The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples.     

The Ig-like domain of Punctin/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1

Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro. We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABA_A receptors at GABAergic synapses in C. elegans. The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.     

The coexistence of multiple receptors in a single nerve terminal provides evidence for pre-synaptic integration

Excitatory synaptic transmission is inhibited by G protein coupled receptors, including the adenosine A₁, GABA_B, and metabotropic glutamate receptor 7. These receptors are present in nerve terminals where they reduce the release of glutamate through activating signaling pathways negatively coupled to Ca²⁺ channels and adenylyl cyclase. However, it is not clear whether these receptors operate in distinct subpopulations of nerve terminals or if they are co-expressed in the same nerve terminals, despite the functional consequences that such distributions may have on synaptic transmission. Applying Ca²⁺ imaging and immunocytochemistry, we show that these three G protein coupled receptors coexist in a subpopulation of cerebrocortical nerve terminals. The three receptors share an intracellular signaling pathway through which their inhibitory responses are integrated and coactivation of these receptors produced an integrated response. Indeed, this response was highly variable, from a synergistic response at subthreshold agonist concentrations to an occluded response at high agonist concentrations. The presence of multiple receptors in a nerve terminal could be responsible for the physiological effects of neurotransmitter spillover from neighboring synapses or alternatively, the co-release of transmitters by the same nerve terminal.     

α7烟碱型乙酰胆碱受体在胆碱能抗炎通路中的作用机制及应用现状

胆碱能抗炎通路是一条神经-免疫调节通路,以中枢神经系统的迷走神经为起点,以神经递质乙酰胆碱为基础,以免疫细胞膜表面的α7烟碱型乙酰胆碱受体(α7 nicotinic acetylcholine receptor, α7 nAChR)为关键作用位点。其中,电信号与化学信号相互转化,激活JAK-STAT3、PI3K-Akt等信号通路,抑制NF-κB的核转位,进而抑制促炎因子的释放,促进抑炎因子的释放,调节和控制机体的炎症,具体的作用机制正在研究中。研究表明,可以通过使用药物激活α7 nAChR和电刺激迷走神经这2种方法,激活胆碱能抗炎通路。其中,激活α7 nAChR具有操作简便、创伤小和效果显著等优势。常用药物有选择性激动剂,例如PNU282987和GTS-21等,非选择性激动剂例如烟碱。多项研究显示,该方法在脓毒症、缺血再灌注、胃肠炎、骨关节炎和自身免疫病等外周器官炎性疾病治疗中都具有一定的效果。α7 nAChR作为胆碱能抗炎通路的关键作用位点,成为许多炎性疾病的潜在治疗靶点,本文对胆碱能抗炎通路中的α7 nAChR所涉及的抗炎机制、通路激活方式,以及其近年来在炎性疾病中的应用进行了综述,为未来研究其具体作用机制和新的治疗靶点提供参考。     

Critical Molecular Determinants of α7 Nicotinic Acetylcholine Receptor Allosteric Activation: SEPARATION OF DIRECT ALLOSTERIC ACTIVATION AND POSITIVE ALLOSTERIC MODULATION*

The α7 nicotinic acetylcholine receptors (nAChRs) are uniquely sensitive to selective positive allosteric modulators (PAMs), which increase the efficiency of channel activation to a level greater than that of other nAChRs. Although PAMs must work in concert with “orthosteric” agonists, compounds such as GAT107 ((3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) have the combined properties of agonists and PAMs (ago-PAM) and produce very effective channel activation (direct allosteric activation (DAA)) by operating at two distinct sites in the absence of added agonist. One site is likely to be the same transmembrane site where PAMs like PNU-120596 function. We show that the other site, required for direct activation, is likely to be solvent-accessible at the extracellular domain vestibule. We identify key attributes of molecules in this family that are able to act at the DAA site through variation at the aryl ring substituent of the tetrahydroquinoline ring system and with two different classes of competitive antagonists of DAA. Analyses of molecular features of effective allosteric agonists allow us to propose a binding model for the DAA site, featuring a largely non-polar pocket accessed from the extracellular vestibule with an important role for Asp-101. This hypothesis is supported with data from site-directed mutants. Future refinement of the model and the characterization of specific GAT107 analogs will allow us to define critical structural elements that can be mapped onto the receptor surface for an improved understanding of this novel way to target α7 nAChR therapeutically.     

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (mu scle‐s pecific k inase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. Results. In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the ‐MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid‐ordered phase by methyl‐β‐cyclodextrin abolished this association. We further show that actin and the actin‐nucleation factors, N‐WASP (neuronal Wiscott—Aldrich syndrome protein) and Arp2/3 (actin‐related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N‐WASP activity perturbed agrin‐elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR‐enriched rafts. Conclusions. The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.     

