Tissue Subcellular Fractions Research Articles

Protein kinase C and dopamine transport—2. Effects of amphetamine in vitro

Synaptoneurosomes, incubated with amphetamine, showed a biphasic dose-response change in activity of PKC and release of DA. Activity of particulate PKC activity and release of DA were decreased at 0.01–10.00 nM, while activity of both was increased at 1–10 μM. The effects of 0.1 nM amphetamine were attenuated by pretreatment with N-ethoxy-carbony1-2-ethoxy-1,2-dihydroquinoline (EEDQ), a peptide-coupling agent which inactivated DA receptors. They persisted if the autoreceptors were protected with sulpiride or apomorphine (0.05 mg/kg), prior to the treatment with EEDQ. In these protected rats, the amphetamine-induced change in activity of PKC was attenuated by sulpiride. In contrast, the effects of the larger doses of amphetamine on the activity of PKC were not affected by pretreatment with EEDQ. Subcellular fractionation of tissues, incubated with 1 μM amphetamine, showed an increase in activity of particulate PKC, only in the synaptic plasma membrane fraction, while tissues incubated with reserpine showed a decrease in activity of particulate PKC, only in the vesicular fraction. Similar results were seen when synaptic plasma membrane or synaptic vesicles were incubated directly with amphetamine or reserpine. These findings suggest the presence of multiple metabolic pools of PKC: a reserpine-sensitive pool, present in synaptic vesicles and an amphetamine-sensitive pool, present in the synaptic plasma membrane. That the latter pool of PKC may be involved in the transport of DA was indicated by the good correlation between the ability of drugs to inhibit the activity of PKC and their ability to inhibit the amphetamine-induced release of DA.

Estrogen sulfatase and steroid sulfatase activities in intrauterine tissues of the pregnant guinea pig

The possible role of intrauterine estrogen sulfatase and steroid sulfatase around the time of parturition in the guinea pig was investigated. [ 3H]Estrone sulfate or [ 3H]pregnenolone sulfate was incubated with intrauterine tissues. Estrogen sulfatase was found in placenta, endometrium, decidua basalis, amnion and chorion. The presence of steroid sulfatase was established in endometrium and decidua basalis but not in placenta or the fetal membranes. Examination of activities in early (days 32–35), mid (days 44–46) and late (within 5 days of parturition) gestation revealed no significant change in estrogen sulfatase specific activity in decidua basalis. However, in chorion and endometrium this activity was seen to increase approx. 12-fold ( P < 0.001) and 2.8-fold ( P < 0.001), respectively, from early to late gestation. In placenta, estrogen sulfatase activity appeared to increase 2.4-fold ( P < 0.001) and in amnion it decreased 2.8-fold ( P < 0.002). Steroid sulfatase activity in decidua basalis did not change during gestation, while activity in endometrium was found to increase by a factor of 5.3 ( P < 0.001), from early to late gestation. The increases, both in estrogen sulfatase activity in chorion, endometrium and placenta and in steroid sulfatase activity in endometrium, occurred primarily within the final 3 weeks of gestation. In contrast, the decrease in estrogen sulfatase activity in amnion occurred principally between the fifth and sixth weeks of gestation. Analysis of radiolabelled metabolites indicated that estradiol and progesterone could be produced via estrogen sulfatase and steroid sulfatase activities in certain tissues. Subcellular fractionation of tissues revealed that the greatest specific activity and total activity in all cases, was associated with the 105,000 g pellet. Significant activity was also detected in the 750 and 10,000 g pellets but not in the 105,000 g supernatant. Radioimmunoassay of endogeneous estradiol-17β (estradiol) in chorion extracts revealed a 6.3-fold increase in the hormone from mid to late gestation. Estradiol levels in endometrium and myometrium did not appear to change during this time. It was concluded that increased estrogen sulfatase activity in guinea pig chorion in late gestation occurs along with elevated levels of the hormone estradiol which may be important for parturition in this species.

