Pharmacology and Therapeutics - Research Publications
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ItemDiacetylbis(N(4)-methylthiosemicarbazonato) Copper(II) (CuII(atsm)) Protects against Peroxynitrite-induced Nitrosative Damage and Prolongs Survival in Amyotrophic Lateral Sclerosis Mouse Model(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-12-23)Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.
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ItemN-Glycosylation Determines Ionic Permeability and Desensitization of the TRPV1 Capsaicin Receptor(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-06-22)The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.
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ItemProteolytic activation of the epithelial sodium channel (ENaC) by the cysteine protease cathepsin-S(SPRINGER HEIDELBERG, 2012-10)Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel activation. Inappropriate ENaC activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating proteolytic channel activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented proteolytic activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.
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ItemPurinergic signalling and diabetes(SPRINGER, 2013-09)The pancreas is an organ with a central role in nutrient breakdown, nutrient sensing and release of hormones regulating whole body nutrient homeostasis. In diabetes mellitus, the balance is broken-cells can be starving in the midst of plenty. There are indications that the incidence of diabetes type 1 and 2, and possibly pancreatogenic diabetes, is rising globally. Events leading to insulin secretion and action are complex, but there is emerging evidence that intracellular nucleotides and nucleotides are not only important as intracellular energy molecules but also as extracellular signalling molecules in purinergic signalling cascades. This signalling takes place at the level of the pancreas, where the close apposition of various cells-endocrine, exocrine, stromal and immune cells-contributes to the integrated function. Following an introduction to diabetes, the pancreas and purinergic signalling, we will focus on the role of purinergic signalling and its changes associated with diabetes in the pancreas and selected tissues/organ systems affected by hyperglycaemia and other stress molecules of diabetes. Since this is the first review of this kind, a comprehensive historical angle is taken, and common and divergent roles of receptors for nucleotides and nucleosides in different organ systems will be given. This integrated picture will aid our understanding of the challenges of the potential and currently used drugs targeted to specific organ/cells or disorders associated with diabetes.
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ItemNo Preview AvailableEffect of Metal Chelators on γ-Secretase Indicates That Calcium and Magnesium Ions Facilitate Cleavage of Alzheimer Amyloid Precursor Substrate.(Hindawi Limited, 2010-12-28)Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβ and the APP intracellular domain (AICD). Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg(2+) stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca(2+) and Mg(2+) stabilize γ-secretase and enhance its activity.
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ItemNo Preview AvailableSexual dimorphism in lung function responses to acute influenza A infection(WILEY, 2011-09)BACKGROUND: Males are generally more susceptible to respiratory infections; however, there are few data on the physiological responses to such infections in males and females. OBJECTIVES: To determine whether sexual dimorphism exists in the physiological/inflammatory responses of weanling and adult BALB/c mice to influenza. METHODS: Weanling and adult mice of both sexes were inoculated with influenza A or appropriate control solution. Respiratory mechanics, responsiveness to methacholine (MCh), viral titre and bronchoalveolar lavage (BAL) cellular inflammation/cytokines were measured 4 (acute) and 21 (resolution) days post-inoculation. RESULTS: Acute infection impaired lung function and induced hyperresponsiveness and cellular inflammation in both sexes at both ages. Males and females responded differently with female mice developing greater abnormalities in tissue damping and elastance and greater MCh responsiveness at both ages. BAL inflammation, cytokines and lung viral titres were similar between the sexes. At resolution, all parameters had returned to baseline levels in adults and weanling males; however, female weanlings had persisting hyperresponsiveness. CONCLUSIONS: We identified significant differences in the physiological responses of male and female mice to infection with influenza A, which occurred in the absence of variation in viral titre and cellular inflammation.
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ItemNo Preview AvailableTargeting Glycogen Synthase Kinase-3β for Therapeutic Benefit against Oxidative Stress in Alzheimer's Disease: Involvement of the Nrf2-ARE Pathway.(Hindawi Limited, 2011)Specific regions of the Alzheimer's disease (AD) brain are burdened with extracellular protein deposits, the accumulation of which is concomitant with a complex cascade of overlapping events. Many of these pathological processes produce oxidative stress. Under normal conditions, oxidative stress leads to the activation of defensive gene expression that promotes cell survival. At the forefront of defence is the nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates a broad spectrum of protective genes. Glycogen synthase kinase-3β (GSK-3β) regulates Nrf2, thus making this kinase a potential target for therapeutic intervention aiming to boost the protective activation of Nrf2. This paper aims to review the neuroprotective role of Nrf2 in AD, with special emphasis on the role of GSK-3β in the regulation of the Nrf2 pathway. We also examine the potential of inducing GSK-3β by small-molecule activators, dithiocarbamates, which potentially exert their beneficial therapeutic effects via the activation of the Nrf2 pathway.