Tetanic failure due to decreased endogenous adenosine A(2A) tonus operating neuronal Ca(v) 1 (L-type) influx in Myasthenia gravis

In healthy motor endplates, tetanic depression is overcome by tonic adenosine A(2A) -receptor-mediated facilitation of transmitter release. The A(2A) receptor operates a coordinated shift from fast-desensitizing Ca(v) 2.1 (P/Q) calcium influx to long-lasting Ca(V) 1 (L) channels on motor nerve terminals. This study aimed at investigating whether A(2A) receptors-operated Ca(2+) influx via Ca(V) 1 (L)-type channels contribute to sustain acetylcholine release evoked by 50 Hz-bursts in toxin-induced Myasthenia gravis (TIMG) rats. In contrast to control animals, inhibition of [(3) H]acetylcholine (ACh) release by the Ca(V) 2.1 (P/Q) channel blocker, ω-Agatoxin IVA (100 nM), in TIMG rats had a higher magnitude than that observed with the Ca(V) 1 (L) channel blocker, nifedipine (1 μM). Adenosine deaminase (0.5 U/mL) and the A(2A) receptor antagonist, ZM 241385 (50 nM), decreased [(3) H]ACh release by a similar amount in control rats, but their effects were smaller in magnitude in myasthenic animals. The adenosine precursor, AMP (100 μM), increased (~40%) ACh release in both control and TIMG animals. Blockade of A(2A) , but not of A(1) , receptors prevented AMP-induced facilitation of transmitter release; nifedipine (1 μM) mimicked the effect of the A(2A) receptor antagonist. Video-microscopy studies designed to measure real-time transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. Thus, impairment of the adaptive shift from Ca(V) 2.1 (P/Q) to Ca(V) 1 (L) channels may contribute to tetanic failure in myasthenic rats. This parallels the reduction of adenosine A(2A) receptor tonus in TIMG animals, which might be restored by exogenous application of AMP.     

Genetic inactivation of the adenosine A(2A) receptor exacerbates brain damage in mice with experimental autoimmune encephalomyelitis

Studies with multiple sclerosis patients and animal models of experimental autoimmune encephalomyelitis (EAE) implicate adenosine and adenosine receptors in modulation of neuroinflammation and brain injury. Although the involvement of the A(1) receptor has been recently demonstrated, the role of the adenosine A(2A) receptor (A(2A) R) in development of EAE pathology is largely unknown. Using mice with genetic inactivation of the A(2A) receptor, we provide direct evidence that loss of the A(2A) R exacerbates EAE pathology in mice. Compared with wild-type mice, A(2A) R knockout mice injected with myelin oligodendroglia glycoprotein peptide had a higher incidence of EAE and exhibited higher neurological deficit scores and greater decrease in body weight. A(2A) R knockout mice displayed increased inflammatory cell infiltration and enhanced microglial cell activation in cortex, brainstem, and spinal cord. In addition, demyelination and axonal damage in brainstem were exacerbated, levels of Th1 cytokines increased, and Th2 cytokines decreased. Collectively, these findings suggest that extracellular adenosine acting at A(2A) Rs triggers an important neuroprotective mechanism. Thus, the A(2A) receptor is a potential target for therapeutic approaches to multiple sclerosis.     

Melatonin attenuated mediators of neuroinflammation and alpha-7 nicotinic acetylcholine receptor mRNA expression in lipopolysaccharide (LPS) stimulated rat astrocytoma cells,C6

Abstract

Melatonin has been known to affect a variety of astrocytes functions in many neurological disorders but its mechanism of action on neuroinflammatory cascade and alpha-7 nicotinic acetylcholine receptor (α7-nAChR) expression are still not properly understood. Present study demonstrated that treatment of C6 cells with melatonin for 24 hours significantly decreased lipopolysaccharide (LPS) induced nitrative and oxidative stress, expressions of cyclooxigenase-2 (COX-2), inducible nitric-oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP). Melatonin also modulated LPS-induced mRNA expressions of α7-nAChR and inflammatory cytokine genes. Furthermore, melatonin reversed LPS-induced changes in C/EBP homologous protein 10 (CHOP), microsomal prostaglandin E synthase-1(mPGES-1) and phosphorylated p38 mitogen activated protein kinase (P-p38). Treatment with pyrrolidine dithiocarbamate (PDTC) inhibited α7-nAChR mRNA expression in LPS-induced C6 cells. Our findings explored anti-neuroinflammatory action of melatonin, which may suggests its beneficial roles in the neuroinflammation associated disorders.     

The spectrum of congenital myasthenic syndromes

The past decade saw remarkable advances in defining the molecular and genetic basis of the congenital myasthenic syndromes. These advances would not have been possible without antecedent clinical observations, electrophysiologic analysis, and careful morphologic studies that pointed to candidate genes or proteins. For example, a kinetic abnormality of the acetylcholine receptor (AChR) detected at the single channel level pointed to a kinetic mutation in an AChR subunit; endplate AChR deficiency suggested mutations residing in an AChR subunit or in rapsyn; absence of acetylcholinesterase (AChE) from the endplate predicted mutations in the catalytic or collagen-tailed subunit of this enzyme; and a history of abrupt episodes of apnea associated with a stimulation dependent decrease of endplate potentials and currents implicated proteins concerned with ACh resynthesis or vesicular filling. Discovery of mutations in endplate-specific proteins also prompted expression studies that afforded proof of pathogenicity, provided clues for rational therapy, lead to precise structure function correlations, and highlighted functionally significant residues or molecular domains that previous systematic mutagenesis studies had failed to detect. An overview of the spectrum of the congenital myasthenic syndromes suggests that most are caused by mutations in AChR subunits, and particularly in the ɛ subunit. Future studies will likely uncover new types of CMS that reside in molecules governing quantal release, organization of the synaptic basal lamina, and expression and aggregation of AChR on the postsynaptic junctional folds.     

