Incorporation and intraparticulate hydrolysis of 131 I-albumin by liver and kidney of an amphibian (Bufo arenarum)

After a single injection of formaldehyde-treated 131 I-albumin into the heart, the incorporation of the labelled protein by liver (% of total injected radioactivity/% of body weight of the organ) was far greater than in other organs. In kidney and spleen it was respectively six and three times greater than in lungs, intestine, testis and fatty body. No radioactivity was found in brain. The radioactivity in liver and kidney reached a peak 30 minutes after the injection, and quickly decreased during the following four hours. In the 27,000 g × ten minute particles recovered from liver homogenates of animals sacrificed at various times after injection, the rate of 131 I-albumin hydrolysis in vitro and the percentage of trichloroacetic acid soluble radioactivity at zero time of incubation showed different stages of intraparticulate hydrolytic activity. The incorporation and intraparticulate hydrolysis in toad kidney was very low if compared with that of toad liver or mouse kidney; however the catheptic specific activity in toad kidney doubles that of mouse kidney. Isolated toad liver was perfused with total blood, containing 131 I-albumin, for five hours at 22°C in a special chamber. In this conditions, 16% of the labelled albumin was hydrolyzed by the liver.     

Intracellular pathway followed by invertase endocytosed by rat liver

Yeast invertase, when injected into rats, is endocytosed by the liver, mainly by sinusoidal cells. The work reported here aims at investigating the organelles involved in the intracellular journey of this protein. Experiments were performed on rats injected with 125I-invertase (25 micrograms/100 g body wt) and killed at various times after injection. Homogenates were fractioned by differential centrifugation, according to de Duve, Pressman, Gianetto, Wattiaux and Appelmans [(1955) Biochem. J. 63, 604-617]. Early after injection the radioactivity was recovered mainly in the microsomal fraction P; later it was found in the mitochondrial fractions (ML). At all times a peak of relative specific activity was observed in the light mitochondrial fraction L. After isopycnic centrifugation in a sucrose gradient, structures bearing 125I-invertase, present in P, exhibited a relatively flattened distribution with a density of around 1.17 g/ml, relatively similar to that of alkaline phosphodiesterase a plasma membrane marker. The organelles located in ML were endowed with a more homogeneous distribution, their median equilibrium density increasing up to 30 min after injection (1.20 g/ml----1.23 g/ml); with time the radioactivity distribution became more closely related to the distribution of arylsulfatase, a lysosomal enzyme. ML fractions, isolated 10 min and 180 min after 125I-invertase injection, were subjected to isopycnic centrifugation in Percoll gradient with, as solvent, 0.25 M, 0.5 M and 0.75 M sucrose. The change of density of the particles bearing 125I-invertase, as a function of the sucrose concentration, paralleled the change of density of the lysosomes as ascertained by the behaviour of arylsulfatase. The distribution of radioactivity and arylsulfatase in a sucrose gradient was established after isopycnic centrifugation of the ML fraction of rats injected with 125I-invertase, the animals having received or not an injection of 900 micrograms/100 g body weight of unlabelled invertase 15 h before killing. In agreement with our previous results, a shift towards higher densities of about 25% or arylsulfatase takes place in rats pretreated with unlabelled invertase. At 10 min, invertase preinjection did not change the radioactivity distribution curve. Later, it caused a progressive shift of the distribution towards higher-density regions of the gradient where the arylsulfatase, which had been shifted, was located.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rate of concentration and digestion of radioactive growth hormone preparations injected in rats,as measured by the amount and nature of radioactivity in the tissues

