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PubMed2003

Reduction of myocardial infarct size by fluvastatin.

Statins have a variety of cardioprotective properties following chronic treatment. In contrast, little is known about the acute effects. Reperfusion acutely injures the heart by activation of neutrophils as well as endothelial cells. Because statins are known to influence the processes pathogenetically involved, we hypothesized that acute application of statins attenuates the sequelae of cardiac reperfusion. In rats, myocardial infarction (MI) was induced by ligature of the left coronary artery followed by reperfusion. Myocardial blood flow (MBF) was determined by H2 clearance and regional myocardial function (fractional thickening, FT) by pulsed Doppler. MI size was measured by triphenyltetrazolium chloride (TTC) staining, neutrophil extravasation by determination of myeloperoxidase (MPO) activity, and nitric oxide generation via measurement of cGMP. Treatment with fluvastatin, administered intravenously 20 min before the onset of ischemia, significantly attenuated the decline of FT and MBF at the end of the reperfusion period and significantly reduced MI size. Furthermore, fluvastatin induced a significant reduction of MPO activity and an increase of cGMP level compared with the control group. The effect of fluvastatin was completely abolished following pretreatment of NG-nitro-l-arginine methyl ester (l-NAME). These findings suggest that acute application of fluvastatin reduces MI size and attenuates reperfusion injury. We propose that the underlying mechanism is at least partially an inhibition of inflammation and endothelial dysfunction by preventing the activation and extravasation of neutrophils.

Animals↗
PubMed2006

Treatment of benign prostatic hyperplasia in patients with cardiovascular disease.

Pharmacological management is the most common therapeutic approach for patients with benign prostatic hyperplasia and alpha-adrenoceptor antagonists are the most commonly prescribed initial treatment. Although all of the alpha-adrenoceptor antagonists produce similar improvements in symptom scores and urinary flow rates, they have different adverse effect profiles, especially with respect to the cardiovascular system. The older alpha-adrenoceptor antagonists, terazosin and doxazosin, were initially approved for the treatment of hypertension and are associated with higher rates of dizziness, syncope and hypotension than the newer agents, tamsulosin and alfuzosin. The older alpha-adrenoceptor antagonists are also involved in more interactions and have a greater number of precautions concerning their usage with other cardiovascular medications. Of the newer alpha-adrenoceptor antagonists, tamsulosin has a lower rate of dizziness than alfuzosin. However, both of these agents are superior to doxazosin and terazosin in that no dose escalation or titration is needed for them.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2002

Involvement of NO in the endothelium-independent relaxing effects of N(omega)-hydroxy-L-arginine and other compounds bearing a C=NOH function in the rat aorta.

The mechanisms of vasorelaxation elicited by N(omega)-hydroxy-L-arginine (L-NOHA) and other compounds bearing a C=NOH function and the structural determinants governing this effect were investigated in rat aorta. L-NOHA, formamidoxime, five aromatic monosubstituted amidoximes, and one aromatic monosubstituted ketoxime elicited relaxation in endothelium-denuded rings. N-Hydroxyguanidine and substituted N-hydroxyguanidines were markedly less active. Relaxations induced by L-NOHA and by the most active studied compound, 4-chlorobenzamidoxime (ClBZA), were unmodified by the presence of endothelium. In endothelium-denuded rings, they were blunted by the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (300 microM) and by the inhibitor of guanylyl-cyclase activation 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (1 microM). In addition, L-NOHA- and ClBZA both caused cGMP accumulation. L-Arginine, but not D-arginine (1 mM), antagonized the effect of L-NOHA but not ClBZA. Both L-NOHA- and ClBZA-induced relaxations were inhibited by the NAD(P)H-dependent enzymes inhibitor diphenyliodonium (30 microM) and the NAD(P)H-dependent reductases inhibitor 7-ethoxyresorufin (10 microM), but they were unmodified by the cytochrome P450 (P450) inhibitor proadifen (10 microM) and by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 300 microM). These results show that L-NOHA and other compounds with a C=NOH function can cause endothelium-independent relaxation in the rat aorta. They suggest that activation of guanylyl cyclase and NO formation is implicated in relaxation and that a 7-ethoxyresorufin-sensitive NAD(P)H-dependent pathway is involved. On one hand, L-NOHA and amidoximes may be useful tools for characterizing this pathway in blood vessels and, on the other, may offer a novel approach for treating vascular diseases with impaired endothelial NO activity.

Animals↗
PubMed2006

Nitric oxide signaling in the cardiovascular system: implications for heart failure.

