Recently, we have described an inositol 1, 4, 5-trisphosphate (IP3) signalling system in cultured rodent skeletal muscle, triggered by membrane depolarization and affecting gene transcription (Jaimovich et al. 2000, Powell et al. 2001). Neonatal rat primary muscle cultures (animals were humanely killed) and muscle cell lines were used to study fluorescence calcium signals after loading with fluo-3. Biochemical measurements, immunocytochemical studies and Western blot analysis were performed on cells before and after depolarising protocols. When myotubes were exposed to tetanic electrical stimulation and the fluorescence calcium signal was monitored, the expected calcium signal sensitive to ryanodine and associated to the E-C coupling was seen during stimulation. A few seconds after the stimulus ended, a long lasting second calcium signal, refractory to ryanodine was evident. The onset kinetics of this slow signal was slightly modified in nominally calcium-free medium, as was by both the frequency and number of pulses during tetanus. The role of the action potential was evidenced since, in the presence of TTX, the signal was abolished. The role of the L-type, voltage dependent calcium channel or dihydropyridine receptor (DHPR) as voltage sensor for this signal (Araya et al. 2003) was assessed by treatment with agonist and antagonist dihydropyridines (Bay K 8644 and nifedipine) showing an enhanced and inhibitory response respectively. When the dysgenic GLT cell line was used, the signal was absent. Transfection of these cells with the α1S subunit, restored the slow signal. The IP3 mass increase induced by electrical pulses was previous in time to the slow calcium signal. Both IP3R blocker and PLC inhibitor (xestospongin C and U73122) dramatically inhibited the slow calcium response. We have shown that K⁺-induced depolarization of rat myotubes elicits a transient increase in the early genes c-fos, c-jun and egr-1 mRNA levels and we can link such increase to calcium signals (Carrasco et al. 2003). Both early genes activation and CREB phosphorylation were inhibited by ERKs phosphorylation blockade.We investigated the possibility that slow calcium signals regulate CREB phosphorylation by a PKC-dependent mechanism. Western blot analysis revealed the presence of seven PKC isoforms (PKC α, -β1, -β2, -δ, –, z{special}, and -u{special}) in rat myotubes. Pre-treatment of myotubes with either bisindolymaleimide I or G÷6976, PKC inhibitors with a preference for calcium-dependent isoforms, blocked CREB phosphorylation. Short-term stimulation with the phorbol ester tetradecanoyl phorbol acetate (TPA) which activates calcium-dependent and -independent isoforms but not atypical PKCs was sufficient to promote CREB phosphorylation and activation. Following chronic exposure to TPA that triggered complete down-regulation of all responsive isoforms except PKCα, CREB was still phosphorylated upon myotube depolarization. Immunocytochemical analysis revealed selective and rapid PKC α translocation to the nucleus following depolarization with kinetics similar to those of IP3 production and calcium release. Such nuclear PKCα translocation, as well as CREB phosphorylation and activation, were blocked by the IP3 receptor inhibitor 2-APB and the phospholipase C inhibitor U73122.Our results agree with a general pattern of intracellular signalling that involves DHPR as voltage sensor, activation of phospholipase C, nuclear calcium increase and translocation of PKCα to the nucleus. Subsequent events include CREB phosphorylation and early gene mRNA expression.

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