title: Ex vivo chemical cytometric analysis of protein tyrosine phosphatase activity in single human airway epithelial cells.
abstract: We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min(-1) mg(-1)) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (76%, 69%, and 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min(-1) mg(-1) and was also effectively inhibited by preincubation of the cells with the inhibitors pervanadate, Zn(2+), and 1,2-naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min(-1) mg(-1) resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34 to 36 pmol min(-1) mg(-1) (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues.
authors.affiliation: Department of Pharmacology, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Phillips
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Dailey
authors.forename: Eric
authors.initials: E
authors.lastname: Bair
authors.forename: James M
authors.initials: JM
authors.lastname: Samet
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002460:Cell Line; D019075:Electrophoresis, Capillary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006207:Half-Life; D006801:Humans; D006868:Hydrolysis; D008845:Microinjections; D009285:Naphthoquinones / Q000494:pharmacology; D010750:Phosphoproteins / Q000008:administration & dosage / Q000378:metabolism*; D061251:Primary Cell Culture; D017027:Protein Tyrosine Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D059010:Single-Cell Analysis; D014638:Vanadates / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D010750:Phosphoproteins; C076715:pervanadate; D014638:Vanadates; C040756:1,2-naphthoquinone; D017027:Protein Tyrosine Phosphatases