Cannabinoid CB2 Receptor Modulation by the Transcription Factor NRF2 is Specific in Microglial Cells.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor that has neuroprotective and anti-inflammatory effects, regulating more than 250 genes. As NRF2, cannabinoid receptor type 2 (CB2) is also implicated in the preservation of neurons against glia-driven inflammation. To this concern, little is known about the regulation pathways implicated in CB2 receptor expression. In this study, we analyze whether NRF2 could modulate the transcription of CB2 in neuronal and microglial cells. Bioinformatics analysis revealed an antioxidant response element in the promoter sequence of the CB2 receptor gene. Further analysis by chemical and genetic manipulations of this transcription factor demonstrated that NRF2 is not able to modulate the expression of CB2 in neurons. On the other hand, at the level of microglia, the expression of CB2 is NRF2-dependent. These results are related to the differential levels of expression of both genes regarding the brain cell type. Since modulation of CB2 receptor signaling may represent a promising therapeutic target with minimal psychotropic effects that can be used to modulate endocannabinoid-based therapeutic approaches and to reduce neurodegeneration, our findings will contribute to disclose the potential of CB2 as a novel target for treating different pathologies.

Hyperkalaemia management and related costs in chronic kidney disease patients with comorbidities in Spain.

BACKGROUND: Hyperkalaemia (HK) is a common electrolyte disorder in patients with chronic kidney disease (CKD) and/or treated with renin-angiotensin-aldosterone system inhibitors (RAASis). The aim of this study is to determine the severity, current management and cost of chronic HK. METHODS: We performed a retrospective cohort study of patients with chronic HK and CKD, heart failure or diabetes mellitus between 2011 and 2018. The study follow-up was 36 months. RESULTS: A total of 1499 patients with chronic HK were analysed: 66.2% presented with mild HK, 23.4% with moderate HK and 10.4% with severe HK. The severity was associated with CKD stage. Most patients (70.4%) were on RAASi therapies, which were frequently discontinued (discontinuation rate was 39.8, 49.8 and 51.8% in mild, moderate and severe HK, respectively). This RAASi discontinuation was similar with or without resin prescription. Overall, ion-exchange resins were prescribed to 42.5% of patients with HK and prescriptions were related to the severity of HK, being 90% for severe HK. Adherence to resin treatment was very low (36.8% in the first year and 17.5% in the third year) and potassium remained elevated in most patients with severe HK. The annual healthcare cost per patient with HK was €5929, reaching €12 705 in severe HK. Costs related to HK represent 31.9% of the annual cost per HK patient and 58.8% of the specialized care cost. CONCLUSIONS: HK was usually managed by RAASi discontinuation and ion-exchange resin treatment. Most patients with HK were non-adherent to resins and those with severe HK remained with high potassium levels, despite bearing elevated healthcare expenditures.

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) inhibited the LPS- and IFN-gamma-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J(2), prostaglandin E(2), or rosiglitazone, a peroxisomal proliferator-activated receptor gamma ligand. Incubation of activated macrophages with 15dPGJ(2) inhibited the degradation of IkappaBalpha and IkappaBbeta and increased their levels in the nuclei. NF-kappaB activity, as well as the transcription of NF-kappaB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IkappaB phosphorylation showed an inhibition of IkappaB kinase by 15dPGJ(2) in cells treated with LPS and IFN-gamma, resulting in an impaired phosphorylation of IkappaBalpha, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IkappaB kinase with 2 microM 15dPGJ(2) reduced by 83% the phosphorylation in serine 32 of IkappaBalpha, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IkappaB kinase complex. These results show rapid actions of 15dPGJ(2), independent of peroxisomal proliferator receptor gamma activation, in macrophages challenged with low doses of LPS and IFN-gamma.

Functional characterization of the L-type pyruvate kinase gene glucose response complex.

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Expression of simple epithelial cytokeratins in mouse epidermal keratinocytes harboring Harvey ras gene alterations.

