Estradiol-17 beta modifies the induction of spermidine/spermine N1-acetyltransferase activity in the liver of lipopolysaccharide-treated mice.

In an attempt to elucidate the effects of estrogen on polyamine metabolism in lipopolysaccharide (LPS)-treated mice, we assayed polyamine content and the activity of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) in some organs. LPS elevated N1-acetylspermidine levels in the liver and lung and putrescine levels in the liver, lung and spleen. LPS increased the activity of ODC at 6 h and that of SAT at 12 h in the liver. When estradiol-17 beta was simultaneously administered with LPS, the maximum increase in hepatic N1-acetylspermidine levels was found 6 h earlier than in the LPS control. Likewise, the peak of the hepatic SAT activity after LPS-treatment was observed 6 h earlier in the estradiol-17 beta-treated mice than in the LPS control. No such effect of estradiol-17 beta was found in the lung and spleen. The LPS-induced ODC activity was not affected by estradiol-17 beta in the liver, lung or spleen. Estrone and 16 beta-ethylestradiol (an anti-estrogen) were also effective in enhancing the LPS-induced elevation of N1-acetyl-spermidine and putrescine in the liver, while both diethylstilbestrol, which has a potent estrogenic activity without steroid structure and estradiol-17 alpha (a non-estrogenic isomer of estradiol-17 beta) were without effect. Tamoxifen (an estrogen receptor antagonist) did not suppress the estrogen-induced increase in hepatic N1-acetylspermidine levels.     

Estradiol-17β modifies the induction of spermidine/spermine N1-acetyltransferase activity in the liver of lipopolysaccharide-treated mice

ABSTRACT— In an attempt to elucidate the effects of estrogen on polyamine metabolism in lipopolysaccharide (LPS)-treated mice, we assayed polyamine content and the activity of spermidine/spermine N¹-acetyltransferase (SAT) and ornithine decarboxylase (ODC) in some organs. LPS elevated N'-acetylspermidine levels in the liver and lung and putrescine levels in the liver, lung and spleen. LPS increased the activity of ODC at 6 h and that of SAT at 12 h in the liver. When estradiol-17β was simultaneously administered with LPS, the maximum increase in hepatic N¹-acetylspermidine levels was found 6 h earlier than in the LPS control. Likewise, the peak of the hepatic SAT activity after LPS-treatment was observed 6 h earlier in the estradiol-17β-treated mice than in the LPS control. No such effect of estradiol-17β was found in the lung and spleen. The LPS-induced ODC activity was not affected by estradiol-17β in the liver, lung or spleen. Estrone and 16β-ethylestradiol (an anti-estrogen) were also effective in enhancing the LPS-induced elevation of N¹-acetyl-spermidine and putrescine in the liver, while both diethylstilbestrol, which has a potent estrogenic activity without steroid structure and estradiol-17α (a non-estrogenic isomer of estradiol-17β) were without effect. Tamoxifen (an estrogen receptor antagonist) did not suppress the estrogen-induced increase in hepatic N¹-acetylspermidine levels.     

Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity.     

Expression pattern and action analysis of genes associated with the responses to chemical stimuli during rat liver regeneration

INTRODUCTION The liver has tremendous capacity to regenerate itself[1,2]. Liver cells proliferate rapidly to compensate the lost liver tissues after liver injury or chemical stimulus, which is called liver regeneration (LR)[1,3]. Generally, based on the c…     

Gossypol activates pancreatic polyamine catabolism in normal rats and induces acute pancreatitis in transgenic rats over-expressing spermidine/spermine N1-acetyltransferase

BACKGROUND: The male antifertility agent gossypol has been reported to induce spermidine/spermine N1-acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity. METHODS: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma 2-amylase activity were determined after different doses of gossypol. RESULTS: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis. CONCLUSIONS: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.     

GTP-binding proteins in rat liver nuclear envelopes.

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.     

Affinity labeling of rat liver thyroid hormone nuclear receptor.

The thyroid hormone receptor from rat liver nuclei has been covalently labeled with the N-bromoacetyl derivatives of L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3). Displacement binding studies showed that, in the presence of 100-fold molar excess of unlabeled N-bromoacetyl-T3 or T4, binding of [125I]T3 or [125I]T4 was nearly totally inhibited. Heat inactivation of the receptor (55 degrees C for 15 min) resulted in parallel losses in the binding of T3 (95%) and N-bromoacetyl-T3 (93%). These results indicated that T3 and T4 and their bromoacetyl derivatives compete for the same binding site. The nuclear receptor showed identical behavior in high-pressure liquid chromatography (HPLC) whether bound to T3 or T4 or covalently labeled with their bromoacetyl derivatives. HPLC provided a single-step 100-fold purification of the nuclear receptor. Na-DodSO4 gel electrophoresis of the nuclear receptor labeled with N-bromoacetyl derivatives of [125I]T3 or [125I]T4 showed one major radioactive component with a molecular weight of 56,000. Furthermore, in the absence of denaturant, the nuclear receptor either bound to [125I]T3 or covalently labeled with N-bromoacetyl-[125I]T3 showed identical mobility. These results suggested that the nuclear receptor is a single polypeptide chain and binds either T3 or T4. Nuclear receptors covalently linked with N-bromoacetyl derivatives of [125I]T3 or [125I]T4 may be useful as a marker for the preparative purification of receptor.     

