APPS November 2002 Meeting Abstract 2421


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 M). 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 M) caused Ca2+ entry (1579% basal at 50 M AA) in the absence of measurable store release. In contrast ATP (1 M) caused significant Ca2+ release (47041%) and Ca2+ entry (26718%). 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 M). 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 M). These data demonstrate multiple Ca2+ entry pathways in ECs that, while reciprocally interacting, cannot simply be ascribed to capacitative and non-capacitative mechanisms.

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