Frontiers in Pharmacology

Published by Frontiers
Online ISSN: 1663-9812
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The mineralocorticoid receptor (MR) antagonists PF-03882845 and eplerenone were evaluated for renal protection against aldosterone-mediated renal disease in uninephrectomized Sprague-Dawley (SD) rats maintained on a high salt diet and receiving aldosterone by osmotic mini-pump for 27 days. Serum K(+) and the urinary albumin to creatinine ratio (UACR) were assessed following 14 and 27 days of treatment. Aldosterone induced renal fibrosis as evidenced by increases in UACR, collagen IV staining in kidney cortex, and expression of pro-fibrotic genes relative to sham-operated controls not receiving aldosterone. While both PF-03882845 and eplerenone elevated serum K(+) levels with similar potencies, PF-03882845 was more potent than eplerenone in suppressing the rise in UACR. PF-03882845 prevented the increase in collagen IV staining at 5, 15 and 50 mg/kg BID while eplerenone was effective only at the highest dose tested (450 mg/kg BID). All doses of PF-03882845 suppressed aldosterone-induced increases in collagen IV, transforming growth factor-β 1 (Tgf-β 1), interleukin-6 (Il-6), intermolecular adhesion molecule-1 (Icam-1) and osteopontin gene expression in kidney while eplerenone was only effective at the highest dose. The therapeutic index (TI), calculated as the ratio of the EC50 for increasing serum K(+) to the EC50 for UACR lowering, was 83.8 for PF-03882845 and 1.47 for eplerenone. Thus, the TI of PF-03882845 against hyperkalemia was 57-fold superior to that of eplerenone indicating that PF-03882845 may present significantly less risk for hyperkalemia compared to eplerenone.
 
Effects of MK-0448 on Kv1.5 currents measured in HK2BN9 cells at 25°C. (A) Voltage clamp and current traces obtained before (black) and after superfusion of cells with 100 nM MK-0448 (green) for 10 min. Rate of clamp pulses as indicated. (B) Onset of blocking effect of MK-0448 (100 nM) on peak Kv1.5 current and recovery from block during 5 min at holding potential of −60 mV. Individual current traces at top were obtained at the time points indicated by small letters in the diary plot at the bottom. (C) Onset of drug effect following exposure during 5 min of continuous clamping at −60 mV. Step frequency: 3 Hz.
Concentration-dependent effects of MK-0448 on action potentials recorded in right atrial trabeculae from patients in sinus rhythm (SR) and permanent atrial fibrillation (AF). Top: Individual tracings for typical experiments, recorded in the presence of increasing concentrations of compound (see color code). Exposure time at each concentration: 20 min; control: pre-drug control. Middle and bottom: Mean values ± s.e.m. from 7 experiments in SR and 5 in AF, of action potential duration at 90, 50, and 20% of repolarization (APD90, APD50, and APD20) and plateau potential (PLT20). Frequency of stimulation: 1 Hz. Please note, that not all preparations were exposed to the full range of concentrations (numbers of preparations indicated in brackets).
Effects of MK-0448 (3 μM, red trace, arrow) on action potentials recorded in right atrial trabeculae from patients in sinus rhythm (SR), intermittent atrial fibrillation (iAF) and chronic atrial fibrillation (AF). Original recordings from one preparation in each group is shown. c, pre-drug control (black trace). Stimulation frequency 1 Hz.
| Effects of MK-0448 (3 µM) on action potential parameters
Selective blockers of the Kv1.5 channel have been developed for the treatment of atrial fibrillation (AF), but little is known how these atrial-selective drugs affect human action potentials (APs). Therefore we have investigated the Kv1.5 blocker MK-0448 (N-{6-[(1S)-1-(4-fluorophenyl)-2,2-di(pyridin-3-yl)ethyl]pyridin-2-yl}methanesulfon- amide) in right atrial trabeculae from patients in sinus rhythm (SR), permanent AF (>6 months), and intermittent AF. MK-0448 blocked Kv1.5 current in an expression system and concentration-dependently elevated the plateau phase of atrial APs. In SR preparations stimulated at 1 Hz, MK-0448 (3 μM) shortened action potential duration at 90% of repolarization (APD90) and effective refractory period (ERP), but in permanent AF preparations, MK-0448 prolonged APD90 and ERP. The effects of MK-0448 in intermittent AF resembled those in SR preparations. Block of IKs is probably more prominent in AF because of reduced repolarization reserve due to AF-induced remodeling.
 
The American Cancer Society estimates that there will be more than 1.5 million new cases of cancer in 2011, underscoring the need for identification of new therapeutic targets and development of novel cancer therapies. Previous studies have implicated the human aldo-ketoreductases AKR1B1 and AKR1B10 in cancer, and therefore we examined AKR1B1 and AKR1B10 expression across all major human cancer types using the Oncomine cancer gene expression database (Compendia Biosciences, www.oncomine.com). Using this database, we found that expression of AKR1B1 and AKR1B10 varies greatly by cancer type and tissue of origin, including agreement with previous reports that AKR1B10 is significantly over-expressed in cancers of the lungs and liver. AKR1B1 is more broadly over-expressed in human cancers than AKR1B10, albeit at a generally lower magnitude. AKR1B1 over-expression was found to be associated with shortened patient survival in acute myelogenous leukemias and multiple myelomas. High AKR1B10 expression tends to predict less aggressive clinical course generally, notably within lung cancers, where it tends to be highly over-expressed compared to normal tissue. These findings suggest that AKR1B1 inhibitors in particular hold great potential as novel cancer therapeutics.
 
Overview of Gq/11-coupled GPCR signal detection by GCaMP sensors. Schematic diagram illustrating the canonical signaling pathway of Gq/11-coupled GPCRs. Heptahelical dimeric receptors bound by agonist can activate the Gq/11 pathway to increase intracellular Ca2+. The signaling intermediaries facilitating Ca2+ elevations include generation of IP3 and DAG from PLC activation, gating of IP3 receptors embedded in the ER and release of intracellular Ca2+ stores. Free Ca2+ can then bind to GCaMP sensors in the cytoplasm, resulting in enhanced fluorescence. Abbreviations: N: amino-terminus C: carboxy-terminus; PLC: phospholipase C; DAG: diacylglycerol, IP3: inositol trisphosphate; ΔF: change in fluorescence.
Genetic identification of cells using cre-lox driver: reporter methods. (A) Mating a gene promotor containing “driver” mouse directed to express cre in a selective fashion to a cre-dependent “reporter” mouse yields offspring which may inherit the driver-reporter combination (red offspring) of genes. Those offspring which do not inherit the combination will not express the identification marker (non-fluorescent offspring). (B) A currently used strategy is to cross a cre driver mouse to a mouse expressing a cre-sensitive element at a ubiquitously expressed locus like rosa26. At this locus is a Stop codon (Stop sign with TGA) flanked by loxP sites (indicated by arrows) designed to be excised by cre recombinase activity allowing expression of genetically encoded fluorescent reporters (red star symbol) in the target cell population. (C) Illustrates a confocal image of a coronal mouse brain section from an animal inheriting the drd1; cre and rosa26; tdTomato genes described above, permitting fluorescent detection of dopamine D1 cells in cortex (left) and striatum (right). (D) Live confocal image of an acute striatal brain slice showing drd2: tdTomato neurons (red, left) and the same section bulk loaded with the calcium sensitive dye Flou-8 (green, center), allowing genetic identification of striatal neurons while imaging calcium (superimposed images, right). See Partridge et al. (2014) for methods. Scale bars in (C) and (D): 100 μm.
Directed expression of genetically encoded calcium sensors to detect Gq/11-GPCR activation. (A) Mating a “driver” mouse directed to express cre in a selective dopamine D2 (drd2) fashion to a cre-dependent “reporter” GCaMP 3 mouse yields offspring which may inherit the driver-reporter combination of genes (green offspring). The offspring which do not inherit this combination will not express the calcium detector (non-fluorescent offspring). (B) Illustrates a confocal image of a coronal mouse brain section from an animal inheriting the drd2; cre and rosa26; GCaMP3 genes, permitting fluorescent detection of calcium changes in striatal dopamine D2 cells. (C) Live confocal image of an acute striatal brain slice showing drd1: GCaMP3 expressing neurons (green, left) with superimposed regions of interest (ROI, red circles) allowing genetically targeted calcium imaging. To the right are shown the time course of changes in [Ca2+]i (ΔF/F) in the ROIs corresponding to cell bodies. Each trace represents a different ROI or cell body. The top horizontal bars above the traces represent the time duration that the drugs DHPG (10 μM, open bars), or NMDA (20 μM, filled bars) were acutely applied. Note the ability to detect GPCR activation following activation of the cells. Scale bars in (B) and (C): 100 μm.
The family of GCaMPs are engineered proteins that contain Ca(2+) binding motifs within a circularly permutated variant of the Aequorea Victoria green fluorescent protein (cp-GFP). The rapidly advancing field of utilizing GCaMP reporter constructs represents a major step forward in our ability to monitor intracellular Ca(2+) dynamics. With the use of these genetically encoded Ca(2+) sensors, investigators have studied activation of endogenous Gq types of G protein-coupled receptors (GPCRs) and subsequent rises in intracellular calcium. Escalations in intracellular Ca(2+) from GPCR activation can be faithfully monitored in space and time as an increase in fluorescent emission from these proteins. Further, transgenic mice are now commercially available that express GCaMPs in a Cre recombinase dependent fashion. These GCaMP reporter mice can be bred to distinct Cre recombinase driver mice to direct expression of this sensor in unique populations of cells. Concerning the central nervous system (CNS), sources of calcium influx, including those arising from Gq activation can be observed in targeted cell types like neurons or astrocytes. This powerful genetic method allows simultaneous monitoring of the activity of dozens of cells upon activation of endogenous Gq-coupled GPCRs. Therefore, in combination with pharmacological tools, this strategy of monitoring GPCR activation is amenable to analysis of orthosteric and allosteric ligands of Gq-coupled receptors in their endogenous environments.
 
