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PubMed1987

Comparison between radiolabelled and endogenous dopamine release from rat striatal slices: effects of electrical field stimulation and regulation by D2-autoreceptors.

Direct comparisons have been made between the release of radiolabelled and endogenous dopamine from superfused rat striatal slices prelabelled with 3H-dopamine. Both spontaneous release and release evoked by electrical field stimulation (3 Hz, 2 min) were measured using a high-sensitivity HPLC system with electrochemical (coulometric) detection, plus scintillation counting of chromatographically separated superfusate fractions. Two periods of electrical stimulation released similar amounts of endogenous dopamine, but the second stimulation released much less 3H-dopamine than did the first, although the levels of spontaneous release immediately before the two stimuli were similar. Substantial increases in endogenous 3,4-dihydroxy phenyl acetic acid (DOPAC) release but only minor increases in 3H-DOPAC release occurred following the two stimuli. The dopamine agonist pergolide (1 microM) reduced the electrically-stimulated release of both 3H-dopamine and endogenous dopamine to a similar extent, whilst the D2-selective antagonist sulpiride (1 microM) produced large increases in both 3H-dopamine and endogenous dopamine electrically-stimulated release. In addition, spontaneous release of both 3H-dopamine and endogenous dopamine were decreased by pergolide and increased by sulpiride. Co-addition of sulpiride and pergolide produced lesser increases than those seen with sulpiride alone. These studies indicate that, despite major differences between 3H-dopamine and endogenous dopamine release in response to various stimuli, their regulation by D2-autoreceptors appears similar; a novel finding being the modulation of spontaneous 3H-dopamine release by autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗

PubMed1993

Different inhibitory effect of etomidate and ketoconazole on the human adrenal steroid biosynthesis.

The narcotic agent etomidate and the antimycotic drug ketoconazole are known to block steroid biosynthesis in man. To study the different effects of these imidazole derivatives on human adrenal steroid biosynthesis we incubated slices of human adrenal glands with 3H-labeled precursors and increasing concentrations of etomidate or ketoconazole (0-2000 microM). After extraction the labeled metabolites were separated by thin-layer chromatography and quantified by scintillation counting. Etomidate inhibited most potently 11 beta-hydroxylase activity by suppressing the formation of corticosterone from 11-deoxycorticosterone to 1% of control [50% inhibitory concentration (IC50) 0.03 microM] while ketoconazole suppressed 11 beta-hydroxylase to only 39% of control activity (IC50 15 microM). Ketoconazole however, most potently blocked the conversion of 17 alpha-hydroxy-progesterone to androstenedione by C17,20-desmolase to about 15% of control activity (IC50 1 microM) while etomidate showed a much weaker effect on this enzyme with a suppression to 50% of C17,20-desmolase control activity at a concentration of 380 microM. Both imidazole drugs showed a similar strong inhibitory effect on the activity of 17 alpha-hydroxylase (IC50 6-18 microM) and 16 alpha-hydroxylase (IC50 4-8 microM) and did not affect 21-hydroxylase. These in vitro data indicate a predominant inhibitory effect of etomidate on corticosteroid biosynthesis by relative selective inhibition of 11 beta-hydroxylase and of ketoconazole on the adrenal androgen biosynthesis by a predominant inhibition of C17,20-desmolase. This differential inhibitory effect of etomidate and ketoconazole on human steroid biosynthesis may be of clinical importance for a possible therapeutic use of these imidazole derivatives in endocrine disorders.

Adrenal Glands↗

PubMed1983

Indole metabolism in the pineal organ of the pigeon with special reference to melatonin-synthesizing cells. In vitro study combining thin layer chromatography, autoradiography and pharmacological treatment.

By means of radioimmunoassay a clear-cut peak of melatonin concentration was found in the pineal organ of the pigeon at the middle of the scotophase (Coisin et al. 1982a). The aim of the present study was to identify the cell type responsible for the nocturnal indole metabolism, including melatonin synthesis, in the pineal of this avian species. After a short-term incubation or organ culture in the presence of [3H]-indolic precursors, [3H]-5-hydroxytryptophan or [3H]-5-hydroxytryptamine, the relative amounts of deaminated and acetylated products occurring in the pineal organ were measured by the use of thin layer chromatography and liquid-scintillation counting. It was possible to modify the relative amounts of deaminated and acetylated indoles by the application of some inhibitors of monoamine oxidase and cyclic nucleotide phosphodiesterase. Irrespective of the experimental conditions, high-resolution autoradiography combined with the above-mentioned radiochemical experiments showed that the cells of the receptor line (modified photoreceptor cells) are responsible for indole storage and metabolism, and very probably also for melatonin biosynthesis. The other cell types of the pineal parenchyma did not display significant labeling.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

PubMed1975

The composition and protein metabolism in the immature rabbit intervertebral disc.

