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Characterization of two distinct P2Y receptors in human tracheal gland cells
M D Merten1, A Saleh, W Kammouni
1Groupe de Recherche sur les Glandes Exocrines, Marseille, France.
Abstract:
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.
Insights
Human tracheal gland cells have specific ATP receptors that influence cyclic AMP (cAMP) levels, crucial for cystic fibrosis research. Understanding these ATP-binding sites aids in developing new therapeutic strategies for the disease.
Area of Science:
- Cell Biology
- Physiology
- Pharmacology
Background:
- Human submucosal tracheal glands are implicated in cystic fibrosis pathophysiology.
- Adenosine triphosphate (ATP) is a therapeutic agent for cystic fibrosis, but its actions on tracheal gland cells are not well understood.
Purpose of the Study:
- To investigate ATP-binding characteristics and ATP-induced cyclic adenosine monophosphate (cAMP) formation in a human tracheal gland cell line (MM39).
- To identify the types of purinergic receptors present on these cells and their signaling pathways.
Main Methods:
- Radiolabeled Adenosine-5'-[35S]thiotriphosphate ([35S]ATP[gammaS]) binding assays with Scatchard analysis.
- Competition binding experiments using various nucleotides and antagonists.
- Measurement of intracellular calcium ([Ca2+]i) mobilization and protein secretion.
- Reverse-transcription PCR (RT-PCR) to detect receptor transcripts.
Main Results:
- MM39 cells possess two classes of ATP-binding sites with distinct affinities (Kd1 = 20 ± 5 µM, Kd2 = 2.5 ± 0.2 µM).
- ATP induced [Ca2+]i mobilization, protein secretion, and increased intracellular cAMP levels, while UTP primarily stimulated secretion and [Ca2+]i mobilization.
- RT-PCR confirmed the presence of P2Y2 and P2Y4 receptor transcripts, suggesting a role for these receptors in ATP signaling.
Conclusions:
- Human tracheal gland cells (MM39) express multiple ATP-sensitive purinergic receptors, including P2Y2, P2Y4, and potentially other ATP-specific receptors.
- These receptors are differentially coupled to intracellular second-messenger systems, influencing cellular responses like cAMP production and protein secretion.
- Findings provide insights into the purinergic signaling mechanisms in tracheal glands relevant to cystic fibrosis therapy.