Uptake of p-alanine and synthesis of carnosine (P-alanyl-histidine) could be demonstrated in primary cell cultures derived from embryonic chick pectoral muscle. Concomitant with the morphological changes, cessation of cell division and the induction of creatine kinase, a rapid increase in the rate of p-alanine uptake and also in the rate of carnosine synthesis could be observed. The uptake of p-alanine is sodium and chloride dependent and obeys Michaelis-Menten kinetics with K, values of about 40 pM that are essentially identical for myoblasts and myotubes. In contrast, V,, increases considerably during differentiation. The p-alanine transport system is highly specific for p-amino acids and exhibits a substantial anion dependency (Cl-> J-> CSN-> SO:-). Stoichiometric studies suggest that the transport of one p-alanine molecule involves two sodium ions and one chloride ion. This ratio is not altered by the process of cell differentiation.Carnosine (P-alanyl-histidine) and structurally related peptides (w-aminoacyl amino acids) are well-known constituents of excitable tissues [l, 21. In some muscles of mammals and birds, these peptides even represent the major nonproteinaceous constituents. Since p-alanine is predominantly synthesized by the liver as a final metabolite of the pyrimidine bases uracil and thymine but not by muscle cells directly [3], muscle cells must be equipped with a highly efficient transport system for this amino acid. So far, however, uptake of p-alanine from the peripheral circulation has been extensively studied in kidney [4], brain [5] and, to some extent, also in other tissues [6, 71, but surprisingly not yet in skeletal muscle. Here, we report on the uptake of p-alanine and the synthesis of carnosine by primary cultures of chick pectoral muscle.
MATERIALS AND METHODSHam's F12 nutrient mixture and horse serum were obtained from Gibco. Cells were grown in cell-culture dishes (Nunc) or flasks (Falcon). 120 Ci/mmol p- Transferrin, bovine serum albumin, phosphoglycerate kinase, hexokinase, glucose-6-phosphate dehydrogenase, creatine phosphate, ADP", NADP", P',P5-diadenosine-5'-pentaphosphate, N-acetyl-L-cysteine, NADH* and glycerate-3-phosphate were obtained from Boehringer Mannheim. Trypsin and Dowex 50 WX2 were from Serva Feinbiochemica. BisCorrespondence to K. Bauer, Max-Planck-Institut fur experimentelle Endokrinologie, Feodor-Lynen-StraBe 7, D-30625 Hannover, Germany benzimide H 33258 fluorochrome was purchased from Calbiochem-Nova-biochem. Scintillator Hydroluma (J. T. Baker) was used for scintillation counting. Glass-fiber filters GF51 were obtained from Schleicher & Schuell. All solvents used were from Merck. Embryo extract was prepared according to the procedure of Konigsberg [8].
Cell culturePrimary cultures of myoblasts were isolated from the pectoral muscle of ll-day-old chick embryos. The muscle tissue obtained from 10 embryos was digested with 5 ml Puck's saline D2 solution (137 mM NaCl, 5.5 mM KCI, 0.22 mM KH2P04, 0.168 mM Na,HPO,, 0.11 mM CaCl, and 5.5 mM glucose) containing ...