Characterization of P2-purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes.
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 microM) and ATP gamma S (100 microM) 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 microM 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 microM-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 microM-1 mM) was insensitive to suramin. 5. The ATP analogue, alpha,beta-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 microM-1 mM) caused a concentration-dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 microM-1 mM) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7. Incubation of the cells with UTP (30 microM- 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 microM-I 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 ATPgammaS, which also activates the formation of cyclic AMP in C2C12 myotubes.
Henning
R H
RH
Department of Pharmacology/Clinical Pharmacology, University of Groningen, The Netherlands.
Duin
M
M
den Hertog
A
A
Nelemans
A
A
D000227:Adenine Nucleotides / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis*; D007293:Inosine Triphosphate / Q000096:biosynthesis; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D018048:Receptors, Purinergic P2 / Q000187:drug effects*; D013268:Stimulation, Chemical; D010738:Type C Phospholipases / Q000378:metabolism
D000227:Adenine Nucleotides; D018048:Receptors, Purinergic P2; D007293:Inosine Triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D010738:Type C Phospholipases