Activation of a Harvey ras (H-ras) protooncogene is a frequent event associated with mouse epidermal carcinogenesis. We report that the transfection of a human H-ras oncogene into an immortalized mouse epidermal cell line (MCA3D) induces the anomalous expression of cytokeratins (CKs) 8 and 18 characteristic of simple epithelia. The comparison of various transfectant cell clones indicated a direct correlation between the levels of CK8 expression and the mutated H-ras p21s. The expression of simple epithelial CKs is also described in cell lines derived from mouse skin carcinomas (HaCa4, CarC) and in keratinocytes transformed in vitro by a chemical carcinogen (PDV, PDVC57), all of which contain altered H-ras genes. The induction of CK8 and CK18 occurs at the mRNA level and, although both CK8 and CK18 mRNAs are expressed, CK18 protein does not accumulate whereas CK8 is incorporated into intermediate filaments. Immunofluorescence studies show that the pattern of CK8 protein expression is heterogeneous; some cells express very low amounts of CK8, whereas others synthesize relatively high levels of this protein. However, selection of strongly CK8-positive cells was found in one case where a more malignant population of cells (PDVC57) was derived by tumor transplantation of PDV. Our results suggest that activation of a H-ras gene can alter the normal differentiation program of epidermal cells and that the ability to synthesize CK8 and CK18 could be related to tumor progression.

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release.

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.

Development of a neuroprotective peptide that preserves survival pathways by preventing Kidins220/ARMS calpain processing induced by excitotoxicity.

Kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), has a central role in the coordination of receptor crosstalk and the integration of signaling pathways essential for neuronal differentiation, survival and function. This protein is a shared downstream effector for neurotrophin- and ephrin-receptors signaling that also interacts with the N-methyl-d-aspartate type of glutamate receptors (NMDARs). Failures in neurotrophic support and glutamate signaling are involved in pathologies related to excitotoxicity and/or neurodegeneration, where different components of these dynamic protein complexes result altered by a combination of mechanisms. In the case of Kidins220/ARMS, overactivation of NMDARs in excitotoxicity and cerebral ischemia triggers its downregulation, which contributes to neuronal death. This key role in neuronal life/death decisions encouraged us to investigate Kidins220/ARMS as a novel therapeutic target for neuroprotection. As the main mechanism of Kidins220/ARMS downregulation in excitotoxicity is proteolysis by calpain, we decided to develop cell-penetrating peptides (CPPs) that could result in neuroprotection by interference of this processing. To this aim, we first analyzed in detail Kidins220/ARMS cleavage produced in vitro and in vivo, identifying a major calpain processing site in its C-terminal region (between amino acids 1669 and 1670) within a sequence motif highly conserved in vertebrates. Then, we designed a 25-amino acids CPP (Tat-K) containing a short Kidins220/ARMS sequence enclosing the identified calpain site (amino acids 1668-1681) fused to the HIV-1 Tat protein basic domain, able to confer membrane permeability to attached cargoes. Transduction of cortical neurons with Tat-K reduced Kidins220/ARMS calpain processing in a dose- and time-dependent manner upon excitotoxic damage and allowed preservation of the activity of pERK1/2 and pCREB, signaling molecules central to neuronal survival and functioning. Importantly, these effects were associated to a significant increase in neuronal viability. This Kidins220/ARMS-derived peptide merits further research to develop novel neuroprotective therapies for excitotoxicity-associated pathologies.

Differential calcium mobilization by vasopressin, angiotensin II, gastrin-releasing peptide, and adenosine triphosphate in adult and fetal hepatocytes. Relevance for the activation of calcium-dependent enzymes.

Early signals elicited after membrane receptor binding of agonists, the transmembrane signaling pathway of which involves activation of phosphoinositide-specific phospholipase C, were compared in fetal (22 days gestation) and adult rat hepatocytes. Free cytosolic calcium changes varied depending on the agonist and type of stimulated cells. Angiotensin II and ATP elicited the maximal responses in both types of cells, whereas the maximal Ca2+ increase produced by vasopressin was twice as much in adult than in fetal hepatocytes. The opposite response was observed for bombesin- or gastrin-releasing peptide-stimulated cells. Triggering of fetal and adult hepatocytes with substances that maximally promote endoplasmic reticulum calcium release or phosphoinositide-specific phospholipase C activation revealed that at least for the actions mediated through the angiotensin II and P2 purinergic receptor, the agonist stimulation was near the maximal response capacity of the signaling pathway. Agreement was observed between the relative number of membrane receptors and the biological responses.

Satellite DNA in the crustacean Artemia.

We have isolated a satellite fraction from the Artemia genome by both restriction endonuclease digestion and equilibrium density centrifugation in CsCl gradients containing ligand dye Hoechst 33258. Satellite DNA was arranged in long stretches (approx. 23 kb) of tandem repeats of a basic unit of 113 bp. The basic unit has been sequenced, showing a G + C content very close to that of total DNA. Different amounts of satellite were present in several populations of Artemia, whereas it was absent from others.

Characterization of two types of rRNA gene repeat units from the crustacean Artemia.

