Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC131

Poster Communications

Role of actin/myosin interaction in control of agonist induced Ca signaling in endothelium of intact rat tail artery

S. Mumtaz1, T. Burdyga1

1. Physiology, University of Liverpool, Liverpool, Merseyside, United Kingdom.


There is strong evidence that Ca release/ Ca entry coupling mechanism plays a crucial role in control of Ca signaling in endothelial cells. However the mechanism controlling sustained Ca entry is not well understood. Studies in cultured endothelial cells have shown that activation of cytoskeletal proteins and several protein kinases [MLCK, PKC, MAP Kinase] may result in Ca entry thereby regulating the barrier function of endothelium [1,2,3]. However, the functional role of cytoskeletal proteins in intact endothelium were not elucidated. Therefore we have investigated the role of actin and myosin interactions in control of agonist induced Ca signaling in intact endothelium of conduit arteries. Rats were humanely killed under CO2 anaesthesia; their tail removed from the ventral grove, cleaned of fat and loaded with Fluo-4 AM (Molecular Probes, 15um) with pluronic. Confocal imaging was done using Nipkow disc based confocal imaging system (Ultraview Perkin Elmer, UK). Minimum of 3 animals were used in each set of experiments. We have found that stimulation of intact endothelial cells by carbachol (0.1µM, 1µM, 10µM) produced activation of a Ca transient which consisted of two components: initial fast - dependent on Ca release and subsequent, sustained dependent on Ca entry. Sustained component of CCh induced Ca transient was 41 ± 0.7 of the peak taken for 100% (n= 372cells, 7vessels). The sustained component was superimposed by Ca oscillations. The frequency of oscillation ranged from 0.05 to 0.3 Hz (n=372cells, 7vessels). Removal of extracellular Ca or SKF-96365 (50µM)abolished Ca oscillations and reduced initial and sustained component induced by CCh. Inhibition of calmodulin by W-7 (10µM) and MLCK by Wortmanin (10µM) strongly inhibited Ca oscillations, reduced the initial fast component of CCh induced Ca transient to 55.3±3% and 57.2±1.8% (n=52cells, 3vessels) and sustained component to 39.9±2.1% and 33±1.2%(n=52cells, 3vessels) of the peak, respectively. Inhibition of MLCP by calyculin A (1µM) abolished Ca oscillations and reduced the initial fast and subsequent sustained component to 52.6±2.9% and 16.9±2.5%(n=26cells, 3vessels) respectively. Inhibitors of myosin 11 and actin polymerization, blebbistatin (50μM) or cytochalasin D (20µM) also blocked Ca oscillations and reduced the initial fast component of CCh induced Ca transient to 51.4±3.9% and 42.8±2.9%(n=37cells, 3vessels) and sustained component to 32.4±2.8% and 20.1±1.9%(n=37cells, 3vessels)of the peak, respectively. These results suggest that store-operated Ca entry plays an important role in control of Ca oscillations in intact endothelial cells of conduit arteries and that protein phosphorylation/dephosphorylation and interactions between actin and myosin fibers are playing crucial role in this process.

Where applicable, experiments conform with Society ethical requirements