Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C102

Oral Communications

Mitochondrial ATP regulates spontaneous calcium signalling in native endothelial cells

C. Wilson1, C. Saunter2, M. D. Lee1, H. Heathcote1, J. Girkin2, J. G. McCarron1

1. SIPBS, University of Strathclyde, Glasgow, United Kingdom. 2. University of Durham, Durham, United Kingdom.

It is generally accepted that endothelial cells are highly glycolytic and do not rely on mitochondria for ATP generation. Rather than providing energy, mitochondria may act as signalling organelles that control cytosolic Ca2+ signalling or modify reactive oxygen species. The majority of studies suggesting endothelial cells are glycolytic have been performed in cell culture systems where ATP production may be significantly altered from the native cell type. This study was designed to investigate the regulation of endothelial function by mitochondria in intact, native arteries. Our results suggest that mitochondrial ATP production is critical in regulating local Ca2+ signalling in intact endothelial cells. Endothelial Ca2+ signalling (Cal-520/AM), endothelial mitochondria (TMRE), and the internal elastic lamina (IEL; auto fluorescence) were visualised by fluorescence microscopy of the vascular wall of small mesenteric arteries from Sprague Dawley rats (1). Under basal conditions, intact endothelial cells exhibited extensive subcellular Ca2+ signalling activity. Spontaneous Ca2+ events occurred in 12.5 ± 1.5% of endothelial cells (N = 27 fields, n = 9). Ca2+ events arose via IP3-mediated release from the endoplasmic reticulum. In support, Ca2+ events were not significantly altered by a Ca2+-free bathing solution, but were abolished by the SERCA inhibitor, CPA, or the IP3R inhibitor, 2-APB (n = 5 for each treatment, paired data, significance assessed at p<0.05 using Student's paired t-test). Spatial maps of the location and distribution of local Ca2+ sites showed the majority occurred close to junctions with smooth muscle cells (holes in the IEL). The distance between Ca2+ events and IEL holes was smaller than would be expected if Ca2+events occurred randomly throughout the cytosol (p < 0.05, paired t-test of real vs randomly permuted data). Mitochondria, are morphologically heterogeneous in the native endothelial cells and occupied a significant proportion (8.7 ± 0.2%) of cell area. However, there was no correlation between sites of Ca2+ event initiation and mitochondrial position (p = 0.86, paired t-test of real vs randomly permuted data). However, endothelial Ca2+ signalling was dependent on a polarised mitochondrial membrane potential. The uncoupler, CCCP, or the complex 1 inhibitor, rotenone, each depolarised the mitochondrial membrane potential and nearly eliminated endothelial Ca2+ events (n = 5 for each, paired data, p < 0.05 using Student's paired t-test). Each drug reduced Ca2+ activity whether applied alone or in combination with the mitochondrial ATP synthase inhibitor, oligomycin, to reversal of the ATP synthase. Oligomycin alone also significantly inhibited endothelial Ca2+ activity. In contrast to studies in cultured endothelial cells, these results suggest that mitochondrial ATP production is critical to the function of the native endothelium and exerts a long-range control of IP3R-mediated intracellular Ca2+ signalling.

Where applicable, experiments conform with Society ethical requirements