Contraction of bladder smooth muscle is predominantly initiated by M(3) muscarinic receptor-mediated activation of the G(q/11)-phospholipase C β-protein kinase C (PKC) and the G(12/13)-RhoGEF-Rho kinase (ROCK) pathways. However, these pathways and their downstream effectors are not well understood in bladder smooth muscle. We used phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DOG), activators of PKC, in this investigation. We were interested in dissecting the role(s) of PKC and to clarify the signaling pathways in bladder smooth muscle contraction, especially the potential cross-talk with ROCK and their downstream effectors in regulating myosin light chain phosphatase activity and force. To achieve this goal, the study was performed in the presence or absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr(38)-CPI-17 and Thr(696)/Thr(850) myosin phosphatase target subunit (MYPT1) were measured during PDBu or DOG stimulation using site specific antibodies. PDBu-induced contraction in bladder smooth muscle involved both activation of PKC and PKC-dependent activation of ROCK. CPI-17 as a major downstream effector, is phosphorylated by PKC and ROCK during PDBu and DOG stimulation. Our results suggest that Thr(696) and Thr(850)-MYPT1 phosphorylation are not involved in the regulation of a PDBu-induced contraction. The results also demonstrate that bladder smooth muscle contains a constitutively active isoform of ROCK that may play an important role in the regulation of bladder smooth muscle basal tone. Together with the results from our previous study, we developed a working model to describe the complex signaling pathways that regulate contraction of bladder smooth muscle.
 
| Experimental protocol for isolating human and mouse peripheral blood mononuclear cells from whole blood. (A) Cartoon representing the different steps for isolating human PBMC from whole circulating blood (for full details of the method, see Blood collection and Peripheral blood mononuclear cells Isolation in human from the Section "Experimental Procedures"). Some elements of this figure were produced
| Chronic fluoxetine treatment (18 mg/kg/day for 28 days) restored β-arrestin 1 levels in the peripheral blood mononuclear cells from anxio/depressive mice treated with chronic corticosterone treatment. (A) Experimental timeline used to measure β-arrestin 1 levels in peripheral blood mononuclear cells from naïve or anxio-depressive mouse submandibular bleeding chronically treated or not with fluoxetine during 28 days (18 mg/kg/day in the drinking water). (B) Representative western blot of β-arrestin 1 levels in peripheral blood mononuclear cells isolated from naïve (vehicle/vehicle, V/V), corticosterone (35 μg/ml/day; corticosterone/ vehicle, C/V) or corticosterone/fluoxetine (18 mg/kg/day; corticosterone/
Experimental protocol for isolating human and mouse peripheral blood mononuclear cells from whole blood. (A) Cartoon representing the different steps for isolating human PBMC from whole circulating blood (for full details of the method, see Blood collection and Peripheral blood mononuclear cells Isolation in human from the Section “Experimental Procedures”). Some elements of this Figure were produced using Servier Medical Art image bank (). (B) Cartoon representing the different steps for isolating mouse PBMC from whole circulating blood (for full details of the method, see Blood collection and Peripheral blood mononuclear cells Isolation in mouse from the Section “Experimental Procedures”). Some elements of this figure were produced using Servier Medical Art image bank ().
β-Arrestin 1 is measurable in both human and mouse peripheral blood mononuclear cells obtained from a low collection volume of fresh blood. Representative western blot of β-arrestin 1 levels in PBMCs isolated either from CD14- human cells, human or naïve mouse whole blood. In each blot, 30 μg of total protein were run. β-Actin was used as a control.
Chronic fluoxetine treatment (18 mg/kg/day for 28 days) restored β-arrestin 1 levels in the peripheral blood mononuclear cells from anxio/depressive mice treated with chronic corticosterone treatment. (A) Experimental timeline used to measure β-arrestin 1 levels in peripheral blood mononuclear cells from naïve or anxio-depressive mouse submandibular bleeding chronically treated or not with fluoxetine during 28 days (18 mg/kg/day in the drinking water). (B) Representative western blot of β-arrestin 1 levels in peripheral blood mononuclear cells isolated from naïve (vehicle/vehicle, V/V), corticosterone (35 μg/ml/day; corticosterone/vehicle, C/V) or corticosterone/fluoxetine (18 mg/kg/day; corticosterone/fluoxetine, C/F) mouse whole blood. In each blot, 30 μg of protein from mouse PBMC were run. The densitometry values for each band allowed the calculation of a ratio: optical density for β-arrestin 1/optical density β-actin value. (C,D) Scatterplot of the individual effects (C) or bar charts of mean ± SEM of the effects (D) of a chronic administration (28 days) treatment with fluoxetine on β-arrestin 1 levels in the peripheral blood mononuclear cells from mice treated with chronic corticosterone in comparison to untreated animals. Data are expressed in percentage normalized to vehicle/vehicle expression; n = 9–10). *p < 0.05, versus control vehicle/vehicle group.
A limited number of biomarkers in the central and peripheral systems which are known may be useful for diagnosing major depressive disorders and predicting the effectiveness of antidepressant (AD) treatments. Since 60% of depressed patients do not respond adequately to medication or are resistant to ADs, it is imperative to delineate more accurate biomarkers. Recent clinical studies suggest that β-arrestin 1 levels in human mononuclear leukocytes may be an efficient biomarker. If potential biomarkers such as β-arrestin 1 could be assessed from a source such as peripheral blood cells, then they could be easily monitored and used to predict therapeutic responses. However, no previous studies have measured β-arrestin 1 levels in peripheral blood mononuclear cells (PBMCs) in anxious/depressive rodents. This study aimed to develop a method to detect β-arrestin protein levels through immunoblot analyses of mouse PBMCs isolated from whole blood. In order to validate the approach, β-arrestin levels were then compared in na\"{\i}ve, anxious/depressed mice, and anxious/depressed mice treated with a selective serotonin reuptake inhibitor (fluoxetine, 18~mg/kg/day in the drinking water). The results demonstrated that mouse whole blood collected by submandibular bleeding permitted isolation of enough PBMCs to assess circulating proteins such as β-arrestin 1. β-Arrestin 1 levels were successfully measured in healthy human subject and na\"{\i}ve mouse PBMCs. Interestingly, PBMCs from anxious/depressed mice showed significantly reduced β-arrestin 1 levels. These decreased β-arrestin 1 expression levels were restored to normal levels with chronic fluoxetine treatment. The results suggest that isolation of PBMCs from mice by submandibular bleeding is a useful technique to screen putative biomarkers of the pathophysiology of mood disorders and the response to ADs. In addition, these results confirm that β-arrestin 1 is a potential biomarker for depression.
 
The market authorization procedure for medicinal products for human use is relying on their demonstrated efficacy, safety, and pharmaceutical quality. This applies to all medicinal products whether of chemical or biological origin. Since October 2009, the first advanced therapy medicinal product (ATMP) has been authorized through the centralized procedure. ATMPs are gene therapy medicinal products, somatic cell therapy medicinal products or tissue-engineered products. An appropriate ATMP - Regulation is dealing with ATMP requirements. Two exemptions are foreseen to the ATMP Regulation: (a) Products, which were legally on the Community market when the Regulation became applicable, should comply to the Regulation by December 30, 2012. (b) The hospital exemption rule for non-routine products for an individual patient. In this work we explored whether the actual application of the Regulation on ATMPs is in line with the aim of the Regulation in terms of guaranteeing the highest level of health protection for patients. Based on the analysis of the relative efficacy of the only EC authorized ATMP and its exempted alternatives, there is evidence against this Regulation 1394/2007 assumption.
 
The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers.
 
The human aldo-keto reductase AKR1B10, originally identified as an aldose reductase-like protein and human small intestine aldose reductase, is a cytosolic NADPH-dependent reductase that metabolizes a variety of endogenous compounds, such as aromatic and aliphatic aldehydes and dicarbonyl compounds, and some drug ketones. The enzyme is highly expressed in solid tumors of several tissues including lung and liver, and as such has received considerable interest as a relevant biomarker for the development of those tumors. In addition, AKR1B10 has been recently reported to be significantly up-regulated in some cancer cell lines (medulloblastoma D341 and colon cancer HT29) acquiring resistance toward chemotherapeutic agents (cyclophosphamide and mitomycin c), suggesting the validity of the enzyme as a chemoresistance marker. Although the detailed information on the AKR1B10-mediated mechanisms leading to the drug resistance process is not well understood so far, the enzyme has been proposed to be involved in functional regulations of cell proliferation and metabolism of drugs and endogenous lipids during the development of chemoresistance. This article reviews the current literature focusing mainly on expression profile and roles of AKR1B10 in the drug resistance of cancer cells. Recent developments of AKR1B10 inhibitors and their usefulness in restoring sensitivity to anticancer drugs are also reviewed.
 
| The proposed model of molecular mechanisms regulating the three choices of tumor cells under stress. Microenvironmental factors regulate the activity of HIF-1α. In addition to normoxia or hypoxia, changes of the proteolytic network can also determine the response of the tumor cell. While normoxia and proteolytic balance favor proliferation (right panel), which reflects the adaption of the tumor cell in situ, hypoxia or alterations in the proteolytic network promote the induction of pro-evasive mechanisms (left panel). Respiration is impaired upon HIF-1α
The proposed model of molecular mechanisms regulating the three choices of tumor cells under stress. Microenvironmental factors regulate the activity of HIF-1α. In addition to normoxia or hypoxia, changes of the proteolytic network can also determine the response of the tumor cell. While normoxia and proteolytic balance favor proliferation (right panel), which reflects the adaption of the tumor cell in situ, hypoxia or alterations in the proteolytic network promote the induction of pro-evasive mechanisms (left panel). Respiration is impaired upon HIF-1α stabilization not only by hypoxia but also in the case of proteolytic imbalance imposed by high TIMP-1 levels or similar environmental stress. It leads to upregulation of miR-210 which directly inhibits expression of the subunit D of the succinate dehydrogenase complex (SDHD) and simultaneously induces cell cycle arrest by inhibition of E2F3. Thus, the tumor cell switches to the evasive metastatic phenotype for which adaptation to hypoxia by their potential of executing anaerobic glycolysis is a pre-requisite.
In contrast to expectations in the past that tumor starvation or unselective inhibition of proteolytic activity would cure cancer, there is accumulating evidence that microenvironmental stress, such as hypoxia or broad-spectrum inhibition of metalloproteinases can promote metastasis. In fact, malignant tumor cells, due to their genetic and epigenetic instability, are predisposed to react to stress by adaptation and, if the stress persists, by escape and formation of metastasis. Recent recognition of the concepts of dynamic evolution as well as population and systems biology is extremely helpful to understand the disappointments of clinical trials with new drugs and may lead to paradigm-shifts in therapy strategies. This must be complemented by an increased understanding of molecular mechanism involved in stress response. Here, we review new roles of Hypoxia-inducible factor-1 (HIF-1), one transcription factor regulating stress response-related gene expression: HIF-1 is crucial for invasion and metastasis, independent from its pro-survival function. In addition, HIF-1 mediates pro-metastatic microenvironmental changes of the proteolytic balance as triggered by high systemic levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), typical for many aggressive cancers, and regulates the metabolic switch to glycolysis, notably via activation of the microRNA miR-210. There is preliminary evidence that TIMP-1 also induces miR-210. Such positive-regulatory co-operation of HIF-1α, miR-210, and TIMP-1, all described to correlate with bad prognosis of cancer patients, opens new perspectives of gaining insight into molecular mechanisms of metastasis-inducing evasion of tumor cells from stress.
 
Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of cystic fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-cell and single patch-clamp recordings showed that RP193 induced a linear, time- and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GP(inh)5a). Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.
 
In this paper we discuss the consensus view on the use of qualifying biomarkers in drug safety, raised within the frame of the XXIV meeting of the Spanish Society of Clinical Pharmacology held in Málaga (Spain) in October, 2011. The widespread use of biomarkers as surrogate endpoints is a goal that scientists have long been pursuing. Thirty years ago, when molecular pharmacogenomics evolved, we anticipated that these genetic biomarkers would soon obviate the routine use of drug therapies in a way that patients should adapt to the therapy rather than the opposite. This expected revolution in routine clinical practice never took place as quickly nor with the intensity as initially expected. The concerted action of operating multicenter networks holds great promise for future studies to identify biomarkers related to drug toxicity and to provide better insight into the underlying pathogenesis. Today some pharmacogenomic advances are already widely accepted, but pharmacogenomics still needs further development to elaborate more precise algorithms and many barriers to implementing individualized medicine exist. We briefly discuss our view about these barriers and we provide suggestions and areas of focus to advance in the field.
 
