Carbohydrate metabolism of hepatocytes from starved Japanese quail

Hepatocytes were isolated from livers of mature male and female starved Japanese quail (Coturnix coturnix japonica). The hepatocytes take up lactate and dihydroxyacetone extensively, and have a very high rate of glucose synthesis from these substrates. Fructose uptake and incorporation into glucose is much less. Pyruvate and alanine are taken up extensively, but form little glucose. There is negligible lipogenesis in cells of starved quail. Alanine increases up to 10-fold incorporation of ³HOH and ¹⁴C from several substrates into fatty acids, but it remains insignificant as compared to lipogenesis by cells of fed quail. There is little utilization of glucose, even in the presence of alanine, in marked contrast to hepatocytes from fed quail. However, glucose is phosphorylated at high rates, but most of the glucose 6-phosphate is recycled to glucose. There is a marked difference in the metabolism of polyols between the sexes. Glycerol, xylitol, and sorbitol are converted nearly quantitatively into glucose by hepatocytes of starved female quail. In cells of starved males, the uptake of polyols is higher, but conversion to glucose less efficient. In cells of starved male quail, alanine markedly stimulates the uptake of glycerol and xylitol and their conversion to glucose, but has no effect on sorbitol metabolism. In cells of female quail, alanine is without a significant effect on polyol metabolism.     

Effects of chronic hemodialysis on thyroid function in chronic renal failure

Thyroid function was studied in 54 patients undergoing chronic hemodialysis. Serum thyroxine, triiodothyronine and free thyroxine and the free thyroxine index were significantly lower than normal. The levels of both serum thyroxine and the free thyroxine index tended to fall progressively the longer the patients were on hemodialysis. These findings, in association with low serum TSH levels and normal increase in radioactive iodine uptake by the thyroid after TSH injection, suggest that a defect in pituitary secretion of TSH may be responsible. Although some patients experienced symptomatic improvement after treatment with L-thyroxine the efficacy of this form of treatment in patients on chronic hemodialysis has not yet been established.     

Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture

Primary cultures of normal astroglia started from the cerebral hemispheres of neonatal rats took up dopamine (DA) and norepinephrine (NE) in the concentration range of 10^?7 to 10^?4M and metabolized each to their respective principal central nervous system products by the actions of both catechol-0-methyl transferase and monoamine oxidase. At 10^?7M, uptake of ³H labelled DA and NE was inhibited by omission of Na⁺, addition of ouabain or lowered temperatures. Uptake at 10^?4M was considerable but was Na⁺-independent. Only Na⁺-independent uptake was seen in primary cultures started from the meninges of neonatal rats. These data suggest that astroglial cells in the CNS have a high affinity uptake system for catecholamines, and such uptake is followed by catecholamine metabolism.     

Controlled Clinical Trial of Combined Triiodothyronine and Thyroxine in the Treatment of Hypothyroidism

A -double-blind crossover trial of a combined preparation of triiodothyronine and thyroxine (1:4 ratio) compared with thyroxine alone was conducted with 99 patients previously stabilized on. thyroxine as treatment for hypothyroidism. Four patients were excluded during the trial and eight afterwards owing to gross deficiencies in taking the tablets. Of the remaining 87 patients 42 (48%) had no -preference for either medication, 29 (33%) preferred thyroxine alone, and 16 (18%) the combination. A high incidence of unpleasant symptoms was experienced during the two months'' treatment with the combined preparation. The serum protein-bound iodine levels were lowered (mean reduction 1·8 μg./100 ml.) on the combination, but the labelled T₃-resin sponge uptake values were not altered and remained in the normal range. on both treatments.     

