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.     

Effect of age on β-adrenergic receptors on cerebral microvessels

The responsiveness of β-adrenergic receptors in cerebral microvessels was studied in aged rats by measuring cAMP formation induced by norepinephrine and [¹²⁵I]iodohydroxybenzylpindolol binding. The density of β-receptor sites is reduced by aging in the rat cerebral microvessels. These results suggest that the changes in brain circulation during aging may be at least partially due to a reduction of the β-adrenergic control of cerebral microvessels.     

Selective interruption of axonal transport of neurofilament proteins in the visual system by β,β′-iminodipropionitrile (IDPN) intoxication

β,β′-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components sucas tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-ricsystem (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system witan intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments witno impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.     

α- and β-adrenergic binding sites in sheep cerebral cortex: Characterisation, effects of photoperiod and treatment with estrogen/progesterone

The radioligands [3H]-dihydroergocryptine and [3H]-dihydroalprenolol were used to characterise alpha- and beta-adrenergic binding sites, respectively, in membrane fractions of sheep cerebral cortex. In terms of affinity, density and specificity these sites possess properties similar to those previously characterised in rat brain. Further, in preliminary studies, these sites also appear to be responsive to treatment with estradiol/progesterone as well as to photoperiod. Thus, estrogen treatment can elevate both alpha- and beta-adrenergic binding sites in cortical tissue of sheep kept in natural light. In contrast, artificial light either has no effect or inhibits binding in response to estrogen.     

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.     

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.     

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.     

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.     

Release of β-lipotropin and β-endorphin from rat hypothalami in vitro

Hypothalamic tissue extracts of rats were chromatographed and β-endorphin immunoreactivity (β-End_i) was measured. The two major peaks of β-End_i co-eluted with β-lipotropin (β-LPH) and β-End respectively. Hypophysectomy caused a local decrease of β-LPH and β-End concentrations in the mediobasal hypothalamus. During superfusion of hypothalamic tissue blocks in vitro, membrane depolarization by electric stimulation or 45 mM K⁺ induced a Ca²⁺-dependent release of both β-LPH and β-End.     

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.     

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.     

Analysis of age dependent changes of Topoisomerase II α and β in rat brain

Eukaryotic Topoisomerase II (Topo II) is present in two isoforms alpha and beta. The alpha isoform is predominantly localized in proliferative tissue, while beta isoform is present in all tissues. In the present study we report the activity and protein levels of Topoisomerase II alpha and beta in rat brains of different age groups viz.: E11 (Embryo day 11), E18 (Embryo day 18), post-natal day 1, young (<10 days), adult (<6 months) and old (>2 years). Topoisomerase II beta isoform is found to be the predominant form in brain tissue but Topoisomerase II alpha is found in embryos up to post-natal day 1. The studies to examine the regional distribution of Topoisomerase II beta in brain showed highest activity in cerebellar region and that too only neuronal cell fraction. There was a significant age-dependent decline in this activity. Hence, Topoisomerase II beta may have some unknown function in cerebellum and the low levels of Topoisomerase II beta activity in ageing cerebellum may contribute to the genomic instability in cerebellar region of ageing brain.     

The effects of α- and β-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia

We have previously shown that certain commercially available lots of α-bungarotoxin block transmission in ciliary and choroid neurons of both pigeon and chicken ciliary ganglia at a concentration of 10 μg/ml (1.2 μM). The blockade is antagonized by pre-incubation with 100 μM tubocurarine.Further evidence that this blockade is produced by a postsynaptic action, as one would expect of an α-neurotoxin, are our findings that: (a) exposure to the toxin prevents the depolarization of ganglion cells normally seen in response to the cholinergic agonist, carbachol; and (b) the blocking activity of the toxin is removed by treatment with membranes purified from Torpedo electric organ containing an excess of α-neurotoxin binding sites.A high affinity binding site for [¹²⁵I]α-bungarotoxin was characterized in the chicken ciliary ganglion. However, since it is labelled equally well by lots of α-bungarotoxin which block transmission and those that do not, this site does not appear to be involved in the blockade of transmission.α-Cobratoxin (fromNaja naja siamensis), the α-neurotoxin L.s. III (fromLaticauda semifasciata) and certain lots of α-bungarotoxin produce a partial blockade of transmission in ciliary neurons of the pigeon ciliary ganglion at a concentration of 10 μg/ml (1.2 μM), but have no effect on transmission in choroid neurons. Two other α-neurotoxins fromLaticauda semifasciata, erabutoxin a and erabutoxin b, have no effect on transmission in either cell population at this concentration. None of the α-neurotoxins tested had any effect on transmission in either the rat superior cervical ganglion or the rat pelvic ganglion at concentrations up to 100 μg/ml (12 μM). Collagenase treatment of these ganglia, in an attempt to increase access of the toxins to ganglion cells, did not alter these negative results.β-Bungarotoxin (0.5 μg/ml, 0.02 μM) produces a complex blockade of transmission in both avian ciliary ganglia and rat superior cervical ganglia. Unlike the action of α-bungarotoxin, the blockade of ciliary ganglion transmission by β-bungarotoxin is irreversible and is not prevented by pretreatment with tubocurarine.     

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background: During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) by presenilin (PS)/γ‐secretase, ε‐cleavages at the membrane‐cytoplasmic border precede γ‐cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε‐cleavages, rather than the γ‐cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42. Methods: Cell‐free γ‐secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI‐TOF MS analysis was carried out to detect the N‐termini of AICD species. Aβ and AICD species were measured by ELISA and immunoblotting techniques. Results: Effects on the ε‐cleavage by AD‐associated pathological mutations around the ε‐cleavage sites (i.e., βAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε‐cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aβ42 production. Only AICDε51, though, and not Aβ42 production, dramatically changed with modifications to the cell‐free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. Conclusion: Our current data show that the generation ratio of AICDε51 and Aβ42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aβ42 generation, measurement of the relative level of Aβ42‐related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε‐cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε‐cleavage as a means for decreasing Aβ42 production.     

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.