Oxidative metabolism of 14C-pyridine by human and rat tissue subcellular fractions.

1. The oxidative metabolism of 14C-pyridine by human and rat microsomal fractions has been studied. Metabolites were separated by h.p.l.c. employing continuous radioactivity monitoring of the column effluent. 2. Human liver microsomal fractions incubated with an NADPH-generating system and oxygen formed pyridine N-oxide (PNO) at an average rate of 275 pmol/min per mg protein, 2-pyridone (2PO) at 207 pmol/min per mg and 4-pyridone (4PO) at 154 pmol/min per mg. One human subject formed 3-hydroxypyridine N-oxide in addition to the other metabolites. 3. Human kidney microsomal fractions formed PNO, 2PO and 4PO at rates similar to those with human liver microsomal fractions, whereas human lung microsomal fractions formed the metabolites at less than half the rate. 4. Metabolism of pyridine by human liver microsomal fractions was inhibited 54% by nitrogen, 34% by 80% carbon monoxide-20% oxygen, and 20% by metyrapone. 2-Diethylaminoethyl-2,2-diphenylvalerate HCl (SKF-525A) did not inhibit pyridine metabolism. 5. Liver microsomal fractions from non-induced rats metabolized pyridine to PNO at a rate of 19 pmol/min per mg protein, 2PO at 17 pmol/min per mg and 4PO at 61 pmol/min per mg. 6. There was no pyridine metabolism by human or rat tissue cytosolic fractions incubated under the same conditions.

Localization of retinal calmodulin kinase

The localization of calmodulin kinase II (CaM kinase) was studied in the retina by light and electron microscopic immunocytochemistry, and by enzymatic and immunoblot assay of cellular and subcellular tissue fractions. By light microscopy, both mono- and polyclonal antibodies revealed CaM kinase-like immunoreactivity in the inner and outer plexiform regions (synaptic layers), retinal pigment epithelium (RPE), and ganglion cells. The inner nuclear layer and photoreceptor outer segments stained much less intensely, and the outer nuclear layer did not stain. Electron microscopy confirmed the high concentration of immunoreactive protein in RPE and minimal outer segment staining. In addition, photoreceptor inner segments also contained CaM kinase-like immunoreactivity. Calcium and calmodulin stimulated phosphate incorporation into proteins of retinal cytosol and of isolated and cultured RPE. Calcium- and calmodulin-dependent kinase activity was present to a lesser degree in crude nuclei and synaptic membranes and was absent in isolated rod outer segments. Immunoblot analyses were consistent with enzymatic assays and immunocytochemistry. These data suggest that retinal CaM kinase is ideally located to play an important role in synaptic transmission and modulation of visual processes. Furthermore, its presence in RPE implies that CaM kinase may have a more ubiquitous role in regulating cellular processes than was previously recognized.

Lung energy metabolism following twenty-four hours exposure to one hundred percent oxygen.

The interstitial edema and endothelial cell damage observed in rat lungs following 48 hours of exposure to 100% oxygen is believed to result from oxygen-derived free-radical damage of cellular membranes, enzyme proteins, and possibly components of the extracellular matrix. Earlier physiological alterations of endothelial function have been demonstrated by depressed serotonin uptake in isolated perfused rat lungs following 18 to 24 hours of oxygen exposure,1 at a time when tissue adenine nucleotide levels have been found not to be affected.2 In vitro experiments using homogenates and isolated subcellular tissue fractions have demonstrated that key enzyme systems involved in energy generation are susceptible to oxygen inactivation. These include the dehydrogenases of glyceraldehyde-3-phosphate, pyruvate, succinate, and oxoglutarate.3–5 The purpose of the present investigation was to use an intact organ preparation to determine whether lung glycolytic and mitochondrial enzyme systems are impaired during the first 24 hours of oxygen exposure when pathological alterations are not observed.Keywords14C02 ProductionOxygen ExposurePyruvate MetabolismPhenazine MethosulfatePyruvate ProductionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Factors influencing the selective toxicity of cis‐ and trans‐cypermethrin in rainbow trout, frog, mouse and quail: Biotransformation in liver, plasma, brain and intestine

