Thomas R. Riggs

Transport of amino acids into the estrogen-primed uterus I. General characteristics of the uptake in vitro☆

Abstract The uptake of free amino acids by the immature rat uterus has been studied in vitro . 1. 1. The uptake of the model amino acid, α-aminoisobutyric acid, showed properties of active transport. It was saturable; it occurred against a gradient of the free amino acid; it could be decreased by metabolic inhibitors such as cyanide, 2,4-dinitrophenol and lack of O 2 ; and it was temperature sensitive. It was also decreased by the presence of high levels of other amino acids or of K + , and by the absence of extracellular Na + . 2. 2. Under appropriate conditions, 1 μg estradiol injected into the rats could increase the uptake in vitro of α-aminoisobutyric acid, l -serine, l -alanine, glycine, l -proline, l -lysine, 1-aminocyclopentanecarboxylic acid, l -valine and dl -norleucine. As short a time as 1 h in vivo was sufficient for this level of the hormone to stimulate uptake of α-aminoisobutyric acid, 1-aminocyclopentanecarboxylic acid, l -alanine and l -proline. A 10 −6 M level of estradiol added in vitro stimulated α-aminoisobutyric acid uptake if incubation periods were extended to 6 h. 3. 3. Estradiol increased uptake at all α-aminoisobutyric acid levels tested (0.3–10 mM). Approximation of kinetic constants showed that the hormone increased the ν max of α-aminoisobutyric acid uptake without altering its K m .

Action of insulin on amino acid uptake by the immature rat uterus in vitro.

SUMMARY 1. The uptake of ~-aminoisobutyric acid by the immature rat uterus was studied kinetically in vitro, and the stimulatory action of inzulin on uptake was characterized. Other amino acids were also studied for comparison. 2. ~-Aminoisobutyric acid uptake was increased if the uteri were pretreated with insulin for 20 rain or more. The V of the saturable transport was increased by 19 ~ by insulin treatment, but no Kr, change was apparent. 3. Puromycin eliminated the insulin action on e-aminoisobutyric acid uptake within 40 min of treatment. 4. The effect of insulin on ~-aminoisobutyric acid uptake was abolished either by a preliminary 10-min incubation of the uterus with a high concentration (1 mM) of N-ethylmaleimide; or by incubation, concurrent or subsequent, with a low concentration of N-ethylmaleimide (0.05 mM). 5. The uptake of L-proline, L-methionine and glycine, but not that of Lleucine or L-valine, was also increased by insulin treatment. Insulin acted on a Na +dependent uptake; it was without effect on the Na+-independent uptake of either e-aminoisobutyric acid or methionine. The evidence suggests that insulin acts on the A system of amino acid transport in the uterus.

Transport of amino acids into the estrogen-primed uterus IV. Effects of sulfhydryl agents on the uptake in vitro

1. n1. Sulfhydryl agents have been found to stimulate uptakes of some amino acids by the immature rat uterus in vitro, and to inhibit the uptakes of others. Those whose uptakes were stimulated can enter the cells well by the L transport system for amino acids, while those inhibited use the A system. Uptake of 1-aminocyclopentanecarboxylic acid (cycloleucine), which probably involves both systems, could be stimulated or inhibited, depending on the conditions. n n2. n2. The results suggest that the uterus contains at least two kinds of groups that react with SH agents and are involved in amino acid transport. N-Ethylmalcimide combines relatively rapidly with one type, apparently involving the L system to produce stimulation of amino acid transport; but it combines more slowly with the other, the A system, to inhibit transport. n n3. n3. The stimulation of amino acid uptake by estradiol, which probably involves the A system, can be inhibited by N-ethylmaleimide. This inhibition, however, is apparently not due to an action of N-ethylmaleimide to prevent a possible binding of the hormone to necessary uterine sites. n n4. n4. The stimulation of amino acid uptake by N-ethylmaleimide appears to be independent of the stimulation by estradiol.

