Ralph K. Rhines

COMPARATIVE UPTAKE OF DL-LYSINE-H3 BY NORMAL AND REGENERATIVE HYPOGLOSSAL NERVE CELLS IN EUTHYROID, HYPOTHYROID AND HYPERTHYROID MALE RATS.

The uptake of DL-lysine-H3 by normal and regenerating hypoglossal nerve cells was studied radioautographically in euthyroid, hypothyroid and hyperthyroid male rats. The radioactive material taken up in the brain tissue was identified by ascending paper chromatography as being lysine. Regenerating cells in all three groups were significantly larger than normal cells and showed a significant increase in the amount of radioactive material present per cell. Since cell enlargement paralleled uptake of lysine, the actual concentration of labeled material in normal and regenerating cells in euthyroid rats was found to be the same. The effect of the hypothyroid state was to increase uptake in both normal and regenerating cells as compared with comparable cells in euthyroid rats. The hyperthyroid state itself had no effect on uptake of lysine on normal cells, but did increase the uptake in regenerating cells. The significance of the increased uptake in regenerating cells and of altered total body metabolic state on lysine uptake is discussed with reference to the blood-brain barrier.

Prenatal exposure to methadone HCL in relationship to body and brain growth in the rat

Offspring from methadone‐treated Wistar rats (last trimester of pregnancy), as compared to ad lib and pair fed controls, showed a reduction of body growth which continued throughout the 28‐day period during which animals were observed after birth. Brain growth, as indicated by weight, cortical thickness and number of cells in the neocortex also showed a reduction in growth, which was apparent only during the first 14 days, after which there were no differences between the groups. However, in the hippocampus, neuronal density changes/unit area continued throughout the 28‐day period in the methadone exposed group and to a lesser extent in the PF group suggesting that this area of brain did not return to normal dimensions or degree of maturation by the end of the third week.

THE UPTAKE OF S35-L-METHIONINE INTO THE BRAIN OF EUTHYROID AND HYPERTHYROID NEONATAL RATS

Thyroid hormone alters the morphology, electrical excitability and integrative activity of the brain of the suckling rat (Eayrs , 1960). The hormone also influences amino acid and protein metabolism in non neural tissue; low doses increasing and high doses decreasing protein synthesis (Hoch, 1962). The experiment reported here reveals that, in ten-day old thyrotoxic male rats, there is an increased incorporation of S35 methionine into both the whole brain and its bound protein fraction.

THE EFFECT OF AGE ON THE UPTAKE AND DEGRADATION OF THYROID HORMONE BY THE BRAIN AND SKELETAL MUSCLE

In recent years a number of investigations have been concerned with the manner in which various factors may influence the uptake and degradation of thyroid hormone by the central nervous system (CNS). Among those factors studied have been the effect of species differences (Ford & Gross 195 th 1958b; Taurog et al . 1956; Ford e t al. 1962), the effect of sex hormones (Ford et al. 1962, 1964), the influence of dysthyroidal states on the metabolism of triiodothyronine (Ts) by brain and other tissues (Ford 1960, 1961; Ford e f at. 1959) and variations in metabolism of T3 by different parts of the brain. (Ford & Gross 1958a, 195813; Ford e f al . 1962). There is evidence suggesting that thyroid hormones may be directly involved in growth processes (Evans et al. 1939; Scow e f al. 1949) and be directly related to protein synthesis (Sokoloff e f al. 1962). Studies comparing young and adult animals have shown that protein synthesis by the microsomal fraction of cells was greater in young rats (Gerin & Tipfon 1965, Grief & Kandemir 1965) and humans (Suzuki e t al. 1964) than in adults. Furthermore, growth and repair of neuronal tissue seems to be augmented by thyroid hormone (Schneck e f al. 1964; Rhodes e f al. 1964). Thus, i t has been concluded that thyroid hormone in some way appears to influence synthesis of protein and growth of neuronal tissue. These observations suggest the possibility that there might be differences in the metabolism of thyroid hormone a t various ages related to the differences in the general metabolic characteristics of the various stages of maturation and development in the rat.

The rate of uptake and radioautographic localization of S35 in the central nervous system, pituitary, and skeletal muscle of the normal male rat after the injection of S35-labeled cystine.

