Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels

A Martelli1, L Testai1, MC Breschi1, K Lawson2, F Miceli3, M Taglialatela4, V Calderone1. 1University of Pisa, Dept. of Psychiatry, Neurobiology, Pharmacology and Biotechnologies, via Bonanno, 6, I-56126 Pisa, Italy, 2Sheffield Hallam University, Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield, S1 1WB, UK, 3University of Naples Federico II, Sect. Pharmacology, Dept. Neuroscience, Naples, Italy, 4University of Molise, Dept. of Health Science, Campobasso, Italy

Hydrogen sulphide (H2S) plays an important role in the homeostasis of the cardiovascular system, where it is biosynthesized from L-cysteine (Martelli et al., 2011). Indeed, H2S is an endogenous vasodilator, acting partially by the activation of ATP-sensitive potassium channels (KATP) (Jiang et al., 2010). Other types of potassium channel, such as members of the Kv7 family, are also known to play an important role in vascular relaxation(Yeung et al., 2007). In this study, the vasorelaxing activity of the H2S-donor NaHS, and its influence on transmembrane rubidium (Rb) efflux were assessed using Wistar rat thoracic aortic rings. The effects of NaHS on the membrane potential of human aortic smooth muscle cells (HASMC) were evaluated using the membrane potential-sensitive fluorescent dye DiBAC4(3), and effects on Kv7.4 channels were assessed using whole-cell configuration patch-clamp techniques in transiently transfected CHO cells. Data are expressed as mean±SEM from n>6 experiments, and have been analyzed by ANOVA and Student t test.

NaHS (10μM-1mM) produced relaxation of KCl (25mM)-precontracted rat thoracic aortic rings (Emax=99±1%, pIC50=3.84±0.02). The response to NaHS was markedly reduced by a higher concentration of KCl (60 mM), which non-specifically inhibits the effects of potassium channel activators (Emax=72±3%, pIC50=3.38±0.04). Glibenclamide 10μM, a KATP channel blocker, partially antagonized the vasorelaxation (Emax=83±1%, pIC50=3.59±0.03). Tetraethylammonium chloride 10mM significantly inhibited the vasorelaxation, indicating the possible involvement of Kv channels (Emax=82±1%, pIC50=3.37±0.01). Consistent with activation of Kv7 channels, linopirdine 10μM and XE-991 10μM (both selective blockers of Kv7), but not margatoxin 10nM (selective blocker of Kv1.3 channels), exhibited significant antagonism of the NaHS-induced vasorelaxation (Emax=91±2%, pIC50=3.61±0.03; Emax=65±5%, pIC50=3.31±0.04 and Emax=99±2%, pIC50=3.85±0.03, respectively). Further, NaHS 1mM induced a marked increase in Rb efflux in rat thoracic aorta rings (136±4% of the basal Rb efflux), which was antagonized by glibenclamide 10μM (96±1%), linopirdine 10μM (100±10%) and XE-991 10μM (114±5%). In HASMCs, NaHS 1mM induced membrane hyperpolarization. This effect was significantly antagonized by XE-991 10μM (49±21% of that induced by NaHS in control conditions) and fully abolished by linopirdine 10μM, but it was unaffected by 4-aminopyridine 3mM (101±25%) and margatoxin 10nM (91±25%). Finally, the H2S-donor NaHS 1 mM activated Kv7.4 channels heterologously expressed in CHO cells, evoking a 10-mV hyperpolarizing shift of the voltage-dependence of the channel activation. These results are consistent with Kv7 potassium channels, in addition to KATP channels, being involved in the direct vasorelaxation induced by H2S.

Acknowledgements

This work is supported by the “Regional Health Research Program 2009” of Regione Toscana, Italy.

References

Jiang, B, Tang, G, Cao, K, Wu, L & Wang, R 2010, ‘Molecular mechanism for H2S-induced activation of K(ATP) channels’, Antioxidant Redox Signal, vol.12, pp. 1167-1178.

Martelli, A, Testai, L, Breschi, MC, Blandizzi, C, Virdis, A, Taddei, S & Calderone, V 2011, ‘Hydrogen sulphide: novel opportunity for drug discovery’, Medicinal Research Review DOI: 10.1002/med.20234.

Yeung, SY, Pucovský, V, Moffatt, JD, Saldanha, L, Schwake, M, Ohya, S & Greenwood, IA 2007, ‘Molecular expression and pharmacological identification of a role for Kv7 channels in murine vascular reactivity’, British Journal Pharmacology, vol.151, pp. 758-770.