ARACHIDONIC ACID-INDUCED Ca²⁺ MOBILIZATION IN CULTURED ENDOTHELIAL CELLS

Nour Bishara, Michael Hill, Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora 3083.

Endothelial cells (ECs) respond to many stimuli with increased intracellular Ca²⁺ which subsequently contributes to production of paracrine factors including nitric oxide. The increase in Ca²⁺ results from intracellular release and entry from the extracellular space. Ca²⁺ entry is thought to occur in response to store depletion (capacitative) and possibly non-capacitative mechanisms including those stimulated by arachidonic acid (AA) release. In several cell lines, evidence has been provided to indicate a reciprocal relationship between such entry mechanisms. The present studies examined AA-mediated Ca²⁺ entry in ECs and whether such pathways are involved in Ca²⁺ entry following agonist stimulation or store depletion. Studies utilized cultured bovine aortic ECs loaded with the Ca²⁺ sensitive dye Fura 2 (2 µM). Changes in fluorescence were determined using a photometry system coupled to an inverted microscope. To demonstrate Ca²⁺ entry, cells were exposed to agents in the absence of extracellular Ca²⁺ after which Ca²⁺ (1 mM) was added to the superfusate. AA (10-50 µM) caused Ca²⁺ entry (157±9% basal at 50 µM AA) in the absence of measurable store release. In contrast ATP (1 µM) caused significant Ca²⁺ release (470±41%) and Ca²⁺ entry (267±18%). 2APB a putative IP₃ receptor blocker and inhibitor of capacitative Ca²⁺ entry both prevented and reversed ATP-mediated Ca²⁺ entry. In contrast, 2APB while inhibiting AA-mediated Ca²⁺ entry when added prior to the stimulus did not reverse established AA-induced Ca²⁺ entry. Consistent with reciprocal interaction between these pathways AA attenuated Ca²⁺ entry induced by ionomycin (1 µM). Multiple Ca²⁺ entry pathways were further demonstrated in studies showing that ionomycin, thapsigargin and AA evoked a residual Ca²⁺ entry component not sensitive to 2APB but attenuated by the non-capacitative Ca²⁺ entry inhibitor LOE-908 (25 µM). These data demonstrate multiple Ca²⁺ entry pathways in ECs that, while reciprocally interacting, cannot simply be ascribed to capacitative and non-capacitative mechanisms.