Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells

• Published in: American Journal of Physiology: Cell Physiology 2002-02, Vol.282 (2), p.C289-C301
• Main Authors: Schwiebert, Lisa M, Rice, William C, Kudlow, Brian A, Taylor, Amanda L, Schwiebert, Erik M
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Adenosine Triphosphate - physiology; Base Sequence - genetics; Calcium - agonists; Calcium - metabolism; Calcium - pharmacology; Cell Membrane - metabolism; Cells, Cultured; Cytosol - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Extracellular Space - metabolism; Humans; Hypotonic Solutions - pharmacology; Ion Channels - metabolism; Ionomycin - pharmacology; Ionophores - pharmacology; Molecular Sequence Data; Protein Isoforms - metabolism; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2X; Receptors, Purinergic P2X4; Receptors, Purinergic P2X5; Receptors, Purinergic P2Y1; Signal Transduction - physiology; Thapsigargin - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.01387.2000

Department of Physiology and Biophysics and Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294 - 0005 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), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X 4 and P2X 5 are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. 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. purinergic receptors; cytosolic calcium; ectoadenosinetriphosphatases; exocytosis; nitric oxide