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.

Animals↗

PubMed1985

Uptake of circulating hyaluronic acid by the rat liver. Cellular localization in situ.

The uptake of [3H]acetyl-labelled hyaluronic acid (HA) was examined in the liver, spleen and kidney of the rat after i.v. injection. 3H-activity was located by light- and electron-microscopic autoradiography after measurement by scintillation counting of tissue digests. In the liver, approximately 90% of the radioactivity was located in the sinusoidal endothelial cells, with autoradiographic grains distributed throughout the cytoplasm; 50% of the grains overlay vacuoles 0.3 to 1.2 micron in diameter. A few grains (4%) were located in Disse's space or nearby in the cytoplasm of hepatocytes. No grains were found in Kupffer cells. The remainder were randomly scattered across the sections in a pattern indicating nonspecific background activity. These observations are in accordance with the selective uptake of HA exhibited by dissociated liver cells in vitro. HA concentrations in the spleen and kidney were too low for detection by autoradiography. Splenic concentrations were much lower than in rabbits or mice; in this respect the uptake of circulating HA in the rat resembles that reported for chondroitin 4-sulphate.

Animals↗

PubMed1981

The regional distribution of d-amphetamine and local glucose utilization in rat brain during continuous amphetamine administration.

The distribution of radioactivity following administration of either [3H]d-ampetamine or [3H]2-deoxy-d-glucose was examined by scintillation counting of 22 microdissected brain regions from rats pretreated with either acute or continuous amphetamine, or continuously administered labeled d-amphetamine. Animals continuously administered drug were sacrificed in behaviorally distinct stages of the continuous amphetamine syndrome, a potential animal model of amphetamine psychosis. Both isotopes were heterogeneously distributed within brain, and their distributions were differentially affected by acute or continuous amphetamine regimens. While the distribution of either isotope in naive rats was characterized by greatest concentrations of counts in rostral rather than caudal regions, and grey-matter rather than white-matter structures, continuous amphetamine administration resulted in progressively increased retention of amphetamine by mesolimbic but not nigrostriatal brain regions; this was accompanied by locally enhanced levels of glucose utilization. This effect was predominantly localized in the nucleus accumbens, which exhibited the greatest retention of amphetamine and greatest relative increase in glucose utilization of any region studied during that stage of the continuous amphetamine syndrome thought to best model amphetamine psychosis. Alterations in amphetamine distribution and local levels of neural activity may reflect a change in the principal locus of control of amphetamine effects within brain as animals progress through the stages of the continuous amphetamine syndrome.

Amphetamine↗

PubMed1987

In vitro stability, plasma protein binding and blood cell partitioning of 14C-carboplatin.

Radiochemically pure 14C-labeled carboplatin, cis-diammine [1,1-cyclobutane (1-14C) dicarboxylato (2-)-0,0'] platinum (II), was added to fresh human, dog and rat plasma, at concentrations ranging from 1 to 100 micrograms 14C-carboplatin/ml. After 10 min incubation at ambient temperature, the plasma was ultrafiltered in Amicon Centrifree micropartition units to generate protein-free plasma ultrafiltrate (PU). Total radioactivity was determined by liquid scintillation counting. A mean (+/- SD) of 102% +/- 2.0%, 99.5% +/- 1.9%, and 99.0% +/- 1.0% of the 14C-carboplatin added to fresh human, dog, and rat plasma respectively was recovered in the PU. 14C-carboplatin was incubated at 37 degrees C with fresh plasma (60 micrograms/ml) and urine (200 micrograms/ml) from humans and dogs for 120 h, and samples were removed at appropriate times for analysis of carboplatin, 1,1-cyclobutane dicarboxylic acid and cyclobutane carboxylic acid. The latter were separated by HPLC on a C-18 column with a mobile phase of H2O/CH3CN/0.3 M tetrabutylammonium phosphate (880:50:20 v/v/v), and the column eluants at the retention time of each compound were collected and counted for total radioactivity. Carboplatin degraded in each of the matrices with a corresponding release of 1,1-cyclobutane dicarboxylic acid. 14C-carboplatin (50 micrograms/ml) was incubated at 37 degrees C with fresh human, dog and rat blood and the distribution of radioactivity into the cellular fraction was determined. Radioactivity did not distribute into the blood cells of humans or dogs, but after 5 h, 44% of the radioactivity in rat blood was associated with the cellular fraction. These results show that carboplatin, at physiological concentrations, does not bind instantaneously and reversibly to the plasma proteins of rat, dog or human, and that the molecule slowly degrades in plasma and urine in vitro with the release of 1,1-cyclobutane dicarboxylic acid. The remaining diammine platinum (II) portion of the molecule therefore accounts for the essentially irreversible protein binding of the platinum from carboplatin.

