Different mechanisms of Ca2+-handling following nicotinic acetylcholine receptor stimulation, P-2U-purinoceptor stimulation and K+-induced depolarization in C2Cl2 myotubes

1 The increase in intracellular Ca2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P-2U-purinoceptor stimulation and K+-induced depolarization was investigated in mouse C2C12 myotubes by use of fura-2 fluorescence to characterize the intracellular organisation of Ca2+ releasing stores and Ca2+-entry process. 2 Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca2+ (EC(50) = 0.85 +/- 0.09 mu M), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 mu M) was completely blocked by the nAChR antagonist, pancuronium (3 mu M), but was not affected by the muscarinic antagonist, atropine (3 mu M), or under conditions when Ca2+ entry was blocked by La3+ (50 mu M) or diltiazem (10 mu M). Addition of pancuronium (3 mu M) during the sustained phase of the carbachol-evoked response did not affect this phase. 3 Stimulation of P-2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC(50) of the rapid phase: 8.72+/-0.08 mu M) than with carbachol. Pretreatment with La3+ abolished the sustained phase of the ATP-induced Ca2+ response, while the response was unaffected by diltiazem or pancuronium. 4 Stimulation of the cells with high K+ (60 mM), producing the same depolarization as with carbachol (10 mu M), induced a rapid monophasic Ca2+ response, insensitive to diltiazem, pancuronium or La3+ 5 Under Ca2+-free conditions, the sustained phase of the carbachol- and ATP-evoked responses were abolished. Pre-emptying of depolarization-sensitive stores by high K+ under Ca2+-free conditions did not affect the carbachol- or ATP-evoked Ca2+ mobilization and vice versa. Preincubation of the cells with ATP in the absence of extracellular Ca2+ decreased the amplitude of the subsequent carbachol-induced Ca2+ response to 11%, while in the reverse procedure the ATP-induced response was decreased to 65%. Ca2+ mobilization evoked by simultaneous addition of optimal concentrations of carbachol and ATP was increased compared to levels obtained with either agonist. 6 Preincubation with high K+ under normal conditions abolished the sustained phase of the ATP-evoked Ca2+ response. The carbachol response consisted only of the sustained phase in the presence of high K+. 7 The carbachol-induced Ca2+ response was completely abolished under low Na+/Ca2+-free conditions, while under low Na+ conditions only a sustained Ca2+ response was observed. The ATP- and K+-induced responses were changed compared to Ca2+-free conditions. 8 ATP (300 mu M) induced the formation of Ins(1,4,5)P-3 under Ca2+-free conditions with a comparable time course to that found for the rise in internal Ca2+. In contrast to ATP, carbachol (10 mu M) did not affect Ins(1,4,5)P-3 levels under Ca2+-free conditions. 9 It is concluded that the Ca2+ release from discrete stores of C2C12 myotubes is induced by stimulation of nAChRs, P-2U-purinoceptors and by high K+. Only the P-2U-purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2+. Sustained Ca2+-entry is activated by stimulation of nAChRs and P-2U-purinoceptors via separate ion-channels, which are different from the skeletal muscle nAChR-coupled cation-channel