INJECTION OF 6-HYDROXYDOPAMINE AND HYDROGEN PEROXIDE INTO THE SUBSTANTIA NIGRA AND LATERAL VENTRICLE OF THE CAT: SPECIFIC AND NONSPECIFIC EFFECTS ON STRIATAL BIOGENIC AMINES1

Attempting to clarify the mechanism by which intracerabral injection of 6-hydroxydopamine (60HDA) reduces catecholamines in the caudate nucleus (CN), we have tested two hypotheses: (1) 60HDA specifically attacks catecholaminergic neurons; (2) 60HDA liberates hydrogen peroxide (H₂O₂) which destroys neurons indiscriminately. To this end, we have injected high or low doses of 60HDA or equimolar amounts of H₂O₂ stereotaxically into the substantia nigra (SN) or the lateral ventricle of cats and have placed electrocoagulative lesions in the SN. We determined the CN levels of dopamine (DA), norepinephrine (NE) and serotonin (5HT) 7-10 days later. Nigral injections of high doses (8 μ mol) of either agent or low doses (80 nmol) of 60HDA decreased both DA and NE and induced similar histologic damage in the SN with neuronal drop-out at the periphery of the lesions. Injection of 80 nmol of H₂O₂ into the SN did not decrease CN amine levels and did not produce histologic damage in the SN. Electrocoagulation of the SN decreased CN DA and NE, but the histologic lesions failed to show any peripheral neuronal drop-out. Ventricular injections of high doses (16 μmol) of 60HDA or H₂O₂ reduced not only DA and NE but also 5HT levels in the ipsilateral CN. Low intraventricular doses (0-16 μmol) of 60HDA decreased only DA and NE without affecting 5HT levels in the CN whereas 0.16 μmol of H₂O₂ had no effect on any of the CN amines. The catecholamine-depleting effects of low doses (80 nmol) of 60HDA were significantly potentiated by inhibiting brain monoamine oxidase by 90 percent or more at the time and site of injection of 60HDA. These results suggest that the extracellular liberation of H₂O₂ from 60HDA could explain some possibly nonspecific effects of high doses of 60HDA; at lower doses, however, 60HDA may act via selective uptake into catecholaminergic neurons with subsequent intracellular release of H₂O₂.     

ALTERATION OF CELLULAR PROLIFERATION IN THE ILEAL EPITHELIUM OF SUCKLING AND WEANED RATS: THE EFFECTS OF ISOPROTERENOL

The purpose of this study was to analyse cell proliferation in the ileum before and after neonatal closure to macromolecules and to determine the effects of the sympathomimetic amine, isoproterenol (IPR), on cell cycle parameters. 8- and 28-day-old rats were employed in this study representing the neonatal periods of pre- and post-closure ileum respectively. The duration of the cell cycle phases was determined by the percentage of labeled metaphases technique (PLM) with computerized analysis of the curves. The generation cycle time was longer in 8-day-old suckling rats (18.63 hr) as compared to the older weaned rats (11.85 hr), and most of this 6.78 hr difference was in the G₁-period (4.5 hr). Other proliferative indices were also lower in the suckling rats—mitotic index (1.9 ± 0.4% as compared to 6.7 ± 0.9%), labelling index (26.8 ± 2.5% versus 44.2 ± 2.6%) and migration rate measured as per cent labeled villus cells (8.9 ± 3.1% versus 45.2 ± 3.4%). IPR was found to inhibit cellular proliferation in the ileal epithelium of both age groups. The cell cycle of the ileal epithelium of 8-day-old rats was lengthened from 18.63 to 21.07 hr and 28-day-old rats from 11.85 to 13.98 hr. IPR produced a decrease in mitotic index from 1.9 ± 0.4% to 1.6 ± 0.4% in pre-closure ileum and from 6.7 ± 0.9% to 5.1 ± 0.6% in post-closure ileum. Labeling index decreased from 26.8 ± 2.5% to 20.0 ± 2.0% in 8-day-old rats and from 44.2 ± 2.6% to 31.1 ± 3.0% in 28-day-old rats after IPR administration. There were also significant differences in growth fraction between age groups and a significant decrease in growth fraction after IPR-treatment. From the results of this study it appears that β-adrenergic stimulation has an inhibitory effect on neonatal ileal epithelium.     