Several studies which demonstrate or suggest an uptake of various amino acids by the central nervous system (CNS) have been reported by a number of workers. Of these, perhaps methionine has been the most thoroughly investigated. Cystine, which may be derived from methionine, has received scant attention despite its presence in the neurosecretory peptides. Therefore, the following investigations were performed to determine the rate and sites of uptake of S35-l-cystine into the CNS, pituitary, and muscle after either intravenous or intraperitoneal injection. Thirty-five adult and six 5-week-old male rats were used. The data were obtained by means of radioautography or by gas-flow couning of tissue samples following various combustion procedures. A definite uptake of S35 into the CNS and other tissues was noted in both adult and young animals, the uptake in the young males being 6.5 times higher than in adults. The S35 activity in the brain was shown by ascending paper chromatographic analysis of several hydrolyzed brain samples to be mainly associated with cystine. The presence of some TCA-precipitable S35 (presumably as cystine) in the brain after intravenous injection suggests that some of this highly insoluble amino acid, which has apparently passed through the blood-brain barrier, has also become incorporated into neural protein.

Accumulation of [131I]triiodothyronine in neurons and other tissues following intravenous injection of the labeled hormone

Abstract The accumulation of 131 I in motor neurons, liver, muscle and spinal cord grey matter after intravenous injection with 131 I-labeled triiodothyronine was determined by liquid scintilattion counting procedures. It was observed that the thyroid hormone which accumulates in the brain (spinal cord) was largely present in the neurons, which had significantly higher accumulations than did masses of spinal grey matter which would contain comparable motor neurons. The nerve cell 131 I activity was also significantly higher than that seen in striated muscle. Liver, which takes up thyroid hormones very rapidly, had levels of radioactivity which were significantly higher than muscle, spinal grey matter, plasma or ventral horn motor neurons at most time intervals investigated. A chromatographic analysis of unfixed, ethanol (pH 8.5) extracted neurons revealed that most of the 131 I detected in the motor neuron samples was associated with triiodothyronine, which is comparable to what has been previously reported for whole brain after intravenous injection of [ 131I ]triiodothyronine.

UPTAKE OF C14 INTO THE BRAIN AND OTHER TISSUES OF NORMAL AND DYSTHYROIDAL MALE RATS AFTER INJECTION OF C14‐L‐GLUTAMINE

Studies dealing with brain protein metabolism have become increasingly frequent in recent years ( H y d i n 1962, Laj tha 1964, IVaelsch & Lajtha 1961). Brain amino acid uptake, while apparently restricted in the total amount taken up when compared with other organs, has been reported to occur for most of the amino acids found in the brain ( C h i rigos et al. 1960, Dingman & Sporn 1959, Ford ct d . 1961, Ford e f al. 1966, Gaitonde 1961, Guroff & Udenfriend 1962, Laj iha 1964, Laj tha & Toth 1961, Kask in & Fishman 1966) and appears to be depcndcnt on sl)ecific transport systems (Chris tensen 1959, Laj tha 1964, Tsukacla et al. 1963). Furthermore, incorporation of amino acids into brain protein seems to be influcmced by local rcquirements for protein synthesis (Ford el al. 1961, Ford et al. 1965, Laj tha 1964, Richter c.1 al. 1959). Indecd, a number of recent investigations suggest that such local biochemical or physiological conditions are more significant in effecting the rate a t which materials penetrate the membranes separating the blood from the neurons than any physical aspect of the membranes or the materials studied ( D e R o p p & Snpdeker 1961, Flexner et al. 1963, Hclkkinen & Kulonen 1961, Rliodes e t al. 1964). Hypothyroidism is one change in physiological state of an animal which has been shown to influence (depresses) protein synthesis in the nervous system of adult animals ( F o r d el al. 1965, Rhodes et al. 1964, Sokoloff 1961). Although Gelber e f al. (1964) have reported that hyperthyroidism has no effect on the total amount of protein formed by the brain of adult animals, hyperthyroidism has been observed to

The uptake of I131-labeled-l-triiodothyronine by the brain, pituitary, and muscle of diestrous and estrous female rats as compared with the uptake in male rats ☆

