Characterization of anionic amino acid transport systems in mouse mammary gland

L-glutamate was transported into mammary tissue via Na(+)-dependent system XAG- that strongly interacted with both D- and L-isomers of aspartate but only with L-isomer of glutamate. Replacement of Cl- by gluconate from the extracellular medium did not affect the uptake of L-glutamate. Although neutral amino acids weakly inhibited the uptake of L-glutamate, there was no evidence for the heterogeneity of anionic amino acid transport system. The XAG- system was inhibited by sulfhydryl group blocking reagent N-ethylmalemide. Low pH (6) partially inhibited the uptake by L-glutamate by mammary tissue. Prior loading of mammary tissue with L-glutamate slightly down regulated its uptake. Culturing pregnant mouse mammary tissue explants in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin) did not affect appreciably the uptake of L-glutamate.     

Control of amino acid transport in the mammary gland of the pregnant mouse

The regulation of the uptake of the amino acid analog α-aminoisobutyric acid was studied in diced mammary glands from pregnant mice. Stimulation of uptake by insulin was not prevented by inhibitors of protein synthesis; protein synthesis inhibitors decreased uptake by 20%; this response occurred more promptly in insulintreated tissues. Elimination of extracellular amino acids led to a substantial increase in transport which was not abolished by inhibitors of protein synthesis. These results indicate that insulin does not increase amino acid transport in this system by altering synthesis and degradation of transport protein. They are consistent with a model in which the activity of the existing amino acid transport protein is subject to negative feedback regulation from the intracellular amino acid pool.     

Effects of arachidonic acid on RNA metabolism in mammary gland explants of mice

Arachidonic acid stimulated ³H-uridine incorporation into RNA in mammary gland explants of mice in a manner similar to that of prolactin. The onset of the effects of both prolactin and arachidonic acid occurred following a 2–4 hour lag period. Further, effects of maximally stimulatory concentrations of these agents were nonadditive. Finally, indomethacin abolished the effects of both prolactin and arachidonic acid. A tenable hypothesis to explain these data is that the action of prolactin in the mammary gland may be mediated by an increased availability of arachidonic acid followed by a subsequent enhanced rate of formation of the prostaglandins.     

Effects of arachidonic acid on RNA metabolism in mammary gland explants of mice

Arachidonic acid stimulated ³H-uridine incorporation into RNA in mammary gland explants of mice in a manner similar to that of prolactin. The onset of the effects of both prolactin and arachidonic acid occurred following a 2–4 hour lag period. Further, effects of maximally stimulatory concentrations of these agents were nonadditive. Finally, indomethacin abolished the effects of both prolactin and arachidonic acid. A tenable hypothesis to explain these data is that the action of prolactin in the mammary gland may be mediated by an increased availability of arachidonic acid followed by a subsequent enhanced rate of formation of the prostaglandins.     

Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland

The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated Mr of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis [Poulose, A.J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 15953-15958]. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.     

Response of brain amino acid metabolism to ketosis

Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.     

Effect of premature weaning on amino acid uptake by the mammary gland of lactating rats.

1. Arteriovenous differences of amino acids across the lactating mammary gland were measured in normal rats and weaned for 4, 5 and 24h. 2. Uptake of amino acids by mammary glands of rats weaned for 5h or more was significantly lower than that of controls. This was not reversed by injection of prolactin. 3. By using 'unilaterally weaned' rats we showed that milk accumulation plays an important role in amino acid uptake by mammary gland. 4. gamma-Glutamyltransferase activity was significantly lower in 'weaned' glands than in 'normal' glands. This provides further support for the hypothesis of the function of the gamma-glutamyl cycle in the mammary gland in vivo.     

Aspects of amino acid metabolism and nutrition in the ectoparasitoid wasp, Exeristes roborator

Exeristes roborator contains a wide variety of free amino acids, and the composition of all developmental stages was quantitatively dominated by proline and glutamic acid. The latter occurred together with lesser amounts of glycine, alanine, valine, leucine, tyrosine, and histidine which varied between developmental stages. Minor and trace amounts of most other commonly occurring amino acids were also found. The percentages or relative amounts of major constituents were not influenced when the parasite was reared on the alternate hosts, Pectinophora gossypiella and Gnorimoschema operculella. Likewise, the major characteristics of the latter hosts' free amino compositions could not be accounted for, in either case, by their diets.Differences in the relative distribution of the major amino acids between the developmental stages of E. roborator indicate large decreases in the percentage of proline occur during development from the larval to the adult stage with corresponding increases in the percentages of glycine in the pupal stage and alanine as well as other amino acids in the adult stage. The results suggest that proline may play an important rôle in E. roborator, probably as an energy reserve.The amino acid compositions of the total proteins of E. roborator and its hosts were similar and all quantitatively dominated by glutamic acid, aspartic acid, and amides. However, the disk gel electropherograms of the proteins of both hosts and parasite were different. Quantitative changes were evident in the protein pattern of the parasite when reared on the alternate hosts.E. roborator incorporated radioactivity from ¹⁴C(U)-glucose into the amino acids, glutamic acid, aspartic acid, serine, glycine, alanine, and proline. Furthermore, ¹⁴C(U)-glutamic acid was incorporated into a wide variety of proteins. The data suggest that the above amino acids may be non-essential dietary components for E. roborator. However, quantitative determinations indicate that the amount of glutamic acid synthesized does not account for the amount incorporated into protein over the same time period.     

The regulation of Na(+)-dependent anionic amino acid transport by the rat mammary gland.

The regulation of anionic amino acid transport, using radiolabelled D-aspartate as a tracer, by rat mammary tissue explants has been examined. Na(+)-dependent D-aspartate uptake by mammary tissue increased between late pregnancy and early lactation and again at peak lactation but thereafter declined during late lactation. In contrast, the Na(+)-independent component of D-aspartate uptake by mammary explants did not change significantly with the physiological state of the donor animals. Premature weaning of rats during peak lactation markedly decreased Na(+)-dependent D-aspartate uptake by mammary tissue. In addition, premature weaning also reduced the effect of reversing the trans-membrane Na(+)-gradient on the fractional loss of D-aspartate from mammary tissue explants. Unilateral weaning of rats during peak lactation revealed that milk accumulation per se reduced the Na(+)-dependent moiety of D-aspartate uptake by mammary tissue suggesting that the transport of anionic amino acids is regulated to match supply with demand. Treating lactating rats with bromocryptine reduced D-aspartate uptake by mammary tissue explants suggesting that the transport of anionic amino acids by the rat mammary gland is regulated by prolactin.