One can argue forever what precise percentage of the world's population use local and traditional medicines. These herbal (or mineral or fungal or occasionally animal) medical products form systems of knowledge and practice that have been transmitted over centuries and which continuously change. However, there can be no doubt that the majority of humans either rely on such products (often due to lack of other alternatives) or that herbal medicines are chosen consciously as an alternative to mainstream medicine. In some cases this knowledge is documented in an extensive historical written body of scholarly and applied writings. Traditions like “Traditional” Chinese, Ayurvedic, Unani, Jamu, Kampo, Iranian, Aztec or various forms of European and Arabic medicine are well known examples. In other regions we rely on the efforts of researchers past and present to document such knowledge and to critically analyze the data. It is now a commonplace that such “traditional knowledge” is disappearing fast, but in fact it is simply changing under the pressures of a globalizing world.
 
Inflammation is a complex set of interactions among soluble factors and cells that can arise in any tissue in response to traumatic, infectious, post-ischaemic, toxic or autoimmune injury (Nathan, 2002; Gilroy et al., 2004). Inflammation, per se, does not cause disease. But unchecked inflammation that spreads possibly due to disruption of the resolution processes can lead to chronic persistent inflammation which plays a role in the progression of many diseases, such as atherosclerosis, asthma, neurodegeneration, inflammatory bowel diseases (IBDs) and cancer. Thus, inflammation is one of the relevant areas in biomedical research. Molecular mechanisms regulating the checkpoints tuning on and off the inflammatory response have been revealed and numerous novel potential therapeutic targets have been uncovered. Humanized antibodies for anti-cytokine therapy have been shown to be effective for the treatment of rheumatoid arthritis and various inflammatory diseases (Maini and Taylor, 2000). However, there is still an important gap to be filled by developing new therapeutics which should be safer and more specific for different inflammation-related diseases. Importantly, we need to dissect the influence of genetic background but also behavioural and psychosocial factors in different socioeconomic groups on increased susceptibility to inflammation. This knowledge will lead to predict and prevent inflammatory-related diseases and hopefully to reduce social inequalities in disease development observed in many populations (Ranjit et al., 2007).
 
Since the advent of molecular cloning, target based screening has become the norm in pharmaceutical drug discovery. A large number of potential drug targets have been cloned and functionally expressed, and enormous progress has been made in the development, miniaturization and automation of cell based assays on target molecules recombinantly expressed in mammalian cell lines. This approach has delivered many clinical candidates but relatively few new drugs. Target based screening is likely to provide very good drug candidates for monogenic diseases, and the following collection of manuscripts is not meant to discourage the use of target based approaches. However, most of the more prevalent human diseases are most likely multifactorial and require interaction with multiple targets to produce clinically meaningful efficacy. In addition, high potency, selective interaction with a single target may increases the risk of adverse events or be limited by redundancies and adaptive resistance. Here, target agnostic approaches using phenotypic assays may offer significant benefit. Making such approaches viable requires addressing a number of challenges. This e-book attempts to discuss some of these challenges and illustrate recent advances.
 
BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASP(Ser239) and VASP(Ser157), respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASP(Ser239) but inhibited phosphorylation at VASP(Ser157). Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASP(Ser239) and VASP(Ser157), whereas PDE5 inhibition potentiated BAY-mediated increases only at VASP(Ser157). In comparison, 8Br-cGMP increased phosphorylation at VASP(Ser239) and VASP(Ser157) which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASP(Ser239) phosphorylation and inhibition of PDE3 increased phosphorylation at VASP(Ser239), while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASP(Ser157) phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders.
 
Peptide Ac2-26 provokes migration of human PMN: comparison to FMLP and SAA. Neutrophils isolated from peripheral blood of healthy volunteers were resuspended at 4 × 106 cells/ml and chemotaxis to peptide Ac2-26 (1–30 μM), SAA (1–30 μM), or FMLP (1–100 nM) was assessed over a 90-min period. Results are ±SEM from three to four cell preparations (**P < 0.01, ***P < 0.001 vs. PBS group).
Peptide Ac2-26, but not FMLP, induces PMN chemokinesis. Peripheral blood neutrophils were incubated with either (A) peptide Ac2-26 (1–30 μM) or PBS prior to loading on to 96 well ChemoTx™ plate where the bottom wells were either loaded with peptide Ac2–26 (10 μM) or (B) FMLP (0.01–1 μM) prior to loading on to a chemotaxis plate in the presence of 1 nM FMLP. (C) Neutrophils were incubated with peptide Ac2-26 (1–30 μM) prior to loading on to the chemotaxis plate. Results are ±SEM from three to four cell preparations [(A,B) *P < 0.05 vs. stimulus alone (dose 0 group); (C) *P < 0.05 vs. PBS].
Both FPR1 and FPR2/ALX mediate peptide Ac2-26 induced PMN migration. (A) Peripheral blood neutrophils were pre-incubated for 5 min at RT with anti-FPR1, FPR2/ALX, or an Isotype control antibody, prior to loading on to a 96 well ChemoTx™ plate in the presence of 10 μM peptide Ac2-26. Chemotaxis was evaluated after a 90-min incubation at 37°C and 5% CO2. Results are ±SEM from three distinct cell preparations (*P < 0.05 vs. isotype control). (B) Ac2-26 (10 μM) was added to peripheral blood neutrophils in the presence of absence of PD98059 (ERK inhibitor; 10 μM) for 5 min prior to protein extraction and assessment of phospho- and total ERK isoforms, as well as beta-actin. Blots are representative of triplicate analyses conducted with two distinct neutrophil preparations.
Peptide Ac2-26 induced PMN migration relies primarily on the ERK signaling pathway. Neutrophils were pre-incubated for 15 min at 37°C with either (A) PD98059 (ERK inhibitor; 3–30 μM), (B) SB203580 (p38 inhibitor; 3–30 μM), or (C) SP600125 (JNK inhibitor; 3–30 μM) prior to loading on to a 96 well ChemoTx™ plate in the presence of PBS or peptide Ac2-26 (3–30 μM). Chemotaxis was evaluated after a 90-min incubation at 37°C and 5% CO2. Results are ±SEM from three distinct cell preparations [*P < 0.05, **P < 0.01 vs. stimulus alone (dose 0 group)].
Peptide Ac2-26 induced human monocytes chemokinesis. Monocytes were isolated from peripheral blood of healthy volunteers as indicated in the Section “Materials and Methods” and resuspended at 4 × 106 cells/ml. (A) Chemotaxis toward peptide Ac2-26 (1–30 μM) was evaluated after a 90-min incubation at 37°C and 5% CO2. FMLP (1 nM) was used as positive control. (B) Chemokinesis induced by peptide Ac2-26 was assessed by incubating the cells with the peptide prior to loading on to the top well of the ChemoTx™ plate, the number of migrated cells to the bottom chamber was evaluated after 90 min incubation at 37°C and 5% CO2. Results are ±SEM of three to four distinct monocyte preparations. (*P < 0.05, **P < 0.01 vs. PBS group).
It is postulated that peptides derived from the N-terminal region of Annexin A1, a glucocorticoid-regulated 37-kDa protein, could act as biomimetics of the parent protein. However, recent evidence, amongst which the ability to interact with distinct receptors other then that described for Annexin A1, suggest that these peptides might fulfill other functions at variance to those reported for the parent protein. Here we tested the ability of peptide Ac2-26 to induce chemotaxis of human neutrophils, showing that this peptide can elicit responses comparable to those produced by the canonical activator formyl-Met-Leu-Phe (or FMLP). However, whilst disruption of the chemical gradient abolished the FMLP response, addition of peptide Ac2-26 in the top well of the chemotaxis chamber did not affect (10 μM) or augmented (at 30 μM) the neutrophil locomotion to the bottom well, as elicited by 10 μM peptide Ac2-26. Intriguingly, the sole addition of peptide Ac2-26 in the top wells produced a marked migration of neutrophils. A similar behavior was observed when human primary monocytes were used. Thus, peptide Ac2-26 is a genuine chemokinetic agent toward human blood leukocytes. Neutralization strategies indicated that engagement of either the GPCR termed FPR1 or its cognate receptor FPR2/ALX was sufficient to sustain peptide Ac2-26 induced neutrophil migration. Similarly, application of pharmacological inhibitors showed that cell locomotion to peptide Ac2-26 was mediated primarily by the ERK, but not the JNK and p38 pathways. In conclusion, we report here novel in vitro properties for peptide Ac2-26, promoting neutrophil and monocyte chemokinesis; a process that may contribute to accelerate the resolution phase of inflammation. We postulate that the generation of Annexin A1 N-terminal peptides at the site of inflammation may expedite the egress of migrated leukocytes thus promoting the return to homeostasis.
 
5-Hydroxytryptamine 2A receptors (5-HT2A-Rs) are G-protein coupled receptors. In agreement with their location in the brain, they have been implicated not only in various central physiological functions including memory, sleep, nociception, eating and reward behaviors, but also in many neuropsychiatric disorders. Interestingly, a bidirectional link between depression and epilepsy is suspected since patients with depression and especially suicide attempters have an increased seizure risk, while a significant percentage of epileptic patients suffer from depression. Such epidemiological data led us to hypothesize that both pathologies may share common anatomical and neurobiological alteration of the 5-HT2A signaling. After a brief presentation of the pharmacological properties of the 5-HT2A-Rs, this review illustrates how these receptors may directly or indirectly control neuronal excitability in most networks involved in depression and epilepsy through interactions with the monoaminergic, GABAergic and glutamatergic neurotransmissions. It also synthetizes the preclinical and clinical evidence demonstrating the role of these receptors in antidepressant and antiepileptic responses.
 
Polymorphic expression of CYP2D6 contributes to the wide range of activity observed for this clinically important drug metabolizing enzyme. In this report we describe novel CYP2D7/2D6 hybrid genes encoding non-functional and functional CYP2D6 protein and a CYP2D7 variant that mimics a CYP2D7/2D6 hybrid gene. Five-kilobyte-long PCR products encompassing the novel genes were entirely sequenced. A quantitative assay probing in different gene regions was employed to determine CYP2D6 and 2D7 copy number variations and the relative position of the hybrid genes within the locus was assessed by long-range PCR. In addition to the previously known CYP2D6*13 and *66 hybrids, we describe three novel non-functional CYP2D7-2D6 hybrids with gene switching in exon 2 (CYP2D6*79), intron 2 (CYP2D6*80), and intron 5 (CYP2D6*67). A CYP2D7-specific T-ins in exon 1 causes a detrimental frame shift. One subject revealed a CYP2D7 conversion in the 5'-flanking region of a CYP2D6*35 allele, was otherwise unaffected (designated CYP2D6*35B). Finally, three DNAs revealed a CYP2D7 gene with a CYP2D6-like region downstream of exon 9 (designated CYP2D7[REP6]). Quantitative copy number determination, sequence analyses, and long-range PCR mapping were in agreement and excluded the presence of additional gene units. Undetected hybrid genes may cause over-estimation of CYP2D6 activity (CYP2D6*1/*1 vs *1/hybrid, etc), but may also cause results that may interfere with the genotype determination. Detection of hybrid events, "single" and tandem, will contribute to more accurate phenotype prediction from genotype data.
 
