Different mechanisms of Ca²⁺‐handling following nicotinic acetylcholine receptor stimulation, P_2U‐purinoceptor stimulation and K⁺‐induced depolarization in C2C12 myotubes

Abstract

1 The increase in intracellular Ca2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P2U-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+ (EC50 = 0.85 + 0.09 gM), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 gM) was completely blocked by the nAChR antagonist, pancuronium (3 gM), but was not affected by the muscarinic antagonist, atropine (3 gM), or under conditions when Ca2+ entry was blocked by La3`(50 gM) or diltiazem (10 gM). Addition of pancuronium (3 gM) during the sustained phase of the carbachol-evoked response did not affect this phase.3 Stimulation of P2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC50 of the rapid phase: 8.72+0.08 gM) 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 gM), 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 carbacholinduced 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 ATPevoked 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 ATPand K+-induced responses were changed compared to Ca2+-free conditions. 8 ATP (300 gM) induced the formation of Ins(1,4,5)P3 under Ca2+-free conditions with a comparable time course to that found for the rise in internal Ca2 . In contrast to ATP, carbachol (10 gM) did not affect Ins(1,4,5)P3 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, P2U-purinoceptors and by high K+. Only the P2U-purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2. Sustained Ca2+-entry is activated by stimulation of nAChRs and P2u-purinoceptors via separate i...

Mitochondrial Calcium Oscillations in C2C12 Myotubes

“…As opposed to KCl-stimulated [Ca 2ϩ ] m oscillations, those produced by ATP were insensitive to CPA but depended on extracellular Ca 2ϩ . The latter is in line with findings on purinergic stimulation in C2C12 myotubes (23).…”

“…The nicotinic blocker ␣-bungarotoxin (100 nM) almost completely attenuated the CCh-induced response, whereas the muscarinic antagonist atropine (10 M) had no effect (Fig. 5, A and B), highlighting that C2C12 myotubes are exclusively sensitive to nicotinic stimulation and confirming the observations by other groups (23,24). Activation of nicotinic receptors causes cell depolarization by Na ϩ entry and, to a much lesser extent, Ca 2ϩ entry via the associated channel.…”

Molecular Cloning and Characterization of a Novel ATP P2X Receptor Subtype from Embryonic Chick Skeletal Muscle

“…The chick P2X 4 and rat P2X 4 receptors share an identity of 75%, suggesting that the difference between avian and mammalian P2X receptor homologues should be close to this percentage. The cP2X 8 receptor shares an identity of 59% with rP2X 5 .…”

“…The cP2X 8 receptor sequence has the two putative transmembrane domains, and the 10 conserved cysteine residues in the extracellular loop are considered as characteristics for this gene family. The identity of the amino acid sequence of cP2X 8 with other cloned rat P2X receptors is 43% for P2X 1 , 39% for P2X 2 , 43% for P2X 3 , 53% for P2X 4 , 59% for P2X 5 , 47% for P2X 6 , and 24% for P2X 7 . The low percentage of identity of cP2X 8 with other P2X receptors indicates this receptor is a new member of the P2X receptor family.…”

Contraction-related stimuli regulate GLUT4 traffic in C2C12-GLUT4mycskeletal muscle cells

“…Although L6 muscle cells have proven extremely useful to dissect out insulin signals and their impact on GLUT4 traffic, C 2 C 12 myotubes may be more suitable to study contraction signal transduction as only they develop a contractile apparatus of sarcomere units (11,43,49). In addition, C 2 C 12 myotubes have abundant levels of nicotinic acetylcholine receptors that are organized into signaling clusters upon ligand binding (21,71). Yet, C 2 C 12 myotubes have not been widely utilized to study insulin-or contraction-stimulated glucose uptake because they express extremely low levels of GLUT4 (37).…”