Characterization of two distinct P2Y receptors in human tracheal gland cells.
Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are poorly known. ATP-binding characteristics, and ATP-induced formation of cAMP were investigated in a cell line (MM39) of human tracheal gland cells. The binding of a radiolabelled non-hydrolysable analogue of ATP Adenosine-5'-[35S]thiotriphosphate: [35S]ATP[gammaS] was rapid (within 30 min at 4 degrees C), stable and reversible. Scatchard analysis revealed two classes of [35S]ATP[gammaS]-binding sites. Low-affinity binding sites had a Kd1 of 20 +/- 5 microM (Bmax = 150 nmol/10(6) cells) and the high-affinity binding sites had a Kd2 of 2.5 +/- 0.2 microM (Bmax = 52 nmol/10(6) cells). Competition experiments showed competition with ATP, ADP and 2-methylthio-ATP but no competition with UTP, AMP and adenosine. UTP stimulates protein secretion as well as it induced [Ca2+]i mobilization but did not affect the intracellular cAMP levels. ATP also caused induced [Ca2+]i mobilization and protein secretion but also caused an increase in cyclicAMP content of the cells, reaching a maximum after 1 min. ATP-induced cAMP formation was concentration dependent and inhibited by the P2-antagonist suramin. Reverse-transcription-PCR amplification revealed the presence of the transcripts of both the P2Y2 and the UTP-specific P2Y4 receptors. In conclusion, P2Y2 receptors, UTP-P2Y4 receptors and unidentified ATP-specific receptors seem to be present in MM39 cells which appear to be coupled differently to intracellular second-messenger systems.
Merten
M D
MD
Groupe de Recherche sur les Glandes Exocrines, Marseille, France.
Saleh
A
A
Kammouni
W
W
Marchand
S
S
Figarella
C
C
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D005088:Exocrine Glands / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D018048:Receptors, Purinergic P2 / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D013462:Sulfur Radioisotopes; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D013462:Sulfur Radioisotopes; C104749:purinoceptor P2Y4; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D014544:Uridine Triphosphate