Interleukin-1beta induces elevation of spermidine/spermine N1-acetyltransferase activity and an increase in the amount of putrescine in synovial adherent cells from patients with rheumatoid arthritis

OBJECTIVE: To investigate polyamine metabolism in rheumatoid synovial adherent cells stimulated by interleukin- 1beta (IL-1beta). METHODS: Synovial adherent cells obtained from patients with rheumatoid arthritis (RA) were cultured and incubated in the presence or absence of human recombinant IL-1beta at a concentration of 10 ng/ml for 24 h. The cellular contents of polyamines as well as the activities of spermidine/spermine N1-acetyltransferase (SAT) and ornithine decarboxylase (ODC) were measured. RESULTS: Polyamines in synovial adherent cells decreased significantly after 24 h incubation in the absence of IL-1beta. However, in the presence of IL-Ibeta, putrescine and N'-acetylspermidine increased significantly. No significant difference was observed between the amount of spermidine in synovial adherent cells incubated with and without IL-1beta. Spermine and N8-acetylspermidine in synovial adherent cells incubated with IL-1beta decreased significantly more than in synovial adherent cells incubated without. SAT activity reached a peak 12 h after the addition of IL-1beta and then decreased, while the ODC activity did not increase. SAT activity was elevated by the addition of IL-1beta in a dose dependent manner. CONCLUSION: An increase in the putrescine level in rheumatoid synovial adherent cells as a result of the elevation of SAT activity induced by IL-1beta may play a role in RA.     

ATP-dependent association of nuclear proteins with isolated rat liver nuclei.

In vitro association of Xenopus nucleoplasmin and mammalian nonhistone chromosomal high mobility group 1 (HMG1) protein with nuclei isolated from rat liver was examined. Efficient association of nuclear proteins with isolated nuclei requires ATP, HCO3-, and Ca2+. Association occurred at 33 degrees C but not at 4 degrees C. ATP could be replaced by adenosine 5'-[alpha,beta-methylene]triphosphate (pp[CH2]pA), a nonhydrolyzable ATP analog. pp[CH2]pA associated with nuclei at 33 degrees C and nucleoplasmin and HMG1 rapidly associated with the pp[CH2]pA-bound nuclei at 4 degrees C. Competition studies showed that these associations at both 33 degrees C and 4 degrees C were specific. More than 80% of the bindings of nuclear proteins to the nuclear surface were blocked by wheat germ agglutinin.     

Putative association between the -1415 T/C polymorphism of spermidine/spermine N1-acetyltransferase (SSAT1) gene and alcohol use disorders in women and men

Background: The activity of N-methyl-D-aspartate (NMDA) glutamate receptor, which responds to the levels of polyamines, modifies the neurotoxicity caused by ethanol. We aimed to investigate if the functionality of the spermidine/spermine N1-acetyltransferase (SSAT1) gene could be associated with a differential risk for alcoholism. Methods: We studied a sample of 586 subjects: 104 alcohol-dependent patients, 273 patients with psychiatric disorders but without substance dependence, and 209 healthy controls. After gender stratification, the allele frequency distribution of the SSAT1 gene was compared between these three groups. Results: In females, the TC genotype was significantly more frequent in alcohol-dependent patients than in non-alcohol-dependent psychiatric controls (χ^2?=?7.509 df?=?2, p?=?0.023). A trend was found when alcohol-dependent females were compared with the healthy control group (χ^2?=?4.897 df?=?2, p?=?0.086). No statistical differences were found among the males. Discussion and conclusion: Gender differences in the regulation of SSAT1 gene expression may possibly be due to gender-specific effects of stress, ethanol toxicity, and/or polyamines levels. Further studies are needed to confirm our findings.     