Uptake and Utilization of Xylem-borne Amino Compounds by Shoot Organs of a Legume

Amino compounds representative of the major N solutes of xylem sap were pulse-fed (10 to 20 minutes) singly in ¹⁴C-labeled form to cut transpiring shoots of white lupin (Lupinus albus L.). ¹⁴C distribution was studied by autoradiography and radioassays of phloem sap, leaflet tissues, and shoot parts harvested at intervals after labeling. Primary distribution of N by xylem was simulated using a 20-minute labeling pulse followed by a 30-minute chase in unlabeled xylem sap. Shoots fed ¹⁴C-labeled asparagine, glutamine, valine, serine, or arginine showed intense labeling of leaflet veins and marked retention (35 to 78%) of ¹⁴C by stem + petioles. Shoots fed ¹⁴C-labeled aspartic acid or glutamic acid showed heaviest ¹⁴C accumulation in interveinal regions of leaflets and low uptake (11 to 20%) of ¹⁴C by stem + petioles. Departing leaf traces were major sites of uptake of all amino compounds, and the implications of this were evaluated. Fruits acquired only 1 to 5% of the fed label directly from xylem, but more than doubled their intake during the period 30 to 160 minutes after feeding through receipt of ¹⁴C transferred from xylem to phloem in stem and leaves. ¹⁴C-Labeled asparagine and valine transferred directly from xylem to phloem, but the ¹⁴C of ¹⁴C-labeled aspartic acid and arginine appeared in phloem mainly as metabolic products of the fed compound. The labeling of the soluble pool of leaflets reflected these differences. The significance of heterogeneity in distribution and metabolism of xylem amino compounds in the shoot was discussed.     

Regulation of H Excretion : EFFECTS OF OSMOTIC SHOCK

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H⁺ excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H⁺ uptake, but osmoregulation can still occur, and enhanced H⁺ uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H⁺ rather than affecting only H⁺ or counter ion uptake.     

UPTAKE OF GUANINE BY THE DIATOM,PHAEODACTYLUM TRICORNUTUM1

Nitrate-cultured cells of Phaeodactylum tricornutum Bohlin lack the ability to take up guanine but can do so after a period of nitrogen deprivation, i.e. photosynthesis in nitrogen-free medium. Maximum rate of uptake occurred after 24 h of nitrogen deprivation. The development of ability to take up guanine required CO₂ fixation and was prevented by cycloheximide, ammonium or nitrate. The guanine taken up accummulated in the cells almost entirely as a compound which is probably methylated hypoxanthine. Guanine uptake was dependent upon metabolism and exhibited Michaelis-Menten like kinetics with a half-saturation value of 0.48 ± 0.05 μM guanine and a maximum uptake rate for guanine of ca. 200 nmol · 10^?8 cells · h^?1. Rate of uptake increased hyperbolically with Na⁺ concentration, with 8.25 mM Na⁺ supporting half-maximal rate, and it was inhibited by K⁺ ions.     

GM2-Ganglioside Metabolism In Situ in Mucolipidosis IV Fibroblasts

Mucolipidosis IV (ML IV) is an inherited lysosomal disorder for which the primary biochemical defect has not been identified. In order to detect any defect in glycosphingolipid metabolism, we have examined the metabolism of sphingosine-labeled (³H)G_M2 in situ in fibroblasts from patients diagnosed with ML IV. Fibroblasts were exposed for 10 days in medium containing (³H)G_M2 (15 uM; Sp. Act. 35000 cpm/nmole), washed, harvested and analyzed for radioactivity in extracted lipids. Control cells metabolized about half of the internalized ganglioside, mostly to ceramide. In ML IV fibroblasts, 70–80% of the cellular radioactivity was present as G_M2 indicating reduced degradation. This is not as severe as in G_M2 gangliosidosis as a small amount of G_M2 was metabolized in ML IV cells to ceramide. Since there is no defect in the lysosomal enzyme profile in these cells, it is possible that an abnormality in the translocation of membrane constituents to the lysosomes may explain the slower ganglioside metabolism.     