AbstractThe biotransformation rates of cis‐ and trans‐cypermethrin, and their routes of metabolism have been studied in subcellular tissue fractions from rainbow trout, frog, mouse and quail to assess the role of detoxification in the selective toxicity of pyrethroid insecticides in these species. The liver was the principal site of biotransformation in mouse and quail, though intestine and plasma (not quail) were also active in hydrolysing cypermethrin isomers. In trout, plasma and intestine were more important sites than liver for the detoxification of tram‐cypermethrin. On the basis of total metabolism of cypermethrin isomers by liver per kilogram of body weight, mouse, quail and frog were respectively 27, 17 and 2 times more active than trout towards the cis‐isomers, and 87, 55 and 6 times more active in metabolising the trans‐isomers. Some of these differences were accounted for by variations in liver/body weight ratios. Extra‐hepatic tissues metabolised cypermethrin principally by ester hydrolysis with maximal activity towards the trans‐isomers, brain and gut contents possessing very low activities. In the presence of NADPH and UDPGA, cis‐ and trans‐cypermethrin were metabolised at very similar rates in frog, mouse and quail liver. Trout liver was more active towards the cis‐isomer forming 4′‐hydroxycypermethrin (aryl hydroxylation) and its glucuronide almost exclusively. Frog and mouse showed either aryl hydroxylation or aliphatic hydroxylation of one of the methyl groups at C‐2 of the cyclopropane ring of cis‐cypermethrin, while quail was unique amongst the four species in metabolising the cis‐isomers largely by oxidative de‐esterification. In all species, except trout, trans‐cypermethrin was rapidly hydrolysed by micro‐somal esterases. The presence of the phase II cofactor UDPGA increased the rate of biotransformation mainly through the glucuronidation of the products of oxidation. This investigation shows that, in addition to differences in target organ sensitivity, biotransformation accounts for some of the observed differences in the susceptibility of these four species to pyrethroids.

Compartmentation of citrate in relation to the regulation of glycolysis and the mitochondrial transmembrane proton electrochemical potential gradient in isolated perfused rat heart

Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The ΔpH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.

PROLACTIN RECEPTORS IN THE MAMMARY GLAND, CORPUS LUTEUM AND OTHER TISSUES OF THE TAMMAR WALLABY, MACROPUS EUGENII

Specific binding of radio-iodinated ovine prolactin to subcellular tissue fractions of the tammar wallaby (Macropus eugenii) was investigated. Specific binding was found, in order of decreasing binding activity, in the lactating mammary gland, corpus luteum, corpus albicans, adrenal gland and ovary. Specific binding was absent in kidney, liver, brain and inactive mammary gland. The mean association constant (Ka at 23 degrees C) was determined as 0.90 x 10(9), 2.20 x 10(9), 2.44 x 10(9), 3.38 x 10(9) and 10.98 x 10(9) l/mol for mammary gland, adrenal, corpus albicans, corpus luteum and ovary respectively. The mean receptor concentration (N) varied from 92.87 x 10(-14) mol/mg protein for the mammary gland to 1.03 x 10(-14) mol/mg protein for the ovary. The concentration in the corpus luteum varied between tissue pools collected at different times of the annual breeding cycle. The specificity for prolactin was shown in the mammary gland and corpus luteum by the failure of ovine FSH, LH, GH and TSH to displace 125I-labelled ovine prolactin, whereas it was displaced readily by both ovine and bovine prolactin.

