CHARACTERIZATION OF 2 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 trache al gland cells are poorly known. ATP-binding characteristics, and ATP- induced formation of cAMP were investigated in a cell line (MM39) of h uman tracheal gland cells. The binding of a radiolabelled non-hydrolys able analogue of ATP denosine-5'-[S-35]thiotriphosphate:[S-35]ATP[gamm a S] was rapid (within 30 min at 4 degrees C), stable and reversible. Scatchard analysis revealed two classes of [S-35]ATP[gamma S]-binding sites. Low-affinity binding sites had a K(d)1 of 20+/-5 mu M (B-max = 150 nmol/10(6) cells) and the high-affinity binding sites had a K(d)2 of 2.5 +/- 0.2 mu M (B-max = 52 nmol/10(6) cells). Competition experim ents showed competition with ATP, ADP and 2-methylthio-ATP but no comp etition with UTP, AMP and adenosine. UTP stimulates protein secretion as well as it induced [Ca2+](i) mobilization but did not affect the in tracellular cAMP levels. ATP also caused induced [Ca2+](i) mobilizatio n and protein secretion but also caused an increase in cyclicAMP conte nt of the cells, reaching a maximum after 1 min. ATP-induced cAMP form ation was concentration dependent and inhibited by the P2-antagonist s uramin. Reverse-transcription-PCR amplification revealed the presence of the transcripts of both the P2Y2 and the UTP-specific P2Y4 receptor s. In conclusion, P2Y2 receptors, UTP-P2Y4 receptors and unidentified ATP-specific receptors seem to be present in MM39 cells which appear t o be coupled differently to intracellular second-messenger systems.