Properties of three distinct pyrimidine transport systems in yeast. Evidence for distinct energy coupling

In Saccharomyces cerevisiae the uptake of cytosine, uracil and uridine is mediated by three permeases. Using mutants blocked in the metabolic utilization of these three compounds we were able to study their specific uptake. Cytosine and uridine show simple saturation kinetics, whereas uracil uptake is a biphasic process. A comparison of the effects of several inhibitors of energy metabolism on these uptake systems was made. Striking differences were found. 2,4-Dinitrophenol (10^?3 M) and NaN₃ (10^?2 M) inhibit the entry of the three compounds to similar extent, but chlorhexidine (10^?5 M) and Dio 9 (50 μg/ml) which are ATPase inhibitors in vitro strongly impaired cytosine and uridine entry and remained without effect on uracil uptake.We provisionally conclude that these systems may be energized by different mechanisms. In the case of cytosine and uridine permease, a membrane ATPase is possibly involved in the process of energetic coupling whereas this does not seem to be so for uracil.     

KINETICS OF GLUCOSE TRANSPORT AND GROWTH OF CYCLOTELLA CRYPTICA REIMANN,LEWIN AND GUILLARD1,2

Growth rate as well as rate of glucose uptake of C. cryptica depends on glucose concentration in the medium according to saturation kinetics. The K _g for growth is 1.9 × 10^?5 M, and the K _t, for glucose transport is 5.8 × 10^?5 M. The maximum growth rate in the dark on glucose is considerably slower than the light-saturated growth rate at the same temperature, and does not appear to be determined by the capacity of the cell for glucose uptake. The glucose transport process is highly specific, and depends on energy metabolism. The Q ¹⁰ for the process is 2.2 (15–2.5 C). Glucose taken up by the cells is almost, quantitatively phosphorylated within 10 min, either through the transport process itself or by a high affinity kinase system in the cells.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

In a previous communication we reported that glucose deprivation from KHRB medium resulted in a marked stimulation of Ca²⁺ uptake by brain tissue, suggesting a relationship between glucose and Ca²⁺ homeostasis in brain tissue [17]. Experiments were carried out to investigate the significance of glucose in Ca²⁺ transport in brain cells. The replacement of glucose with either D-methylglucoside or 2-deoxyglucose, non-metabolizable analogues of glucose, resulted in stimulation of Ca²⁺ uptake just as by glucose deprivation. These data show that glucose metabolism rather than glucose transfer was necessary to stimulate Ca²⁺ uptake in brain tissue. Inhibition of glucose metabolism with either NaF, NaCN, or iodoacetate resulted in stimulation of Ca²⁺ uptake similar to that produced by glucose deprivation. These results lend further support for the concept that glucose metabolism is essential for Ca²⁺ homeostasis in brain. Anoxia promotes glucose metabolism through glycolytic pathway to keep up with the demand for ATP by cellular processes (the Pasteur effect). Incubation of brain slices under nitrogen gas did not alter Ca²⁺ uptake by brain tissue, as did glucose deprivation and the inhibitors of glucose metabolism. We conclude that glucose metabolism resulting in the synthesis of ATP is essential for Ca²⁺ homeostasis in brain. Verapamil and nifedipine which block voltage-gated Ca²⁺ channels, did not alter Ca²⁺ uptake stimulated by glucose deprivation, indicating that glucose deprivation-enhanced Ca²⁺ uptake was not mediated by Ca²⁺ channels. Tetrodotoxin which specifically blocks Na⁺ channels, abolished Ca²⁺ uptake enhanced by glucose deprivation, but had no effect on Ca²⁺ uptake in presence of glucose (controls). These results suggest that stimulation of Ca²⁺ uptake by glucose deprivation may be related to Na⁺ transfer via Na-Ca exchange in brain.     

Effect of sugars,Na+-, and K+-ions on biopterin transport in Crithidia fasciculata

Biopterin uptake by Crithidia fasciculata is pH dependent with optimum at pH 6 and is strongly inhibited by 0.5 mM NAA and DNP,respectively. Both inhibitors also reduce respiration by 40% (NAA) and 97% (DNP). K⁺-ions (1.1%) and K⁺/Na⁺ (0.5% each) stimulate biopterin uptake to the same high extent, but ouabain has no effect, thereby ruling out involvement of Na⁺/K⁺ pump. In absence of these ions, even in 5% glucose solution biopterin uptake is reduced to minimum. Proton excretion seems to be linked to sugar uptake. Both these sugars seem to have the same site of entry, demonstrated by competitive uptake, though D-glucose is taken up much faster by Crithidia than D-galactose. DNP (0.5 mM) causes greater proton excretion in glucose than in galactose medium. With NAA (0.5 mM) proton excretion is inhibited in both glucose and galactose media. D-glucose promotes greater biopterin uptake than D-galactose.     

