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Molecular and Cellular Biochemistry

, Volume 158, Issue 2, pp 179–188 | Cite as

The regulation of total creatine content in a myoblast cell line

  • Joseph E. Odoom
  • Graham J. Kemp
  • George K. Radda
Article

Abstract

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 μM) and largely dependent on extracellular [Na+]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na+,K+-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed α- and β-adrenergic agonist noradrenaline, the β-adrenergic agonist isoproterenol, the β2-agonist clenbuterol and the cAMP analogue N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate, but was unaffected by the α1 adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed α- and β-antagonist labetalol and by the β-antagonist propranolol, but was unaffected by the α2 antagonist phentolamine; greater inhibition was caused by the β2 antagonist butoxamine than the β1 antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3′,5-triiodothyronine (at 70 μM) and to 220 ± 35% of control by amylin (60 nM). As 3,3′,5-triiodothyronine, amylin and isoproterenol all stimulate the Na+,K+-ATPase, we suggest that they stimulate Na+-creatine cotransport indirectly by increasing the transmembrane [Na+] concentration gradient and membrane potential.

Key words

amylin creatine insulin-like growth factor 1 Na+,K+-ATPase triiodiothyronine 

Abbreviations

IGF-I

insulin-like growth factor I

IGF-II

insulin-like growth factor II

T3

3,3′,5-triiodothyronine

CGRP

calcitonin gene-related peptide

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Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Joseph E. Odoom
    • 1
    • 2
  • Graham J. Kemp
    • 1
    • 2
  • George K. Radda
    • 1
    • 2
  1. 1.Department of BiochemistryUniversity of OxfordOxfordUK
  2. 2.MRC Biochemical and Clinical Magnetic Resonance UnitOxford Radcliffe HospitalOxfordUK

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