Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells.
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.
Horvath
Gabor
G
Div. of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, PO Box 016960 R-47 Miami, FL 33101, USA.
Sutto
Zoltan
Z
Torbati
Aliza
A
Conner
Gregory E
GE
Salathe
Matthias
M
Wanner
Adam
A
D001158:Arteries / Q000166:cytology / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D001980:Bronchi / Q000098:blood supply*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003345:Corticosterone / Q000378:metabolism / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009479:Neuropeptides; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D027701:Organic Cation Transport Proteins / Q000235:genetics; D027702:Organic Cation Transporter 1 / Q000235:genetics; D000075102:Organic Cation Transporter 2; D012333:RNA, Messenger / Q000378:metabolism; D027981:Symporters / Q000235:genetics; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D005938:Glucocorticoids; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D050484:Norepinephrine Plasma Membrane Transport Proteins; D027701:Organic Cation Transport Proteins; D027702:Organic Cation Transporter 1; D000075102:Organic Cation Transporter 2; D012333:RNA, Messenger; C493449:SLC18A1 protein, human; C491013:SLC22A2 protein, human; C493227:SLC6A2 protein, human; D027981:Symporters; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D003345:Corticosterone; D009638:Norepinephrine