Assessment of [125I]WYE-230949 as a Novel Histamine H3 Receptor Radiopharmaceutical

Histamine H₃ receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer''s disease and obesity. We set out to evaluate the novel compound, [¹²⁵I]WYE-230949, as a potential radionuclide imaging agent for the histamine H₃ receptor in brain. [¹²⁵I]WYE-230949 had a high in vitro affinity for the rat histamine H₃ receptor (K_d of 6.9 nM). The regional distribution of [¹²⁵I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H₃ receptor. Rat brain uptake of intravenously injected [¹²⁵I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [¹²⁵I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [¹²⁵I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [¹²⁵I]WYE230949 is not a potential radiotracer for imaging rat histamine H₃ receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.     

The Uptake of Cytokinins by Protoplasts and Root Sections of Brassica campestris

The effect of cytokinins was studied on the incorporation of ¹⁴C-labelled precursors into the nucleic acid fraction of protoplasts isolated from callus or roots of Brassica campestris. Protoplasts from callus and roots took up ¹⁴C-uridine from the incubation medium and incorporated this precursor into the ribonucleic acid fraction during the experimental period of 16 h. Low concentrations of kinetin (10^?8-5 × 10^?6M) did not stimulate the incorporation, and kinetin inhibited this process at higher concentrations (5 × 10^?5M). This result led to an investigation on the uptake of cytokinins by protoplasts of roots. In contrast to a rapid uptake of radio-actively labelled adenine and uridine. protoplasts from roots took up only small amounts of labelled kinetin. zeatin, zeatin riboside and zeatin nucleotides from the incubation medium. Root sections took up far more adenine and kinetin than protoplasts from roots. The ratio between the amount of kinetin taken up and applied was much higher for the sections than for protoplasts, indicating that intact root cells took up kinetin far more rapidly than protoplasts. It is suggested that the plasmalemma and cell wall play an essential role in the uptake of cytokinins or that the differences in the uptake rates are related to differences between the rates of metabolism of cytokinins in root sections and in protoplasts.     

Examination of Reptilian Erythrocytes as Models of the Progenitor of Mammalian Red Blood Cells

Among the reptile species examined, only loggerhead turtle RBC with their high capacity of anaerobic metabolism and low oxygen uptake possess all the suitable metabolic characteristics as a model for transition from aerobic to anaerobic metabolism of mammalian erythrocytes (RBC). Neither the alligator RBC, which lack a significant level of anaerobic metabolism, nor the savannah monitor lizard RBC with their higher level of temperature-dependent aerobic metabolism, possess all the characteristics suitable as a model for the metabolic evolution of mammalian RBC. In the formation of this metabolic model, no phylogenetic relationships are implied or inferred. The metabolic similarity of loggerhead turtle RBC to mammalian RBC is further indicated by the high activity of the pentose phosphate (PPO₄) pathway, as evidenced by the low thermal sensitivity of their oxygen uptake and by their low ¹⁴C⁶O₂/¹⁴C¹O₂ ratios. By comparison, although the ¹⁴C⁶O₂/¹⁴C¹O₂ ratios of both alligator and monitor lizard RBC are low as compared to loggerhead turtle RBC, only alligator RBC share with loggerhead turtle RBC a low thermal sensitivity of their oxygen uptake. A comparison of hemoglobin concentrations relative to hematocrit for loggerhead turtle, alligator and monitor lizard RBC indicates that RBC hemoglobin concentrations are approximately the same for each of these species. Apart from this similarity, RBC from these three species of reptiles were differentiated in this study with respect to their density and osmotic fragility.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-d-glucosamine

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[¹⁸F]fluoroacetyl-d-glucosamine (¹⁸F-GlcNFAc). The uptake of ¹⁸F-GlcNFAc was slightly higher (P < 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. ¹⁸F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-d-[l-¹⁴C]glucosamine, 2-deoxy-d-[l-¹⁴C]glucose and [6-³H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.     