We have previously described that Artemia rRNA genes are organized with a basic repeat unit of 16.5 kb [Cruces et al., Biochem. Biophys. Res. Commun. 98 (1981) 404-409]. Here we describe the organization of the DNA coding for rRNA of a different population of this crustacean that has a repeat unit of 12.2 kb. Both types of repeat units have been cloned and the organization of the external spacers studied by restriction analysis. Both external spacers contain repeated sequences, but they are not homologous to each other. Sequences from the external spacer of the 16.5 kb repeat are also found elsewhere in the genome, within sequences not related to rRNA genes.

Substrate-dependent inhibition of protein kinase C by specific inhibitors.

Protein kinase C (PKC) and its proteolysis-derived protein kinase independent of Ca2+ and phospholipids (PKM), were purified from rat brain. By using histone H1 and protamine as substrates, we assayed the effect of several inhibitors of PKC and PKM. The inhibition turned out to be dependent on both the nature of the kinase and the type of substrate assayed. These results may help to interpret the different responses elicited by PKC inhibitors in vivo.

Full activation of RNaseL in animal cells requires binding of 2-5A within ankyrin repeats 6 to 9 of this interferon-inducible enzyme.

To define protein domains important for activation of the interferon (IFN)-induced enzyme 2-5A-dependent RNaseL, we have generated vaccinia virus (VV) recombinants able to express in cultured cells truncated forms of this protein and compared their biologic activities with those producing the wild-type enzyme, with and without coexpression of 2-5A synthetase. Our results show that full activation of RNaseL requires binding of 2-5A oligonucleotides within amino acid positions 212-339, corresponding to ankyrin repeats 6 to 9. The protein kinase and ribonuclease domains of RNaseL, amino acids 340-741, are sufficient for a constitutively active enzyme that is unresponsive to excess 2-5A. These results demonstrate in vivo the importance of the ankyrin domains in the biologic function of RNaseL. We suggest that ankyrin repeats act as key modulators of RNaseL activity.

Mechanisms of the neuroprotective effect of aspirin after oxygen and glucose deprivation in rat forebrain slices.

Acetylsalicylic acid (ASA, Aspirin) is an anti-inflammatory drug with a wide spectrum of pharmacological activities and multiple sites of action. Apart from its preventive actions against stroke due to its antithrombotic properties, recent data in the literature suggest that high concentrations of ASA also exert direct neuroprotective effects. We have used an in vitro model of brain ischaemia using rat forebrain slices deprived of oxygen and glucose to test ASA neuroprotective properties. We have found that ASA inhibits neuronal damage at concentrations lower than those previously reported (0.1-0.5 mM), and that these effects correlate with the inhibition of excitatory amino acid release, of NF-kappaB translocation to the nucleus and iNOS expression caused by ASA. All of these three mechanisms may mediate the neuroprotective effects of this drug. Our results also show that the effects of ASA are independent of COX inhibition. Taken together, our present findings show that ASA is neuroprotective in an in vitro model of brain ischaemia at doses close to those recommended for its antithrombotic effects.

Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines.

Cyclooxygenase-2 (COX-2) is involved in the biosynthesis of prostanoids in the course of inflammatory reactions. This isoenzyme is regulated at the transcription level and many cells express COX-2 upon challenge with lipopolysaccharide (LPS) or pro-inflammatory cytokines. Since hepatocytes respond to LPS and pro-inflammatory stimuli, we investigated the expression of COX-2 in foetal and adult hepatocytes upon challenge with these substances. COX-2 was expressed in foetal hepatocytes incubated with LPS, tumour necrosis factor-alpha and interleukin-1beta. This response rapidly decreased after birth and was absent in hepatocytes from animals aged 2 days or more and treated under identical conditions. The expression of COX-2 was determined at the mRNA, protein and enzyme activity levels using Northern and Western blot, and following the synthesis of prostaglandin E2, respectively. The use of NS 398, a specific pharmacological inhibitor of COX-2, confirmed the expression of this isoenzyme in activated foetal hepatocytes. Synergism in COX-2 expression was observed between LPS, tumour necrosis factor-alpha and interleukin-1beta. Interleukin-6 and permeant analogues of cyclic AMP failed to induce COX-2 or to synergize with LPS. Also, transforming growth factor-beta inhibited the LPS- and pro-inflammatory cytokines-dependent expression of COX-2. These results indicate that foetal hepatocytes are competent to express COX-2 upon challenge with pro-inflammatory stimuli, a process lost completely in hepatocytes isolated from animals aged 2 days.

Inhibition of NOS-2 expression in macrophages through the inactivation of NF-kappaB by andalusol.

