known to effect antinociception in the spinal cord dorsal horn. Such evidence suggests that LH neurons may activate A7 neurons to produce antinociception. To test this hypothesis, the cholinergic agonist carbachol was microinjected into the LH at doses of 63, 125 and 250 nmol and the resulting effects on tail-flick and nociceptive foot-withdrawal latencies were measured. All three doses significantly increased response latencies on both tests, with the 125-nmol dose providing the optimal effect. Intrathecal injection of the opioid antagonist naltrexone (97 nmol) partially reversed antinociception, but neither the α₂-adrenoceptor antagonist yohimbine nor the α₁-adrenoceptor antagonist WB4101 altered latencies. However, two sequential doses of yohimbine blocked LH-induced antinociception on both tests. In contrast, two sequential doses of WB4101 increased nociceptive responses on both the tail-flick and foot-withdrawal tests. These findings, and those of published reports, suggest that neurons in the LH activate spinally projecting methionine enkephalin neurons, as well as two populations of A7 noradrenergic neurons that exert a bidirectional effect on nociception. One of these populations increases nociception through the action of α₁-adrenoceptors and the other inhibits nociception through the action of α₂-adrenoceptors in the spinal cord dorsal horn.     

Alpha 2- and beta-adrenoceptors differentially modulate GABAA- and GABAB-mediated inhibition of red nucleus neuronal firing

In mesencephalic red nucleus (RN), GABA-induced inhibition of neuronal firing is modulated by noradrenaline acting on alpha2- and beta-adrenoceptors. Since both GABAA and GABAB receptors are present in the rat RN, we have recorded the firing activity of RN neurons in vivo from anaesthetized rats to study how GABAA- and GABAB-mediated effects are modulated by either alpha2- or beta-adrenoceptor activation. Both the GABAA agonist isoguvacine and the GABAB agonist baclofen depressed the firing of RN neurons. During simultaneous application of clonidine, an alpha2-adrenoceptor agonist, half of the isoguvacine- and baclofen-mediated responses were modified: isoguvacine-mediated inhibition was enhanced by 97% without any change in effect duration, whereas baclofen responses were either increased or slightly reduced in the same number of cases. Application of isoprenaline, a beta-adrenoceptor agonist, increased isoguvacine effect in 66% of neurons without modifying effect duration; the amount of increase (43%) was significantly lower than that induced by clonidine. On the other hand, in the presence of isoprenaline, baclofen response was reduced in 72% of neurons with respect to both the amount (52%) and the duration (34%) of effect. Taken together, these results indicate that alpha2-adrenoceptors mainly enhance GABAA-induced inhibition and induce mixed effects on GABAB response; on the other side, beta-adrenoceptors exert an opposite modulation on GABA effects, respectively, enhancing and depressing GABAA- and GABAB-mediated responses.