Improved methods for determination of guanylyl cyclase activity and synthesis of [α-32P]GTP

A modification of the adenylyl cyclase assay method of Y. Salomon, C. Londos, and M. Rodbell (1974, Anal. Biochem. 48 , 541–548) is described. It makes the method applicable to the determination of guanylyl cyclase in subcellular tissue fractions. The modification consists of performing the Dowex 50 chromatography at acid pH in a column that has bed volume that is three times as large. This modification results in low blanks and allows for reuse of both Dowex 50 and aluminum oxide columns. A modification of the method of Symons [1974, Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29, pp. 105–115, Academic Press, New York] for synthesis of [α- 32 P]nucleoside triphosphates is also presented. It allows all steps to be carried out within 5 h in a single reaction vessel. Synthesized NTPs are then purified by DEAE-Sephadex A-25 (HCO 3 − form) using a linear ammonium bicarbonate gradient. Its applicability to synthesis of [α- 32 P]GTP, [α- 32 P]dGTP, and [α- 32 P]ATP having specific activities of up to 75 Ci/mmol is shown.

Improved methods for determination of guanylyl cyclase activity and synthesis of [α- 32P]GTP

A modification of the adenylyl cyclase assay method of Y. Salomon, C. Londos, and M. Rodbell (1974, Anal. Biochem. 48, 541–548) is described. It makes the method applicable to the determination of guanylyl cyclase in subcellular tissue fractions. The modification consists of performing the Dowex 50 chromatography at acid pH in a column that has bed volume that is three times as large. This modification results in low blanks and allows for reuse of both Dowex 50 and aluminum oxide columns. A modification of the method of Symons [1974, Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 29, pp. 105–115, Academic Press, New York] for synthesis of [α- 32P]nucleoside triphosphates is also presented. It allows all steps to be carried out within 5 h in a single reaction vessel. Synthesized NTPs are then purified by DEAE-Sephadex A-25 (HCO 3 −form) using a linear ammonium bicarbonate gradient. Its applicability to synthesis of [α- 32P]GTP, [α- 32P]dGTP, and [α- 32P]ATP having specific activities of up to 75 Ci/mmol is shown.

An automated procedure for the extraction and fluorimetric determination of 5-hydroxytryptamine in platelet subcellular fractions

A fully automated procedure for the determination of 5-hydroxytryptamine is described. The specificity and sensitivity of the assay depend upon a differential solvent extraction procedure to remove interfering 5-hydroxy-indoles and the measurement of the fluorescence of the amine after reaction with ninhydrin. The method is highly suitable for the analysis of tissue subcellular fractions since there is no interference by sucrose, and after a preliminary deproteinisation of the sample with ZnSO 4 NaOH , performed manually, the rest of the procedure is fully automated. The sensitivity of the assay depends upon the quality of the fluorimeter used but is better than 5 ng/ml of sample with a double monochromator spectrofluorimeter. There is no interference by 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, or 5-hydroxytryptophol. The suitability of the procedure for the routine analysis of 5-hydroxytryptamine in platelet subcellular fractions prepared by zonal gradient centrifugation has been explored and the soluble phase and particulate bound amine stores can be identified.

Binding of basic and acidic drugs to rat tissue subcellular fractions

Summary Binding of drugs to rat tissue subcellular fractions has been studied using equilibrium dialysis and/or centrifuge sedimentation, and Scatchard analysis of the experimental data. Chlorpromazine and imipramine are shown to be reversibly bound to liver microsomes and mitochondria, revealing regular Scatchard-type binding with two classes of binding sites—one site of high affinity/low capacity and the second site of low affinity/high capacity. The association constants for the two sites for chlorpromazine, for example, are 1.4 · 10 5 and 3.9 · 10 3 M −1 respectively and the total binding capacity is 715 nmoles per mg microsomal protein. Near identical values are obtained with microsomal fractions from liver, lung, kidney, brain and skeletal muscle. The binding to nuclei and soluble cell constituents of liver is much weaker. Comparative binding studies with a range of drugs were also carried out using liver microsomes. Chlororomazine and imipramine and their demethylated derivatives, 3-chloroimipramine and methadone all exhibit binding to two classes of sites, with high affinity and high but variable capacity values. The more polar tricyclic compounds show less binding. Among the acidic drugs chosen, salicylic acid and sulphadimethoxine show no detectable binding while binding parameters for phenylbutazone and warfarin are low. Some of the weak binding compounds such as imipramine- N -oxide, a glucuronide of an imipramine metabolite and warfarin did not fulfill the Scatchard binding model.

