Functional and molecular evidence for P2X receptors in LLC-PK1 cells.
Extracellular ATP affects a wide variety of cells via purinergic membrane receptors. One class of purinergic receptors, P2X, consists of ATP-gated, calcium-permeable, cation-selective channels. We performed whole cell patch-clamp studies, intracellular calcium concentration ([Ca2+]i) measurements, and reverse transcription-polymerase chain reaction (RT-PCR) to determine whether P2X receptors are expressed in LLC-PK1 cells. First, in patch-clamp studies, 100 microM ATP depolarized the cell membrane and increased the whole cell conductance of LLC-PK1 cells. This response was dose dependent and inhibited by 100 microM suramin, a P2 receptor antagonist. The ATP-induced conductance was cation selective but did not discriminate between Na+ and K+. ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP had no effect on the whole cell conductance. Next, 10 microM ATP caused a rapid rise in [Ca2+]i in LLC-PK1 cells. This effect of ATP was inhibited by the absence of extracellular calcium and by suramin but not by pretreatment with pertussis toxin. ADP and beta,gamma-methylene-ATP had little or no effect on [Ca2+]i. Finally, RT-PCR produced a 330-bp fragment from LLC-PK1 cell RNA, whose sequence was 80% identical to the rat P2X1 receptor. We conclude that LLC-PK1 cells express purinergic receptors of the P2X class, which mediate depolarization and calcium entry when activated.
Filipovic
D M
DM
Division of Nephrology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Adebanjo
O A
OA
Zaidi
M
M
Reeves
W B
WB
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects* / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D013552:Swine
D018048:Receptors, Purinergic P2; D013498:Suramin; D000255:Adenosine Triphosphate; D002118:Calcium