Summary The distribution of radioactivity in the tissues of the rat has been established after the administration of radioactive bovine growth hormone preparations.Bovine growth hormone was used either transformed in to a¹⁴C-guanidinated derivative, which was fully active, of labeled with less than 1 mole per mole of¹²⁵I.The tissue radioactivity distribution curves obtained belong to two different categories: in kidney, liver and spleen there is an early concentration which attains a maximum in 15 minutes after the injection of the hormone, and rapidly declines. In heart, skeletal muscle, pancreas, intestine, bone and fat, the radioactivity increases gradually and a steady-state is reached after 30 to 60 minutes.Kidney is the organ where the highest concentration of radioactivity occurs. However, muscle accumulates more than 60% of the initial doses after 2 hours. Very little radioactivity appears in the urine, in this period.Similar results have been obtained with pharmacological or physiological doses of the labeled hormones.Blood plasma does not degrade the injected hormone but kidney, liver and muscle rapidly produce radioactive fragments soluble in 10% trichloro-acetic acid.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70^th birthday.     

Glycoprotein catabolism in rat liver. Lysosomal digestion of iodinated asialo-fetuin

(125)I-labelled asialo-fetuin, administered intravenously, rapidly accumulates in rat liver and the radioactivity is subsequently cleared from the liver within 60min. Plasma radioactivity reaches a minimum between 10 and 15 min after injection and rises slightly during the period of liver clearance. Free iodide is the only radioactive compound found in plasma during this latter period. Fractionation of rat liver at 5 and 13min after injection of (125)I-labelled asialo-fetuin supports the hypothesis that asialo-glycoprotein is taken into liver by pinocytosis after binding to the plasma membrane and is then hydrolysed by lysosomal enzymes. At 5min, radioactivity was concentrated 23-fold in a membrane fraction similarly enriched in phosphodiesterase I, a plasma-membrane marker enzyme, whereas at 13min the radioactivity appeared to be localized within lysosomes. Separation of three liver fractions (heavy mitochondrial, light mitochondrial and microsomal) on sucrose gradients revealed the presence of two populations of radioactive particles. One population banded in a region coincident with a lysosomal marker enzyme. The other, more abundant, population of radioactive particles had a density of 1.13 and contained some phosphodiesterase, but very little lysosomal enzyme. These latter particles appear to be pinocytotic vesicles produced after uptake of the asialo-fetuin bound by the plasma membrane. Lysosomal extracts extensively hydrolyse asialo-fetuin during incubation in vitro at pH4.7 and iodotyrosine is completely released from the iodinated glycoprotein. Protein digestion within lysosomes was demonstrated by incubating intact lysosomes containing (125)I-labelled asialo-fetuin in iso-osmotic sucrose, pH7.2. The radioactive hydrolysis product, iodotyrosine, readily passed through the lysosomal membrane and was found in the external medium. These results are not sufficient to account for the presence of free iodide in plasma, but this was explained by the observation that iodotyrosines are deiodinated by microsomal enzymes in the presence of NADPH.     

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.     

Cold shock hemolysis in human erythrocytes studied by spin probe method and freeze-fracture electron microscopy.

When human erythrocytes are osmotically stressed or chemically treated, they hemolyze on cooling below 10 degrees C (called cold shock). We have studied the effects of osmotic stress and cooling on the state of membrane by the spin-probe method and freeze-fracture electron microscopy. At room temperature, the membrane fluidity detected by 12-doxyl stearate spin probe showed a steady decrease with osmolality in hypertonic NaCl solutions up to 900 mOsm/kg, above which it remained unchanged. In hypertonic sucrose solutions, the electron paramagnetic resonance spectra showed an additional pair of absorptions, indicating development of regions, in the membrane, further immobilized than in NaCl solutions. Mobility of a cholesterol analogue probe, androstane, did not show change by hypertonicity, but the spectral intensity dropped at 1,200 mOsm/kg, probably due to formation of loose aggregates in the cholesterol phase. On cooling the osmotically stressed cells in NaCl solution, the isotropic rotational correlation time vs. inverse temperature plot of 12-doxyl stearate probe exhibited a step-wise discontinuity at approximately 10 degrees C, suggestive of a drastic transition in the state of the membrane. At about the same temperature, the freeze-fracture pattern of osmotically stressed cells revealed the development of large wrinkles and aggregation of membrane particles, in contrast to the case of the cells in isotonicity. Significance of these findings in understanding cold shock hemolysis is discussed.     