Animals↗

PubMed1976

Patterns of incorporation of tritiated thymidine by the dorsal polytene foot-pad nuclei of Sarcophaga bullata (Sarcophagidae:Diptera).

The incorporation of tritiated thymidine into the DNA of the dorsal foot-pad nuclei of Sarcophaga bullata, during pharate adult development, was studied by scintillation counting and autoradiography. Incorporation was maximal on day 4 and showed a progressive temporal decline on days 5 to 8. Autoradiographs of chromosomal arms A1, A2 and D1, from late stages of the prolonged S period, showed discontinuous incorporation. On days 7 and 8 loci which incorporated the radio-labeled precursor were almost exclusively associated with DNA granules. DNA granules were associated with 8 specific loci in chromosomal arm A1, 10 in A2, and 8 in D1. The relationship between patterns of incorporation of tritiated thymidine and DNA granules is discussed.

Animals↗

PubMed1980

Effects of diethyldithiocarbamate and penicillamine on the tissue distribution of 63NiCl2 in mice.

The effects of sodium diethyldithiocarbamate and DL-penicillamine on the fate of 63Ni2+ in mice injected with 63NiCl2 were studied by whole body autoradiography and liquid scintillation counting. Sodium diethyldithiocarbamate caused a retention and redistribution of 63Ni2+ in the tissues. DL-penicillamine decreased the 63Ni2+-concentration in the tissues. The nickel-diethyldithiocarbamate complex is lipophilic, whereas the nickel-DL-penicillamine complex is hydrophilic, and these differences in chemical properties may determine the different effects which these chelating agents have on the fate of Ni2+.

Animals↗

PubMed1976

Studies on in vitro carcinogenesis. I. Autoradiographic and biochemical study on the incorporation of labelled methylcholantrene into mouse embryonic cells.

The incorporation of radioactive labelled Methylcholanthrene into CBA/T6T6 mouse embryonic fibroblast cultures was studied by using light and electronmicroscopic autoradiographic and also liquid scintillation counting techniques. During 24 hrs treatment time at the applied Methylcholanthrene concentrations (0.01-2.5 mug/ml) the proportion of cells labelled with 3H-Methylcholanthrene and the number of grains above the cells showed a relationship with the duration of treatment and also with the doses. Up to 24 hrs all cells were labelled after treatment with 2.5 mug/ml whereas at 0.01 and at 0.1 mug/ml concentrations Methylcholanthrene could have been detected only 2 and 10% of the cell population resp. The presented results suggested that labelling index reached saturation level earlier than the average number of grains. Electronmicroscopic autoradiography revealed a rather even distribution of grains and there were no signs for preferential binding site of Methylcholanthrene. Biochemical separation showed that 59 and 44% of the radioactivity was bound to macromolecules at 1 and 24 hrs after commencing treatment resp.

Animals↗

PubMed1988

Effects of B-aminopropionitrile on mineralization during endochondral ossification in chick tibia.

