RESUMO
Human lactoperoxidase (LPO) exists as two distinct molecules independent of glycosylation. The N-terminus of one form is blocked and has not been identified while the other is proteolytically processed at the N-terminus similar to myeloperoxidase. Our analysis identified alternatively spliced human LPO mRNAs that may explain the observed molecular heterogeneity of LPO. Two mRNAs omit propeptide encoding exons while retaining the 5' exon encoding the secretion signal, consistent with the heterogeneity and suggesting a possible functional role for the propeptide. Two LPO forms were expressed using baculovirus and both showed similar enzyme activity. LC/MS/MS analysis of trypsin digested, partially purified, salivary LPO confirmed the larger unprocessed LPO is present in saliva. To compare variant expression patterns, antisera were raised against purified recombinant (rhLPO) as well as against an antigenic peptide sequence within the exons encoding the propeptide region. Immunohistochemistry demonstrated proLPO was differently localized within gland cells compared to other forms of LPO. The data suggested splice variants may contribute to LPO molecular heterogeneity and its regulation by intracellular compartmental localization.
Assuntos
Processamento Alternativo , Lactoperoxidase/química , Lactoperoxidase/genética , Células Cultivadas , Clonagem Molecular , Éxons , Variação Genética , Humanos , Íntrons , Pulmão/enzimologia , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia/enzimologiaRESUMO
Inhaled glucocorticosteroids (GSs) cause acute, alpha1-adrenoreceptor (AR)-mediated bronchial vasoconstriction. After release from sympathetic nerves, norepinephrine (NE) must be taken up into cells for deactivation by intracellular enzymes. Because postsynaptic cellular NE uptake is steroid sensitive, GSs could increase NE concentrations at alpha1-AR, causing vasoconstriction. We therefore evaluated mRNA expression of different NE transporters in human bronchial arterial smooth muscle and pharmacologically characterized NE uptake into these cells. RT-PCR demonstrated mRNA expression of the extraneuronal monoamine transporter (EMT) and organic cation transporter 1 (OCT-1). Fluorometric uptake assay showed time (within minutes)- and concentration-dependent NE uptake by freshly isolated bronchial arterial smooth muscle cells (SMC) with an estimated Km of 240 microM. Corticosterone and O-methylisoprenaline (1 microM each), but not desipramine, inhibited NE uptake, a profile indicative of NE uptake by EMT, but not OCT-1. Budesonide and methylprednisolone inhibited uptake with IC50 values of 0.9 and 5.6 microM, respectively. Corticosterone's action was reversible and not sensitive to RU-486 (GS receptor antagonist), actinomycin D (transcription inhibitor), or cycloheximide (protein synthesis inhibitor). Corticosterone made membrane impermeant by coupling to BSA also blocked NE uptake. Immunocytochemistry indicated a specific membrane binding site for corticosterone on bronchial arterial SMC. These data demonstrate that although human bronchial arterial SMC express OCT-1 and EMT, EMT is the predominant plasma membrane transporter for NE uptake. This process can be inhibited by GSs, likely via a specific membrane binding site. This nongenomic GS action (increasing NE concentrations at alpha1-AR) could explain acute bronchial vasoconstriction caused by inhaled GSs.
Assuntos
Brônquios/irrigação sanguínea , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neuropeptídeos , Norepinefrina/metabolismo , Artérias/citologia , Artérias/metabolismo , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Corticosterona/metabolismo , Corticosterona/farmacologia , Glucocorticoides/farmacologia , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Músculo Liso Vascular/citologia , Norepinefrina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico , RNA Mensageiro/metabolismo , Simportadores/genética , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Proteínas Vesiculares de Transporte de MonoaminaRESUMO
Norepinephrine (NE) concentration at alpha-adrenergic receptors is partially regulated by steroid-sensitive, extraneuronal catecholamine uptake (uptake-2). Because alpha(1)-adrenergic agonist- and glucocorticosteroid (GS)-induced bronchial vasoconstriction is enhanced in individuals with asthma, atopy could be associated with decreased uptake-2 by vascular smooth muscle cells (SMCs). We therefore evaluated whether NE uptake and its specific transporter messenger RNA (mRNA) were reduced in aortic SMCs of rabbits systemically sensitized with ovalbumin (OVA). NE uptake was measured using a semiquantitative fluorescence microscopic method. Corticosterone and O-methyl-isoprenaline, but not desipramine, co-incubation (1 micro M each) for 20 min decreased NE uptake into SMCs, an inhibitor profile indicative of extraneuronal monoamine transporter (EMT). In OVA-sensitized rabbits, NE uptake was 25.9 +/- 4.5% (mean +/- SEM) lower than in control animals (P < 0.05). Sensitized serum had no effect on NE uptake into naive SMCs. EMT mRNA expression was measured in aortic smooth muscle, using multiplex reverse transcriptase-polymerase chain reaction. In OVA-sensitized rabbits, expression was 61.1 +/- 16.4% lower than in control animals (P < 0.05). These data demonstrate that NE uptake by aortic SMCs is impaired in atopic rabbits, and associated with a decreased transporter mRNA expression. The same mechanism may operate in bronchial arteries in individuals with asthma.