Selenium triggers Nrf2-AMPK crosstalk to alleviate cadmium-induced autophagy in rabbit cerebrum.

Cadmium (Cd) is a toxic heavy metal that accumulates in the brain and causes a series of histopathological changes. Selenium (Se) exerts a crucial function in protecting damage caused by toxic heavy metals, but its potential mechanism is rarely studied. The main purpose of this study is to explore the protective effects of Se on Cd-induced oxidative stress and autophagy in rabbit cerebrum. Forty rabbits were randomly divided into four groups and treated as follows: Control group, Cd (1 mg/kg⋅BW) group, Se (0.5 mg/kg⋅BW) group and Cd (1 mg/kg⋅BW)+Se (0.5 mg/kg⋅BW) group, with 30 days feeding management. Our results suggested that Se treatment significantly suppressed the Cd-induced degenerative changes including cell necrosis, vacuolization, and atrophic neurons. In addition, Se decreased the contents of MDA and H2O2 and increased the activities of CAT, SOD, GST, GSH and GSH-Px, alleviating the imbalance of the redox system induced by Cd. Furthermore, Cd caused the up-regulation of the mRNA levels of autophagy-related genes (ATG3, ATG5, ATG7, ATG12 and p62), AMPK (Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag2, Prkag3) and Nrf2 (Nrf2, HO-1 and NQO1) signaling pathway, and the expression levels of LC3II/LC3I, p-AMPK/AMPK, Beclin-1, Nrf2 and HO-1 proteins, which were alleviated by Se, indicated that Se inhibited Cd-induced autophagy and Nrf2 signaling pathway activation. In conclusion, our study found that Se antagonized Cd-induced oxidative stress and autophagy in the brain by generating crosstalk between AMPK and Nrf2 signaling pathway.

Pharmacological mechanisms leading to synergy in fixed-dose dual bronchodilator therapy.

Long-acting ß2 adrenoceptor agonists (LABAs) in combination with long-acting muscarinic antagonists (LAMAs) can elicit functional and clinical benefits in chronic obstructive pulmonary disease (COPD). LABA/LAMA combinations synergistically relax human isolated airways at the level of the medium and small bronchi. LABAs and LAMAs both modulate the bronchial tone via different pathways localized at the level of presynaptic parasympathetic fibers and airway smooth muscle cells. The exact nature of the interactions between these pathways is not completely understood, but there is cross-talk at many levels in airway smooth muscle cells that is also regulated by the activity of calcium-activated potassium channels and protein tyrosine kinases. While the synergy between LABAs and LAMAs is a class effect, some of the currently available fixed-dose combinations (FDCs) do not induce synergistic interaction because the individual components are not appropriately balanced in the combination. Concerns remain on the cardiovascular safety profile of LABA/LAMA FDCs.

Progress with the use of monoclonal antibodies for the treatment of systemic lupus erythematosus.

In recent years, significant progress has been made in the use of monoclonal antibodies in the treatment of systemic lupus erythematosus (SLE). Advances in our understanding of the complexity of SLE immunopathogenesis have led to the testing of several biologic agents in clinical trials. Monoclonal therapies currently emerging or under development include B-cell depletion therapies, agents targeting B-cell survival factors, blockade of T-cell co-stimulation and anticytokine therapies. Issues remain, however, regarding clinical trial design and outcome measures in SLE which need to be addressed to optimize translation of these promising therapies into clinical practice.

The risks of targeting co-inhibitory pathways to modulate pathogen-directed T cell responses.

The identification of T cell co-inhibition as a central mechanism in the regulation of adaptive immunity during infectious diseases provides new opportunities for immunotherapeutic interventions. However, the fact that T cell activity is frequently downregulated during pathogen-directed responses suggests a pivotal physiological role of co-inhibitory pathways during infectious disease. Reports of exacerbated immunopathology in conditions of impaired co-inhibition foster the view that downregulation of T cell activity is an essential negative feedback mechanism that protects from excessive pathogen-directed immunity. Thus, targeting co-inhibitory pathways can bear detrimental potential through the deregulation of physiological processes. Here, we summarize recent preclinical and clinical interventions that report immune-related adverse events after targeting co-inhibitory pathways.