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.     

Biological evidence for a precursor protein of serum albumin.

Radioactive leucine was injected into the portal vein of rats followed after 15 seconds by a 180 fold excess of nonradioactive leucine. An albumin-like protein in the liver became highly labelled within 15 minutes after injection. After 150 minutes, the radioactivity in the albumin-like protein had decreased to one tenth. In the serum, radioactively labelled albumin started to appear after 15 minutes and increased there-after up to 150 minutes after injection. Radioactivity in albumin within the liver remained constant at a low level. These results suggest that the albumin-like protein is a biological precursor protein of serum albumin, i.e. a proalbumin.     

The uptake and distribution of radiolabelled thyroxine by isolated cardiac myocytes.

The uptake and distribution of ¹⁴C and ¹²⁵I-labelled thyroxine was studied in ventricular myocytes, isolated from the hearts of male Sprague-Dawley rats. Equilibrium was established between the radioactivity of the incubation medium and the cells within 15 minutes. At equilibrium the concentration of ¹⁴C-thyroxine in the cells was approximately 50 times the concentration in the incubation medium. Fractionation of the cells revealed that the equilibrium had been attained for all fractions except the nuclear. The radioactivity of the nuclear fraction showed an increase for at least 60 minutes of incubation. At equilibrium the distribution of radioactivity was: Soluble fraction 51.3%, Mitochondria 33.6%, Microsomal 7.0% and Nuclear 7.0%. When the values for these fractions were corrected for mitochondrial contamination the specific activity (CPM/MG protein) of the mitochondrial fraction was by far the highest, exceeding the next highest fraction (the supernatant) by an order of magnitude. The presence of equimolar amounts of triiodothyronine produced little change in the pattern of uptake of the label by any of the cell fractions. The uptake of labelled thyroxine was profoundly affected by the presence of calcium in the media. The uptake of ¹⁴C-thyroxine by cells incubated in media containing 1.25mM calcium was less after 60 minutes than in cells incubated in calcium free buffer. Fractionation of the cells revealed that the amount of label bound to the mitochondria of cells in calcium containing medium was significantly increased while the radioactivity bound to the other cellular fractions was decreased. The data indicate that the cell fraction with the highest specific activity was the mitochondria. The relation of these findings to some of the current theories of thyroid hormone action is discussed.     

The effect of cobalt on the uptake of plasma iron by the liver.

1. After an intraperitoneal injection of 59Fe the recovery of radioactivity in the liver, but not in other tissues, was increased in cobalt-pretreated rats. There was no proportional increase in the radioactivity recovered from liver haem. 2. Rats were injected intravenously with serum containing protein-bound 59Fe and 125I-labelled albumin as a marker. At various times after injection the specific radioactivities of iron in plasma and of non-haem iron in liver were determined; corrections were applied for the content of plasma in samples of liver. In cobalt-pretreated rats there was a more rapid loss of 59Fe radioactivity from the plasma and a corresponding increase in the uptake of 59Fe into liver non-haem iron. 3. The results are discussed in relation to the possible sites of action of cobalt, and the possibility is considered that only a fraction of the liver non-haem iron may be involved.     

Characterization of water-soluble products of palmitic acid beta-oxidation by a rat brain preparation