1. Andalusol, ent-6alpha,8alpha,18-trihydroxy-13(16),14-labdadiene, is a naturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo models of paw and ear inflammation (Navarro et al., 1997: Z. Naturforsch., 52, 844-849). The pharmacological effects of this diterpene have been analysed on the activation of the macrophage cell line J774 with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). 2. Incubation of J774 macrophages with andalusol (0.1 - 100 microM) inhibited the synthesis of nitrite caused by LPS (1 microg ml-1) in concentration and time-dependent manners. The maximal inhibition was observed when andalusol was added 30 min before LPS stimulation and decreased progressively as the interval between andalusol and LPS challenge increased up to 14 h. 3. Incubation of J774 cells with LPS resulted in the expression of NOS-2 protein (130 kDa) as identified by Western blot analysis. The levels of this enzyme decreased significantly in the presence of andalusol (IC50=10.5 microM), suggesting that this diterpene inhibited NOS-2 expression. 4. Andalusol inhibited nuclear factor kappaB activation, a transcription factor necessary for NOS-2 expression in response to LPS and IFN-gamma. This compound also inhibited the degradation of IkappaBalpha favouring the retention of the inactive NF-kappaB complexes in the cytosol. 5. Related compounds to andalusol but lacking the polyol groups were less effective inhibiting NOS-2 expression in LPS-activated macrophages. The present findings provide a mechanism by which the anti-inflammatory properties of this diterpene could be mediated.

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L.

Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted significant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kappaB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10-1000 ng/ml specifically promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound.

Growth of Escherichia coli in acetate as a sole carbon source is inhibited by ankyrin-like repeats present in the 2',5'-linked oligoadenylate-dependent human RNase L enzyme.

Expression of low levels of the 2',5'-linked oligoadenylate-dependent human RNase L, an enzyme induced by interferons, is highly toxic in Escherichia coli. This protein contains an ankyrin domain responsible for RNase L toxicity. The only known ORF in E. coli containing ankyrin repeats is yjaC in the acetate metabolic cluster. We have investigated if expression of mutant forms of RNase L interfere with metabolism of acetate in E. coli. Our findings demonstrate that E. coli expressing RNase L ankyrin repeats is unable to grow in medium containing acetate as the sole carbon source, while it can grow when expressing other domains of the protein. This defect correlates with a severe decrease in the levels of induction of enzymes in the glyoxylate bypass.

Structure and expression of a polyubiquitin gene from the crustacean Artemia.

We have characterized two polyubiquitin genes from the crustacean Artemia franciscana. One of them, Ubi1, has nine ubiquitin units and an intron of a minimum size of 3.5 kb that ends 7 bp before the initiator ATG. The 5' end of the transcript from this gene has been identified by anchored PCR. The existence of the other gene (Ubi2) was inferred from several cDNA clones that differ from Ubi1 in the C-terminal extension and in the 3' untranslated region as well as in the nucleotide sequence of the coding region. We find two transcripts of ubiquitin genes, of 2.7 and 3.3 kb. Hybridization of RNA blots with an oligonucleotide specific for Ubi2 gene demonstrates that this gene codes for the 3.3 kb transcript. Ubiquitin messenger RNAs are present in the dormant embryos and their steady-state levels are maximum at 8 h after resumption of development, declining thereafter. The Ubi2 gene transcripts are less abundant but its proportion in relation to the other transcript does not vary with development.

Characterization of nitric oxide dependent changes in carbohydrate hepatic metabolism during septic shock.

The role of nitric oxide in the alterations of liver carbohydrate metabolism during septic shock has been studied in fed and starved animals injected with bacterial lipopolysaccharide (LPS). One h after LPS injection an hyperglycemic peak was observed followed by hypoglycemia when the plasma nitric oxide concentration increased. However, in animals pharmacologically treated with nitric oxide donors only hypoglycemia was observed. In isolated hepatocytes from LPS treated rats an impairment of the gluconeogenic flux was observed accompanied by a decrease in the mRNA levels of the glucose transporter GLUT-2 and phosphoenolpyruvate carboxykinase, at the time that increased the mRNA levels of the inducible form of nitric oxide synthase. These results suggest that part of the effects observed in response to LPS challenge are due to early signaling molecules (cytokines and other factors molecules) whereas other effects can be attributed to nitric oxide synthesis which in turn has specific effects on hepatic metabolism.

Imbalance of neurotrophin receptor isoforms TrkB-FL/TrkB-T1 induces neuronal death in excitotoxicity.

A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies.