Elsevier

European Journal of Pharmacology

Volume 791, 15 November 2016, Pages 179-184
European Journal of Pharmacology

Cardiovascular pharmacology
Novel α1-adrenoceptor antagonism by the fluroquinolone antibiotic trovafloxacin

https://doi.org/10.1016/j.ejphar.2016.08.035Get rights and content

Abstract

Trovafloxacin, a fluroquinolone antibiotic, was recently found to be an inhibitor of pannexin-1 channels through which ATP is released as “find-me” signals in apoptotic Jurkat cells. Our interest in the role of pannexin-1 channels in α1-adrenoceptor-mediated vasoconstriction led us to the novel finding reported here. Concentration-response curves to methoxamine and phenylephrine were competitively antagonised by trovafloxacin (1–30 µM) with a pKB of 5.54 and 5.32, respectively, in rat mesenteric small arteries isolated for myography. In comparison, prazosin (1–10 nM) antagonised methoxamine concentration-response curves with a pKB of 9.76. Trovafloxacin (1–30 µM) had no effect on either the thromboxane mimetic (U46619) or endothelin-1 concentration-contraction curves. Interestingly, the concentration range is similar for trovafloxacin antagonising the 3 distinct pharmacological targets: (i) fourth generation fluroquinolone antibiotic, (ii) pannexin-1 channel inhibitor in apoptotic cells, and now (iii) as an α1-adrenoceptor antagonist. When trovafloxacin was in use clinically, CNS side effects of dizziness, flushing and headache consistent with α1-adrenoceptor antagonism were common. We conclude that trovafloxacin with its quinolone moiety is a weak α1-adrenoceptor competitive antagonist in comparison with prazosin.

Introduction

Pannexin-1 plasma membrane hemichannels are the most widely expressed of the 3 pannexin family members and, when activated, allow intracellular ATP to be released into the extracellular space to then activate or modulate purinoceptors (Penuela et al., 2013). Pannexin-1 channels are implicated in many physiological processes such as (i) apoptosis (Chekeni et al., 2010), (ii) airway inflammation (Seminario-Vidal et al., 2011) and (iii) the regulation of vasoconstriction (Billaud et al., 2011). In the first physiological process, caspase mediates the pannexin-1 channel opening in apoptotic Jurkat cells (human immortalised T-lymphocytes) to allow the release of ATP as “find me” signals to attract phagocytes. Poon et al. (2014) optimised a flow cytometry-based entry of fluorescent dye TO-PRO-3, when the pannexin channel was open, to screen for potential inhibition of pannexin-1. Amongst a small molecule library (LOPAC1280) they found the quinolone-based antibiotic trovafloxacin caused inhibition of the fluorescent dye entry into Jurkat cells. The IC50 for trovafloxacin in this assay was ≈4 µM, consistent with the inhibition of ATP release from Jurkat cells (Fig. 1d in Poon et al. (2014)).

Our interest in pannexin-1 channels in resistance arteries was stimulated by the report from Billaud et al., 2014, Billaud et al., 2011, Billaud et al., 2012 that there was a direct link between α1-adrenoceptor activation and release of ATP via pannexin-1 channels. Their evidence rested on mefloquine (20 µM) and a range of other drugs that blocked the contraction to phenylephrine completely but not to K+ depolarisation in mouse arteries. If their conclusions were correct then all α1-adrenoceptor activity would be mediated by ATP release from pannexin-1 channels to act via purinoceptors to cause contraction. However, we showed that mefloquine (3 or 20 µM) not only inhibited the contraction to phenylephrine but also to a range of constrictor agents and K+ 40 mM in small resistance arteries (Angus et al., 2015). We concluded that when mefloquine (<1 µM) was tested in an appropriate concentration range for blocking pannexin-1 channels there was no evidence for a role of those channels in small artery contraction (Angus et al., 2015).

In a recent study, we found that ATP concentration-contraction curves in arteries of the rat or mouse were competitively antagonised by the selective P2X1 purinoceptor antagonist NF449 (1–10 µM) (Angus and Wright, 2015). However, concentration-contraction curves to phenylephrine were completely unaltered in the presence of NF449 (10 µM). We concluded that if ATP is released, through pannexin-1 channels, during α1-adrenoceptor stimulation then it plays no material role in the subsequent contraction of vascular smooth muscle (Angus and Wright, 2015).

Given the surprising finding that the antibiotic trovafloxacin was an inhibitor of pannexin-1 channels in apoptotic Jurkat cells, we tested this drug on vasoconstrictor agents in small resistance arteries. To our surprise, we show here that trovafloxacin is a selective α1-adrenoceptor competitive antagonist with a similar potency as its antibiotic activity and pannexin-1 inhibitory action in Jurkat cells. To our knowledge this α1-adrenoceptor property has not been described previously. For comparison, we also tested the potency of prazosin in this assay.

Section snippets

Materials and methods

The Ethics Committee of the University of Melbourne approved experiments in accordance with The Australian Code for the care and use of animals for scientific purposes (8th edition, 2013, National Health and Medical Research Council, Canberra). Male Sprague-Dawley rats (357±7 g) were deeply anaesthetised by inhalation of 5% isoflurane in oxygen and killed by rapid excision of the heart. The abdomen was opened and a small length (≈10 cm) of jejunum and its attached vascular fan was removed, pinned

Trovafloxacin

Rat small mesenteric arteries of 364.5±9.5 µm internal diameter when normalised to D100 contracted to increasing concentrations of methoxamine to give a pEC50 of 5.99±0.07 and Emax of 110.9±3.9% of the contraction to KPSS (Fig. 1a); the second agonist phenylephrine was similar in potency (pEC50 6.10±0.14) and Emax (108.7±3.4% KPSS) (Fig. 1b). Both U46619 and endothelin-1 concentration-response curves were significantly more potent than the α1-adrenoceptor agonists with pEC50 values of 7.38±0.30

Discussion

Selectivity in pharmacology has been defined in various ways; we refer to Kenakin's definition: “in general, the concentration of a drug at which a particular activity is observed is critical to characterization of the event, and selective labels must always be qualified by quantitative limits” (Kenakin, 1993). Here we show that over a similar concentration range the fluroquinolone trovafloxacin with known antibiotic and pannexin-1 channel blocking activities is also an α1-adrenoceptor

Conflicts of interest

None.

Acknowledgement

We thank Mr Mark Ross-Smith for expert technical support and Dr Susan Northfield for drawing the chemical structures of prazosin and trovafloxacin.

References (21)

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