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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Pharmacol. | doi: 10.3389/fphar.2018.01055


  • 1Department of Biological Sciences, University of Padova, Italy
  • 2Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy
  • 3Department of Medicine, University of Padova, Italy
  • 4School of Chemical Sciences, Faculty of Science, University of Auckland, New Zealand
  • 5Landcare Research New Zealand, New Zealand

Background. Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabelled compound. We found that NRB-AF12 labelled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: A) to verify if NRB distribution could be influenced by the attached fluorophore; B) to improve the fluorescent performance of NRB-AF12. Methods. MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results. In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labelling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions. This study demonstrates that the distribution of fluorescently labelled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labelling of intracellular structures in both living and fixed cells.

Keywords: Fluorescent probes, live cell imaging, Norbormide, BODIPY, vascular smooth muscle cells, LX2 cells

Received: 25 Jun 2018; Accepted: 03 Sep 2018.

Edited by:

Ajay Sharma, Chapman University, United States

Reviewed by:

Kyeongsoon Park, Chung-Ang University, South Korea
Lin Yuan, Hunan University, China  

Copyright: © 2018 D'Amore, Orso, Forgiarini, Ceolotto, Rennison, Ribaudo, Smith, Hopkins, Brimble and Bova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
PhD. Claudio D'Amore, Department of Biological Sciences, University of Padova, Padova, Italy,
Prof. Sergio Bova, Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, 35131, Italy,