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Decline of the calcium response on successive stimulation of a rat brain endothelial cell P2U purinoceptor

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A microfluorimetric method using Fura-2 as calcium indicator was used to study the mechanism of desensitization of the calcium response evoked by activation of a brain endothelial cell P2U receptor. The study was mainly carried out on an immortalized rat brain endothelial cell line (RBE4), with some additional experiments on primary cultured rat brain microvascular endothelial cells. As previously described ( Nobles et al. 1995 ), ATP (100 M, 20 s) caused a transient increase in intracellular calcium levels ([Ca2+]i). This effect was dependent on the rate of filling of intracellular calcium stores, since a large inhibition of the ATP-mediated response was seen in the presence of cyclopiazonic acid, an inhibitor of the store Ca2+-ATPase. Application of repeated pulses of extracellular ATP led to a desensitization of the response, as measured by a decline in the release of intracellular calcium ( Nobles et al. 1995 ). This desensitization was partially reversed after 300 s of incubation in agonist-free medium. Extracellular phosphorylation of the purinergic receptor appeared not to be involved in the desensitization process, since a similar rate of desensitization was obtained with the non-hydrolysable ATP analogue ATP gammaS. Oxidation of the purinergic receptor cannot account for the desensitization, since the decline of the ATP-mediated response was unchanged in the presence of 3 mMdithiothreitol. In the presence of ATP together with UTP, two equally potent activators of the P2U receptor, the desensitization was less than in the presence of only one of the agonists. The desensitization was greater when ATP was applied for longer (150 s) periods. Although these results do not exclude the participation of post-receptor events in the desensitization process, they suggest that desensitization is governed at least in part by agonist–receptor interaction.

Keywords: P2 purinoceptors; desensitization; endothelial cells; intracellular calcium

Document Type: Original Article


Affiliations: King's College London, Physiology Group, Biomedical Sciences Division, Strand, London, UK

Publication date: January 1, 1998

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