Glial uptake of monoamines in primary cultures of rat median raphe nucleus and cerebellum

Summary Glial uptake of serotonin and dopamine was studied in primary cultures of the median raphe nucleus and cerebellum by using consecutive demonstration of monoamine fluorescence and glial fibrillary acidic protein immunofluorescence. Most of the glial cells taking up monoamines were glial fibrillary acidic protein positive. Astrocytes with a strong immunoreactivity exhibited monoamine fluorescence only occasionally, although such cells did take up L-dopa readily. Glial fibrillary acidic protein negative cells — morphologically identified as astrocytes — were seen to exhibit monoamine fluorescence after exposure. Glial uptake of serotonin at a concentration of 10^–4 M was detected in cerebellar cultures but not in cultures from the median raphe nucleus. When the concentration was 10^–3 M uptake of serotonin took place in both the areas but was weaker in cultures from the median raphe nucleus. At concentrations greater than 10^–5 M glial uptake of dopamine was detected in cultures from both the regions studied. No region dependent differences in glial uptake of dopamine was demonstrated, however. Based on these observations astrocytes and astrocyte-like glial cells take up dopamine and serotonin. Also glial cells with a remarkably high content of the glial fibrillary acidic protein are more resistant to monoamine uptake than cells exhibiting less intense or no glial fibrillary acidic protein immunofluorescence. The existence of regional differences in uptake of serotonin between the median raphe nucleus and cerebellum suggests that glial uptake of monoamines is not an entirely passive mechanism but may be actively controlled by glial cells in a region dependent manner.     

Uptake and metabolism of indole-3-butyric acid and indole-3-acetic acid by Petunia cell suspension culture

The uptake and metabolism of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were studied in suspension cell cultures of Petunia hybrida. The initial uptake of ³H-IBA was much higher than that of ³H-IAA, and after 10 min of incubation with labeled IBA and IAA, 4.6 pM vs 0.35 (39% vs 12% of total applied radioactivity) respectively, were found in the cell extracts. The uptake of IBA reached a plateau of 6.0 pM (62%) after 2 h while that of IAA increased continuously up to 1.5 pM (46%) after 24 h. Following the addition of 40 µM of unlabeled auxin more IBA was taken in initially than IAA (39% vs 12%), but the level almost equalized after 24 h of incubation when IBA uptake reached 890 nM (55%) and IAA 840 nM (46%).IBA was metabolized very rapidly by Petunia cell suspension to new compounds. HPLC of the cell extracts demonstrated a new metabolite after only 2 min of incubation, and after 30 min 60% of the radioactivity was in the new metabolite vs 10% in the IBA. The new compound was resolved by autofluorography to two metabolites but after 24 h only one metabolite was present. The IBA metabolites were identified tentatively as IBA aspartic acid (IBAasp) and IBA glucose (IBAglu). In the medium IBA disappeared at a fast rate and after 24h most of the radioactivity was present in the new metabolite, probably IBAasp. IAA was also converted rapidly to two new metabolites and both were still present after 24 h. No attempt was made to identify the metabolites of IAA. IAA metabolism proceeded at a slower rate, and autofluorography showed that while free IBA disappeared after 0.5 h, free IAA was still present after 1 h of incubation. We postulate that Petunia cells conjugate IBA rapidly to IBAglu which in turn is converted to form IBAasp which probably acts as a slow release hormone. Only intact cells were able to metabolize IBA and the reaction was affected by low temperature and anaerobic conditions. The fast rate of IBA uptake, the need for whole cells for the metabolism to proceed, and the fast change of IBA to a new metabolite in the medium, all suggest that both uptake and metabolism of IBA in Petunia cells occur on the cell surface.