Investigation of procedures for subcellular fractionation of rabbit aortic tissue

A procedure has been developed to obtain an improved subcellular fractionation of rabbit aortic tissue. Treatment of rabbit aortic tissue with collagenase and elastase resulted in the removal of the bulk of the collagen and elastin. This procedure facilitated subsequent homogenization and subcellular fractionation of the tissue.

The primary immune response in mice. 3. Retention of sheep red blood cell immunogens by the spleen and liver.

The antibody-inducing activities of foreign red blood cell immunogens sequestered in the spleens and livers of mice injected with sheep erythrocytes were evaluated during the early periods of the immune response. Estimates of immunogenicity, obtained from the magnitudes of anti-sheep red blood cell hemolysin responses evoked in sensitized recipient mice by subcellular tissue fractions prepared from these phagocytic organs, showed that the liver and spleen differ greatly in their handling of this particulate antigen. The liver, functioning primarily as a scavenger organ, destroys completely the immunogenicity of the heterologous erythrocytes in 12 hr. In contrast, the spleen handles foreign erythrocyte immunogens in at least two different ways: approximately 90% of the initially sequestered activity is rapidly destroyed by the spleen in 6 hr, but the remaining activity, associated chiefly with a tissue fraction possessing the sedimentation properties of "light mitochondria," is retained at a significant level for 3 days, and then progressively decreases to a low level until the 7th day. A correlation of the observed changes in the properties of the immunogenic tissue fraction with known cellular events in the spleen stimulated by antigens indicates that the retention of the degradable erythrocyte immunogen is essential for stimulating and maintaining immune reactions in this antibody-producing organ.

Cadmium and zinc binding in mammalian liver and kidneys.

Rats and mice were injected subcutaneousty with cadmium chloride Cd 109 and zinc chloride Zn 65. Both isotopes concentrated primarily in liver and kidneys. Tissue subcellular fractionation showed 70% to 80% 109Cd and 65Zn associated with cytoplasmic soluble fraction. Soluble fraction gel filtration revealed most 109Cd was bound to proteins of 11,000 to 12,000 molecular weight; 65Zn was associated mainly with larger sized molecules. In rats, two weeks following isotope dose, depletion of 65Zn from cytoplasmic macromolecules was demonstrated; 109Cd remained fixed to cadmium-binding proteins (Cd-BP). By ion-exchange chromatography, Cd-BP was resolved into major components. Similar components were separated from human kidney Cd-BP. Presence of Cd-BP was noted in humans and monkey liver tissues. It is suggested that mammalian organism intracellular Cd-BP apparently acts as biochemical mechanism for sequestration of toxic Cd2+ ions.

The uptake and distribution of 65Zn in oysters

Over a period of 6 weeks in aquaria, Portuguese oysters Crassostrea angulata, accumulate 65Zn to a greater extent than do native Ostrea edulis, although intake rates for any particular organ in either species are quite similar. The general distribution pattern of radioactivity in the tissues is similar to that observed for stable zine and 65Zn in other oyster species, concentration occurring to the greatest extent in gills and mantle, and least in muscle. The observed equilibrium concentrations and biological half-lives are considerably less than those measured in the natural environment, and the significance of this and its bearing on the mechanism of uptake is discussed. Cobalt and iron depress the rate of 65Zn uptake by both oyster soft tissues and the shell. The limiting effect in soft tissues is probably due to competition for sites at the actual point of uptake. The distribution of 65Zn in tissue subcellular fractions separated by centrifugation shows the greatest concentration of the radioisotope in the insoluble tissue components of gills, mantle and heart. Appreciable amounts of 65Zn are associated with tissue proteins.