Characterization of Methylaminoisobutyric Acid Transport by System A in Rat Mammary Gland Armando R. Tovar, Euclides Avila, Soledad DeSantiago, and Nimbe Torres During lactation, the mammary gland has a large demand for amino acids for the synthesis of milk proteins and fatty acids. Arteriovenous differences in amino acids across the mammary gland show an elevated uptake of small neutral amino acids that are mainly transported via system A. The purpose of this study w as to characterize the transport of methylaminoisobu- tyric acid (M eAIB), an amino acid analog used to model transport by system A in lactating rat mammary gland explants. M eAIB accumulation in mammary gland cells increased steadily, and after 3 hours of incubation, the intracellular concentration of the analog w as 8-fold higher than the concentration in the medium. M eAIB transport into mammary gland explants show ed a K m of 3.3  0.4 mmol/ L and a maximal velocity (Vmax) of 555  23 pmol/ L intracellular fluid (ICF) · min, indicating a system with high capacity but low affinity for its substrate. M eAIB transport into mammary tissue depended highly on Na  , and the uptake w as inhibited by addition of natural and analog small neutral amino acids. Cationic, anionic, and large neutral amino acids did not reduce M eAIB transport into mammary gland explants. Preincubation of mammary gland explants in an amino acid–free medium stimulated MeAIB transport, suggesting an adaptive regulation. The addition of an equimolar mixture of alanine, glycine, and serine to the preincubation medium inhibited stimulation of M eAIB transport. Furthermore, stimulation of M eAIB uptake by amino acid starvation was also prevented by the addition of actinomycin D, cycloheximide, tunicamycin, and colchicine. Dibutyryl cyclic adenosine monophosphate (cAMP) increased MeAIB uptake, whereas phorbol 12-myristate 13-acetate (PM A) did not stimulate M eAIB transport. During the first postw eaning days, kinetic analyses show ed a decrease of 27% in the Vmax. Injection of rat lactating mammary gland mRNA into Xenopus laevis oocytes induced expression of the MeAIB transport system; however, the induction was only 83% above background MeAIB uptake. The results of this study provide a partial explanation for the formation of the metabolic pool of small neutral amino acids in the lactating mammary gland. Copyright  2000 by W.B. Saunders Company T HE LACTATING MAMMARYgland has a large demand for amino acids to sustain the synthesis of milk protein, oxidation of amino acids (mainly branched-chain amino acids), 1 and synthesis of fatty acids. 2 Arteriovenous amino acid concen- tration differences across the mammary gland indicate that amino acids are extracted from the blood to the mammary gland. Amino acid uptake into the mammary gland depends on the type of amino acid and the species. 3-5 The capacity of amino acid extraction in the mammary gland depends on specific transport systems localized in mammary cells. 4,6 Identification and characterization of the amino acid transport systems in the mammary gland are required to understand how the supply of amino acids, in part, may regulate the process of milk-protein synthesis and the formation of other milk components. Arteriovenous differences in amino acids across the mam- mary gland of the rat indicate uptake of all amino acids, with alanine and glutamine being the most actively transported. 5 These amino acids are preferentially transported by system A in several tissues. 7 This system has been identified in mouse, rat, and bovine mammary tissue. 3,8,9 System A preferentially trans- ports short straight-chain neutral amino acids, as well as the amino acid analog methylaminoisobutyric acid (MeAIB), which is therefore used as the model to identify amino acid transport via system A. 10 Amino acid transport via system A into mammary gland tissue has been characterized by measuring -amino isobutyric acid (AIB) uptake in the presence of saturating amounts of MeAIB or 2-aminobicyclo (2,2,1) heptane- 2-carboxylic acid (BCH), using the latter as a model for the L system. AIB uptake in many tissues, especially the liver, is achieved via the A and alanine, serine, cysteine (ASC) system. 11 AIB in the mammary gland is transported proportionally by the A and L systems. 9 Therefore, it is important to further character- ize transport system A of the lactating mammary gland using MeAIB as its specific amino acid probe. The aim of the present study was to determine the kinetic and ion-dependent parameters of MeAIB transport, as well as the response of system A in the mammary gland to amino acid deprivation and to cyclic adenosine monophosphate (cAMP). The metabolic role of system A is fundamental for the entry of small neutral amino acids that are required not only to sustain adequate rates of protein synthesis but also as a source of precursor molecules for energy and as osmolytes. 7,12 The molecular analysis of system A has been difficult, since several distinct proteins probably contribute to the entire system A transport activity. 13-15 In this study, we also identify system A activity from rat mammary gland by functional expression in Xenopus oocytes. MATERIALS AND METHODS Animals Lactating Wistar rats (200 to 250 g) at 12 days postpartum with 8 to 10 suckling pups were used throughout the study. The animals were maintained on a 12-hour light/dark cycle and allowed free access to water and a chow diet. Rats at the end of lactation (21st day postpartum) were separated from their young by removing the pups from the mother’s cage, and are referred to as ‘‘postweaning dams.’’ Nonpreg- nant nonlactating rats were used as control rats, and are referred to as From the Departamento de Fisiologı ´a de la Nutricio ´n, Instituto Nacional de la Nutricio ´n ‘‘Salvador Zubira ´n,’’ Me ´xico, D.F. Submitted August 9, 1999; accepted December 20, 1999. Supported by the National Council of Science and Technology (0593P-M) of Me ´xico. Address reprint requests to Nimbe Torres y Torres, PhD, Departa- mento de Fisiologı ´a de la Nutricio ´ n, Instituto Nacional de la Nutricio ´n, Vasco de Quiroga No. 15, Tlalpan, Me ´xico, D.F., 14000 Me ´xico. Copyright  2000 by W.B. Saunders Company 0026-0495/00/4907-0001$10.00/0 doi:10.1053/mt.2000.6731 Metabolism, Vol 49, No 7 (July), 2000: pp 873-879 873