Coordination of Intercellular Ca²⁺ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries

Abstract

Objective To test the hypothesis that Ca2+ responses to G-protein coupled receptor (GPCR) activation are coordinated between neighboring endothelial cells of resistance arteries. Methods Endothelial cell tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca2+ responses to ACh were imaged with confocal microscopy. Results Cell-to-cell transfer of propidium iodide confirmed functional gap junction channels. 1 μM ACh evoked Ca2+ responses [9.8±0.8/min, (F/F0)=3.11±0.2] which pseudo-linescan analysis revealed to be composed of Ca2+ waves and spatially-restricted Ca2+ release events. 100 nM ACh induced Ca2+ responses of lower frequency (4.5±0.7/min) and amplitude (F/F0=1.95±0.11) composed primarily of spatially-restricted events. The interval between Ca2+ waves in Adjacent cells (0.79±0.12 s) was shorter (P<0.05) than between Nonadjacent cells (1.56±0.25 s). Spatially-restricted Ca2+ release events had similar frequencies and latencies between Adjacent and Nonadjacent cells. Inhibiting intracellular Ca2+ release with 2-APB, Xestospongin C or thapsigargin eliminated Ca2+ responses. Conclusions With moderate GPCR stimulation, localized Ca2+ release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca2+ waves via the endoplasmic reticulum. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

“…The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions 6,7 . In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.…”

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

“…The physiological significance of Ca 2+ waves is still unclear however it has been suggested that oscillatory Ca wave signaling in the EC can regulate vasomotor tone (Socha et al 2012) however the exact physiological roles of individual signaling mechanisms still need to be discerned.…”

“…Agonist-induced oscillatory Ca 2+ waves have been observed in both the SMCs Iino, Kasai, and Yamazawa 1994;Boittin et al 1999;Jaggar and Nelson 2000;Dai et al 2007;Dai et al 2006;Lee et al 2001;Syyong et al 2009) and ECs (Beliveau, Lapointe, and Guillemette 2011;Socha et al 2012) (Keener and Sneyd 1998). In another scenario, the whole cell is oscillatory, and spatial cellular heterogeneity can cause the wave like pattern appearance due to phase differences in oscillatory activity in different region of the cells (Jafri and Keizer 1994).…”

“…Moreover, SMC Ca 2+ waves may synchronize in neighboring cells to initiate the phenomenon of vasomotion . On the contrary it has also been suggested that Ca 2+ waves have no significant impact on global cytosolic Ca 2+ levels and are insignificant to force generation in the myogenic response (Jaggar 2001 (Jacob et al 1988;Socha et al 2012;Moccia et al 2003) and SMCs (Jaggar 2001;Lee et al 2002;Mauban et al 2001;Iino, Kasai, and Yamazawa 1994 oscillations in variety of cells have been studied for nearly few decades, ambiguity and controversy still exist on their exact cellular mechanisms. A number of reviews have discussed these issues in detail (Berridge and Galione 1988;Dupont et al 2011;Dupont, Combettes, and Leybaert 2007;Falcke 2004;Schuster, Marhl, and Hofer 2002 oscillations have been discussed in detail in (Dupont et al 2011).…”

“…Diverse cellular events like contraction, relaxation, gene expression, cell death and proliferation are associated with cytoplasmic calcium (Ca 2+ ) mobilization (Foskett et al 2007 waves (Halidi et al 2011;Socha et al 2012). Intracellular Ca higher density of these receptors on one side of cell compared to other was shown to be essential for generation and propagation of Ca…”