title: Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells.
abstract: Receptor sites, specific for guanosine 5'-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca2+ concentration ([Ca2+]i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (Kd = 15.4 +/- 4.6 microM; Bmax = 1.7 +/- 0.5 nmol mg(-1) protein); and a low affinity site (Kd = 170 +/- 94.5 microM; Bmax = 14.2 +/- 3.9 nmol mg(-1) protein). In myotube membranes only a low affinity binding site for GTP (Kd = 169 +/- 39 microM; Bmax = 12.3 +/- 1.4 nmol mg(-1) protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca2+]i. The patterns of Ca2+ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca2+ responses: (1) a fast increase in [Ca2+]i, followed by a sustained [Ca2+]i elevation, and (2) a slow raising and steady prolonged increase in [Ca2+]i. In myotubes, however only fast Ca2+ responses were observed following the addition of 500 microM GTP. In the myoblasts and myotubes GTP-stimulated [Ca2+]i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca2+ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle.
authors.affiliation: Dipartimento di Scienze del Farmaco, Università G. d'Annunzio, Chieti, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Pietrangelo
authors.forename: Maria A
authors.initials: MA
authors.lastname: Mariggiò
authors.forename: Paola
authors.initials: P
authors.lastname: Lorenzon
authors.forename: Stefania
authors.initials: S
authors.lastname: Fulle
authors.forename: Feliciano
authors.initials: F
authors.lastname: Protasi
authors.forename: Michel
authors.initials: M
authors.lastname: Rathbone
authors.forename: Eva
authors.initials: E
authors.lastname: Werstiuk
authors.forename: Giorgio
authors.initials: G
authors.lastname: Fanò
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D032448:Myoblasts, Skeletal / Q000187:drug effects / Q000378:metabolism*
chemical_list: D006160:Guanosine Triphosphate; D002118:Calcium