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ItemNo Preview AvailableOseltamivir treatment of mice before or after mild influenza infection reduced cellular and cytokine inflammation in the lung(WILEY, 2011-09)BACKGROUND: Lung inflammation is a critical determinant of influenza infection outcomes but is seldom evaluated in animal studies of oseltamivir (OS), which have focused on viral titre and survival. OBJECTIVES: To study the effects of pre- and post-infection dosing with OS on viral replication and inflammation in a mouse model of non-lethal influenza infection. METHODS: BALB/c mice were infected with a laboratory-adapted H3N1 strain of influenza. In pre-dosing studies, OS was gavaged twice daily (1 and 10 mg/kg/day) from 4 hours prior to infection and continuing for 5 days (d) post-infection (p.i). In the second post-infection dosing study, dosing at 10 mg/kg/day began at 24-48 hours p.i. Mice were dissected at d3, d5 and d7 p.i. (pre-dosing study) and d5 p.i. (post-dosing study). Lung viral titres were determined by plaque assay. Bronchoalveolar lavage fluid (BALF) was collected and used for the quantitation of inflammatory cells and mediators. RESULTS: Pre-infection dosing of OS reduced total cells, neutrophils and macrophages in BALF. With pre- or post-infection dosing, the pro-inflammatory mediators TNF-α, IL-1β, IL-6 and granulocyte-macrophage colony-stimulating factor, the neutrophil chemokines keratinocyte-derived chemokine and MIP-1α and the macrophage chemokine MCP-1 were reduced in BALF. Pre-dosing with 1 mg/kg OS did not reduce viral titres, while 10 mg/kg slightly reduced viral titres at d3 and d5 p.i. CONCLUSIONS: Oseltamivir reduced the inflammatory response to influenza when given pre- or post-infection. This anti-inflammatory effect may contribute to the clinical benefit of OS.
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ItemNeuroinflammation and copper in Alzheimer's disease.(Hindawi Limited, 2013)Inflammation is the innate immune response to infection or tissue damage. Initiation of proinflammatory cascades in the central nervous system (CNS) occurs through recognition of danger associated molecular patterns by cognate immune receptors expressed on inflammatory cells and leads to rapid responses to remove the danger stimulus. The presence of activated microglia and astrocytes in the vicinity of amyloid plaques in the brains of Alzheimer's disease (AD) patients and mouse models implicates inflammation as a contributor to AD pathogenesis. Activated microglia play a critical role in amyloid clearance, but chronic deregulation of CNS inflammatory pathways results in secretion of neurotoxic mediators that ultimately contribute to neurodegeneration in AD. Copper (Cu) homeostasis is profoundly affected in AD, and accumulated extracellular Cu drives A β aggregation, while intracellular Cu deficiency limits bioavailable Cu required for CNS functions. This review presents an overview of inflammatory events that occur in AD in response to A β and highlights recent advances on the role of Cu in modulation of beneficial and detrimental inflammatory responses in AD.
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ItemIntroduction and perspective, historical note(FRONTIERS MEDIA SA, 2013-11-21)P2 nucleotide receptors were proposed to consist of two subfamilies based on pharmacology in 1985, named P2X and P2Y receptors. Later, this was confirmed following cloning of the receptors for nucleotides and studies of transduction mechanisms in the early 1990s. P2X receptors are ion channels and seven subtypes are recognized that form trimeric homomultimers or heteromultimers. P2X receptors are involved in neuromuscular and synaptic neurotransmission and neuromodulation. They are also expressed on many types of non-neuronal cells to mediate smooth muscle contraction, secretion, and immune modulation. The emphasis in this review will be on the pathophysiology of P2X receptors and therapeutic potential of P2X receptor agonists and antagonists for neurodegenerative and inflammatory disorders, visceral and neuropathic pain, irritable bowel syndrome, diabetes, kidney failure, bladder incontinence and cancer, as well as disorders if the special senses, airways, skin, cardiovascular, and musculoskeletal systems.