SYNCHRONIZATION OF BABY RAT HEPATOCYTES: A NEW TEST FOR THE DETECTION OF FACTORS CONTROLLING DNA SYNTHESIS IN RAT HEPATIC CELLS

The subcutaneous injection of irritating substances to baby rats results in a very reproducible wave of synchronized S phase DNA synthesis in hepatic cell involving 20% of the total population. Use has been made of this reaction to detect factors affecting DNA synthesis in hepatic cells. It enables substances to be tested during precise periods of the cell cycle. Two activities which were detected in normal adult rat serum, could not be found in the serum of the baby rat or of the partially hepatectomized adult rat: an activity inhibiting the progression of hepatocytes through the cell cycle in the late G₁ phase, and an activity inducing the production of binucleate hepatocytes, effective in the late G₁ and in the S phase.     

HYDROXYUREA: USE IN DETERMINING THE DURATION OF G2 IN TETRAHYMENA

Observation of division of individual cells in microdrops, plus autoradiographic studies using tritiated thymidine and standard cell cycle analysis techniques, reveal that hydroxyurea (10 DIM) reversibly arrests the normal progression of exponentially growing Tetrahymena pyriformis through the initial 92 % of S-phase while not affecting cells in the terminal 8 % and in G₂ and division. Thus the fraction of the population of cells that is in G₂ can be approximately determined by the fraction of the population able to divide in the presence of hydroxyurea. This fraction can be related to the approximate duration of G₂ by calculations which compensate for the age gradient.     

Epidermis Extracts (Chalone) Inhibit Cell Flux At the G1-S, S-G2 and G2-M Transitions In Mouse Epidermis

Epidermal cell flux at the G₁-S, S-G₂ and G₂-M transition was examined during the first 4 hr after injection of epidermis extract. the flux parameters were estimated by a combination of several methods. the G₁-S and S-G₂ transit rates were calculated on the basis of a double labelling technique with [³H]TdR, the G₂-M flux by means of colcemid and the relative proportion of cells in the S or G₂ phase by means of flow cytometry. All experiments were performed both in early morning and late evening, corresponding to maximum and minimum rates of epidermal cell proliferation in the hairless mouse. the epidermis extract inhibited the S-G and G₂-M transit rates to the same degree, while the inhibition of cell flux at the G₁-S transit was consistently stronger. In general, the inhibition of cell flux at the different transitions was most pronounced when the rate of cell proliferation was low and vice versa.     

PROPERTIES OF G0 CELLS: VARIATIONS IN THE PROLIFERATIVE RESPONSE FOLLOWING ISOPRENALINE

The proliferative behaviour induced in the acinar cells of the rat submaxillary gland in response to isoprenaline has been used to examine the transit time of cells from a quiescent (G₀) state into the S phase. Cumulative ³H-TdR labelling index curves were constructed to determine the mean time interval (G_is time) between stimulation with isoprenaline and entry into the S phase. Data were collected for the proliferative wave induced by three sequential injections of isoprenaline, and the effects of varying the interval between the second and third injections of isoprenaline, and of changing the dose of the drug, were examined. Intervals of 28, 52 and 76 hr between isoprenaline injections resulted in mean G_is times of 16-2, 20-9 and 25-6 hr respectively. It was concluded that the G_is time depended on the recent history of cells with respect to stimulation, but not division. The results are considered in terms of two models, in one of which the time to leave G₀ is variable, whilst in the other the cells leave G₀ immediately the stimulus is applied.     

ON THE EXISTENCE OF 'ARRESTED G2 CELLS' IN MOUSE EPIDERMIS

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G₁-S boundary and the other between G₂ and mitosis. the ‘arrested G₂ cells’ were estimated, by the labelled mitosis method, to comprise 510% of the epidermal population and presumed to function as a ‘reserve pool’ which could be activated by wounding. A comprehensive search has now been carried out for arrested G₂ cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G₂ cells were not lost during the isolation of epidermal nuclei.     

