Transport of β‐alanine and biosynthesis of carnosine by skeletal muscle cells in primary culture

Abstract

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 ...

Role of Glutamate Decarboxylase-like Protein 1 (GADL1) in Taurine Biosynthesis

“…All the currently known ␤-alanine-synthesizing enzymes have low expression levels in muscles, where the demand for ␤-alanine is great; accordingly, it is generally considered that the intramuscular ␤-alanine is transported from elsewhere to make carnosine (13). A study with chicken embryonic muscle cell culture indicated that a ␤-amino acid transporter is present in the cells (45); hence the sarcoplasmic ␤-alanine delivery is the rate-limiting factor for muscle carnosine synthesis. ␤-Alanine supplementation has been exploited to boost the blood ␤-alanine concentration, drive the carnosine synthesis in muscles, and improve muscle performance (46).…”

Carnosine-related dipeptides in the mammalian brain

“…Carnosine synthetase (EC 6.3.2.11), whose activity is 98% cytosolic (Harding and O'Fallon, 1979), is an enzyme characterized by a broad substrate specificity, capable of synthesizing different aminoacyl-histidine dipeptides (e.g., carnosine, homocarnosine, anserine; Winnick and Winnick, 1959;Kalyankar and Meister, 1959;Ng and Marshall, 1978;Horinishi et al, 1978). The synthesis has been demonstrated to occur in different tissues, including muscle (Bakardjiev and Bauer, 1994;Bauer and Schulz, 1994) and brain (Horinishi et al, 1978). A carnosinase (EC 3.4.13.3) and an homocarnosinase responsible for the degradation of these compounds have also been identified in different tissues (Rosemberg, 1960;Harding and Margolis, 1976;Lenney, 1976;Lenney et al, 1977;Ng et al, 1977;Ng and Marshall, 1978;Harding and O'Fallon, 1979;Margolis et al, 1983;Margolis and Grillo, 1984b).…”

“…Several lines of evidence suggest that carnosine synthetase is not saturated with its substrate in physiological conditions and that the immediate precursor β-alanine may play a role in regulating tissue carnosine levels (Margolis et al, 1985). In cell cultures, biosynthesis of carnosine has been demonstrated to occur in muscle cells and in glial cells (Bauer et al, 1982;Bakardjiev and Bauer, 1994;Hoffmann et al, 1996;Bakardjiev, 1997). By contrast, no significant synthesis of homocarnosine was described, probably due to the fact that glial cells rapidly degrade γ-aminobutyric acid, a precursor of homocarnosine.…”

Muscle Carnosine Metabolism and β-Alanine Supplementation in Relation to Exercise and Training

“…β-alanine can be obtained through the hydrolysis of dipeptides extracted from dietary meat or fish and through the degradation of uracil in the liver [10]. Bakardjiev and Bauer [11] have shown that the transsarcolemmal transport of one molecule of β-alanine requires two sodium ions and one chloride ion.…”