Biomedical subjects
Publications and source records attributed to M A Grillo.
1. Okadaic acid inhibited basal ODC activity in rat hepatocytes in culture and prevented any increase in ODC activity and in the rate of spermidine uptake promoted by both insulin and hypotonicity. 2. The increase promoted by AIB was not counteracted by okadaic acid.
1. 1 mM 2-amino isobutyric acid (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake. 2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB. 3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB. 4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine. 5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide. 6. Okadaic acid had no effect on this increase, whereas it prevented the increase of ODC activity.
1. In human lymphocytes alkalinization of the cytoplasm with monensin or NH4Cl promotes release of polyamines. The effect of NH4Cl is abolished by EGTA and diltiazem. 2. Concanavalin A also promotes an increase of the efflux, counteracted again by EGTA and diltiazem. 3. By effect of TPA, polyamine efflux is decreased in the first 90 min, and later increased. The activation is partially prevented by H7 and by sphingosine. 4. In contrast with human lymphocytes, L 1210 cells release actively endogenous polyamines, but slowly radioactive polyamines. 5. Concanavalin A does not activate the latter process; A 23187 and NH4Cl on the contrary promote a much higher increase in the efflux rate than in normal lymphocytes. EGTA and diltiazem partially counteract the effect of NH4Cl on the release of radioactivity.
Preparations enriched in human blood B lymphocytes are able to take up polyamines efficiently. Uptake by T cells is barely detectable. Human non-circulating B cells (from tonsils) have a much lower ability to take up polyamines, as do mixed populations of bovine lymph nodes. B cells contain a higher amount of endogenous polyamines and show higher ornithine decarboxylase activity than T cells.
Human lymphocytes in culture loaded with radioactive polyamines slowly release radioactivity into the medium. N1-Acetylspermidine is mostly released from spermidine and spermine. Both ouabain and calcium ionophore A23187 increase the outward transport, but by different mechanisms. Ouabain inhibits the acetylation of spermidine, and free spermidine is released, whereas A23187 increases both acetylation of spermidine and the efflux of N1-acetylspermidine.
In rat hepatocytes cultured for 120 h polyamine content was markedly modified. Putrescine concentration reached a maximum at 48 h, spermidine increased for 48 h and then remained constant, spermine after a decrease returned to its initial values. Total polyamine amount was increased by 75%. Both ornithine decarboxylase and the retroconversion pathway were responsible for these modifications. The possible correlation between polyamine metabolism and retrodifferentiation process was investigated by studying them in conditions which are known to preserve differentiated functions.
1. Polyamines are taken up by human peripheral lymphocytes in a concentration, time and pH dependent manner, with an energy-dependent transport system. 2. Each polyamine inhibits the uptake of the others, with the exception of putrescine. Spermine appears to have the highest affinity for the transporter/s. 3. Inhibition by ouabain, amiloride and vanadate suggests that the transport is dependent on Na+. 4. Polyamine content inside the cells increases by ca 6 and 3 times respectively during incubation with spermidine or spermine. 5. The incorporated polyamines are partially transformed into each other.
1. In vivo, adrenergic agonists promote an increase of ornithine decarboxylase activity (ODC) in chicken spleen, as opposed to a decrease in thymus and bursa of Fabricius. The increase is not due to the cell fraction separated on Lymphoprep, i.e. the spleen cells, but it could be due to the macrophages. 2. With spleen cells in culture, a marked increase of ODC activity is observed during the first 3 hr, followed by a decrease. 3. cAMP drastically decreases after 10 min in culture. 4. Adrenergic agonists promote a decrease of activity, both alpha and beta receptors being involved in these modifications. TPA promotes partial desensitization. 5. Selenite, which in vivo has the same effect as epinephrine, enhances ODC activity in culture. Propranolol partially counteracts this effect, while prazosin has a synergistic effect. TPA partially desensitizes spleen cells to selenite.
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The effect of turpentine, a chemical inflammatory agent, on polyamine synthesis has been studied. Ornithine decarboxylase activity is markedly increased in liver 6 hrs after subcutaneous injection of turpentine, and then decreases. No significant modification is observed in S-adenosylmethionine decarboxylase. Putrescine injected prior to turpentine prevents this increase. Putrescine, spermidine and spermine concentrations are all increased following turpentine, but with different patterns: spermidine alone keeps increasing for 50 hours. Putrescine and spermidine injected prior to turpentine partially counteract the increase of serum alpha 2-macroglobulin, which is believed to be a marker of inflammation.
