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Articles in PresS, published online ahead of print September 5, 2001
Am J Physiol Cell Physiol, 10.1152/ajpcell.01387.2000
Submitted on December 22, 2000
Accepted on December 31, 1969
1 Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA; Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA
2 Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA
3 Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA
* To whom correspondence should be addressed. E-mail: eschwiebert{at}physiology.uab.edu.
ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists, ionomycin and thapsigargin, stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), degenerate RT-PCR and sequencing of the degenerate P2XR product and immunoblotting with P2XR subtype-specific antibodies were performed. Results revealed that P2X4 and P2X5 are expressed predominantly by endothelial cell primary cultures derived from multiple blood vessels. Taken together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for "self-regulation" of endothelial cell function and modulation of vascular tone.
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