Abstract: (1) [1-¹⁴C]Palmitic acid was oxidized to CO₂ and a water-soluble material by a rat brain preparation. The radioactive CO₂ and water-soluble material were produced in a ratio of 1.0:1.3 when the mitochondrial fraction was used, and 1.0:10 or more with the postnuclear fraction. There was a lag period of 10 min for CO₂ production. These conversions were stimulated by carnitine and inhibited by cyanide. (2) Of the total radioactivity in the water-soluble material obtained with the mitochondrial fraction, 65% after 10 min of incubation and 80% thereafter were associated with amino acids, mostly with aspartate and glutamate. The remaining radioactivity, 35 and 20%, respectively, was associated with organic acids, 60–65% in citrate. The water-soluble material obtained with the postnuclear fraction contained an equal amount of radioactivity in organic and amino acids during the course of the experiment. In the organic acids, succinate was the highest labeled product during 10–40 min of incubation, whereas citrate was the highest labeled at the end of 60 min of incubation. After 60 min, the radioactivity in the amino acids was markedly associated with glutamate, and its radioactivity was 10 times greater with the postnuclear fraction than with the mitochondrial one. (3) An experiment with rat liver preparations was also camed out. The liver mitochondrial fraction showed an accumulation of radioactive organic acids within 10 min of incubation, which was followed by a linear production of ¹⁴CO₂. With the liver postnuclear fraction, the radioactivity was found mostly in the organic acids during the course of the experiment. In the liver system, the radioactive amino acids accounted for only 25% or less of the total radioactivity in the water-soluble material.     

Distribution of tritium labeled 12(S) hydroxy-eicosatetraenoic acid (12-HETE) in the rat.

The in vivo metabolism of 12-(S)-Hydroxy-eicosatetraenoic acid (12-HETE), the end-lipoxygenase product of arachidonic acid in platelets, has been investigated in the rat. Fifty microcuries of 5,6-[3H]-12-HETE (50 Ci/mmol) were injected to anesthetized rats and the radioactivity was followed in plasma. At the end of the experiment, various organs of the animal were removed and the radioactivity attached to them was determined. The label of the plasma plateaued to approximately one third of the initial radioactivity ten minutes after the injection. Among the various organs tested (brain, heart, intestine, kidney, liver, lungs, spleen, testis/uterus) the kidney was far the most active to accumulate 12-HETE and/or its labeled metabolites, and no radioactivity could be detected in urine during the course of the experiment. The analysis of lipid extracts from the various tissues revealed that 12-HETE was not accumulating in its unesterified form but was likely bound to phospholipids. We conclude that, although the label providing from the initial 12-HETE did not completely disappear from plasma, circulating 12-HETE cannot be considered as a circulating marker of cell activation.     

Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells

We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.     

Tissue distribution of retinol and its metabolites after administration of double-labelled retinol.

The tissue concentrations and distribution of radioactivity present in retinol and its metabolites were investigated in vitamin A-deficient rats 24h after injection of physiological doses (10mug) of [6, 7-14C2, 11,12-3H2] retinol. The highest concentration of radioactivity was observed in the adrenals, followed by kidney, spleen, liver, intestine and blood. The total radioactivity was greatest in urine, followed in descending order by liver, kidney, blood and intestine. The 14C/3H ratios of crude light-petroleum extracts in the liver, intestines, lungs, heart and faeces were similar to the ratio of the injected retinol dispersion. However, the 14C/3H ratios in the adrenals, kidney, spleen, blood, brain and urine were quite different from that of injected retinol. Alumina chromatography of the kidney and intestinal extracts demonstrated that retinol and retinyl palmitate are the principal forms of vitamin A present. However, alumina chromatography of the liver extract did not reveal the presence of retinol but yielded a major compound with a low 14C/3H ratio. That this compound was not retinol was shown by its inability to react with ethanolic HC1 to yield anhydroretinol. The distribution of radioactivity in ether-soluble, acidic and water-soluble fractions of urine indicated that most of the radioactivity was present in the acidic and water-soluble fractions. The 14C/3H ratios in ether-soluble and acidic fractions were higher than that of injected retinol, whereas in the water-soluble fraction the ratio was similar to the injected material.     