Astrocytes modulate a postsynaptic NMDA-GABAA-receptor crosstalk in hypothalamic neurosecretory neurons.

A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABA(A)ergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABA(A) receptors (GABA(A)Rs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABA(A)Rs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR-GABA(A)R crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate-GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate-GABA interactions in MNCs, which involves crosstalk between NMDA and GABA(A) postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABA(A)Rs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure.

Thrombopoietin receptor down-modulation by JAK2 V617F: restoration of receptor levels by inhibitors of pathologic JAK2 signaling and of proteasomes.

The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.

Effects of prochloraz or propylthiouracil on the cross-talk between the HPG, HPA, and HPT axes in zebrafish.

The objective of this study was to assess chemical-induced effects on cross-talk among the hypothalamic-pituitary-gonad (HPG), hypothalamic-pituitary-adrenal (HPA), and hypothalamic-pituitary-thyroid (HPT) axes of fish. Adult female zebrafish were exposed to 300 µg/L prochloraz (PCZ) or 100 mg/L propylthiouracil (PTU), and the transcriptional profiles of the HPG, HPA, and HPT axes were examined. Exposure to PCZ decreased plasma testosterone (T) and 17ß-estradiol (E2) concentrations and affected HPA and HPT axes by down-regulating corticotrophin-releasing hormone (CRH) after 12 and 48 h. By using correlation analyses, it was found that the decrease in E2 plasma concentrations caused by PCZ was correlated with the down-regulation of CRH mRNA expression. Exposure to PTU resulted in lesser concentrations of thyroxine (T4) and triiodothyronine (T3), greater concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) peptides, and increase in steroidogenic gene expression after 12 and 48 h. Concentrations of FSH and LH were negatively correlated with concentrations of T4 and T3. These results are consistent with the hypothesis that increased steroidogenic gene expression after PTU exposure resulted from a reduction in T4 and T3 concentrations, which resulted in greater secretion of FSH and LH.

Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways.

Apo-1 (Fas/CD95), a cell surface receptor, triggers apoptosis after binding to its physiological ligand, Apo-1L (FasL/CD95L). This study reports that mahanine, purified from the leaves of Murraya koenigii, has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34(+) cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.

Retinoic acid cross-talk with calcitriol activity in Atlantic salmon (Salmo salar).

Vitamins A (VA) and D (VD) are metabolised by vertebrates to bioactive retinoic acid (RA) and calcitriol (CTR). RA and CTR involvement in bone metabolism requires fine-tuned regulation of their synthesis and breakdown. In mammals antagonism of VA and VD is observed, but the mechanism of interaction is unknown. We investigated VA-VD interactions in Atlantic salmon (Salmo salar L.) following i.p. injection of RA and/or CTR. VA metabolites, CTR, calcium (Ca), magnesium (Mg) and phosphorus (P) were determined in plasma. Expression of bone matrix Gla protein (mgp), collagen 1 alpha2 chain (col1a2) and alkaline phosphatase (alp) mRNA was quantified to reflect osteogenesis. Branchial epithelial Ca channel (ecac listed as trpv6 in ZFIN Database) mRNA levels and intestinal Ca and P influx were determined to study Ca/P handling targets of RA and CTR. RA-injection (with or without CTR) decreased plasma CTR-levels three- to sixfold. CTR injection did not affect RA metabolites, but lowered CTR in plasma 3 and 5 days after injection. Lowered plasma CTR correlated with decreased mgp and col1a2 expression in all groups and with decreased alp in CTR-injected fish. RA-treated salmon had enhanced alp expression, irrespective of reduced plasma CTR. Expression of ecac and unidirectional intestinal influx of Ca were stimulated following RA-CTR treatment. Plasma Ca, Mg and P were not affected by any treatment. The results suggest cross-talk of RA with the VD endocrine system in Atlantic salmon. Enhanced Ca flux and osteogenesis (alp transcription) in RA-treated fish and inhibition of mgp expression revealed unprecedented disturbance of Ca physiology in hypervitaminosis A.

