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Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells
Tiziana Pietrangelo1, Maria A Mariggiò, Paola Lorenzon
1Dipartimento di Scienze del Farmaco, Università G. d'Annunzio, Chieti, Italy.
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.
Insights
C2C12 mouse skeletal muscle cells exhibit two guanosine 5'-triphosphate (GTP) binding sites in myoblasts but only one in myotubes. GTP also influences intracellular calcium (Ca2+) levels differently in these cell types.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Guanosine 5 -triphosphate (GTP) plays crucial roles in cellular signaling pathways.
- Understanding GTP receptor sites in skeletal muscle cells is important for elucidating muscle development and function.
- C2C12 cells, a well-established mouse skeletal muscle cell line, provide a valuable model for studying muscle differentiation.
Purpose of the Study:
- To characterize guanosine 5 -triphosphate (GTP) receptor sites in C2C12 myoblasts and myotubes.
- To investigate the effect of GTP on intracellular calcium ([Ca2+]i) concentrations in these cells.
- To explore the potential role of GTP binding sites in skeletal muscle development.
Main Methods:
- Binding experiments were performed to identify and quantify GTP binding sites.
- Intracellular calcium ([Ca2+]i) concentrations were measured using fluorescence indicators.
- The effects of GTP, ATP, UTP, and purine receptor antagonists (suramin, Reactive Blue 2) were assessed.
Main Results:
- Two distinct GTP binding sites (high and low affinity) were identified in myoblasts, while only a low affinity site was found in myotubes.
- GTP addition induced intracellular calcium ([Ca2+]i) increases in both myoblasts and myotubes, with different response patterns observed.
- GTP-induced calcium responses were specifically abolished by suramin or Reactive Blue 2, suggesting a purinergic receptor involvement.
Conclusions:
- C2C12 cells undergo changes in GTP binding site characteristics during differentiation from myoblasts to myotubes.
- A high-affinity GTP binding site may play a significant role in the early stages of skeletal muscle development.
- Purinergic signaling involving GTP is implicated in calcium regulation during skeletal muscle cell differentiation.