MINOR COMPONENTS OF THE DNA OF PHYSARUM POLYCEPHALUM : Cellular Location and Metabolism

DNA metabolism in the slime mold Physarum polycephalum was studied by centrifugation in CsCl of lysates of cultures labeled with radioactive thymidine at various times in the cell cycle. During the G₂ (premitotic) phase of the cell cycle, two components of the DNA are labeled. One component is lighter (buoyant density 1.686 g/cc) than the mean of the principal DNA (1.700 g/cc), and one is heavier (approximately 1.706 g/cc). The labeled light DNA was identified chemically by its denaturability, its susceptibility to DNase, and the recovery of its radioactivity in thymine. Cell fractionation studies showed that the heavy and the principal DNA components are located in the nucleus and that the light DNA is in the cytoplasm. The light DNA comprises approximately 10% of the DNA. About ⅓–½ of the light DNA is synthesized during the S period, and the remainder is synthesized throughout G₂ (there is no G₁ in Physarum). The light DNA is metabolically stable. A low, variable level of incorporation of radioactive thymidine into the principal, nuclear DNA component was observed during G₂.     

THE DISTRIBUTION OF LABELED NOREPINEPHRINE WITHIN SYMPATHETIC NERVE TERMINALS STUDIED WITH ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The distribution of radioactivity in association with sympathetic nerve terminals and intraneuronal organelles 30 min after the administration of tritiated norepinephrine (NE-³H) was studied by electron microscope radioautography with recently developed quantitative methods of analysis reported in the accompanying paper (Salpeter et al., 1969). Nerves from the pineal body and the adrenal capsule were examined. It was found that nerve terminals containing vesicles were heavily labeled. (These terminals were not necessarily in contact with some innervated structure.) There was no selective labeling of either the intraneuronal mitochondria or the relatively small population of large (~1000 A) dense core granules. Small vesicles (~500 A), some of which have a dense internal granule, could not be analysed separately because they are closely packed and occupy ~60% of the volume in terminals. Because of the extensive distribution of these small granular and agranular vesicles in the radioactive terminals, they remain the most likely site for norepinephrine binding. Yet although the vesicles were uniformly distributed within the nerve terminals, it appears that the radioactivity was not. There appeared to be a somewhat higher concentration of radioactivity at the periphery of the nerve terminals than in the center. The usefulness of the method of analysis used in this study for determining the location of bound H³NE pools in the nerve is discussed.     

RENEWAL OF GLYCEROL IN THE VISUAL CELLS AND PIGMENT EPITHELIUM OF THE FROG RETINA

The renewal of glycerol in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with [2-³H]glycerol. Assay of chloroform:methanol extracts showed that the labeled precursor was used mainly in lipid synthesis, although there was also some utilization in the formation of protein. Radioactive glycerol was initially concentrated in the myoid portion of rods and cones, indicating that this is the site of phospholipid synthesis in visual cells. The glycogen bodies (paraboloids) of accessory cones were also heavily labeled, suggesting the diversion of some glycerol into glycogenic pathways. In the pigment epithelium, only the oil droplets became significantly radioactive. The outer plexiform layer (which contains the visual cell synaptic bodies) and the cone oil droplets gradually accumulated considerable amounts of labeled material. Within 1–4 h, labeled molecules began to appear in the visual cell outer segments, evidently having been transported there from the myoid portion of the inner segment. Most of these were phospholipid molecules which became distributed throughout the outer segments, presumably replacing comparable constituents in existing membranes. In rods only, there was also an aggregation of labeled material at the base of the outer segment due to membrane biogenesis. These highly radioactive membranes, containing labeled molecules of lipid and protein, were subsequently displaced along the rod outer segments due to repeated membrane assembly at the base. The distribution of radioactivity supported the conclusion that membrane renewal by molecular replacement is more rapid for lipid than it is for protein.     

