P2X purinergic receptor channel expression and function in bovine aortic endothelium.
We examined bovine aortic endothelial cells (BAECs) for the functional expression of P2X receptors, the ATP-gated cation channels. We identified the P2X subtypes present in BAECs using RT-PCR. mRNA was present for only three of seven family members: P2X4, P2X5, and P2X7. We then characterized agonist-activated currents in whole cell and outside-out patch recordings using 2-methyl-thio-ATP (MeSATP) as a P2X4 and P2X5 receptor agonist and 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP) as a P2X7 receptor agonist. MeSATP (10-20 microM) produced current with characteristics of P2X4 receptors. The current was an inwardly rectifying current, reversed near 0 mV, slowly desensitized, was not blocked by suramin (300 microM) or reactive blue (60 microM), and had a single channel conductance of 36 pS. BzATP (10-100 microM), on the other hand, activated a 9-pS channel with sustained activity in the continued presence of the agonist. BzATP-activated current was blocked by reactive blue (60 microM) and by suramin (approximately 50% block at 300 microM). We confirmed, by immunocytochemistry, the presence of P2X4 and P2X7 protein. The agonists failed, however, to induce significant uptake of the large molecule YO-PRO, indicating the lack of pore development that has been demonstrated for P2X7 and P2X4 in response to agonist in some cell types.
Ramirez
Angelina N
AN
Rammelkamp Center for Education and Research, MetroHealth Systems and Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44109-1998, USA. arnavarro@metrohealth.org
Kunze
Diana L
DL
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D001583:Benzoxazoles; D015153:Blotting, Western; D001923:Brain Chemistry; D002417:Cattle; D002531:Cerebellum / Q000737:chemistry; D004553:Electric Conductivity; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000737:chemistry / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression; D006624:Hippocampus / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D008564:Membrane Potentials; D009620:Nodose Ganglion / Q000737:chemistry; D011806:Quinolinium Compounds; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D058469:Receptors, Purinergic P2X; D058848:Receptors, Purinergic P2X4; D058485:Receptors, Purinergic P2X5; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013498:Suramin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D001583:Benzoxazoles; D005456:Fluorescent Dyes; C548226:P2RX4 protein, human; C546465:P2RX5 protein, human; C546468:P2RX7 protein, human; D011806:Quinolinium Compounds; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058848:Receptors, Purinergic P2X4; D058485:Receptors, Purinergic P2X5; D058486:Receptors, Purinergic P2X7; D013873:Thionucleotides; C089813:YO-PRO 1; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D013498:Suramin; D000255:Adenosine Triphosphate; C008501:2-methylthio-ATP