The orexin system regulates feeding, nutrient metabolism and energy homeostasis. Acute pharmacological blockade of orexin receptor 1 (OXR-1) in rodents induces satiety and reduces normal and palatable food intake. Genetic OXR-1 deletion in mice improves hyperglycemia under high-fat (HF) diet conditions. Here we investigated the effects of chronic treatment with the novel selective OXR-1 antagonist ACT-335827 in a rat model of diet-induced obesity (DIO) associated with metabolic syndrome (MetS). Rats were fed either standard chow (SC) or a cafeteria (CAF) diet comprised of intermittent human snacks and a constant free choice between a HF/sweet (HF/S) diet and SC for 13 weeks. Thereafter the SC group was treated with vehicle (for 4 weeks) and the CAF group was divided into a vehicle and an ACT-335827 treatment group. Energy and water intake, food preference, and indicators of MetS (abdominal obesity, glucose homeostasis, plasma lipids, and blood pressure) were monitored. Hippocampus-dependent memory, which can be impaired by DIO, was assessed. CAF diet fed rats treated with ACT-335827 consumed less of the HF/S diet and more of the SC, but did not change their snack or total kcal intake compared to vehicle-treated rats. ACT-335827 increased water intake and the high-density lipoprotein associated cholesterol proportion of total circulating cholesterol. ACT-335827 slightly increased body weight gain (4% vs. controls) and feed efficiency in the absence of hyperphagia. These effects were not associated with significant changes in the elevated fasting glucose and triglyceride (TG) plasma levels, glucose intolerance, elevated blood pressure, and adiposity due to CAF diet consumption. Neither CAF diet consumption alone nor ACT-335827 affected memory. In conclusion, the main metabolic characteristics associated with DIO and MetS in rats remained unaffected by chronic ACT-335827 treatment, suggesting that pharmacological OXR-1 blockade has minimal impact in this model.
 
Background: Cytochrome P450s (CYPs) are mono-oxygenases that metabolize endogenous compounds, such as fatty acids and lipid signaling molecules, and furthermore have a role in metabolism of xenobiotics. In order to investigate the role of CYP genes in fat metabolism at the molecular level, four Caenorhabditis elegans mutants lacking functional CYP-35A1, CYP-35A2, CYP-35A4, and CYP-35A5 were characterized. Relative amounts of fatty acids, as well as endocannabinoids, which regulate weight gain and accumulation of fats in mammals, were measured while fat contents in worms were visualized using Oil-Red-O staining. Results: The cyp-35A1 and cyp-35A5 mutants had a significantly lower intestinal fat content than wild-type animals, whereas cyp-35A2 and cyp-35A4 mutants appeared normal. The overall fatty acid compositions of CYP mutants did not alter dramatically, although modest but significant changes were observed. cyp-35A1 and cyp-35A5 mutants had significantly higher levels of C18:1n7 and lower C18:2n6c. All four mutants had higher relative amounts of C18:1n7 than the wild-type. In the cyp-35A5 mutant, the levels of the endocannabinoid anandamide were found to be 4.6-fold higher than in wild-type. Several fatty acid synthesis genes were over-expressed in cyp-35A1 including fat-2. Feeding oleic or elaidic triglycerides to wild-type animals demonstrated that cyp-35A1 transcriptional levels are insensitive to environmental exposure of these fats, while cyp-35A2, cyp-35A4, and cyp-35A5 were significantly down regulated. Conclusion: These results demonstrate a dynamic role for CYP-35A subfamily members in maintaining the diversity of fatty acid profiles in C. elegans, and more generally highlight the importance of CYPs in generating both structural and signaling fatty acid functions in other organisms.
 
P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB1 (P-gp) expression was evaluated by microarray for these cell lines. P-gp expression was determined by western blot and activity determined by rhodamine efflux assay. Knock down of P-gp and pharmacologic inhibition of P-gp to restore PF-309 activity was performed in vitro. PF-309 activity was evaluated in vivo in cell line xenograft models and in primary patient derived tumor xenografts (PDTX). Mice were treated with 25 mg/kg PF-309 orally, twice daily. On the last day of treatment, tumor and plasma were collected for PF-309 analysis. Here we show that ABCB1 gene expression correlates with resistance to PF-309 treatment in vitro and the expression and activity of P-gp was verified in a panel of resistant cells. Furthermore, inhibition of P-gp increased the sensitivity of resistant cells, resulting in a 4-100-fold decrease in the IC50s. Eleven cell line xenografts and 12 PDTX models were treated with PF-309. From the cell line xenografts, we found a significant correlation between ABCB1 gene expression profiles and tumor response. We evaluated tumor and plasma concentrations for eight tumor models (three cell line xenografts and five PDTX models) and a significant correlation was found between tumor concentration and response. Additionally, we show that tumor concentration is approximately fourfold lower in tumors that express P-gp, verified by western blot. Our in vitro and in vivo data strongly suggests that PF-309 efficacy is influenced by the expression of tumor P-gp.
 
The p21-activated kinase (PAK) family of serine/threonine kinases, which are overexpressed in several cancer types, are critical mediators of cell survival, motility, mitosis, transcription, and translation. In the study presented here, we utilized a panel of colorectal cancer (CRC) cell lines to identify potential biomarkers of sensitivity or resistance that may be used to individualize therapy to the PAK inhibitor PF-03758309. We observed a wide range of proliferative responses in the CRC cell lines exposed to PF-03758309, this response was recapitulated in other phenotypic assays such as anchorage-independent growth, three-dimensional (3D) tumor spheroid formation, and migration. Interestingly, we observed that cells most sensitive to PF-03758309 exhibited up-regulation of genes associated with a mesenchymal phenotype (CALD1, VIM, ZEB1) and cells more resistant had an up-regulation of genes associated with an epithelial phenotype (CLDN2, CDH1, CLDN3, CDH17) allowing us to derive an epithelial-to-mesenchymal transition (EMT) gene signature for this agent. We assessed the functional role of EMT-associated genes in mediating responsiveness to PF-3758309, by targeting known genes and transcriptional regulators of EMT. We observed that suppression of genes associated with the mesenchymal phenotype conferred resistance to PF-3758309, in vitro and in vivo. These results indicate that PAK inhibition is associated with a unique response phenotype in CRC and that further studies should be conducted to facilitate both patient selection and rational combination strategies with these agents.
 
Transient receptor potential canonical (TRPC) channels receptor-operated Ca2+ currents (ROCs). (A) Schematic representation of ROCs. Binding of an agonist to a Gq-protein-coupled receptor leads to phospholipase C (PLC) activation. The activated PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to produce of diacylglycerol (DAG) and IP3. DAG and the reduction of PI(4,5)P2 levels directly contribute to TRPC channels activation, while IP3 triggers Ca2+ release from intracellular stores. (B) ROCs through TRPC7 channels. TRPC7 currents were induced using carbachol, a muscarinic receptor agonist (gray line, 100 μM). Left and right panels respectively display currents observed with low and high levels of muscarinic receptor expression (data from Itsuki et al., 2014). The vertical and horizontal gray scale bars indicate 200 pA and 15 s, respectively.
The inhibition upon the voltage-sensing phosphatases (VSPs) activation. (A) TRPC6 currents induced by a DAG analog (OAG) are transiently inhibited by Dr-VSP activation (Imai et al., 2012). (B) Inhibitory effects of VSPs on TRPC6 and KCNQ2/3 channels. Data for TRPC6 channels are from Imai et al. (2012). Parenthesizes indicate values for KCNQ2/3 channels (Falkenburger et al., 2010). *Unpublished data from experiments in which equal amounts of plasmid harboring cDNA encoding Ci-, Dr-, or Gg-VSP, and TRPC6 were co-transfected into HEK293 cells. Currents were evoked using 50 μM OAG.
Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.
 
| Genetic ablation of Cx43 reduces mitochondrial potassium uptake. (A) Western blot analysis was performed for Cx43 and the mitochondrial marker protein MnSOD on proteins isolated from SSM of Cx43 fl/fl and Cx43 Cre-ER(T)fl + 4-OHT mice. (B) Bar graphs represent the mitochondrial Cx43 content normalized to MnSOD in
Mitochondrial potassium uptake is decreased by Gap19. (A) Original traces showing PBFI fluorescence in arbitrary units (a.u.) before and after addition of 140 mM KCl of control- or Gap19-treated SSM at 340 and 380 nm excitation and 500 nm emission, respectively. (B) Bar graphs represent the maximal slope of the PBFI fluorescence at 340 nm excitation and 500 nm emission of control and Gap19-treated mitochondria. *p < 0.05.
Genetic ablation of Cx43 reduces mitochondrial potassium uptake. (A) Western blot analysis was performed for Cx43 and the mitochondrial marker protein MnSOD on proteins isolated from SSM of Cx43fl/fl and Cx43Cre-ER(T)fl + 4-OHT mice. (B) Bar graphs represent the mitochondrial Cx43 content normalized to MnSOD in Cx43fl/fl control mice, which were set to 100%, and 4-OHT-treated Cx43Cre-ER(T)/fl mice. *p < 0.05. (C) Bar graphs represent the maximal slope of the PBFI fluorescence at 340 nm excitation and 500 nm emission of 4-OHT-treated Cx43fl/fl and Cx43Cre-ER(T)/fl mitochondria. *p < 0.05.
In cardiomyocytes, connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43(Cre-ER(T)/fl) + 4-OHT). The mitochondria of the Cx43(Cre-ER(T)/fl) + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.
 
The membrane and channel protein connexin-43 (Cx43), as well as the cytokine transforming growth factor (TGF) β, suppress proliferative growth in cardiomyocytes and other cell types. Previously we showed that the inhibitory effect of Cx43 is canceled when Cx43 becomes phosphorylated at serine (S) 262 in response to mitogen stimulation. We have now asked if the TGFβ-triggered inhibition of DNA synthesis is associated with changes in Cx43 phosphorylation at S262. Conversely, we investigated if inhibition of DNA synthesis by overexpressed Cx43 is dependent on engaging TGFβ signal transduction. We report that TGFβ acutely prevented mitogen-induced Cx43 phosphorylation at S262, while chronic inhibition of TGFβ signal transduction raised baseline levels of endogenous phospho-S262-Cx43 without affecting total Cx43. Inhibition of baseline TGFβ signal transduction through (a) inhibiting TGFβ receptor I (TGFβRI) with SB431542, (b) inhibiting TGFβ receptor II (TGFβRII) by overexpressing dominant-negative (DN) TGFβRII, (c) inhibiting the downstream signaling mediator Smad2 by overexpressing DN Smad2, each separately increased baseline cardiomyocyte DNA synthesis, but could not reverse DNA synthesis inhibition by overexpressed Cx43. It is suggested that inhibition of cardiomyocyte DNA synthesis by TGFβ/TGFβRI/II/phospho-Smad2 signaling is mediated, at least in part, by reducing endogenous phospho-S262-Cx43 levels.
 