The Phosphorylation of Ribosomal Protein in Lemna minor

Sterile cultures of Lemna minor have been labeled with ³²P₁, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with ³²P₁, calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome.     

THE USE OF RADIOACTIVE IODINE IN THE TREATMENT OF GRAVES' DISEASE

Fifty patients with uncomplicated Graves'' disease were treated with radioactive iodine (I₁₃₁). Twenty-six patients who were followed for one year or longer are the basis of this report. Twenty-five are now euthyroid; only one is not completely well.The total dose of radioiodine administered varied from 0.5 to 10 millicuries. The average length of time necessary for return to a euthyroid state was from three to four months.Hypometabolism developed in three patients, and in one the signs and symptoms of myxedema developed. No other complications ensued. One patient who apparently relapsed had complete return to normal after further iodine administration.The determination of the uptake of radioactive iodine by the thyroid gland is a useful diagnostic procedure in differentiating conditions simulating hyperthyroidism.Following treatment with radioactive iodine, the thyroid gland becomes smaller, the uptake of iodine by the gland is reduced, and the level of organic iodine in the plasma becomes normal.In acute thyroiditis, in spite of a high basal metabolic rate, high content of organic iodine in the plasma and other evidences of “hyperthyroidism,” the uptake of I₁₃₁ has been very low.     

Central noradrenergic activity and the formation of glycol sulfate metabolites of brain norepinephrine

The present study utilized intraventricular injection of Na₂³⁵SO₄ to detect drug induced changes in the $\text{in vivo}$ formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihyd (DOPEG-SO₄). Assays involved the hypothalamus only. Rats pretreated with clonidine showed a reduced formation of both MOPEG-³⁵SO₄ and DOPEG-³⁵SO₄ after intraventricular Na₂³⁵SO₄ as well as reduced synthesis of ³H-NE from intraventricular ³H-tyrosine. Phenoxybenzamine (POB) produced increases in the synthesis of both ³⁵S-labeled conjugates and ³H-NE. Neither drug altered the loss of exogenous ³H-MOPEG-SO₄ but clonidine increased both the accumulation of labeled sulfate and the sulfation of exogenous MOPEG in pheniprazine treated rats. These results show that the rates of formation of the labeled glycol sulfates are sensitive indicators of changes in brain NE turnover but can also be influenced by factors involved in sulfation that are unrelated to NE turnover. Blockade of NE synthesis with alpha methyltyrosine did not affect resting or POB-elevated levels of the labeled conjugates until stores of NE were reduced by 40%. The latter findings suggest that central noradrenergic neurons can release and metabolize NE at a normal rate despite synthesis blockade so long as adequate stores of NE are available.     

DISTRIBUTION OF LABELED CHROMATIN : I. M1 and M2 Anaphases of Diploid and Tetraploid Cultured Mammalian Cells

The question of whether distribution of chromatids to daughter cells in mitosis is a random or nonrandom process was investigated by study of the distribution of labeled chromatin in anaphase pairs at M₁ and M₂ after a pulse of tritiated thymidine. Diploid and tetraploid rat and diploid human fibroblast-like cells in serial monlayer culture were synchronized by two different methods to "purify" M₁ and M₂ anaphases: metaphase shake, and FUdR block to DNA synthesis followed by exogenous thymidine. Exposed grains of NTB-2 emulsion were counted over M₁ and M₂ anaphase pairs. An analysis (by pair) of diploid M₂ anaphase grain counts showed two discrete populations of daughters with less and with more radioactivity. A similar analysis of diploid M₁ and tetrapolid M₂ anaphases showed a single grain-count distribution. These findings may support a nonrandom model of chromatid segregation for diploid mammalian cells but do not rule out random segregation until sound mathematical models are formulated for expected random grain distributions in M₂ anaphases of cells with differing numbers of chromosomes.     