Two-week-old white leghorn chicks were fed a diet containing BAPN (0.05%) for three weeks. Thirty-six hours before sacrifice, the controls and BAPN fed chicks were dosed with 35S. The zone of provisional calcification was isolated, and 35S incorporation was estimated by liquid scintillation counting. Alkaline phosphatase and Ca+2-ATPase were biochemically analyzed. Microdensitometry, to assess the level of mineralization, was done on epiphysis and the metaphysis. Morphometry was performed on the various zones of growth plate. 35S incorporation was significantly lower in the bones of BAPN treated chicks as compared to the controls. The enzymatic studies showed a significant inhibition of alkaline phosphatase and Ca+2-ATPase. The microdensitometric studies showed a smaller area of highly mineralized bone in the zones of provisional calcification of the BAPN treated chicks as compared to the controls. Morphometry showed a reduction in the width of the zone of calcification in BAPN treated chicks as compared to the controls. On the basis of the above data, it is suggested that BAPN induced inhibition of mineralization during endochondral ossification may be the result of a lower synthesis of sulfur containing GAG's, the inhibition of enzymes alkaline phosphatase and Ca+2-ATPase and the derangement of cellular zones of the growth plate. The implications of these results lie in the fact that mineralization is dependent on multifactorial control of the microenvironment of bone and cartilage.

Alkaline Phosphatase↗

PubMed1993

The influence of fluoride on proteoglycan structure using a rat odontoblast in vitro system.

Using an in vitro rat incisor odontoblast system, the effect of fluoride on proteoglycans was investigated at both the metabolic and structural level. Incisors were removed from 4-week-old rats, split longitudinally, and the pulps removed. Teeth were incubated at 37 degrees C, 5% CO2 in Eagle's Minimum Essential Medium containing 35S-sulfate for 7 hours in the presence of 0 mM, 3 mM, or 6 mM sodium fluoride. Teeth were demineralized in EDTA, proteoglycan was extracted from the residue with 4 M guanidinium chloride, and further purified by anion exchange chromatography. Uptake of radiolabel was monitored by liquid scintillation counting. The resultant products were examined by cellulose acetate electrophoresis, SDS-PAGE, chondroitinase digestion, and amino acid analysis. Differential effects of fluoride were observed in both metabolism and biochemical characterization of proteoglycans following incubation at the two concentrations. Fluoride decreased uptake of the radiolabel but led to an accumulation of glycosaminoglycan within the proteoglycan of the matrix. Chondroitin sulfate was the predominant glycosaminoglycan identified, with the additional presence of dermatan sulfate and heparan sulfate identified. Dermatan sulfate levels increased in 3 mM-treated teeth. Fluoride-treated proteoglycans had a reduced molecular weight (200-90K to 180-79K); this reduction is primarily a result of smaller glycosaminoglycan chains, with limited reduction in the size of the core protein of 6 mM-treated teeth occurring. Such alterations in the biochemical metabolism and hence structure and function of proteoglycan may be implicated in the hypomineralization seen in fluorosis.

Amino Acids↗

PubMed1979

The half-life of mRNA in Saccharomyces cerevisiae.

The decay kinetics of mRNA was studied in a yeast temperature-sensitive mutant, ts136, which is defective in cytoplasmic RNA production at 37 degree C. The disappearance of the synthetic capacity of mRNA was determined by withdrawing equal volumes of ts136 cell culture and pulse-labelling with [35S]methionine at various time intervals after the shift to 37 degrees C from 23 degrees C. The synthesized proteins were separated on a two-dimensional gel electrophoretic system and then quantitatively analyzed for theri incorporated radioactivities by scintillation counting. Our results show that yeast mRNAs have divergent functional half-lives ranging from 4.5 to 41 min, with an average value of 22 min. Each mRNA exhibits a simple exponential decay with its own characteristic dacay pattern. Of the approximately 500 major polypeptides made by yeast cells, which are detectable on autoradiograms of the gels, 80 were arbitrarily selected and the mRNAs coding for those polypeptides were examined for their decay kinetics.

Electrophoresis↗

PubMed1975

Distribution of 2,2(1),4,4(1),5,5(1)-hexachlorobiphenyl in mice and Chinese hamsters: dose dependent accumulation in the mouse bronchial mucosa.