1. When injected i.p., sodium selenite promoted a marked increase of rat liver ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities; when administered with the diet for 6 weeks, a less marked increase in liver ODC was observed, whereas SAMDC was not significantly changed. 2. Protein synthesis was involved in the observed modifications. The rate of ODC inactivation was also changed. 3. ODC increase was accompanied by an enhanced putrescine concentration in liver. 4. A marked increase of ODC, accompanied by an enhancement of putrescine, was promoted by selenite (i.p.) also in chicken liver, together with an enhancement of glutathione concentration. Spermidine acetyltransferase (SAT) was also increased. 5. In the bursa of Fabricius, SAT activity was also increased, whereas ODC was decreased. However the expected modifications in polyamine concentration were not observed. 6. Decrease of ODC activity in the bursa was not due to an antizyme. 7. In vitro, selenite concentrations known to inhibit cell proliferation (greater than 1 microgram/ml) inhibited both ODC and SAT activities; at lower concentration, SAT activity was enhanced.
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Vanadate in the presence of pyridoxal phosphate promotes the decarboxylation of S-adenosylmethionine. Pyridoxal has a lower effect; pyridoxine none. The rate of decarboxylation depends on pyridoxal phosphate and vanadate concentration. Vanadate as low as 10(-7) M gives significant decarboxylation. The reaction seems to occur through the formation of a Schiff base. The spectral shift elicited by S-adenosylmethionine on pyridoxal phosphate due to the presence of the sulfonium function is influenced by vanadate. Orthovanadate is a little less effective then metavanadate; vanadyl sulfate is even less efficient, and the effect of Cu2+ at the same concentration is still lower. Bleomycin partially prevents the vanadium effect. In vivo, vanadate promotes a marked increase in chicken liver S-adenosylmethionine and S-adenosylhomocysteine concentration, whereas the polyamine concentration is unaffected.
The effect of diabetes on some enzymes of polyamine metabolism was studied in male rats 1-12 days after administration of streptozotocin. Hepatic ornithine decarboxylase activity decreased in the first days after the administration, but increased thereafter. The decrease was not due to an alteration of the ODC-antizyme concentration, nor to a posttranslational modification catalyzed by transglutaminase. S-adenosylmethionine decarboxylase and ornithine transaminase were both increased. Spermidine acetyltransferase activity was practically unchanged, while its inactivating factor was markedly decreased.
Both spermidine and spermine are acetylated in chicken brain and retina. From spermidine, more N1-acetylspermidine than N8-acetylspermidine is formed by both the brain and the retinal cytosol. Km for spermidine is similar with the enzyme preparation of the two tissues, but that for spermine is lower with the retinal preparation. Both tissues contain an activity able to reduce spermidine acetyltransferase activity. Both alkaline phosphatase and cAMP-dependent protein kinase (catalytic subunit) are able to inactivate the spermidine acetyltransferase activity of both tissues. Spermidine acetyltransferase activity and polyamine levels have been measured in both brain and retina during embryonic life. Only in the last part of the development can enzyme activity be correlated with the retina spermidine and spermine concentration.
Muscarinic receptor distribution in smooth muscle, exocrine glands, and epithelium of the ferret trachea was determined using [3H]propylbenzilylcholine mustard ([3H]PrBCM) binding and autoradiography. Specific, atropine-sensitive [3H]PrBCM binding was quantified autoradiographically in the trachealis muscle (approximately 21 binding sites/microns2), surface epithelium (approximately 6 binding sites/microns2), and submucosal glands (approximately 5 binding sites/microns2). Serous and mucous cells in the glands did not differ in receptor density. Binding sites on gland and epithelial cells were associated with basolateral membranes. In the trachealis muscle, a gradient in receptor density was observed, with outer layers of muscle containing 3 to 10 times more receptors per unit area than inner layers. Receptor distribution in both glands and muscle paralleled the distribution of cholinergic axons. However, at the light microscope level, there was no evidence for the presence of receptor "hot spots" related to the position of individual axons. The parallelism in the distribution of axons and receptors suggests the possibility of neural control of the genesis and/or maintenance of receptor distribution in these tissues.
Transglutaminase activity measurements in the liver of insulin- and diaminopropane treated chickens showed that insulin enhances activity in the supernatant, but has no effect on the nuclear fraction. In the absence of exogenous Ca++ ions, both fractions were more active, showing that insulin promotes an increase in the available Ca++ ions. No modification was observed after diaminopropane. It follows that changes in ornithine decarboxylase activity previously observed under these conditions are not dependent on transglutaminase activity.
Arginase, ornithine decarboxylase and S-adenosylmethionine decarboxylase are active in both retina and brain. Activity is higher in cerebellum than in the cerebral hemispheres and optical lobes. Arginase and ornithine decarboxylase are very active in the retina of very young chicks, while S-adenosylmethionine decarboxylase is poorly active. By contrast, S-adenosylmethionine decarboxylase is much more active in brain. The pattern of activity during development is different; only ornithine decarboxylase is very active during embryonal life; S-adenosylmethionine decarboxylase, at all events in brain, is more active in adult life. Ornithine decarboxylase is inhibited in vitro by alpha-difluoromethylornithine, but not in vivo. Diaminopropane inhibits brain ornithine decarboxylase, but does not induce an ornithine decarboxylase-antizyme. Methylglyoxal bis(guanylhydrazone) promotes an increase of S-adenosylmethionine decarboxylase activity in both the brain and the retina in vivo.