Proceedings of The Physiological Society

University College London 2006 (2006) Proc Physiol Soc 3, C80

Research Symposium

Calcium signals mediated by IP3-gated channels in endothelial cells in pre-capillary arterioles

Ludmila Borisova1, David Eisner2, Susan Wray1, Ted Burdyga1

1. The Physiological Laboratory, University of Liverpool, Liverpool, United Kingdom. 2. Unit of Cardiac Physiology, University of Manchester, Manchester, United Kingdom.

Although localized Ca2+-release signals (puffs) arising from the opening of IP3 channels on the sarcoplasmic reticulum (SR) have been described in endothelial cells, to date there are no descriptions of these events in native cells in intact preparations of terminal arterioles. Given the expected importance of cell to cell contact, and interactions with neighbouring smooth muscle cells in modulating local Ca2+ signals and propagated Ca2+ waves, we have characterized these events. Fluo-4 was loaded into in situ endothelial cells in pre-capillary arterioles contained in small bundles of rat ureter. Both spontaneous and agonist-induced events were recorded using wide-field, real time confocal imaging. Both spontaneous and carbachol–induced Ca2+ signals in the endothelial cells were resistant to Ca2+-free (2mM EGTA) solutions, nifedipine and ryanodine (n=17). In contrast, the Ca2+ events were completely and reversibly blocked by cyclopiazonic acid (Ca2+-ATPase inhibitor), 2-APB (IP3Rs antagonist) or U73122 (phospholipase C inhibitor) (n=11). There was significant variability in frequency, amplitude, time course and spatial spread of Ca2+ puffs not only among different discharging sites but also for the same site. These data suggest that IP3-gated channels in the endothelial cells exist in clusters containing variable numbers of channels and that a variable number of channels can be recruited within these clusters. The number of discharging sites per cell varied between 2-3 (n=30). The main discharging site, which showed the highest frequency of Ca2+ puffs (0.33±0.06 Hz, n=22 sites), was normally located at the edge of the cell membrane adjacent to a smooth muscle cell in the thicker part of the endothelial cell, several microns away from the nucleus. Additional discharging sites were located in the distal narrow regions of the cell. The frequency, amplitude, time course and spatial spread of Ca2+ puffs as well as the number of the discharging sites per cell were increased in the presence of low concentrations of carbachol (50-100 nM) (n=27). Higher concentrations of carbachol (1-10 μM) induced oscillatory propagating Ca2+ waves that originated from several sites (n=27). These Ca2+ waves could have different orientation and spatial spread and very often when initiated at distal ends of the cells at the same time could annihilate each other when reaching central parts of the cell. These data suggest that in the endothelial cells propagating Ca2+ waves are mediated exclusively by IP3 receptor gated channels which can be spatially recruited to induce a regenerative Ca2+ wave.

Where applicable, experiments conform with Society ethical requirements