title: An NBD Derivative of the Selective Rat Toxicant Norbormide as a New Probe for Living Cell Imaging.
abstract: Norbormide (NRB) is a unique compound that acts directly on rat vascular myocytes to trigger a contractile process, through an as yet unknown mechanism, which results in the selective contraction of rat peripheral arteries. To gain insight into the mechanisms involved in NRB rat-selective activity, we investigated the subcellular distribution of NRB-AF12, a nitrobenzoxadiazole (NBD)-derivative of NRB, in living NRB-sensitive and NRB-insensitive cells. In both cell types, NRB-AF12 localized to the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and endosomes; however, in NRB-sensitive cells, the fluorescence also extended to the plasma membrane. NRB-AF12 was rapidly internalized into the cells, could easily be washed out and then reloaded back into the same cells, all with a high degree of reproducibility. Cells exposed for 24 h to NRB-AF12 did not show apparent signs of toxicity, even at concentrations of the dye (10 μM) much higher than those required for fluorescence labeling (500 ηM). The distribution pattern of NRB-AF12 fluorescence was near identical to that of ER-Tracker® (Er-Tr), a fluorescent derivative of glibenclamide, a known KATPchannel blocker. Displacement tests did not demonstrate, but at the same time did not rule out the possibility of a common target for ER-Tr, NRB-AF12, NRB, and glibenclamide. On the basis of these results we hypothesize a common target site for NRB-AF12 and ER-Tr, and a similar target profile for NRB and glibenclamide, and propose NRB-AF12 as an alternative fluorescence probe to ER-Tracker. Furthermore, NRB-based fluorescence derivatives could be designed to selectively label single cellular structures.
authors.affiliation: Department of Surgical and Biomedical Sciences, University of Perugia Perugia, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: D'Amore
authors.affiliation: IRCCS "E. Medea" Conegliano, Italy.
authors.forename: Genny
authors.initials: G
authors.lastname: Orso
authors.affiliation: Department of Life Sciences, University of Siena Siena, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Fusi
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua Padua, Italy.
authors.forename: Mario A
authors.initials: MA
authors.lastname: Pagano
authors.affiliation: Department of Molecular Medicine, University of Padua Padua, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Miotto
authors.affiliation: Istituto di Ricerca Pediatrica Città della Speranza Padua, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Forgiarini
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua Padua, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: De Martin
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua Padua, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Castellani
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua Padua, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Ribaudo
authors.affiliation: School of Chemical Sciences, University of Auckland Auckland, New Zealand.
authors.forename: David
authors.initials: D
authors.lastname: Rennison
authors.affiliation: School of Chemical Sciences, University of Auckland Auckland, New Zealand.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Brimble
authors.affiliation: School of Chemical Sciences, University of AucklandAuckland, New Zealand; Landcare ResearchLincoln, New Zealand.
authors.forename: Brian
authors.initials: B
authors.lastname: Hopkins
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua Padua, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ferrarese
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua Padua, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Bova
keywords: ER tracker; LX2 cells; fluorescent probes; norbormide; sulphonylurea receptors