The metabolism of dl-(1,6-14C)lipoic acid in the rat

dl-[1,6-¹⁴C]Lipoic acid was synthesized and administered to rats or incubated in vitro with rat liver systems. The urinary excretion of radioactivity after labeled lipoate was administered intraperitoneally at a level of 0.5 mg/100 g body weight was maximal at 3–6 hr, with 60% of the injected radioactivity recovered within 24 hr. Respiratory ¹⁴CO₂ from the same animals is maximal at 3 hr, after which it falls off markedly. Approximately 30% of the injected radioactivity was recovered as ¹⁴CO₂ within 24 hr. The excretion of radioactivity after lipoate was administered by stomach tube was similar to that after intraperitoneal injection. Localization of radioactivity in the body was greatest in liver, intestinal contents, and muscle in all cases. Ionexchange and paper chromatographies of 24-hr pooled urine revealed several watersoluble radioactive metabolites. Incubation of [¹⁴C]lipoate with homogenates or mitochondrial preparations in vitro resulted in the production of ¹⁴CO₂, which was decreased by incubation with unlabeled fatty acids and unaffected by the addition of carnitine or (+)-decanoylcarnitine. The rat, like certain bacteria, metabolizes lipoate via β-oxidation of the valeric acid side chain and by other metabolic reactions on the dithiolane ring, which render the molecule more water soluble.     

Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase

Rats were injected intravenously with 125I-labeled bovine lipoprotein lipase. The lipase disappeared within minutes from the blood due to uptake both in the liver (about 50% of the injected dose) and in extrahepatic tissues. Lipase enzyme activity disappeared in parallel to the 125I radioactivity. Thus, there was no inactivation of lipase in the circulating blood. Similar results were obtained when lipoprotein lipase purified from guinea pigs was injected into guinea pigs. Using supradiphragmatic rats we could show that the extrahepatic uptake was saturable and that the amounts of lipase that could be bound far exceeded the amounts of endogenous lipase expected to be present on the endothelium. When the lipase was denatured before injection, its removal in supradiaphragmatic rats became slower, and in intact rats the fraction of the uptake that occurred in extrahepatic tissues was much decreased. It is concluded that recognition by the extrahepatic receptors depends on the native conformation of the lipase. The extrahepatic uptake was strongly impeded by injection of heparin prior to injection of the lipase, and the uptake could to a large extent be reversed by injection of heparin after the lipase. Even after 1 h lipase that had been taken up by extrahepatic tissues reappeared immediately in the blood on injection of heparin. This was true both for enzyme activity and for enzyme radioactivity. Thus, internalization-inactivation-degradation occur only slowly in extrahepatic tissues. It is possible that the extrahepatic binding occurs to the enzyme's physiological receptors. The hepatic uptake was not dependent on the native conformation of the lipase, was less sensitive to heparin, could not be reversed by heparin and was not saturable. The enzyme was not rapidly inactivated after uptake; its activity could be detected in liver homogenates even after 1 h. Degradation to acid-soluble products in the liver was relatively slow; the t1/2 for native lipase was about 1 h. In comparison, in parallel experiments asialofetuin was degraded with a t1/2 of about 15 min.     

The sialic acid residue of exogenous GM1 ganglioside is recycled for biosynthesis of sialoglycoconjugates in rat liver.