Inhibition of the Extraneuronal Uptake of Catecholamine in the Isolated Rat Heart by Cholesterol

EARLIER work has demonstrated that the removal of catecholamines from plasma involves several different mechanisms: uptake into sympathetic nerve terminals¹; extraneuronal uptake (“Uptake₂”) into cardiac and smooth muscle and other tissues², a process which is subsequently followed by metabolism of the amine^3,4 and active transport across kidney tubules followed by excretion⁵. Various steroids, including corticosterone, 17-β-oestradiol and deoxycorticosterone, can selectively inhibit the extraneuronal uptake of noradrenaline (Uptake₂) in the isolated perfused rat heart^6,7. The inhibition of Uptake₂ occurred at concentrations of steroid which exceeded those normally found in the plasma and is thus unlikely to be of physiological significance under normal conditions. We have now found, however, that cholesterol which is present in plasma at much higher concentrations than any other steroid^8,9 also acts as an inhibitor of the Uptake₂ mechanism.     

PREFERENTIAL LABELING OF RAT LYMPHOCYTES WITH A RAPID RATE OF TURNOVER BY TRITIATED DEOXYCYTIDINE

Lymphocytes in thymic cortex and germinal centers of lymphoid tissues are labeled intensely with generally labeled tritiated deoxycytidine [G-³H]dCyd whereas they are weakly labeled with methyl tritiated deoxythymidine [methyl-³H]dThd of the same specific activity, not only by single injection but also by an intensive injection schedule. [G-³H]dCyd can be used to label short-lived lymphocytes strongly, although not specifically. The distribution patterns of labeled lymphocytes were different depending on the injection schedules of [G-³H]dCyd. [G-³H]dCyd can be used as a precursor molecule for cytosine and also thymine found in DNA. The ratios of radioactive thymine to cytosine measured biochemically on DNA extracted from radioactive lymphocytes labeled by the various schedules indicate strongly that short- and long-lived lymphocyte populations have different abilities to utilize pyrimidine nucleosides for DNA synthesis.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

Effects of catecholamines on fetal rat cardiocytes in vitro

Norepinephrine stimulates the growth in size of non-dividing, neonatal cardiac muscle cells, and it can stimulate the growth in numbers of dividing hepatocytes and endothelial cells in culture. The objective of this study was to test the hypothesis that in dividing fetal cardiocytes, norepinephrine would stimulate growth in cell number rather than in cell size. Fourteen-day fetal heart cells were placed in serum-free or serum-supplemented cultures in the presence or absence of norepinephrine (NE), NE plus propranolol, or isoproterenol for 4 days. Almost 90% of the cardiocytes in serum-supplemented medium were in the cell cycle as determined by proliferating cell nuclear antigen (PCNA) antibody staining during this period. In addition, between days 2 and 4 of culture, 35% and 40% of these cardiocytes were labeled with 3H-thymidine. After 4 days the cardiocytes increased in cell number in the serum-supplemented NE cultures as compared to serum-free cultures. In contrast, there was no significant change in cardiocyte volume between any of the groups examined. It was concluded that in dividing muscle cell populations the effect of norepinephrine was to enhance cell proliferation rather than to stimulate cell growth in size.     

ON THE SITE OF SULFATION IN COLONIC GOBLET CELLS

The location of bound S³⁵ in the goblet cell of the rat colon at time points from 2 to 60 minutes after administration of S³⁵ as sodium sulfate has been observed in vivo and in vitro by radioautographic techniques. Grains were first observed by electron microscopy over the stacked lamellae of the paranuclear part of the Golgi apparatus. The label was subsequently found associated with the supranuclear Golgi lamellae and was then seen associated with the smooth membranes limiting the mucin granules in the goblet. Finally, between ½ and 1 hour, the secreted mucus product in the crypts became radioactive. Neither mitochondria nor the endoplasmic reticulum was labeled. It is concluded that the Golgi apparatus is the organelle in which sulfation occurs.     

THE CYTONUCLEOPROTEINS OF AMEBAE : I. Some Chemical Properties and Intracellular Distribution

Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.     

