Abstract
ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca(2+) concentration, with an EC(50) value of 7.8 +/- 3.1 microm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 mum suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y(2) receptor and pannexin-1. As reported previously for electrical stimulation, 500 mum ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca(2+) homeostasis and muscle physiology.
Highlights
(DHPR),2 located in transverse (T) tubules of the plasma membrane, and the ryanodine receptor (RyR), a Ca2ϩ channel present in the sarcoplasmic reticulum
In this work we have found evidence that ATP is released by the physiological activity of skeletal muscle cells and is metabolized extracellularly, that P2Y/P2X functional receptors are expressed in this system, and that calcium transients can be evoked upon nucleotide exposure
In the present work we have demonstrated that nucleotides released to the extracellular medium are the essential mediators in promoting slow calcium signals and can significantly affect fast calcium transients evoked by tetanic stimulation in skeletal muscle cells
Summary
In differentiated cells derived from human skeletal muscle, the activation of P2Y1 receptors by exogenous agonists promotes calcium transients dependent on the IP3 pathway but independent of RyRs, which leads to ERK1/ERK2 activation [40]. Considering all of this background, extracellular ATP and its metabolites could be proposed as muscle plasticity regulators, which opens some very interesting questions about the interaction of nucleotide receptors with proteins such as DHPR, relevant to excitation-contraction and excitation-transcription events. We have postulated that extracellular nucleotides can be crucial mediators between electrical stimulation and both the fast and slow calcium transients involved, respectively, in muscle activity and plasticity
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