Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2-purinoceptors in C2C12 myotubes

1 The presence of a nucleotide receptor and a discrete ATP-sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP-sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+.

2 The nucleotides ATP and UTP caused a concentration-dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 +/- 2 muM, UTP 80 +/- 4 muM). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1-, P2X- and P2Y-purinoceptors, adenosine, alpha,beta-methylene ATP and 2-methylthio ATP, appeared to be ineffective.

3 Under Ca2+ -free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP- and UTP-induced formation of Ins(1,4,5)P3 was diminished.

4 The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP- and UTP-induced Ins(1,4,5)P3 formation showed cross-desensitization, whereas cross-desensitization was absent in responses elicited by one of the nucleotides and bradykinin.

5 The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP- and UTP-stimulated myotubes showed slopes greater than unity (1.63 +/- 0.09 and 1.37 +/- 0.11, respectively). Apparent pA2 values were 4.50 +/- 0.48 and 4.41 +/- 0.63 for ATP and UTP, respectively.

6 Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+. The ATP and UTP-evoked increase in intracellular Ca2+ was not additive and showed cross-desensitization. Cross-desensitization was absent in myotubes stimulated with one of the nucleotides and bradykinin.

7 These results show that ATP- and UTP-induced formation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+-influx from the extracellular space are mediated exclusively via the nucleotide type P2-purinoceptor in mouse C2C12 myotubes.