INTRANEURAL DISTRIBUTION OF EXOGENOUS NOREPINEPHRINE IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

Catecholaminergic neurons, which take up and retain exogenous norepinephrine labeled with tritium, were studied by means of high resolution radioautography, in the substantia nigra, the substantia grisea periventricularis, and the locus coeruleus of the rat. Under the conditions required for the radioautographic detection of exogenous norepinephrine-³H, it was established that (1) glutaraldehyde was the most suitable fixative for preserving the labeled amine in situ; (2) norepinephrine-³H itself, rather than metabolites, accounted for most of the reactions detected in catecholaminergic neurons. At various time intervals after an intraventricular injection of norepinephrine-³H, the tracer reached a concentration 15–100 times higher, and disappeared at a slower rate, in presynaptic axons (t½:4 hr) than in nerve cell bodies (t½:0.8–1.3 hr). After pretreatment with a monoamine oxidase inhibitor, the radioautographic reactions increased and persisted longer, especially in the preterminal axons. Within neurons, the labeled amine was ubiquitously distributed in the nerve cell body and concentrated in presynaptic axons and synaptic terminals of various morphological types. Although large granular vesicles were usually present in the labeled axonal bulbs, no structural characteristic could be specifically ascribed to catecholaminergic neurons. It is suggested that exogenous norepinephrine bound to macromolecular complexes is present in all parts of catecholaminergic neurons and mainly concentrated within presynaptic axons.     

Ultrastructure and Atrial Natriuretic Peptide(ANP)-like Immunoreactivity of Cardiocytes in the Larval, Metamorphosing and Adult Specimens of the Japanese Toad, Bufo japonicus formosus

The distribution of an atrial natriuretic peptide (ANP)-like material in the cardiocytes of larval, metamorphosing, and adult specimens (both breeding and non-breeding) of the toad, Bufo japonicus formosus , was studied immunohistochemically, ultrastructurally and immunocytochemically. Histochemically, ANP-immunoreaction was positive in the atrium and ventricle in stage 33 larvae, while negative in the ventricle in stage 40 larvae. In adult toads, the reaction was stronger in the right than in the left atrium but quite weak in the ventricles, particularly those of non-breeding specimens. Electron microscopy indicated a very small number of secretory granules in the atrial and ventricular cardiocytes of embryos as early as the limb-bud stage (stage 28), and as development proceeded, the number of these granules increased rapidly in atrial but not in ventricular cardiocytes. In metamorphosing animals, a small population of larger granules (200–250 nm) was noted next to those of ordinary size (the median, 110 nm) in the same cell. In adult toads, granules of about 120 nm and 200 nm in median size were found in the same cell. Postembedding immunogold staining consistently indicated ANP-immunoreactivity in these granules in atrial and ventricular cardiocytes. The plasma content of immunoreactive ANP was considerably higher in breeding (20.5 ± 5.9 pg/ml) than in non-breeding toads (5.4 ± 1.7 pg/ml). These results are discussed in relation to presently available data on the physiological role of ANP.     

ON THE SPATIAL ARRANGEMENT OF PROTEINS IN MICROSOMAL MEMBRANES FROM RAT LIVER

Rat liver rough microsomes were labeled enzymatically with ¹²⁵I using lactoperoxidase and glucose oxidase. In intact microsomes only proteins exposed on the outside face of the microsomal membrane were iodinated. Low concentrations of detergent (0.049% deoxycholate) were used to allow entrance of the iodination system into the vesicles without disassembling the membranes. This led to iodination of the soluble content proteins and to an increased labeling of the membrane proteins. The distribution of radioactivity in microsomal proteins was analyzed after separation by sodium dodecyl sulfate acrylamide gel electrophoresis. Most membrane proteins were labeled when intact microsomes were iodinated. No major membrane proteins were exclusively labeled in the presence of low detergent concentrations or after complete membrane disassembly. Therefore it is unlikely that there are major membrane proteins, other than glycoproteins, present only on the inner membrane face or completely embedded within the microsomal membrane. Microsomal proteins were also labeled by incubating rough microsomes with [³H]-NaBH₄ after reaction with pyridoxal phosphate. Microsomal membranes were permeable to these small molecular weight reagents as shown by the fact that proteins in the vesicular cavity as well as membrane proteins were labeled with this system.