The effect of the estrous cycle and estrogen treatment on the uptake of intravenously injected I131-labeled-triiodothyronine (T3) by the brain, pituitary, thyroid, and muscle of the female rat was investigated. No alteration in uptake or degradation rate of I131-T3 in brain, pituitary, or muscle was observed which could be attributed to estrogenic hormones. There was, however, a slightly increased rate of uptake of I131 by the thyroid glands of estrogen treated animals. The possibility that this is related to increased TSH production is discussed. While there was no difference in I131 uptake between groups of diestrous and estrous female rats, it was observed that uptake and degradation of T3 in female rats was in general somewhat different than in normal male rats. Thus, the mean total uptake of I131 by brain, pituitary, and muscle tissues was higher in the females, degradation rate appeared faster, and there was some evidence that formation of the T3-glucuronide complex by the liver and kidney in female be different. The possibility that the change in uptake of T3 by tissues in female rats may be related to differences in the formation of the T3 glucuronide complex, or to the release of T3 from the complex is discussed.

3H) LYSINE ACCUMULATION IN MOTONEURONS IN RATS OF DIFFERENT AGE COMPARED WITH THE ACCUMULATION IN OTHER TISSUES

In recent years the accumulation of most amino acids by the brain has been well demonstrated by both i n uiuo (Altiszan 1964, Chirigos et al. 1964, Roberts et al. 1959, Roberts & Morelos 1965, and Schain et al. 1967) and in vitro (Adams & Lini 1966, Blasberg & Lajtha 1966, Lajtha & Toth 1962, Leui et al. 1965, Murthy & Rappoport 1964, Neaine 1961, Yoniogaini e t al. 1966) experiments. A rapid efflux of amino acids has also been demonstrated (Laj tha & Toth 1964, Levi et al. 1965). This is sufficiently large to suggest that no significant net uptake occurs in slices or in blocks of tissue taken from whoie brain following intravenous injection into intact animals. The failure to observe a large net accumulation of non-labeled or isotopically labeled amino acids by the brain from the blood has most often been attributed to the restrictive effects of a blood-brain barrier (Laj tha 1962). However, recent reports (Ford et a1 1965, Ford & Rhines 1967) demonstrating sizeable accumulations of labeled amino acids specifically into neurons following intravenous injection suggest that the restriction of transport of an amino acid from blood into brain may not be as marked as previously believed. These studies dcmonstrate a high level of accumulation of lysine, tyrosine, and glutamine, csscntially only into neurons. This accumulation would to a considerable extent be masked by the very low accumulation occurring in ihe large bulk of the surrounding neuropi1 and while matter if the analyses were performed on the very heterogenous blocks of tissue which contain these cells. The determination of the amount of labeled material actually present in these neurons in distinction from that occurring in blocks of brain discloses the significantly high levels of uptake which occur in neurons.

ADENINE-H3 UPTAKE IN NERVOUS TISSUE, INCLUDING REGENERATING NERVE CELLS, AS COMPARED WITH OTHER TISSUES IN EUTHYROID, HYPO- AND HYPERTHYROID MALE RATS

In recent years, the number of studies on the biochemistry of the brain has become quite large. Numerous original and review articles have dealt with the presence, uptake and incorporation of amino acids into brain protein and with the turnover rates of brain protein (Abadom & Scholefield 1962, Bert & Waelsch 1958, Brattgdrd et al. 1957, Chirigos et al. 1960, Flexner et al. 1962, Gaitonde 1961, Koenig 1958, Lajtha 1964, Rhodes et al. 1964, Schneck et al. 1964). The metabolism of lipids (Folch-Pi & LeBaron 1958, Rossiter 1957) and uptake and utilization of various inorganic ions (Keynes 1957, Shanes 1962) have been extensively reported. Interest in nucleic acids has also been widespread, particularly in relation to its roIe in protein synthesis. Morphologic studies (Einarson 1957, La-Velle 1958, Tewari & Bourne 1962) have demonstrated a relationship between Nissl substance (ribonucleoprotein (RNA-P) ) and the nucleolus with the functional state of the cell, particularly in regard to synthetic processes. These studies have been amplified by the investigations of HydCn and cu-workers (Brattgdrd et ~ l . 1957, Hamberger 1963, HgdCn 1961) which illustrate changes in the RNA content of neurons with changing physiologic states. This has also been indicated by Pakkenberg (19631, using different techniques. Thc presence and nature of the free nucleotides and nucleosides in brain tissue have also been investigated to a considerable degree (McZZwain 1957). Reports dealing with the uptake and incorporation of nucleic acid precursors are much less frequently encountered. Koenig (1958) observed an uptake of adenine-8-C14 and orotic acid-6-C14 into neurons