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 r eceptor on C2C12 myotubes has been shown by electrophysiological exper iments. In this study, the ATP-sensitive receptors of C2C12 myotubes w ere 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 con tent 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 t he cells, while selective agonists for P1-, P2X- and P2Y-purinoceptors , adenosine, alpha,beta-methylene ATP and 2-methylthio ATP, appeared t o be ineffective. 3 Under Ca2+ -free conditions, the basal level of In s(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP- and UT P-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 addit ive. ATP- and UTP-induced Ins(1,4,5)P3 formation showed cross-desensit ization, whereas cross-desensitization was absent in responses elicite d 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 Stimulatio n of the cells with ATP or UTP induced a rapid increase in intracellul ar Ca2+, followed by a slow decline to basal levels. Ca2+ responses re ached lower maximal values and did not show the slow phase in the abse nce of extracellular Ca2+. The ATP and UTP-evoked increase in intracel lular Ca2+ was not additive and showed cross-desensitization. Cross-de sensitization was absent in myotubes stimulated with one of the nucleo tides and bradykinin. 7 These results show that ATP- and UTP-induced f ormation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+-i nflux from the extracellular space are mediated exclusively via the nu cleotide type P2-purinoceptor in mouse C2C12 myotubes.