Mitochondria are one of the major oxygen sensors triggering hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen unique. Various mitochondrial factors including reactive oxygen species, cytochrome c, ATP and magnesium, are also potent modulator of voltage-gated K+ (Kv) channels in plasmalemmal membrane of pulmonary arterial smooth muscle cells (PASMCs). The main aim of this investigation was to determine whether differences between mitochondria localization in art PASMCs and systemic mesenteric arterial SMCs (MASMCs) could be a contributing factor to the divergent oxygen sensitivity in two different circulations. Myocytes were enzymatically isolated from arteries dissected from male Wistar rats (250-300g)(Smirnov et al., 2002; Firth et al., 2008); experiments were performed at room temperature. Cellular localization of mitochondria was compared using confocal imaging and immunofluorescent labelling (Gordienko et al., 2001; Gordienko & Zholos, 2004). It was found that mitochondria (labelled with MitoTracker Green) were located significantly closer to the plasmalemmal membrane (labelled with Di-8-ANEPPS) in PASMCs compared to MASMCs. On the other hand, sarcoplasmic reticulum (labelled with Brefeldin A BODIPY) was more closer to the plasmalemmal membrane in MASMCs than in PASMCs. Differences in the functional coupling between mitochondria and Kv channels were then evaluated with the patch-clamp technique and two specific mitochondrial inhibitors, antimycin A (a Complex III inhibitor) and oligomycin (an ATP synthase inhibitor)(Smirnov et al., 2002; Firth et al., 2008). These inhibitors were chosen as they have most pronounced effect on IKv amplitude and steady-state activation in rat PASMCs (Firth et al., 2008). Electrophysiological results showed that relative changes in the steady-state activation, inactivation of IKv (Fig. 1) and IKv block (not shown) induced by antimycin A and oligomycin were significantly greater in PASMCs than in MASCMs. These findings suggest a greater structural and functional coupling between mitochondria and Kv channels specifically exists in PASMCs, which could play an important role in HPV.
King's College London (2008) Proc Physiol Soc 13, C4
Oral Communications: Cellular localization of mitochondria contributes to the regulation of voltage-dependent potassium currents in the rat pulmonary, but not mesenteric arterial myocytes
A. L. Firth2, D. V. Gordienko3,4, K. H. Yuill1, S. V. Smirnov1
1. Pharmacy & Pharmacology, University of Bath, Bath, United Kingdom. 2. Department of Medicine, University of California, San Diego, California, USA. 3. Division of Basic Medical Sciences, Ion Channels and Cell Signalling Centre, St George's University of London, London, United Kingdom. 4. Laboratory of Molecular Pharmacology and Biophysics of Cell signalling, Bogomoletz Institute of Physiology, Kiev, Ukraine.
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Mitochondrial inhibition has significantly greater effects on IKv in PASMCs than in MASMCs. A and B Comparison of antimycin A and oligomycin induced changes in the half activation and half inactivation potentials respectively. Open and filled bars represent the relative changes in the half activation and half inactivation potential in PASMCs (n=8-12) and MASMCs (n=9-11) for antimycin A and oligomycin respectively. * P <0.05 and *** P <0.001. * compares the difference in the parameters between the control condition and in the presence of an inhibitor measured in the same cell using paired statistical test. # P<0.05 ## P <0.01 ### P <0.001. # compares the difference between the two cell types using non-paired statistical test.
Where applicable, experiments conform with Society ethical requirements.