The distribution of 2,2(1),4,4(1),5,5(1)-hexachlorobiphenyl-14C was studied in mice and Chinese Hamsters using whole body autoradiography and liquid scintillation counting. The mice exhibited a strong and persistent accumulation of radioactivity in the bronchial mucosa, and this accumulation was not fully developed until about 24 hours after an intravenous injection. The labelled substance passed to the fetuses of pregnant mice and was also concentrated in the fetal bronchi. Mice pretreated with a large dose of unlabelled PCB per os in peanut oil showed a completely different distribution pattern in the lungs--only traces of label being taken up by the bronchi. Quantitative measurements revealed a concomitant reduction of the total radioactivity retained by the lungs. Except for the lungs, however, no major differences in the distribution pattern were found at the various dose levels. The distribution in the Chinese hamsters equaled approximately that of the mice, but a very weak accumulation of label was observed in the hamster bronchi. The radioactivity in the mouse bronchi was considered as perhaps representing metabolized PCB.

Adipose Tissue↗

PubMed1980

Unscheduled DNA synthesis in the testis, a secondary test for the evaluation of chemical mutagens.

DNA damage induced by methylmethane sulfonate, cyclophosphamide, doxorubicin and procarbazine in male germ cells was assessed in rabbits by the demonstration of unscheduled DNA synthesis (UDS), in meiotic and postmeiotic phases of maturation. Immediately after treatment by the intravenous route tritiated thymidine was injected into both testicles. Subsequently, rabbits were ejaculated serially, sperm heads were isolated and assayed for radioactivity by liquid scintillation counting. Dose-dependent UDS was demonstrated in late spermatocytes and early spermatids. High doses of hycanthone also induced UDS, but isoniazid and metronidazole had no effect. The rabbit testis UDS test takes into account metabolic and pharmacokinetic aspects of the test substances and provides information about their penetration through the blood-testicular barrier. It is therefore useful for secondary evaluation of potential mutagens. UDS induced by procarbazine was abolished by simultaneous treatment with Ara-C. Thus, the test also recognizes substances that inhibit DNA repair synthesis.

Animals↗

PubMed1976

Babesia hylomysci in mice: Preference for erythrocytes of a particular age-group and pathogenesis of the anaemia.

Scintillation counting on 59Fe-labelled haemoglobin in erythrocytes, serum and urine during B. hylomysci infection in mice demonstrated that the parasite had a predilection to older mature erythrocytes and that the anaemia produced during the infection was mainly due to the direct destruction of infected erythrocytes. An autoimmune reaction has also been incriminated as a factor in the pathogenesis of the anaemia, but was of a lesser magnitude and its manifestations were masked by the direct destruction of infected erythrocytes.

Anemia, Hemolytic↗

PubMed1981

Ocular kinetics of 8-methoxypsoralen. An experimental study with pigmented and albino rabbits without UVA-stimulation.

Tritiated 8-methoxypsoralen (8-MOP), with radioactivity in the ring system, was injected into both pigmented rabbits and albino rabbits; at 0.5, 1, 2, 4, 6, and 24 h after 8-MOP injection the animals' plasma and ocular radioactivities were determined by liquid scintillation counting. The experiment was done without photostimulation but in incomplete darkness. The plasma radioactivity declined rapidly during the first hour, peak radioactivity occurring in the first samples of vascularized ocular structures taken 0.5 h after the injection. The radioactivity of these tissues generally declined rapidly and similarly in both animal groups. The 1, 2, and 4 h samples of pigmented animals formed an exception; in these the radioactivities in the iris-ciliary body and the retina-choroid of the pigmented animals showed increased activities that were statistically significant, and the plasma radioactivity was exceeded by two to three times. The radioactivities in the internal parts of the eye were lower than in the vascular parts. The radioactivity in the lens reached a peak in the specimens taken 1-2 h after injection and remained at the level of the plasma radioactivity for the rest of the 24 h.

Animals↗

PubMed1978

Penetration of atenolol in the rabbit eye.

The ocular penetration of topically applied [14C]-atenolol in the rabbit was determined by means of liquid scintillation counting. Only one eye was treated, the fellow eye serving as a control. Blood plasma levels were measured as well. The absolute amount of atenolol which penetrated the eye was very low, but a relatively high concentration was achieved in the tissues of the nictitating membrane. We could only detect an increase in the amount of atenolol with time in the aqueous humor. In all other ocular tissues, including iris and ciliary body, the atenolol level remained constant with time. Hardly any atenolol could be detected in the untreated eye and none in the blood plasma. These findings suggest that the ocular penetration of atenolol administered as an eye drop is very poor. Ocular penetration, therefore, hardly seems to play a part in the antiglaucomatous effect of atenolol.

Animals↗