| Ro15-4513 antagonism of ethanol-induced motor ataxia. (A) Performance of WT and GABA A-Rα4 subunit KO mice on fixed speed (8 rpm) rotarod following pretreatment with 2 g/kg ethanol 5 min prior to treatment with 10 mg/kg Ro15-4513 or vehicle.*p ≤ 0.01-WT ethanol/vehicle vs WT ethanol/Ro15-4513. (B) AUCs of response curves in (A). Effect of genotype, treatment, and the interaction were not significant. (C) Performance on fixed
Ro15-4513 antagonism of ethanol-induced motor ataxia. (A) Performance of WT and GABAA-Rα4 subunit KO mice on fixed speed (8 rpm) rotarod following pretreatment with 2 g/kg ethanol 5 min prior to treatment with 10 mg/kg Ro15-4513 or vehicle.*p ≤ 0.01 – WT ethanol/vehicle vs WT ethanol/Ro15-4513. (B) AUCs of response curves in (A). Effect of genotype, treatment, and the interaction were not significant. (C) Performance on fixed speed (12 rpm) rotarod following treatment with 1.5 g/kg ethanol ±3 mg/kg Ro15-4513. *p < 0.005, **p < 0.0001 – WT ethanol vs WT ethanol + Ro15-4513. (D) AUCs of response curves in (C). WT and KO mice did not differ following injection of ethanol alone. Ro15-4513 antagonized ethanol-induced motor ataxia in WT, but not in KO mice. All values are expressed as mean ± SEM.
Antagonism of ethanol (3. 5 g/kg)-induced LORR by Ro15-4513 (20 mg/kg). (A) In male mice, Ro15-4513 antagonized the duration of ethanol-induced LORR in WT, but not in KO. (B) In female mice, Ro15-4513 antagonized ethanol-induced LORR (p < 0.0001) irrespective of genotype. All values are expressed as mean ± SEM.
Alcohol (ethanol) is widely consumed for its desirable effects but unfortunately has strong addiction potential. Some imidazobenzodiazepines such as Ro15-4513 are able to antagonize many ethanol-induced behaviors. Controversial biochemical and pharmacological evidence suggest that the effects of these ethanol antagonists and ethanol are mediated specifically via overlapping binding sites on α4/δ-containing GABA(A)-Rs. To investigate the requirement of α4-containing GABA(A)-Rs in the mechanism of action of Ro15-4513 on behavior, wildtype (WT) and α4 knockout (KO) mice were compared for antagonism of ethanol-induced motor incoordination and hypnosis. Motor effects of ethanol were tested in two different fixed speed rotarod assays. In the first experiment, mice were injected with 2.0 g/kg ethanol followed 5 min later by 10 mg/kg Ro15-4513 (or vehicle) and tested on a rotarod at 8 rpm. In the second experiment, mice received a single injection of 1.5 g/kg ethanol ± 3 mg/kg Ro15-4513 and were tested on a rotarod at 12 rpm. In both experiments, the robust Ro15-4513 antagonism of ethanol-induced motor ataxia that was observed in WT mice was absent in KO mice. A loss of righting reflex (LORR) assay was used to test Ro15-4513 (20 mg/kg) antagonism of ethanol (3.5 g/kg)-induced hypnosis. An effect of sex was observed on the LORR assay, so males and females were analyzed separately. In male mice, Ro15-4513 markedly reduced ethanol-induced LORR in WT controls, but α4 KO mice were insensitive to this effect of Ro15-4513. In contrast, female KO mice did not differ from WT controls in the antagonistic effects of Ro15-4513 on ethanol-induced LORR. We conclude that Ro15-4513 requires α4-containing receptors for antagonism of ethanol-induced LORR (in males) and motor ataxia.
 
Schematic of CK2-mediated signaling pathways inhibited by CX-4945.
Anti-cancer drugs for potential combination therapy with CX-4945 in treatment of human hematological cancers.
The casein kinase 2 (CK2) protein kinase is a pro-survival kinase and therapeutic target in treatment of various human cancers. CK2 overexpression has been demonstrated in hematological malignancies, including chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, and multiple myeloma. CX-4945, also known as Silmitasertib, is an orally administered, highly specific, ATP-competitive inhibitor of CK2. CX-4945 induces cytotoxicity and apoptosis and is currently being evaluated in clinical trials for treatment of many cancer types. In the past 2 years, the focus on the therapeutic potential of CX-4945 has shifted from solid tumors to hematological malignancies. CX-4945 exerts anti-proliferative effects in hematological tumors by downregulating CK2 expression and suppressing activation of CK2-mediated PI3K/Akt/mTOR signaling pathways. Furthermore, combination of CX-4945 with other inhibitors yielded synergistic effects in cell death induction. These new findings demonstrate that CK2 overexpression contributes to blood cancer cell survival and resistance to chemotherapy. Combinatorial use of CX-4945 is a promising therapeutic tool for treatment of hematological malignancies.
 
A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.
 
Liquid chromatography-mass spectrometry (LC-MS) with generic gradient elution for a large number of chemically different compounds is a common approach in drug development, used to acquire a large amount of data in a short time frame for drug candidates. The analysis with non-optimized parameters however may lead to a poor method performance for many compounds, and contains a risk of losing important information. Here, generic electrospray time of flight (ESI-TOF) MS methods in various pH conditions were tested for 55 chemically diverse compounds (10 acids, 25 bases, 17 neutrals, and 3 amphoterics), aiming to find best analytical conditions for each compound, for studies of in vitro metabolic properties in liver preparations. The effect of eluent pH and elution gradient strength on chromatographic performance and electrospray MS ionization efficiency were examined for each compound. The data are evaluated how well the best generic approach could cover the analysis of test compounds and how many compounds would still need completely different analytical conditions after that. Aqueous mobile phase consisting of 0.05% acetic acid and 5 mM ammonium acetate (pH 4.4) showed the best general suitability for the analyses, showing adequate performance for metabolite profiling for 41 out of 55 compounds either in positive or negative ion mode. In positive ion mode, the main limitation of performance in various pH conditions was generally not the lack of ionization, but rather the poor chromatographic performance (inadequate retention or poor peak shape), suggesting that more emphasis should be put in finding conditions providing best chromatographic performance, rather than highest ionization properties. However, a single generic approach for a large number of different compounds is not likely to produce good results for all compounds. Preferably, at least two or three different conditions are needed for the coverage of a larger number of structurally diverse compounds.
 
Depression is a commonly reported co-morbidity during rehabilitation from alcohol use disorders and its presence is associated with an increased likelihood of relapse. Interventions which impede the development of depression could be of potential benefit if incorporated into treatment programs. We previously demonstrated an ameliorative effect of physical exercise on depressive behaviors in a mouse model of alcohol abstinence. Here, we show that environmental enrichment (cognitive and social stimulation) has a similar beneficial effect. The hypothalamic-pituitary-adrenal (HPA) axis is a key physiological system regulating stress responses and its dysregulation has been separably implicated in the pathophysiology of depression and addiction disorders. We performed a series of dexamethasone challenges and found that mice undergoing 2 weeks of alcohol abstinence had significantly greater corticosterone and ACTH levels following a DEX-CRH challenge compared to water controls. Environmental enrichment during alcohol abstinence corrected the abnormal DEX-CRH corticosterone response despite a further elevation of ACTH levels. Examination of gene expression revealed abstinence-associated alterations in glucocorticoid receptor (Gr), corticotrophin releasing hormone (Crh) and pro-opiomelanocortin (Pomc1) mRNA levels which were differentially modulated by environmental enrichment. Overall, our study demonstrates a benefit of environmental enrichment on alcohol abstinence-associated depressive behaviors and HPA axis dysregulation.
 
Structures of selected E1312 agonists and antagonists, and their activities. Key compound activities and associated publications were listed.
EBI2, aka GPR183, is a G-couple receptor originally identified in 1993 as one of main genes induced in Burkitt's lymphoma cell line BL41 by Epstein-Barr virus (EBV) infection. After it was reported in 2009 that the receptor played a key role in regulating B cell migration and responses, we initiated an effort in looking for its endogenous ligand. In 2011 we and another group reported the identification of 7α, 25-dihydroxyxcholesterol (7α, 25-OHC), an oxysterol, as the likely physiological ligand of EBI2. A few subsequently published studies further elucidated how 7α, 25-OHC bound to EBI2, and how a gradient of 7α, 25-OHC could be generated in vivo and regulated migration, activation, and functions of B cells, T cells, dendritic cells (DCs), monocytes/macrophages, and astrocytes. The identification of 7α, 25-OHC as a G protein-coupled receptor ligand revealed a previously unknown signaling system of oxysterols, a class of molecules which exert profound biological functions. Dysregulation of the synthesis or functions of these molecules is believed to contribute to inflammation and autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, cancer as well as metabolic diseases such as diabetes, obesity, and dyslipidemia. Therefore EBI2 may represent a promising target for therapeutic interventions for human diseases.
 
| Spatial patterns of locomotor hyperactivity shown by representative, individual sham-operated (top) and DHI (bottom) animals. Plots show activity traces in the 60-90 min time block following
Effect of 5,7-DHT-lesions targeting the dorsal or ventral hippocampus on locomotor hyperactivity induced by 0.5 and 2.5 mg/kg PCP and 0.1 mg/kg MK-801. Panels show mean distance moved (cm) in 5 min intervals ± SEM for DHI (n = 16), VHI (n = 8) and sham-operated rats (n = 17) treated with (A) saline (B) 0.5 and (C) 2.5 mg/kg PCP and (D) 0.1 mg/kg MK-801. Total distance moved following 0.5 and 2.5 mg/kg PCP injection was significantly higher in DHI rats compared to controls (see text for details).
Effect of 5,7-DHT-lesions targeting the dorsal hippocampus on qualitative aspects of locomotor hyperactivity induced by 2.5 mg/kg PCP and 0.1 mg/kg MK-801. Panels show mean (A) distance moved (cm), (B) spatial d, and (C) entropy in 30 min intervals ± SEM for DHI (n = 12) and sham-operated rats (n = 11) treated with saline (top), 2.5 mg/kg PCP (middle), and 0.1 mg/kg MK-801(bottom). Total distance moved following 2.5 mg/kg PCP injection was, again, significantly higher in DHI rats compared to controls (see text for details). ##p < 0.01, ###p < 0.001 for comparison with baseline. **p < 0.01, ***p < 0.001 for comparison with saline in respective time block.
Spatial patterns of locomotor hyperactivity shown by representative, individual sham-operated (top) and DHI (bottom) animals. Plots show activity traces in the 60–90 min time block following treatment with (A) saline (B) 2.5 mg/kg PCP and (C) 0.1 mg MK-801. Enhanced PCP-induced locomotor hyperactivity in DHI rats is clearly depicted by the increased density of tracings in the chamber (panel B, bottom).
Antagonism of N-methyl-D-aspartate (NMDA) receptors by phencyclidine (PCP) is thought to underlie its ability to induce a schizophrenia-like syndrome in humans, yet evidence indicates it has a broader pharmacological profile. Our previous lesion studies highlighted a role for serotonergic projections from the median, but not dorsal, raphe nucleus in mediating the hyperlocomotor effects of PCP, without changing the action of the more selective NMDA receptor antagonist, MK-801. Here we compared locomotor responses to PCP and MK-801 in rats that were administered 5,7-dihydroxytryptamine (5,7-DHT) into either the dorsal or ventral hippocampus, which are preferentially innervated by median and dorsal raphe, respectively. Dorsal hippocampus lesions potentiated PCP-induced hyperlocomotion (0.5, 2.5 mg/kg), but not the effect of MK-801 (0.1 mg/kg). Ventral hippocampus lesions did not alter the hyperlocomotion elicited by either compound. Given that PCP and MK-801 may induce different spatiotemporal patterns of locomotor behavior, together with the known role of the dorsal hippocampus in spatial processing, we also assessed whether the 5,7-DHT-lesions caused any qualitative differences in locomotor responses. Treatment with PCP or MK-801 increased the smoothness of the path traveled (reduced spatial d) and decreased the predictability of locomotor patterns within the chambers (increased entropy). 5,7-DHT-lesions of the dorsal hippocampus did not alter the effects of PCP on spatial d or entropy - despite potentiating total distance moved - but caused a slight reduction in levels of MK-801-induced entropy. Taken together, serotonergic lesions targeting the dorsal hippocampus unmask a functional differentiation of the hyperlocomotor effects of PCP and MK-801. These findings have implications for studies utilizing NMDA receptor antagonists in modeling glutamatergic dysfunction in schizophrenia.
 