LOCATION OF THE G0-PHASE IN THE CELL CYCLE OF THE MOUSE HAEMOPOIETIC SPLEEN COLONY FORMING CELLS

Experiments in mice on the fraction of haemopoietic stem cells in S-phase after irradiation indicated that a large fraction of the cells resting in G₀ will enter S-phase after a very short interval of time.
After excluding alternative explanations it must be concluded that cells in G₀ have completed all preparations for going into S-phase or, in other words, that the localization of these G₀ cells in relation to other phases of the cell cycle must be between G₁ and S-phase.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

STUDIES ON LIVER REGENERATION: I. 131IODODEOXYURIDINE AS A PRECURSOR of DNA IN NORMAL and REGENERATING RAT LIVER

Abstract. ¹³¹Iododeoxyuridine (¹³¹IDU) was injected into normal and partially hepatectomized rats, and the specificity of incorporation of this thymidine analogue into liver DNA was determined 2, 24 and 48 hr following intramuscular injection. At 2 and 24 hr after ¹³¹DU injection, a major proportion of radioactivity in the liver was in the acid-soluble fraction, whereas 48 hr after injection the label in the acid-soluble fraction had decreased considerably. In liver obtained 2 hr after injection of ¹³¹IDU, only 1.8–16.6% of the total radioactivity were in DNA. If, however, the tissue was subjected to formalin fixation, the acid-soluble label was extracted selectively, and of the remaining radioactivity 64–88% was in DNA. Therefore, the radioactivity that is not extracted by formalin may be used as a measure of DNA synthesis at the time of injection of ¹³¹IDU, thus obviating time-consuming biochemical fractionation procedures.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

TRANSPORT OF 14C INCORPORATED PROTEIN IN THE CORTICOSPINAL TRACT OF THE RAT BRAIN

Abstract— The origin of fibres of a corticospinal pathway in rat brain was located by cortical ablation and Marchi staining and also by electrical stimulation of the motor cortex.
Enzyme changes investigated histochemically over 0–14 days post-injection of 5 μ10.15 m NaCl into the neocortex indicated that very little apparent disturbance of nerve cell metabolism beyond a narrow band adjacent to the path of the microneedle within the cortex had occurred. [¹⁴ C]Leucine as a precursor of protein synthesis was used to study incorporation of the amino acid into protein. At the site of injection the maximum level of labelled protein was recorded at 30 min post-injection, the level decreasing to less than 2 per cent of this at 6 hr.
The subsequent axonal flow of labelled protein along the corticospinal pathway was investigated during the period 15 min to 21 days post-injection. Within 24 hr increasing amounts of labelled proteins were measured caudally, but not more than 6 per cent had migrated beyond 5 mm from the site of injection. At 3 days this percentage had increased to 14.6 per cent, the labelled proteins being distributed in progressively decreasing amounts to a further 13 mm caudal. Very little change from this position was seen during the following 18 days.     

HISTAMINE-SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5-HT) and drugs such as isoprenaline (ISP), 2-(2-pyridyl)ethylamine (H₂ stimulant—H_ls), 4-methyl-histamine (H₂ stimulant H_2s), mepyramine (H₁ antagonistp H_la), cimetidine (H₂ antagonist—H_2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.
The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H_2s, induced a greater stimulation of adenylate cyclase than H_ls. On the other hand, the H_2a inhibited HA stimulation to a greater extent than the H_la, H_la and H_2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans-mitter role in the hypothalamus, and in this area there appears to be a mixed population of H₁ and H₂ receptors, with a majority of H₂ receptors.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

BRAIN TYROSINE HYDROXYLATION: ALTERATION OF OXYGEN AFFINITY IN VIVO BY IMMOBILIZATION OR ELECTROSHOCK IN THE RAT

Abstract— The rates of brain tyrosine and tryptophan hydroxylation, estimated in vivo from the accumulation of DOPA and 5-hydroxytryptophan after the administration of a decarboxylase inhibitor, appear dependent on the availability of oxygen as a substrate. During two types of physical stress, electroshock and curare-immobilization, the rate of brain tyrosine hydroxylation was greater than in unstressed controls and was not significantly decreased when the stresssed animals were made hypoxic. The loss of oxygen dependence by brain tyrosine hydroxylation during stress was observed in several brain regions and was not associated with alterations in the concentrations of brain tyrosine. tryptophan, serotonin, dopamine or norepinephrine. The rate of brain tryptophan hydroxylation was not affected by stress and remained oxygen dependent. The increase in catecholamine synthesis during stress appears to be the result of increased catecholaminergic nerve impulse flow. These experiments are consistent with the hypothesis that during neuronal stimulation an allosteric change in tyrosine hydroxylase increases the affinity of the enzyme for oxygen allowing greater catecholamine synthesis despite limiting concentrations of this substrate.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.