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Different mechanisms of Ca 2+ ‐handling following nicotinic acetylcholine receptor stimulation, P 2U ‐purinoceptor stimulation and K + ‐induced depolarization in C2C12 myotubes
Author(s) -
Henning R.H.,
Duin M.,
Popta J.P.,
Nelemans A.,
Hertog A.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15355.x
Subject(s) - carbachol , muscarinic acetylcholine receptor , chemistry , depolarization , stimulation , endocrinology , medicine , acetylcholine , oxotremorine , muscarinic antagonist , purinergic receptor , receptor , biology , biochemistry
1 The increase in intracellular Ca 2+ 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 Ca 2+ releasing stores and Ca 2+ ‐entry process. 2 Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca 2+ (EC 50 = 0.85±0.09 μ m ), followed by a sustained phase. The Ca 2+ response evoked by carbachol (10 μ m ) was completely blocked by the nAChR antagonist, pancuronium (3 μ m ), but was not affected by the muscarinic antagonist, atropine (3 μ m ), or under conditions when Ca 2+ entry was blocked by La 3+ (50 μ m ) or diltiazem (10 μ m ). Addition of pancuronium (3 μ 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 Ca 2+ response (EC 50 of the rapid phase: 8.72±0.08 μ m ) than with carbachol. Pretreatment with La 3+ abolished the sustained phase of the ATP‐induced Ca 2+ 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 μ m ), induced a rapid monophasic Ca 2+ response, insensitive to diltiazem, pancuronium or La 3+ . 5 Under Ca 2+ ‐free conditions, the sustained phase of the carbachol‐ and ATP‐evoked responses were abolished. Pre‐emptying of depolarization‐sensitive stores by high K + under Ca 2+ ‐free conditions did not affect the carbachol‐ or ATP‐evoked Ca 2+ mobilization and vice versa . Preincubation of the cells with ATP in the absence of extracellular Ca 2+ decreased the amplitude of the subsequent carbachol‐induced Ca 2+ response to 11%, while in the reverse procedure the ATP‐induced response was decreased to 65%. Ca 2+ 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 Ca 2+ response. The carbachol response consisted only of the sustained phase in the presence of high K + . 7 The carbachol‐induced Ca 2+ response was completely abolished under low Na + /Ca 2+ ‐free conditions, while under low Na + conditions only a sustained Ca 2+ response was observed. The ATP‐ and K + ‐induced responses were changed compared to Ca 2+ ‐free conditions. 8 ATP (300 μ m ) induced the formation of Ins(1,4,5)P 3 under Ca 2+ ‐free conditions with a comparable time course to that found for the rise in internal Ca 2+ . In contrast to ATP, carbachol (10 μ m ) did not affect Ins(1,4,5)P 3 levels under Ca 2+ ‐free conditions. 9 It is concluded that the Ca 2+ 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 Ca 2+ . Sustained Ca 2+ ‐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.