This study evaluated the in vitro and in vivo pharmacological properties of TD-8954, a potent and selective 5-HT(4) receptor agonist. TD-8954 had high affinity (pK(i) = 9.4) for human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptors, and selectivity (>2,000-fold) over all other 5-hydroxytryptamine (5-HT) receptors and non-5-HT receptors, ion channels, enzymes and transporters tested (n = 78). TD-8954 produced an elevation of cAMP in HEK-293 cells expressing the h5-HT(4(c)) receptor (pEC(50) = 9.3), and contracted the guinea pig colonic longitudinal muscle/myenteric plexus preparation (pEC(50) = 8.6). TD-8954 had moderate intrinsic activity in the in vitro assays. In conscious guinea pigs, subcutaneous administration of TD-8954 (0.03-3 mg/kg) increased the colonic transit of carmine red dye, reducing the time taken for its excretion. Following intraduodenal dosing to anesthetized rats, TD-8954 (0.03-10 mg/kg) evoked a dose-dependent relaxation of the esophagus. Following oral administration to conscious dogs, TD-8954 (10 and 30 μg/kg) produced an increase in contractility of the antrum, duodenum, and jejunum. In a single ascending oral dose study in healthy human subjects, TD-8954 (0.1-20 mg) increased bowel movement frequency and reduced the time to first stool. It is concluded that TD-8954 is a potent and selective 5-HT(4) receptor agonist in vitro, with robust in vivo stimulatory activity in the gastrointestinal (GI) tract of guinea pigs, rats, dogs, and humans. TD-8954 may have clinical utility in patients with disorders of reduced GI motility.
 
Pan-adenosine receptor agonist (NECA) down regulates p53 and Rb level in HSC through adenosine receptor A2A signaling. Morphological characteristics of primary rat HSC were recorded for 2–21 days in the culture and shown (A) at day-2 in quiescent stage, (B) at day-10 in activated stage, or (C) at day-21 in highly activated stage. (D) Western blot shows expression of α-smooth muscle actin (α-SMA) at day-2 and at day-21 as a marker of activated primary HSC. Human HSC line LX-2 cells were treated for 2 days with or without 10 μM of NECA in presence or absence of either an A2A receptor antagonist ZM241385 (10 μM) or agonist CGS21680 (10 μM). The control cells (CTL) were treated with DMSO. Primary rat HSC were similarly treated with NECA, ZM241385 and, or CGS 21680 for 5 days after 10 days normal culture (at the activated stage). (E–G) Western blots of p53 and Rb are shown for LX-2 (F–H) and primary rat HSC.
NECA enhances BrdU incorporation and decreases H2O2-induced cell death in HSC through activation of adenosine receptor A2A. LX-2 cells or 10 days in-vitro cultured primary rat HSC were treated with pan adenosine receptor agonist NECA (10 μM), an A2A receptor antagonist (ZM241385 10 μM), or an A2A receptor agonist (CGS21680 10 μM) for 24 h and measured BrdU incorporation. The level of BrdU incorporation was shown in (A) LX-2 cells (* = 0.0126, § = 0.00099, ¶ = 0.00428) and in (B) primary rat HSC (* = <0.001, § = <0.001, ¶= <0.001). LX-2 cells were treated (C) with vehicles DMSO, (D) NECA (10 μM), (E) an A2A receptor antagonist (ZM241385 10 μM), or, (F) ZM241385 (10 μM) plus NECA (10 μM), or, (G) with an A2A receptor agonist CGS21680 (10 μM) for 24 h followed by 100 μM of H2O2 for 5 h. (H) Viable and dead cells were quantified by both Annexin V and PI staining flow-cytometry (paired symbols mark comparisons with significance greater than P of 0.05). (* = 0.0084, § = 0.0017, ¶ = 0.0017, # = 0.0084).
NECA suppresses senescence by down regulating p53 and Rb in the primary rat HSC. Cells were cultured for 2–21 days and senescence detected using SA-β-Gal staining (A) at day-2 or (B) at day-21, and (C) the number of senescent cells quantified (* = significant but p value is not applicable because first column is totally zero). (D) Western blot analysis of p53 and Rb level in the normal culture at the day-2 or, day-21. Similarly senescence was detected in the HSC treated (E) with vehicle DMSO, or (F) with NECA for 21 days. (G) The number of senescence cells was quantified in the cells treated with DMSO (CTL) or, with NECA for 21 days using Image-J cell count software (* = <0.001). (H) Western blot analysis of p53 and Rb level in the cells treated with DMSO (CTL) or, with NECA for 21 days.
A cAMP analog, or an activator of adenylyl cyclase Forskolin, or NECA down regulates p53 and Rb through down regulation of Rac1 and decreased activation of p38 MAPK in the HSC. LX-2 cells were treated for 2 days, and 10 days cultured activated primary rat HSC were treated for 5 days with 8-Bromo-cAMP (1mM) or, Forskolin (50 μM) or NECA (10 μM) in presence or absence of PKA inhibitor 12–14 amide (myristoylated; 10 μM) or H-89 dihydrochloride hydrate (25 μM). (A) Western blot analysis of p53, Rb, Rac1, and p44/42 MAPK levels in the 2 days treated LX-2 cells. (B) Western blot analysis of p53, Rb, Rac1, and p44/42 MAPK levels in the 5 days treated activated rat primary HSC. (C) Summary of adenosine receptor A2A mediated signaling in the process of senescence in HSC.
Background and Aims: During fibrosis hepatic stellate cells (HSC) undergo activation, proliferation, and senescence but the regulation of these important processes is poorly understood. The adenosine A2A receptor (A2A) is known to be present on HSC, and its activation results in liver fibrosis. In this study, we tested if A2A has a role in the regulation of HSC proliferation, apoptosis, senescence, and the relevant molecular mechanism. Methods: The ability of adenosine to regulate p53 and Rb protein levels, proliferation, apoptosis and senescence was tested in the human HSC cell line LX-2 and rat primary HSC. Results: Adenosine receptor activation down-regulates p53 and Rb protein levels, increases BrdU incorporation and increases cell survival in LX-2 cells and in primary rat HSC. These effects of NECA were reproduced by an adenosine A2A receptor specific agonist (CGS21680) and blocked by a specific antagonist (ZM241385). By day twenty-one of culture primary rat HSC entered senescence and expressed β-gal which was significantly inhibited by NECA. Furthermore, NECA induced down regulation of p53 and Rb and Rac1, and decreased phosphorylation of p44-42 MAP Kinase in LX-2 cells and primary rat HSC. These effects were reproduced by the cAMP analog 8-Bromo-cAMP, and the adenylyl cyclase activator forskolin, and were blocked by PKA inhibitors. Conclusions: These results demonstrate that A2A receptor regulates a number of HSC fate decisions and induces greater HSC proliferation, reduces apoptosis and senescence by decreasing p53 and Rb through cAMP-PKA/Rac1/p38 MAPK pathway. This provides a mechanism for adenosine induced HSC regulation and liver fibrosis.
 
Possible relationships in diabetes, blood brain barrier (BBB) function, β-amyloid and Alzheimer's disease (AD).
Diabetes is a systematic metabolic disease, which often develops a number of well-recognized vascular complications including brain complications which may partly result from the dysfunction of blood-brain barrier (BBB). BBB is generally considered as a mechanism for protecting the brain from unwanted actions resulting from substances in the blood and maintaining brain homeostasis via monitoring the entry or efflux of compounds. ATP-binding cassette (ABC) family of transporters including P-glycoprotein (P-GP) and breast cancer-related protein (BCRP), widely expressed in the luminal membrane of the microvessel endothelium and in the apical membrane of the choroids plexus epithelium, play important roles in the function of BBB. However, these transporters are easily altered by some diseases. The present article was focused on the alteration in expression and function of both P-GP and BCRP at BBB by diabetes and the clinical significances.
 
ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce "collateral sensitivity" leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and (3)H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded.
 
Chemical structure of compounds investigated in this study.
Interactions of EGFRIs with BCRP ATPase. (A) Activation of BCRP-ATPase in Sf9 membrane preparations containing BCRP. Membranes were incubated with ATP and the tested compounds at 400 nM in the presence and absence of sodium orthovanadate. (B) Inhibition of sulfasalazine-stimulated BCRP-ATPase. The effect of EGFRIs was evaluated in sulfasalazine (10 μM)-stimulated membranes. Data are presented as means ± SD of the vanadate-sensitive ATPase activity from two experiments in duplicates.
Effects of BCRP overexpression on the cytotoxicity of EGFRIs by the MTT assay. Stable MDCKII transfectants of vector alone or vectors expressing wild-type BCRP were incubated for 72 h with various concentrations of the indicated compounds. Results are shown as experimental findings (circles) and predicted model (line) in MDCK-CT cells (red), MDCK-BCRP cells (black), and MDCK-BCRP cells treated with 20 μM verapamil, to block endogenous P-gp activity (green). Data represent the means ± SD of six replicates.
Effect of EGFRIs on BODIPY-prazosin accumulation. Cell protein concentration-normalized BODIPY prazosin fluorescence in MDCK-BCRP cells were evaluated in the presence and the absence of 400 nM of the tested compounds following 1 h incubation. Results are presented as means ± SD. *Significantly different from vehicle-treated cells, P < 0.01; **Significantly different from FTC-treated cells, P < 0.01.
BCRP protein expression in HCC827 and A549 cells. (A) Representative image demonstrating the intensity of BCRP expression. (B) Relative β-actin-normalized BCRP expression. MDCK-BCRP and MDCK-CT cells were used as positive and negative controls, respectively. n = 4 for each cell type.
The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal.
 