Inhibitory effect of salvinorin A, from Salvia divinorum, on ileitis-induced hypermotility: cross-talk between kappa-opioid and cannabinoid CB(1) receptors.

BACKGROUND AND PURPOSE: Salvinorin A, the active component of the hallucinogenic herb Salvia divinorum, inhibits intestinal motility through activation of kappa-opioid receptors (KORs). However, this compound may have target(s) other than the KORs in the inflamed gut. Because intestinal inflammation upregulates cannabinoid receptors and endogenous cannabinoids, in the present study we investigated the possible involvement of the endogenous cannabinoid system in salvinorin A-induced delay in motility in the inflamed gut. EXPERIMENTAL APPROACH: Motility in vivo was measured by evaluating the distribution of a fluorescent marker along the small intestine; intestinal inflammation was induced by the irritant croton oil; direct or indirect activity at cannabinoid receptors was evaluated by means of binding, enzymic and cellular uptake assays. KEY RESULTS: Salvinorin A as well as the KOR agonist U-50488 reduced motility in croton oil treated mice. The inhibitory effect of both salvinorin A and U-50488 was counteracted by the KOR antagonist nor-binaltorphimine and by the cannabinoid CB(1) receptor antagonist rimonabant. Rimonabant, however, did not counteract the inhibitory effect of salvinorin A on motility in control mice. Binding experiments showed very weak affinity of salvinorin A for cannabinoid CB(1) and CB(2) and no inhibitory effect on 2-arachidonoylglycerol and anandamide hydrolysis and cellular uptake. CONCLUSIONS AND IMPLICATIONS: The inhibitory effect of salvinorin A on motility reveals a functional interaction between cannabinoid CB(1) receptors and KORs in the inflamed--but not in the normal--gut in vivo.

Novel omega -- 3-derived local mediators in anti-inflammation and resolution.

The integrated inflammatory response of the host is essential in health and disease. Hence, it is important to achieve a more complete understanding of the local cellular and molecular events that govern the formation and actions of local mediators that can serve as endogenous local mediators of resolution. Because these compounds in experimental animal models of inflammation can control the duration and magnitude of inflammation, knowledge of their formation and actions may provide new avenues for appreciating the molecular basis of many inflammatory diseases. The first of these endogenous local counterregulators recognized were the lipoxins, which are trihydroxytetraene-containing mediators generated from arachidonic acid during cell-cell interactions via transcellular biosynthesis. Because this circuit of lipoxin formation appears to be of physiological relevance in resolution, therapeutic modalities targeting this and related systems should allow for the development of novel therapeutic agents (i.e., agonists of the important cellular and physiological responses required for timely resolution). This review offers a general overview of recent advances from studies by the author and colleagues on the biosynthesis and bioactions of the novel anti-inflammatory lipid mediators, resolvins, docosatrienes, and neuroprotectins as well as their endogenous aspirin-triggered epimeric counterparts. These previously unappreciated families of lipid-derived mediators were originally isolated from experimental murine models of acute inflammation captured during the natural spontaneous resolution phase. They possess anti-inflammatory, pro-resolving, and protective properties. Inappropriate resolution mechanism(s) may underlie our current appreciation of the inflammatory phenotype(s) that characterizes many prevalent human diseases where inflammation is now acknowledged to play an important role in the disease process. Moreover, these new pathways give opportunities to appreciate the complex roles of neutrophils in the generation of potent host protective lipid mediators that may be harnessed for the design of novel treatments for a wide range of diseases where inflammation contributes to the pathophysiology of the disorder.

Investigation of the effect of the farnesyl protein transferase inhibitor R115777 on isoprenylation and intracellular signalling by the prostacyclin receptor.

