Characterization of P2‐purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes

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Abstract
1 The formation of adenosine 3′:5′-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2C12 myotubes. 2 ATP (100 μm) and ATP7S (100 μm) caused a sustained increase in cyclic AMP content of the cells, reaching a maximum after 10 min. The cyclic AMP content reached a maximum in the presence of 100 μm ATP, followed by a decline at higher ATP concentrations. ATP-induced cyclic AMP formation was inhibited by the P2-purinoceptor antagonist, suramin. 3 Myotubes hydrolysed ATP to ADP at a rate of 9.7 ± 1.0 nmol mg−1 protein min−1. However, further hydrolysis of ADP to AMP and adenosine was negligible. 4 The cyclic AMP formation induced by ADP (10 μm–1 mm) showed similar characteristics to that induced by ATP, but a less pronounced decline was observed than with ATP. ADP-induced cyclic AMP formation was blocked by suramin, while cyclic AMP formation elicited by adenosine (10 μm–1 mm) was insensitive to suramin. 5 The ATP analogue, α,β-methylene-ATP also induced a suramin-sensitive cyclic AMP formation, while 2-methylthio-ATP and the pyrimidine, UTP, did not affect cyclic AMP levels. 6 Stimulation of the myotubes with ATP or UTP (10 μm–1 mm) caused a concentration-dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 μm–1 mm) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7 Incubation of the cells with UTP (30 μm–1 mm) inhibited the ATP- and ADP-induced cyclic AMP formation, suggesting that stimulation of the ‘nucleotide’ type P2-receptor inhibits P2-purinoceptor mediated cyclic AMP formation in C2C12 myotubes. In contrast, UTP (30 μm–1 mm) enhanced adenosine-induced cyclic AMP formation. 8 Adenosine-sensitive P1-purinoceptors activating cyclic AMP formation were found in C2C12 myotubes. Further, a novel P2-purinoceptor is postulated, sensitive to ATP, ADP and ATPγS, which also activates the formation of cyclic AMP in C2C12 myotubes.