An abdominal aortic aneurysm (AAA) is an enlargement of the greatest artery in the body defined as an increase in diameter of 1.5-fold. AAAs are common in the elderly population and thousands die each year from their complications. The most commonly used mouse model to study the pathogenesis of AAA is the angiotensin II (Ang II) infusion method delivered via osmotic mini-pump for 28 days. Here, we studied the site-specificity and onset of aortic rupture, characterized three-dimensional (3D) images and flow patterns in developing AAAs by ultrasound imaging, and examined macrophage infiltration in the Ang II model using 65 apolipoprotein E-deficient mice. Aortic rupture occurred in 16 mice (25%) and was nearly as prevalent at the aortic arch (44%) as it was in the suprarenal region (56%) and was most common within the first 7 days after Ang II infusion (12 of 16; 75%). Longitudinal ultrasound screening was found to correlate nicely with histological analysis and AAA volume renderings showed a significant relationship with AAA severity index. Aortic dissection preceded altered flow patterns and macrophage infiltration was a prominent characteristic of developing AAAs. Targeting the inflammatory component of AAA disease with novel therapeutics will hopefully lead to new strategies to attenuate aneurysm growth and aortic rupture.
 
| Participant demographics and clinical measures. 
for additional demographics and clinical measures. 
| Genotype frequency for BDNF Val66Met, COMT Val158Met. 
| Associations between neurocognitive domains 
Chronic cigarette smoking and polymorphisms in brain-derived neurotrophic factor (BDNF) and catechol-o-methyltransferase (COMT) are associated with neurocognition in normal controls and those with various neuropsychiatric conditions. The influence of these polymorphisms on neurocognition in alcohol dependence is unclear. The goal of this report was to investigate the associations of single nucleotide polymorphisms (SNP) in BDNF Val66Met and COMT Val158Met with neurocognition in a treatment-seeking alcohol dependent cohort and determine if neurocognitive differences between non-smokers and smokers previously observed in this cohort persist when controlled for these functional SNPs. Genotyping was conducted on 70 primarily male treatment-seeking alcohol dependent participants (ALC) who completed a comprehensive neuropsychological battery after 33 ± 9 days of monitored abstinence. Smoking ALC performed significantly worse than non-smoking ALC on the domains of auditory-verbal and visuospatial learning and memory, cognitive efficiency, general intelligence, processing speed and global neurocognition. In smoking ALC, greater number of years of smoking over lifetime was related to poorer performance on multiple domains. COMT Met homozygotes were superior to Val homozygotes on measures of executive skills and showed trends for higher general intelligence and visuospatial skills, while COMT Val/Met heterozygotes showed significantly better general intelligence than Val homozygotes. COMT Val homozygotes performed better than heterozygotes on auditory-verbal memory. BDNF genotype was not related to any neurocognitive domain. The findings are consistent with studies in normal controls and neuropsychiatric cohorts that observed COMT Met carriers showed better performance on measures of executive skills and general intelligence. Overall, the findings support to the expanding clinical movement to make smoking cessation programs available at the inception of treatment.
 
Cocaine, similarly to most drugs of abuse, produces long lasting brain changes, that relie on gene expression and new protein synthesis. These long-term neuronal adaptations take place in majority in DA-D1R expressing MSNs that belong to the direct pathway. The ERK pathway is central in this striatal plasticity, and detects a combination of glutamate and DA signals that are essential for long lasting modifications. Cocaine-induced ERK activation depends on complex cascade of phosphorylation events downstream D1-R. Importantly, the triggering event in ERK activation depends on a non-canonical signaling pathway that associates Fyn-induced phosphorylation of GluN2B and increases in calcium influx into D1-R MSNs. The duration and maintenance of ERK activation occurs via cAMP/PKA pathway. The cAMP/PKA pathway, downstream D1 receptors (left panel) triggers deactivation of phosphatases, PP1 on one hand via DARPP-32, and STEP on the other hand (Valjent et al., 2005). By controling the activity of the tyrosine kinase Fyn and the dual specificity protein kinase MEK, this signaling cascade intervenes in the state of phosphorylation of ERK downstream DA-D1R stimulation. However, we propose that triggering ERK activation depends on a “non-canonical signaling” pathway downstream D1-R (right panel). A cAMP-independent activation of Fyn produces tyrosine phosphorylation of GluB2B subunit of NMDA-Rs that in turn facilitates calcium influx (Pascoli et al., 2011a), and activation of the calcium-sensitive Ras-guanine releasing factor (Ras-GRF1) that activates the MEK/ERK pathway (Fasano and Brambilla, 2011). Upon cocaine stimulation ERK translocates to the nucleus where it controls epigenetic and genetic programs. Among the latter, the transcription factor Elk-1 is a component of the ternary complex factor, which binds to SRE (Besnard et al., 2011a). ERK1/2 also phosphorylates the nuclear kinase MSK1which appears to play a prominent role in phosphorylation of histone H3 and cAMP-response element-binding (CREB) protein (Brami-Cherrier et al., 2005). This leads to the expression of immediate-early genes, which are particularly sensitive to CREB (e.g., cFos) or ternary complex factor (e.g., Zif268 a.k.a. Egr-1). These two nuclear pathways downstream from ERK have distinct roles in long-term behavioral adaptations (see text). DA-GPCR and NMDAR, in particular D1R and GluN1, oligomerization has been documented in heterologous systems, hippocampal, and striatal tissues after cocaine exposure. The role of D1R-GluN1 interaction in ERK activation and downstream molecular events remains to be established.
Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse.
 
The overview of our integrated DA domain-specific cloud computing and sourcing platform (CloudDA) with illustration of a friendly user-interfaced query (A) and output (B) and server backend (C) of the constructed computational chemogenomics database as well as the implemented computing tools/programs (D) for DA research. CloudDA provides the DA chemogenomics knowledgebase, polydrug addiction/polypharmacology prediction tools, and additional online services.
| List of drug-abuse related GPCRs in the DA-KB database.
Tissue distribution of 85 drug-abuse related GPCRs. Red lines indicate that these tissues are located in the central nervous system (CNS). For example, 84 of these 85 GPCRs are expressed in the nucleus accumbens, where most drugs of abuse act.
Illustration of our cloud-based HTDocking server to predict potential targets (pink nodes) and cross-targets (yellow, green and blue nodes) of compounds and to explore possible mechanisms. (A) The predicted targets of six known abused/approved drugs (opioids: codeine and DB01532; benzodiazepines: bromazepam and alprazolam; barbiturates: secobarbital and pentobarbital). (B) The predicted targets of 3 approved drugs for DA treatments (methadone, naltrexone, and buprenorphine). MAPK10, Mitogen-Activated Protein Kinase 10; CYP2A6, cytochrome P450, family 2, subfamily A, polypeptide 6; RAB6A, RAB6A, member RAS oncogene family; PBRM1, polybromo 1; PDE5A, phosphodiesterase 5A, cGMP-specific; A4, amyloid beta protein; RB1, retinoblastoma 1; MAPK14, mitogen-activated protein kinase 14; ALDR, Aldose reductase; PPARg, Peroxisome proliferator-activated receptor gamma; GABRA(1-6):Gamma-aminobutyric acid receptor subunit alpha-(1-6); MAOB, Monoamine Oxidase B; MAP2K1(5), Mitogen-Activated Protein Kinase 1(5); LCK, Tyrosine-protein kinase Lck; ESR1, Estrogen receptor; CTDS2, Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 2; GRB14, Growth factor receptor-bound protein 14; GLR2, AMPA-selective glutamate receptor 2; ALDOA, Fructose-bisphosphate aldolase A; CA2, Carbonic anhydrase II; OPRD (OPRM,OPRK), Delta (Mu, Kappa)-type opioid receptor; AA2AR, Adenosine receptor A2a; MDR1, Multidrug resistance protein 1; GARS, Glycine-tRNA ligase.
Illustration of TargetHunter webserver (www.cbligand.org/TargetHunter) for polypharmacology DA research. (A) TargetHunter mapping literature reported (abused) drugs-targets interactions. The large (yellow, green, and red) nodes represent targets. The small nodes (pink and blue) represent drugs, among which, seven, designated by medicine bottles (blue nodes), are approved/clinical trial medicine for DA treatments: (i) methadone, naltrexone, oxycodone, and buprenorphine targeting mu-opiate receptor (OPRM, large yellow nodes); (ii) Nabilone and Cannabidiol targeting cannabinoid receptor 1(CNR1 or CB1, large green nodes); and (iii) Adrogolide targeting dopamine receptor 1(DRD1, red). (B) TargetHunter prediction for possible cross-talk interactions of cannabinoid (CB) receptors (CNR1 and CNR2; green nodes), opiate receptors (OPRK, OPRD and OPRM; yellow) and dopamine receptors (DRD1, DRD2, DRD3, DRD4, and DRD5; red). Small blue and pink nodes in the center suggest potential cross-talk. (C,D) Pharmacophore models showing the shared common pharmacophoric features of the ligands of cannabinoid receptor 1 (CNR1), dopamine receptor D1 (DRD1) and mu-opioid receptor (OPRM).
Drug abuse (DA) and addiction is a complex illness, broadly viewed as a neurobiological impairment with genetic and environmental factors that influence its development and manifestation. Abused substances can disrupt the activity of neurons by interacting with many proteins, particularly G-protein coupled receptors (GPCRs). A few medicines that target the central nervous system (CNS) can also modulate DA related proteins, such as GPCRs, which can act in conjunction with the controlled psychoactive substance(s) and increase side effects. To fully explore the molecular interaction networks that underlie DA and to effectively modulate the GPCRs in these networks with small molecules for DA treatment, we built a drug-abuse domain specific chemogenomics knowledgebase (DA-KB) to centralize the reported chemogenomics research information related to DA and CNS disorders in an effort to benefit researchers across a broad range of disciplines. We then focus on the analysis of GPCRs as many of them are closely related with DA. Their distribution in human tissues was also analyzed for the study of side effects caused by abused drugs. We further implement our computational algorithms/tools to explore DA targets, DA mechanisms and pathways involved in polydrug addiction and to explore polypharmacological effects of the GPCR ligands. Finally, the polypharmacology effects of GPCRs-targeted medicines for DA treatment were investigated and such effects can be exploited for the development of drugs with polypharmacophore for DA intervention. The chemogenomics database and the analysis tools will help us better understand the mechanism of drugs abuse and facilitate to design new medications for system pharmacotherapy of DA.
 
| Schematic representation of relevant intracellular pathways of BDNF and dopamine dependent structural plasticity in dopaminergic neurons. TrkB, tropomyosin-related kinase B; Src, proto-oncogene tyrosine protein kinase; MEK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase; D3R, dopamine D3  
Mesencephalic dopaminergic neurons were suggested to be a critical physiopathology substrate for addiction disorders. Among neuroadaptive processes to addictive drugs, structural plasticity has attracted attention. While structural plasticity occurs at both pre- and post-synaptic levels in the mesolimbic dopaminergic system, the present review focuses only on dopaminergic neurons. Exposures to addictive drugs determine two opposite structural responses, hypothrophic plasticity produced by opioids and cannabinoids (in particular during the early withdrawal phase) and hypertrophic plasticity, mostly driven by psychostimulants and nicotine. In vitro and in vivo studies identified BDNF and extracellular dopamine as two critical factors in determining structural plasticity, the two molecules sharing similar intracellular pathways involved in cell soma and dendrite growth, the MEK-ERK1/2 and the PI3K-Akt-mTOR, via preferential activation of TrkB and dopamine D3 receptors, respectively. At present information regarding specific structural changes associated to the various stages of the addiction cycle is incomplete. Encouraging neuroimaging data in humans indirectly support the preclinical evidence of hypotrophic and hypertrophic effects, suggesting a possible differential engagement of dopamine neurons in parallel and partially converging circuits controlling motivation, stress, and emotions.
 