The human (h) and mouse (m) prostacyclin receptors (IPs) undergo isoprenylation through attachment of a C-15 farnesyl moiety within their conserved carboxyl terminal -CSLC sequences. Herein, the effects of a novel farnesyl transferase inhibitor R115777 on signalling by the hIP and mIP, overexpressed in human embryonic kidney 293 cells, and by the hIP endogenously expressed in human erythroleukaemia cells were investigated. R115777 significantly impaired IP-mediated cyclic AMP generation (IC(50) 0.37-0.60 nm) and intracellular calcium ([Ca(2+)](i)) mobilization (IC(50) 37-65 nm), but had no effect on signalling by the control nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Additionally, R115777 significantly reduced IP-mediated cross-desensitization of signalling by the TP alpha, but not by the TP beta, isoform of the human TP and impaired the farnesylation-dependent processing of the chaperone HDJ-2 protein (IC(50) 4.5 nm). Furthermore, R115777 fully impaired isoprenylation of both the Ha-Ras(WT) and Ha-Ras(CSLC) in vitro and in whole cells confirming that, unlike N-Ras and Ki-Ras, the -CSLC motif associated with the IP cannot support alternative geranylgeranylation in the presence of R115777 and does not act as a substrate for geranylgeranyl transferase 1 in vitro or in whole cells. In conclusion, these data confirm that R115777 potently impairs IP isoprenylation and signalling, and suggest that clinically it may not only target Ras proteins but may also disrupt IP isoprenylation, events which could impact on physiologic processes in which prostacyclin and its receptor are implicated.

Effect of the statin atorvastatin on intracellular signalling by the prostacyclin receptor in vitro and in vivo.

Prostacyclin plays a central role within the vasculature. We have previously established that the prostacyclin receptor (IP) undergoes isoprenylation, a lipid modification obligate for its function. The aim of the current study was to investigate the effect of the hydroxy methyl glutaryl co-enzyme A reductase inhibitor atorvastatin on signalling and function of the IP expressed in mammalian whole cells and in platelets isolated from patients undergoing therapeutic intervention with atorvastatin. Initially, the effect of atorvastatin on signalling by the human (h) and mouse (m) IP overexpressed in human embryonic kidney 293 cells and the hIP endogenously expressed in human erythroleukaemic 92.1.7 cells was investigated. Atorvastatin significantly reduced IP-mediated cAMP generation (IC(50) 6.6-11.1 microm) and [Ca(2+)](i) mobilization (IC(50) 7.2-16.4 microm) in a concentration-dependent manner, but had no effect on signalling by the nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Moreover, atorvastatin significantly reduced IP-mediated crossdesensitization of signalling by TP alpha (IC(50) 10.4 microm), but not by TP beta. In contrast to the whole-cell data, atorvastatin therapy did not interfere with IP-mediated cAMP generation or IP-induced inhibition of TP-mediated aggregation of platelets isolated from human volunteers undergoing therapeutic intervention with atorvastatin (10-80 mg per daily dose). In conclusion, while data generated in whole cells indicated that atorvastatin significantly impairs signalling by both the hIP and mP, the in vivo clinical data indicated that, at the administered therapeutic dose, atorvastatin does not significantly compromise IP signalling and function in humans.

NR2B-containing receptors mediate cross talk among hippocampal synapses.

Under some conditions, synaptically released glutamate can exert long-range actions in the cortical microcircuitry. To what extent glutamate spillover leads to direct cross talk among individual synapses remains unclear. We recorded NMDAR-mediated EPSCs in acute hippocampal slices at 35 degrees C by stimulating two independent pathways that converge on the same CA1 pyramidal cell. Activation of a conditioning pathway in the presence of the use-dependent blocker dizocilpine maleate (MK801) resulted in partial NMDA receptor (NMDAR) blockade in the other, silent pathway. This was accompanied by an increase in the rise time of the EPSCs in the conditioning (although not the silent) pathway, implying an increase in diffusional distance from release site to NMDARs. We estimated that up to approximately 30% of NMDARs contributing to EPSCs were activated by glutamate released from multiple synaptic sources; however, NMDAR-mediated synaptic cross talk was undetectable when NR2B subunit-containing receptors were blocked (but could be rescued by blocking glutamate uptake). We propose that NR2B-containing NMDARs can detect glutamate arising from multiple synapses, whereas NR2A-containing NMDARs only normally mediate direct synaptic transmission. These NMDAR isoforms thus play complementary roles in sensing global and local glutamate signals, respectively.