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

Abstract

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 (EC5o: ATP 33 ± 2 JAM, UTP 80 ± 4 jaM). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for PI-, P2X-and P2Y-purinoceptors, adenosine, a,3-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. ATPand 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.

Different mechanisms of Ca2+‐handling following nicotinic acetylcholine receptor stimulation, P2U‐purinoceptor stimulation and K+‐induced depolarization in C2C12 myotubes

“…At least three different P2-purinoceptors are present in mouse C2C12 myotubes: a P2u-purinoceptor coupled to phospholipase C (Hemming et al, 1992;1993a) and two unclassified P2-purinoceptors activating Na+-influx (Henning et al, 1992) and the formation of cyclic AMP (Henning et al, 1993b), respectively. Experiments using different purinoceptor agonists have shown that the ATP-evoked increase in cytoplasmic Ca2" in these cells is exclusively mediated by the nucleotide type P2U-purinoceptor (Henning et al, 1993a).…”

“…Ins(1,4,5)P3 measurement Ins(1,4,5)P3 contents of the myotubes was assessed by mass measurement with a radioligand binding assay as described by Henning et al (1993a). Before the experiment, the cells were washed two times in the standard buffer and a final wash was performed with the Ca2"-free buffer.…”

“…The depolarization caused by extracellular application of high K+ to the myotubes is thought to cause Ca2+ release from the ryanodinesensitive store via calcium-induced calcium release following the influx of Ca2+ via voltage-sensitive L-type Ca2+-channels and through a direct molecular interaction between the L-type Ca2+ channel and the ryanodine receptor (Schneider & Chandler, 1973;Meissner, 1994). It has been proposed that both nAChRs (Giovannelli et al, 1991;Grassi et al, 1993) and P2u-purinoceptors (Henning et al, 1993a) activate the phosphoinositide pathway to mobilize Ca2+ from internal stores in C2C12 myotubes, which would be remarkable as both types represent receptors from very different families (Noda et al, 1983;Webb et al, 1993;Lustig et al, 1993). The similarities of the signal transduction pathway activated by nicotinic and P2-purinoceptor agonists or high K+ were characterized and their interaction was studied by measuring cytoplasmic Ca2+ in C2C12 myotubes.…”

“…In myotubes of different origin, the ACh-sensitive receptor which is involved in augmenting Ca2" has invariably been characterized ____________________ as the muscle type nicotinic receptor (Giovannelli et al, 1991; ceptors (P2-purinoceptors) is still largely based on their sensitivity to ATP analogues (Cusack & Hourani, 1990), because of the lack of a specific antagonist at cloned receptors (Webb et al, 1993;Lustig et al, 1993). With the exception of mouse C2C12 myotubes, in which the augmentation of Ca2+ is mediated by a P2u-purinoceptor (Henning et al, 1993a), the P2-purinoceptors involved in other types of myotubes do not fit the proposed subclassification (Cusack & Hourani, 1990).…”

“…C2C12 myotubes possess both a ryanodine-sensitive Ca21 store (sarcoplasmic reticulum) and inositol-(1,4,5) trisphosphate (Ins(1,4,5)P3)-sensitive Ca stores, as judged by functional responses (Grassi et al, 1993;Henning et al, 1993a) and expression of ryanodine and Ins(1,4,5)P3 receptors (Arai et al, 1992;De Smedt et al, 1994). Stimulation of both nAChR and P2u-purinoceptors in C2C12 myotubes increases intracellular Ca2 , due to mobilizing Ca2+ from internal stores as well as initiating Ca2+-entry from the extracellular space.…”