Recent studies have implicated glutamate neurotransmission as an important substrate for the extinction of conditioned behaviors, including responding for drug reinforcement. Positive allosteric modulation of the type-5 metabotropic glutamate receptor (mGluR5) in particular has emerged as a treatment strategy for the enhancement of extinction of drug-motivated behaviors. Here, we investigated the effects of the mGluR5 positive allosteric modulator CDPPB, a compound known for its cognitive enhancing effects in rodents, on extinction learning in rats with different histories of methamphetamine (METH) training. Rats were trained to self-administer METH under two conditions: 16 daily sessions of short access (90 min/day, ShA), or eight daily sessions of short access followed by eight sessions of long access (6 h/day, LgA). Control rats self-administered sucrose pellets in daily 30 min sessions. Next, rats were administered vehicle or 30 mg/kg CDPPB prior to seven consecutive daily extinction sessions, subjected to additional extinction sessions to re-establish a post-treatment baseline, and then tested for reinstatement of behavior in the presence of METH- or sucrose-paired cues. Rats were then subjected to a second series of extinction sessions, preceded by vehicle or 30 mg/kg CDPPB, and an additional test for cue-triggered reinstatement. CDPPB treatment resulted in a more rapid extinction of responding on the active lever, especially in the early sessions of the first extinction sequence. However, treatment effects were minimal during subsequent cue reinstatement tests and non-existent during the second series of extinction sessions. Rats with histories of ShA, LgA, and sucrose training expressed similar behavioral sensitivities to CDPPB, with LgA rats demonstrating a modestly higher treatment effect. Positive allosteric modulation of mGluR5 may therefore have some beneficial effects on efforts to facilitate extinction learning and reduce methamphetamine seeking.
 
| Assessment of innovative medicines in the European Union.
This Perspective describes (a) the current situation, (b) challenges and initiatives, (c) and formulates recommendations to valorize and create access to innovative medicines in the EU. We are currently still far away from optimal assessment of value for money in the EU. On the one hand, valorizing innovative medicines involves a local appraisal by health technology assessment (HTA) bodies and competent authorities about the value for money, the budget impact, and the local medical need that can be filled with new medicines. Therefore, local priorities and national health care policy environments should be reflected in the processes and criteria used for assessing value for money and ultimately for reimbursement decisions. On the other hand, a pan-European assessment of both relative effectiveness and medical need (including general ethical and social considerations) should be envisaged in order to feed part of the data needed for the local decisions in an efficient way. This could be the task of the European Medicines Agency, HTA bodies, and competent authorities together.
 
Ethanol-induced behavior and metabolic adaptations are not altered by Homer2 KO. (A) Neither WT nor KO mice developed metabolic tolerance to ethanol after the final IP injection and there was no effect of genotype on ethanol metabolism. (B) Wild-type and KO mouse weights were not different from each other and did not change during the course of the chronic intermittent ethanol IP injection procedure. (C) There was no effect of genotype on loss of righting reflex in response to a single 5 g/kg IP injection of ethanol.
Repeated chronic intermittent injections of ethanol induced NMDA receptor plasticity independent of genotype. (A) Representative images of western blots of the PSD-enriched tissue samples. There was no effect of ethanol treatment or genotype on the expression of (B) PSD-95, (C) GluA1, (D) GluN1, or (F) GluN2A. (E) Repeated intermittent injections of ethanol resulted in increased expression of GluN2B in both WT and Homer2 KO mice (*p < 0.05).
Deletion of Homer2 increased long spine density compared to WT mice. (A) Representative images of diolistically labeled dendritic segments from WT saline and Homer2 KO saline treated mice. The dendritic shaft is shown in gray and spines are shown in light blue. Classified spines are b color (dark blue = long, thin; pink = filopodia; red = stubby; green = mushroom). (B) Dendritic diameter and (C) total spine density were not altered by genotype. (D) Analysis of spine subclasses revealed that long thin spine density is increased in KO mice compared WT (*p < 0.05). (E) There were no genotypic differences in spine length.
Deletion of Homer2 is associated with increased long spine density compared to WT mice that is unaffected by repeated ethanol exposure. (A) Representative images of WT and Homer2 KO saline (sal) and ethanol (EtOH) treated mice showing a diolistically labeled dendrite segment (top), a labeled dendrite with a computer-generated filament (middle), and the filament with classified spines (blue = long thin, pink = filopodia, red = stubby, green = mushroom; bottom). (B) Total spine density was unaffected by genotype or treatment. (C) Class analyses of spine density showed no effect of treatment or genotype on stubby spine density. (D) There was a main effect of genotype on thin spine density (*p = 0.0122), but no effect of treatment. Similar analyses showed no effect of treatment or genotype on the density of (E) mushroom spines or (F) filopodia. Representative images for WT Sal and KO Sal are the same as those used in
Repeated exposure to ethanol followed by withdrawal leads to alterations in glutamatergic signaling and impaired synaptic plasticity in the nucleus accumbens (NAc) in both clinical and preclinical models of ethanol exposure. Homer2 is a member of a family of postsynaptic density (PSD) scaffolding proteins that functions in part to cluster N-methyl-D-aspartate (NMDA) signaling complexes in the PSD, and has been shown to be critically important for plasticity in multiple models of drug and alcohol abuse. Here we used Homer2 knockout (KO) mice and a chronic intermittent intraperitoneal (IP) ethanol injection model to investigate a potential role for the protein in ethanol-induced adaptations in dendritic spine morphology and PSD protein expression. While deletion of Homer2 was associated with increased density of long spines on medium spiny neurons of the NAc core of saline treated mice, ethanol exposure had no effect on dendritic spine morphology in either wild-type (WT) or Homer2 KO mice. Western blot analysis of tissue samples from the NAc enriched for PSD proteins revealed a main effect of ethanol treatment on the expression of GluN2B, but there was no effect of genotype or treatment on the expression other glutamate receptor subunits or PSD95. These data indicate that the global deletion of Homer2 leads to aberrant regulation of dendritic spine morphology in the NAc core that is associated with an increased density of long, thin spines. Unexpectedly, intermittent IP ethanol did not affect spine morphology in either WT or KO mice. Together these data implicate Homer2 in the formation of long, thin spines and further supports its role in neuronal structure.
 
Glutamate quantitative microdialysis in the nucleus accumbens (NAc) of ethanol-dependent (EtOH) and non-dependent (CTL) mice (n = 5–7/group). (A) The group means and linear regressions on the dialysate glutamate levels are plotted for the no net flux function and indicate that the functions are parallel and there is a right shift for the ethanol-dependent mice, indicating higher basal levels of glutamate in the NAc of these mice. (B) After calculating the x-intercept for each mouse to determine basal glutamate concentrations use and comparison of the group means indicate that the basal glutamate concentrations were twofold higher (*p < 0.05). (C) The slopes of the no net function were calculated and the statistical comparison indicated no difference, consistent with the observation that the functions are parallel. (D) Probe placements for the mice included in the analyses. Data are means ± SEM.
Glutamate transport in NAc tissue from ethanol-dependent (EtOH) and non-dependent (CTL) mice (n = 6–7/group). (A) Na+-dependent glutamate transport, which reflects the function of excitatory amino acid transporters in the cell membrane, was not influenced by ethanol dependence. (B) Na+-independent glutamate transport, which reflects the function of System Xc– in glial membranes, also was not affected by ethanol dependence. Data are means ± SEM.
Repeated cycles of chronic intermittent ethanol (CIE) exposure increase voluntary consumption of ethanol in mice. Previous work has shown that extracellular glutamate in the nucleus accumbens (NAc) is significantly elevated in ethanol-dependent mice and that pharmacologically manipulating glutamate concentrations in the NAc will alter ethanol drinking, indicating that glutamate homeostasis plays a crucial role in ethanol drinking in this model. The present studies were designed to measure extracellular glutamate at a time point in which mice would ordinarily be allowed voluntary access to ethanol in the CIE model and, additionally, to measure glutamate transport capacity in the NAc at the same time point. Extracellular glutamate was measured using quantitative microdialysis procedures. Glutamate transport capacity was measured under Na(+)-dependent and Na(+)-independent conditions to determine whether the function of excitatory amino acid transporters (also known as system XAG) or of system Xc (-) (glial cysteine-glutamate exchanger) was influenced by CIE exposure. The results of the quantitative microdialysis experiment confirm increased extracellular glutamate (approximately twofold) in the NAc of CIE exposed mice (i.e., ethanol-dependent) compared to non-dependent mice in the NAc, consistent with earlier work. However, the increase in extracellular glutamate was not due to altered transporter function in the NAc of ethanol-dependent mice, because neither Na(+)-dependent nor Na(+)-independent glutamate transport was significantly altered by CIE exposure. These findings point to the possibility that hyperexcitability of cortical-striatal pathways underlies the increases in extracellular glutamate found in the ethanol-dependent mice.
 
| A positive feedback loop in the death of neurons in PD. Inhibition of mitochondrial complex I by endogenous or exogenous toxins or mutations in PD genes Parkin, Pink 1, Alpha-synuclein, DJ-1 or LRRK2 generates a multifactorial positive feedback loop. In this loop, complex I inhibition results in iron accumulation driven by decreased Fe-S cluster synthesis, IRP1 activation, increased DMT1 and TfR1 expression and decreased FPN1 expression, increased ROS levels and decreased
Inflammation causes ROS/RNS production, mitochondrial dysfunction, and iron accumulation. Inflammation, oxidative damage, and mitochondrial dysfunction are common features of neurodegenerative diseases. A complex net of relationships connect these features, which through feedback mechanisms contribute to the evolvement of neuronal death (see text for details).
A positive feedback loop in the death of neurons in PD. Inhibition of mitochondrial complex I by endogenous or exogenous toxins or mutations in PD genes Parkin, Pink 1, Alpha-synuclein, DJ-1 or LRRK2 generates a multifactorial positive feedback loop. In this loop, complex I inhibition results in iron accumulation driven by decreased Fe-S cluster synthesis, IRP1 activation, increased DMT1 and TfR1 expression and decreased FPN1 expression, increased ROS levels and decreased glutathione levels. Both increased oxidative stress and low GSH levels further inhibit complex I activity. Another input to this cycle is contributed by inflammatory cytokines that through self-feeding cycles induce mitochondrial dysfunction, increased ROS/RNS production and iron accumulation mediated by the transcriptional regulation of DMT1 and FPN1 (see text). The cumulative oxidative damage finally results in apoptotic death (see text for details).
A growing set of observations points to mitochondrial dysfunction, iron accumulation, oxidative damage and chronic inflammation as common pathognomonic signs of a number of neurodegenerative diseases that includes Alzheimer's disease, Huntington disease, amyotrophic lateral sclerosis, Friedrich's ataxia and Parkinson's disease. Particularly relevant for neurodegenerative processes is the relationship between mitochondria and iron. The mitochondrion upholds the synthesis of iron-sulfur clusters and heme, the most abundant iron-containing prosthetic groups in a large variety of proteins, so a fraction of incoming iron must go through this organelle before reaching its final destination. In turn, the mitochondrial respiratory chain is the source of reactive oxygen species (ROS) derived from leaks in the electron transport chain. The co-existence of both iron and ROS in the secluded space of the mitochondrion makes this organelle particularly prone to hydroxyl radical-mediated damage. In addition, a connection between the loss of iron homeostasis and inflammation is starting to emerge; thus, inflammatory cytokines like TNF-alpha and IL-6 induce the synthesis of the divalent metal transporter 1 and promote iron accumulation in neurons and microglia. Here, we review the recent literature on mitochondrial iron homeostasis and the role of inflammation on mitochondria dysfunction and iron accumulation on the neurodegenerative process that lead to cell death in Parkinson's disease. We also put forward the hypothesis that mitochondrial dysfunction, iron accumulation and inflammation are part of a synergistic self-feeding cycle that ends in apoptotic cell death, once the antioxidant cellular defense systems are finally overwhelmed.
 
Top-cited authors
Martins Ekor
  • University of Cape Coast
Brian Godman
  • University of Strathclyde
Fatemeh Moheimani
  • Manchester Metropolitan University
Teja Veerati
  • Rice University
Lisa Sercombe
  • University of Newcastle