Hemodynamic and metabolic responses to leukotriene C4 in isolated perfused rat liver

Responses of isolated perfused rat liver to leukotriene C4 were studied in order to assess the mechanisms involved in leukotriene-mediated liver injury. Infusion of leukotriene C4 (11 and 44 pmoles per min per gm liver weight) into the portal vein resulted in a rise in portal pressure, a decrease in oxygen consumption, an increase in hepatic glucose and lactate efflux and lactate/pyruvate ratio in the perfusate and a small decrease in bile flow. Isoproterenol (1 microM) counteracted the effects of leukotriene C4 on respiration and portal pressure, whereas bile flow and glucose efflux were reversibly stimulated. The same changes were observed upon withdrawal of leukotriene C4. The release of glucose was correlated with the increase in oxygen consumption upon both isoproterenol addition and withdrawal of leukotriene C4. These results are indicative of leukotriene C4-induced microcirculatory redistribution of perfusate flow. Since, in the presence of nitroprusside (50 microM), both the effects of leukotriene C4 and their reversal by isoproterenol were diminished, a vascular site of action can be assumed. Accordingly, the accompanying metabolic responses can be explained by gradual changes in oxygen supply to parts of the liver. Reversibility of the leukotriene C4 effects and lack of short-term impairment of viability of the isolated liver suggest that leukotriene-mediated liver injury is a long-term effect related to events subsequent to microcirculatory changes.     

Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys

We examined effects of hypercholesterolemia and atherosclerosis on vasoconstrictor responses to norepinephrine and serotonin. Responses were compared in normal, atherosclerotic, and hypercholesterolemic but non-atherosclerotic cynomolgus monkeys. The hindlimb was perfused at constant flow so that changes in perfusion pressure indicated changes in vascular resistance. We measured the pressure gradient from the iliac to the dorsal pedal artery so that responses of the large artery segment could be determined. Serotonin decreased total hindlimb resistance in normal and hypercholesterolemic monkeys, but increased total resistance in atherosclerotic monkeys. There was a greater than 10-fold increase in constrictor responses of large arteries to serotonin in atherosclerotic monkeys, compared with normal and hypercholesterolemic monkeys. In contrast, we found that vasoconstrictor responses to norepinephrine are normal in atherosclerotic monkeys and increased in hypercholesterolemic monkeys prior to development of atherosclerosis. Hypercholesterolemia augmented responses of small vessels to norepinephrine. We conclude that, during early stages of hypercholesterolemia in cynomolgus monkeys, vasoconstrictor responses to norepinephrine are increased in small vessels. At a later stage, as atherosclerosis develops, responses to norepinephrine return to normal, but vasoconstrictor effects of large arteries to serotonin are greatly potentiated.     

Left ventricular diastolic function and responses to adrenergic stimuli in borderline arterial hypertension.

OBJECTIVE: To detect the existence of a possible relationship between arterial hypertension and adrenergic reactivity to pressure stimuli, and changes in left ventricular diastolic function (LVDF). PATIENTS: Fifty-nine young subjects with borderline arterial hypertension and ten sex- and age-matched controls were investigated. After three medical examinations, the subjects were divided into hypertensive and borderline groups on the basis of the blood pressure reading at visit 3. A complete echocardiographic study was performed in 25 of the 59 subjects. DESIGN: Blood pressure was measured in baseline conditions and during pressure stimuli (mental stress, handgrip and cold pressor tests). LVDF was evaluated primarily by means of filling velocities during diastolic phases taken from the left ventricular volume curve (obtained from a complete echocardiographic study). RESULTS: No significant changes in blood pressure responses were observed for the borderline or hypertensive groups during the adrenergic test. The echocardiographic indices of diastolic function were statistically different for the two groups when compared with the control group. The LVDF parameters correlated significantly with systolic blood pressure and diastolic blood pressure measured at the time of the echocardiogram, but not with blood pressure measured occasionally. CONCLUSIONS: Blood pressure increases similarly during adrenergic stimuli in both the hypertensive and borderline groups. The correlation between systolic blood pressure, diastolic blood pressure and LVDF parameters may indicate a very early onset of reduced compliance of the left ventricle, even in a preclinical phase of hypertension.     

Sodium-dependent regenerative responses in dendrites of axotomized motoneurons in the cat.

Ten days after extradural axotomy, partial spikes are found in greater than 20% of cat L7 motoneurons, while 15-21 days after axotomy the incidence increases to 60%. These responses are produced in excitable (hot) spots in the dendrites by synaptic excitation. Intracellular injection of QX-314, a lidocaine derivative and effective blocker of Na+ channels from within neurons, results in elimination of partial spikes before blocking somadendritic spikes. The action of QX-314 does not depend on changes in passive membrane properties or on changes in synaptic properties. Injections of Cs+ and Cl- ions rule out any major role for calcium, potassium, or chloride currents in the production of partial spikes. The partial spikes represent an unusual Na+-dependent dendritic phenomenon induced by axotomy when carried out relatively near the soma. It is reasonable to postulate that the partial spikes result from a higher concentration of Na+ channels in the dendrites. This may be the consequence of a high rate of production of Na+-channel proteins that are intended for the cut end of the axon; alternatively, they may result from the reflection from the cut end of such proteins produced at either a normal or an increased rate. These aberrant channels are inserted into somatic and dendritic membranes in higher concentrations than normal and, as well as producing local dendritic regions of low safety factor responsible for the partial spikes, also produce somadendritic spikes of unusually fast rise time and lower than usual threshold, which are relatively resistant to QX-314.