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Effects of extracellular ATP on freshly isolated mouse skeletal muscle cells during pre-natal and post-natal
Claude Collet1, Caroline Strube, László Csernoch
1Laboratoire de Physiologie des Eléments Excitables, Université Claude Bernard Lyon 1, ERS CNRS 2019, 43 boulevard du 11 novembre 1918, 69622 Villeurbanne, France.
Abstract:
Extracellular adenosine 5'-triphosphate (ATP) has profound effects on membrane conductance and on the intracellular free [Ca(2+)] ([Ca(2+)](i)) in cultured skeletal muscle cells. The aim of the present study was to examine the occurrence and to characterize the properties of such responses during mammalian muscle development in vivo. The effect of ATP (0.2 mM) was tested on membrane current and [Ca(2+)](i) in freshly isolated pre- and post-natal mouse skeletal muscle cells. Pre-natal cells were from 14- to 19-day-old fetuses. In pre- and early post-natal cells, very small elevations of [Ca(2+)](i) (<50 nM) following ATP application could be detected with the fluorescent indicator fura-2. A clear subsarcolemmal rise in [Ca(2+)] was however associated to the presence of ATP, as demonstrated by increased activity of plasma membrane Ca(2+)-activated K(+) channels in cells bathed in a depolarizing, high-calcium-containing solution. In cells voltage-clamped at -80 mV in external Tyrode, ATP induced an inward current associated with an increased membrane conductance. The mean maximal amplitude of the ATP-induced current was -0.84 +/- 0.07 A/F ( n=39). The response to ATP was still present after birth, although its amplitude tended to decrease with post-natal development and was completely absent in muscle cells from 3- to 6-month-old mice. The ATP-induced current could be abolished reversibly by suramin. Our results suggest that, over the range of developmental stages examined, skeletal muscle cells display an ionotropic purinergic signalling pathway with functional properties qualitatively consistent with what is observed in cultured myotubes.
Insights
Extracellular adenosine 5'-triphosphate (ATP) influences membrane conductance and intracellular calcium in developing mouse skeletal muscle. This purinergic signaling pathway is present in pre- and postnatal cells but diminishes with age.
Area of Science:
- Physiology
- Neuroscience
- Cell Biology
Background:
- Extracellular adenosine 5 -triphosphate (ATP) is known to affect membrane conductance and intracellular calcium levels ([Ca(2+)](i)) in cultured skeletal muscle cells.
- However, the presence and characteristics of these ATP-induced responses in developing mammalian skeletal muscle in vivo remain largely uncharacterized.
Purpose of the Study:
- To investigate the occurrence and properties of extracellular ATP-induced responses in freshly isolated mouse skeletal muscle cells during different developmental stages in vivo.
- To characterize the developmental changes in purinergic signaling in skeletal muscle.
Main Methods:
- Studied membrane current and intracellular calcium ([Ca(2+)](i)) in pre- and post-natal mouse skeletal muscle cells (14-19 day fetuses to 6-month-old mice).
- Utilized fluorescent indicator fura-2 for [Ca(2+)](i) measurements and electrophysiological techniques (voltage-clamping) to assess membrane currents and conductance.
- Investigated the effect of suramin on ATP-induced currents.
Main Results:
- ATP application in pre- and early post-natal cells caused minor elevations in [Ca(2+)](i) but clearly increased subsarcolemmal calcium, evidenced by enhanced Ca(2+)-activated K(+) channel activity.
- ATP induced an inward current and increased membrane conductance in voltage-clamped cells, with a mean maximal amplitude of -0.84 +/- 0.07 A/F.
- This ATP response persisted after birth but decreased with postnatal development, becoming undetectable in adult mice (3-6 months old).
- The ATP-induced current was reversibly inhibited by suramin.
Conclusions:
- Skeletal muscle cells exhibit an ionotropic purinergic signaling pathway during development.
- The functional properties of this pathway are consistent with those observed in cultured myotubes.
- The developmental profile suggests a transient role for this specific purinergic signaling in skeletal muscle maturation.