Developmental Changes in the Neutral α-Amino Acid Transport Systems of Rat Brain Over the First Three Weeks After Birth

Abstract: Transport of seven different amino acids into brain slices increased as donor rats aged from 1 to 6 days. Uptakes of 2‐aminoisobutyric acid, 2‐(methyl‐amino)isobutyric acid, and L‐alanine then decreased by day 14, while uptakes of other amino acids continued to increase or remained fairly constant. Neutral α‐amino acid transport systems were characterized by measuring inhibition of uptakes and kinetics for representative amino acids at different ages. Results indicate that 2‐ami‐noisobutyrate and 2‐(methylamino)isobutyrate used only one (and the same) system in brain slices from 6‐day‐old rats, with characteristics of system A (the major sodium‐dependent system in most mammalian cells). They used at least two systems at ages 1, 14, and 23 days, but, of these, only at 1 day did they use the same systems in the same proportions. Alanine and leucine used more than one system at all four ages, and somewhat different combinations than used by each other or by 2‐aminoisobu‐tyrate or 2‐(methylamino)isobutyrate. Their transport characteristics showed they used mostly system ASC (a sodium‐dependent system distinguished from A) and/or system L (sodium‐independent). We conclude that system A increases as the brain ages from 1 to 6 days and declines thereafter. System L probably increases with aging from 1 to 23 days.

Thyroxine-Induced Changes in the Development of Neutral Α-Amino Acid Transport Systems of Rat Brain

Abstract: Transport of representative neutral α‐amino acids was measured in brain slices after injecting thy‐roxine into donor rats of various ages from 1 to 23 days old. The hormone did not alter uptake in slices from 1‐day‐old rats even when treatment was begun on pregnant rats as much as 10 days before delivery. Injecting thy‐roxine until age 6 days, however, decreased the activity of transport system A (the major sodium‐dependent system in most mammalian cells) and caused appearance of a new transport system used by the model amino acids, 2‐aminoisobutyric acid and 2‐(methylamino)isobutyric acid. Uptake at 6 days was similar to that found in slices from older, untreated rats (e.g., those 14 days old). These results strongly suggest that one action of thyroxine is to accelerate the development of neutral α‐amino acid transport systems of brain over the first six days after birth. Thyroxine treatment of rats from birth to age 14 days also appears to increase the activities of both system A and the second transport system used by the two model amino acids in brains from 14‐day‐old rats.

Use of labeled nonmetabolized amino acids in biochemical research.

The biochemical investigator usually is pleased to find that a large quantity of C14 or other tracer atom has been transferred from his starting compound to another substance. This article will consider a group of compounds for which metabolic modification to other substances can scarcely be detected, and the experimental applications which this property may permit.

Distribution of α-aminoisobutyric-C14 acid in rats deficient in thiamine, pantothenate, fatty acids, or potassium

A nutritional deficiency of fatty acids in young rats resulted in a marked elevation of the steady-state serum level of an injected dose of ol-aminoisclbutyric acid, and in a decrease in the transport of this model amino acid into skeletal muscle, heart, and intestine. The liver level was greatly increased. Adrenalectomy of the deficient rats reduced the uptake in both liver and kidney. These changes were similar to those reported earlier for rats deficient in vitamin Be. No consistent pattern of change in distribution of the model amino acid was found in rats deficient in thiamine, pantothenate, or potassium. The extremely high liver levels of the amino acid seen in thiamine-deficient rats could be returned toward normal by removing the adrenal glands. All of the changes found in the K+-deficient animals were brought essentially to normal by bilateral adrenalectomy. No consistent relationship was found between the uptake of the amino acid and the Kf level in the heart or skeletal muscle of the rats in the control, K+-deficient, and the Kc-deficientadrenalectomized groups. The accumulation of amino acids by taken from vitamin Be-deficient mice (l), Ehrlich mouse ascites tumor cells in vitro and for normal uptake of the model amino has been found to be increased by a number acid, a-aminoisobutyric acid, by all tissues of unrelated biological agents, including examined in rats in vivo (5; cf. Ref. (6) for a pyridoxal and pyridoxal phosphate (l), more detailed discussion of the relations e&radio1 disulfate and estrone sulfate (2), between vitamin Be and amino acid transand several auxins (1). In addition, the port). Estradiol-170, on the other hand, accumulation is dependent upon the normal alters the distribution of model amino acids K+ and Na+ content of the tumor cells (3). in vivo only in those tissues that are suscepti