ABCA1调控与动脉粥样硬化

ATP结合盒转运体A1(ABCA1)在细胞内胆固醇流出中起着重要作用,增加它的表达可促进细胞内胆固醇流出,降低动脉粥样硬化风险.ABCA1的表达受到多种因素的调控,包括肝X受体、过氧化物酶体增殖物激活受体、钙蛋白酶抑制剂和miRNA等.本文主要综述ABCA1的表达与调控,以及它对动脉粥样硬化性心血管疾病发生发展的影响,...     

Differential α4(+)/(?)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)₂(β2)₃ and (α4)₃(β2)₂ subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with ¹²⁵I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.     

A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)₂β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs.     

Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin

Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended.     

An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)₂ concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets.     

Modulation of electrically evoked acetylcholine release in cultured rat septal neurones

The electrically evoked release of acetylcholine and its modulation via auto- and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D-lysine-coated glass coverslips. They developed a dense network of non-neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre-incubated with [3H]choline (0.1 micromol/L), superfused with modified Krebs-Henseleit buffer at 25 degrees C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90-100 mA). The electrically evoked overflow of [3H] from the cells consisted of approximately 80% of authentic [3H]Ach, was largely Ca2+-dependent and tetrodotoxin sensitive, and hence represents an action potential-evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonist added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1- and opioid mu-receptors, could be detected, whereas delta-opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2-week-old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and mu-opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones.     

ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis

Patients with pulmonary alveolar proteinosis (PAP) display impaired surfactant clearance, foamy, lipid-filled alveolar macrophages, and increased cholesterol metabolites within the lung. Neutralizing autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) are also present, resulting in virtual GM-CSF deficiency. We investigated ABCG1 and ABCA1 expression in alveolar macrophages of PAP patients and GM-CSF knockout (KO) mice, which exhibit PAP-like pulmonary pathology and increased pulmonary cholesterol. Alveolar macrophages from both sources displayed a striking similarity in transporter gene dysregulation, consisting of deficient ABCG1 accompanied by highly increased ABCA1. Peroxisome proliferator-activated receptor gamma (PPARgamma), a known regulator of both transporters, was deficient, as reported previously. In contrast, the liver X receptor alpha, which also upregulates both transporters, was highly increased. GM-CSF treatment increased ABCG1 expression in macrophages in vitro and in PAP patients in vivo. Overexpression of PPARgamma by lentivirus-PPARgamma transduction of primary alveolar macrophages, or activation by rosiglitazone, also increased ABCG1 expression. These results suggest that ABCG1 deficiency in PAP and GM-CSF KO alveolar macrophages is attributable to the absence of a GM-CSF-mediated PPARgamma pathway. These findings document the existence of ABCG1 deficiency in human lung disease and highlight a critical role for ABCG1 in surfactant homeostasis.     

Protoporphyrin IX regulates peripheral benzodiazepine receptor associated protein 7 (PAP7) and divalent metal transporter 1 (DMT1) in K562 cells

BackgroundProtoporphyrin IX (PP IX), the immediate precursor to heme, combines with ferrous iron to make this product. The effects of exogenous PP IX on iron metabolism remain to be elucidated. Peripheral-type benzodiazepine receptor (PBR) is implicated in the transport of coproporphyrinogen into the mitochondria for conversion to PP IX. We have demonstrated that PBR-Associated Protein 7 (PAP7) bound to the Iron Responsive Element (IRE) isoform of divalent metal transporter 1 (DMT1). PP IX and PAP7 are ligands for PBR, thus, we hypothesized that PAP7 interact with PP IX via PBR.MethodsWe have examined in K562 cells, which can be induced to undergo erythroid differentiation by PP IX and hemin, the effects of PP IX on the expression of PAP7 and other proteins involved in cellular iron metabolism, transferrin receptor 1 (TfR1), DMT1, ferritin heavy chain (FTH), c-Myc and C/EBPα by western blot and quantitative real time PCR analyses.ResultsPP IX significantly decreased mRNA levels of DMT1 (IRE) and (non-IRE) from 4 h. PP IX markedly decreased protein levels of C/EBPα, PAP7 and DMT1. In contrast, hemin, which like PP IX also induces K562 cell differentiation, had no effect on PAP7 or DMT1 expression.ConclusionWe hypothesize that PP IX binds to PBR displacing PAP7 protein, which is then degraded, decreasing the interaction of PAP7 with DMT1 (IRE) and resulting in increased turnover of DMT1.General significanceThese results suggest that exogenous PP IX disrupts iron metabolism by decreasing the protein expression levels of PAP7, DMT1 and C/EBPα.