Regulation of GTP Cyclohydrolase I Gene Expression and Tetrahydrobiopterin Content in Cultured Sympathetic Neurons by Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor

Abstract: Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3–7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78–96% reduction in GTPCH mRNA content relative to β-actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

[125I]RTI-55 Binding to Cocaine-Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain

[¹²⁵I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [¹²⁵I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [¹²⁵I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [¹²⁵I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with K_D= 66 ± 35 pM and S_max= 13.2 ± 10.1 pmol/g of tissue and a low-affinity site with K_D= 1.52 ± 0.55 nM and B_max of 47.5 ± 11-2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > mazindol > WIN 35428 > = methylphenidate > (?)-cocaine > buproprion > (±)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with K_D= 370 ± 84 pM. It appears that [¹²⁵I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.     

The distribution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassembled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one or more actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however, we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length. Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in their distributions. Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division.     

血清肌钙蛋白I、N端前体脑钠肽和高敏C反应蛋白水平在充血性心力衰竭中的临床意义

目的：探讨血清肌钙蛋白I（cTnI）、N端前体脑钠肽（NT-proBNP）和高敏c反应蛋白（hs—CRP）水平在充血性心力衰竭（CHF）中的临床意义。方法：选取2010年1月-2012年12月在我院诊治的CHF患者210例（CHF组）,心功能按纽约心脏病协会（NYHA）分级：Ⅱ级88例、Ⅲ级96例和Ⅳ级26例。同时选取同期在我院行健康体检的自愿者30例为健康对照组。观察和比较两组血清hs．CRP、cTnI和NT．proBNP水平的差异及其与CHF患者心功能的心功能分级的相关系。结果：CHF组患者的血清hs-CRP．cTnI和NT．proBNP水平均较对照组明显增高,差异均有统计学意义（P〈0．0t）,且CHF患者的血清hs—CRP、cTnI和NT—proBNP水平随着心功能分级的增加而升高,差异有统计学意义（P〈0．01）。血浆hs—cI冲（F0．694,P〈0．01）水平,cTnI（r=0．782,P〈0．01）水平均与NT—proBNP水平呈显著正相关,血浆hs．CRP和cTnI水平也呈显著正相关（r==0．649,P〈0．01）。结论：CHF患者的血清hs—CRP、cTnI和NT-proBNP水平与其心功能分级显著相关,有助于评估CHF患者心功能不全的严重程度,可能与CHF患者的预后相关。     

New vistas for P-aminobenzoate participation in the biosynthesis of dihydro folate: a tentative model of the tetrahydro folate multienzyme complex.

Figures 6a and 6b and Table 2 show the united pattern of possible pathways of THFA biosynthesis with the substrates and the enzymes involved. With substrate selection ten different individual enzyme activities can be distinguished, but A2′ is identical with A2, and their apparent molecular weight is 28,000 daltons ±7%, and similarly c1–4 are the same enzymes with an apparent molecular weight of 40,000 daltons ±5% (Tóth-Martinez et al., 1974a). The identity of these enzymes has preliminary been shown, and construction of the THFA-MEC model was partly based on these findings.So, no distinction can be made among functioning MEC-es. The different pathways, mentioned in the introductory part of this paper, can be a product of the separated study of the individual enzymic steps of DHFA (THFA) biosynthesis. By all means it is an important approach to understand the dynamics of the integrated process what we tentatively suggest in this paper for further elucidation.     

Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal

Background

Activation of adenylyl cyclase (AC) by prolonged exposure of mammalian organism to morphine was demonstrated in previous studies of mechanism of action of this drug. However, expression level of individual AC isoforms was not analyzed in crucial cell structure, plasma membrane (PM).

Methods

Rats were adapted to morphine for 10 days and sacrificed 24 h (group + M10) or 20 days (+ M10/−M20) after the last dose. Control animals were sacrificed in parallel with morphine-treated (groups − M10 and (− M10/−M20)). Percoll®-purified PM were isolated from brain cortex and analyzed by immunoblotting and specific radioligand binding.

Results

ACI (ACII) was increased 8× (2.5×) in morphine-adapted rats (+ M10) when compared with controls (− M10). Increase of ACI and II by long-term adaptation to increasing doses of morphine represented a specific effect as the amount of ACIII–ACIX, of prototypical PM marker, Na, K-ATPase and of trimeric G protein α and β subunits was unchanged. Increase of ACI and II was not detected in PM isolated from group (+ M10/−M20). Thus, the marked increase of ACI and ACII faded away 20 days since the last dose of morphine.

Conclusions

We assume that the specific increase in expression level of ACI and ACII in brain cortex of morphine-adapted rats proceeds as a compensatory, homeostatic response to prolonged exposure to inhibitory drug, morphine.

General significance

Our findings demonstrate that the dramatic and specific change of the crucial component of the opioid receptor cascade in brain cortex, manifested as an increase in PM level of ACI and II, is reversible.