In order to assess metabolic recycling of sialic acid, GM1 ganglioside [nomenclature of Svennerholm (1964) J. Lipid. Res. 5, 145-155; IUPAC-IUB Recommendations (1977) Lipids 12, 455-468], 14C-radiolabelled at the acetyl group of sialic acid, was intravenously injected into Wistar rats, and the presence of radioactive sialic acid in liver sialoglycolipids (gangliosides) and sialoglycoproteins was ascertained. A time-course study (20 min-72 h) showed that the radioactivity present in the liver distributed in the following fractions, with reciprocal proportion varying with time: the protein (glycoprotein) fraction, the ganglioside fraction and the diffusible fraction, which contained low-Mr compounds, including sialic acid. Ganglioside-linked radioactivity gradually decreased with time; protein-linked radioactivity appeared soon after injection (20 min), reached a maximum around 20 h, then slowly diminished; diffusible radioactivity provided a sharp peak at 4 h, then rapidly decreased till disappearing after 40 h. The behaviour of bound radioactivity in the individual liver gangliosides was as follows: (a) rapid diminution with time in GM1, although with a lower rate at the longer times after injection; (b) early appearance (20 min) with a peak at 1 h, followed by continuous diminution, in GM2; (c) early appearance (20 min), peak at 1 h, diminution till 4 h, followed by a plateau, in GM3; (d) appearance at 60 min, maximum around 40 h and slow diminution thereafter, in GD1a, GD1b and GT1b. A detailed study, accomplished at 40 h after injection, demonstrated that almost all radioactivity present in the protein fraction was released by mild acid treatment and recovered in purified sialic acid; most of radioactive glycoprotein-bound sialic acid was releasable by sialidase action. In addition, the radioactivity present in the different gangliosides was exclusively carried by sialic acid and present in both sialidase-resistant and sialidase-labile residues. Only in the case of GD1a was the specific radioactivity of sialidase-resistant sialic acid superior to that of sialidase-releasable sialic acid. The results obtained lead to the following conclusions: (a) radioactive GM3 and GM2 were produced by degradation of GM1 taken up; GM3 originated partly by a process of neosynthesis; (b) radioactive GM1 consisted in part of residual exogenous GM1 and in part of a neosynthetized product; (c) radioactive GD1a originated in part by direct sialylation of GM1 taken up and in part by a neosynthetic process; (d) radioactive GD1b and GT1b resulted only from neosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nuclear and cytosolic distribution of conjugated cholic acid and radiolabelled glycocholic acid in rat liver.

1. Normally fed and cholestyramine-treated rats were injected through the superior mesenteric vein with different amounts of radiolabelled glycoholic acid and the appearance of radioactivity in bile was measured. 2. In normally fed rats radioactivity appeared in bile within 30 s of injection and reached a maximum after 2 1/2 min; in the cholestyramine-treated animals the appearance of radioactivity was slower and less of the injected material was excreted into bile. 3. At 10 min after injection, livers were removed and the amounts of radioactive glycoholic acid and endogenous cholic acid conjugates in nuclei and cytosol were determined; most of the bile acid was found in the cytosol, only small amounts being found in nuclei. 4. Nuclear preparations from both normally fed and cholestyramine-fed rats were extracted with KCl (0.4 M) in an attempt to identify a putative bile acid receptor, but no such receptor was found. 5. Regulation of bile acid synthesis does not involve nuclear binding of bile acids.     

Sodium-Stimulated Glutamate Transport in Osmotically Shocked Cells and Membrane Vesicles of Escherichia coli

Three phenotypically distinct strains of Escherichia coli B were studied: one in which the transport of glutamate was strongly stimulated by sodium, one in which the transport was relatively independent of sodium, and one which did not transport glutamate. Membrane vesicle preparations from the three strains followed the behavior of whole cells with respect to sodium-stimulated transport. Although glutamate-binding material could be released from cells by osmotic shock, its affinity for glutamate was not significantly influenced by sodium. Furthermore, the shocked cells retained sodium-stimulated transport. The accumulated results suggest that the sodium-activated glutamate transport system resides in the cytoplasmic membrane and that releasable binding protein(s) is not intimately involved in its function.     

Distribution and persistence of kainic acid in brain.

The distribution of ³H-kainic acid in rat brain was studied as a function of time after injections of 5 nmoles into the neostriatum, substantia nigra or cerebellum. More than half of the injected material had disappeared from the injection site and the brain by 1/2 hour post injection. Under the conditions used very small amounts of radioactivity (corresponding to less than 7 pmol/ mg of tissue) were found in areas other than the injection site, suggesting that the histological damage reported in the hippocampus and pyriform cortex after striatal injections may be due to a secondary process not dependent on the presence of toxic concentrations of kainic acid in those areas. No radioactivity was found in the TCA-insoluble material nor did it appear that there was rapid metabolism of the bulk of the kainic acid.