Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC354
Comparison of TMEM16A and Best 3 expression and function in murine thoracic aorta.
A. Davis1, N. Pakroo1, Z. Yao2, D. Baines1, I. Greenwood1
1. Biomedical Sciences, St George's, University of London, London, United Kingdom. 2. Department of Physiology & Medicine, University of California, San Francisco, California, United States.
Introduction: Opening of Ca2+-activated Cl- channels (CACC) produces smooth muscle depolarization and increases contractility. Yet, the functional impact of these channels has always been difficult to determine because of the relatively ineffective pharmacological agents. However, since the original identification in 2008 of TMEM16A as a strong candidate for the molecular correlate of CACCs, there has been renewed interest in deciphering the functional role of these channels in a variety of cell types (see reviews Galietta, 2009; Kunzelmann et al., 2009). Moreover, an earlier CACC candidate, Bestrophin 3 (Best 3), has been shown to be important for cGMP-dependent Ca2+-activated Cl- conductance in rat mesenteric arteries (Matchkov et al., 2008). The aim of this study was to ascertain the relative abundance of Best 3 and TMEM16A in murine thoracic aorta and to determine the effectiveness of two novel TMEM16A inhibitors, tannic acid and T16inh-01 (Namkung et al., 2010 & 2011), as well as the cGMP blocker Zn2+ on β-adrenoceptor-induced contractions of mouse thoracic aorta. Methods: Female BALB/c mice (6-8 week) were killed by overdose of pentobarbitone in accordance with Schedule 1 of the Animal (Scientific Procedures) Act 1986. Vessels were dissected and utilised in either mRNA analysis or isometric tension studies. Results: Quantitative PCR revealed relative abundance of these genes as: Best 3 approximately equal to TMEM16A >>TMEM16B (n=3). Similar expression was observed in mouse portal vein and carotid artery. In addition, all blood vessels studied expressed a Best 3 splice variant that appeared to be specific to the vasculature (n=3). Application of ZnCl2 (100 µM) had no effect on methoxamine-induced contractions. In contrast, the T16inh-01 relaxed methoxamine-induced contractions effectively (67 ± 9% relaxation at 10 µM (n=4) although the effects were relatively slow (time to maximum relaxation = 18 ± 4 min). Tannic acid had variable effects on methoxamine contractions either producing a rapidly relaxation of ~75% within 5 mins or having no effect at all. Conclusion: TMEM16A, but not Best 3, appears to contribute to β-adrenoceptor-induced contractions.
Where applicable, experiments conform with Society ethical requirements