PURPOSE OF REVIEW: The role played by nitric oxide (NO) in cardiovascular physiology remains highly controversial. Following the discovery that NO is the prototypic endothelium-derived relaxing factor, this signaling molecule was implicated as possessing many other biological actions within the cardiovascular system, including effects on cardiac contraction, relaxation, and energetics. Here, we discuss new concepts regarding NO signaling, its effector pathways, and interactions between NO and the redox milieu within a framework of cardiac physiology and pathophysiology. RECENT FINDINGS: Major recent insights that have advanced understanding of the mechanisms of NO bioactivity include the following. (1) NO acts in subcellular signaling compartments or modules. (2) S-nitrosylation (covalent modification of cysteine thiol moieties) of proteins represents a prototypic second messenger signaling mode in biologic systems. (3) Reactive oxygen and nitrogen species work together to facilitate signaling. (4) Disruption of physiologic signaling can occur by either increased formation of reactive oxygen species or decreased production of reactive nitrogen species, a situation of nitroso-redox imbalance. SUMMARY: These insights, which challenge classically held views that NO acts as a freely diffusible molecule regulated primarily by concentration and exerting signaling primarily through cyclic GMP production, offer a new perspective on the pathophysiology and treatment of congestive heart failure.

Alcohol Dehydrogenase↗
PubMed1976

Distribution of enzyme activities in subcellular fractions of bovine retina.

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2004

Relaxant effect of oxime derivatives in isolated rat aorta: role of nitric oxide (NO) formation in smooth muscle.

Various oxime derivatives were evaluated as nitric oxide (NO) donors in arteries. Relaxation of rat aortic rings was used for bioassay of NO production, and electron paramagnetic resonance spectroscopy for demonstration of NO elevation. In rings with or without endothelium or adventitia, hydroxyguanidine and hydroxyurea were almost inactive, whereas formamidoxime, acetaldoxime, acetone oxime, acetohydroxamic acid and formaldoxime elicited relaxation. Active compounds increased NO levels in endothelium-denuded rings. Formaldoxime was the most potent agent for both relaxation and NO elevation in aortic rings, and it also increased NO in human aortic smooth muscle cells. In endothelium-denuded rings, relaxation was inhibited by a NO scavenger (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) and by inhibitors of soluble guanylyl-cyclase (1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one) or cyclic GMP-dependent protein kinases (Rp-8-bromo cyclic GMP monophosphorothioate). Neither N(omega)-nitro-l-arginine methylester (a NO synthases inhibitor) nor proadifen (a cytochrome P450 inhibitor) decreased the effect of oxime derivatives. However, 7-ethoxyresorufin (7-ER, an inhibitor of P4501A(1) which can also inhibit various NADPH-dependent reductases) abolished the relaxant effect of these compounds, without affecting the one of glyceryl trinitrate (GTN) or 2-(N,N-diethylamino)-diazenolate-2-oxide. 7-ER also abolished formaldoxime-induced NO increase in aortic rings. In rings tolerant to GTN, formaldoxime-induced relaxation and NO elevation were not different from those obtained in control rings. In conclusion, some oxime derivatives release NO by 7-ER-sensitive pathways in aortic smooth muscle, thus eliciting vasorelaxation. Pathways of NO formation are likely distinct from NO synthases and from those responsible for GTN biotransformation. Oxime derivatives could be useful for NO delivery in arteries in which endothelial NO synthase activity is impaired.

Animals↗
PubMed2004

Depletion of deoxyribonucleoside triphosphate pools in tumor cells by nitric oxide.

Nitric oxide displays pro- and anti-tumor activities, prompting further studies to better understand its precise role. Nitric oxide inhibits ribonucleotide reductase (RnR), the limiting enzyme for de novo dNTP synthesis. We report here the first detailed analysis of dNTP variations induced in tumor cells by NO. NO prodrugs induced a depletion in dNTP pools and an activation of the pyrimidine salvage pathway, as did hydroxyurea, the prototypic RnR inhibitor. In the presence of dipyridamole, which blocked salvaged dNTP synthesis, depletion of dNTP pools was also observed in tumor cells cocultured with macrophages expressing the high-output iNOS activity. This effect was rapid, reversible, blocked by NO scavengers, and cGMP independent. It was quantitatively correlated to iNOS activity. In the absence of dipyridamole, NO still induced a decrease in dATP concentration in tumor cells cocultured with macrophages, whereas surprisingly, concentrations of dCTP and dTTP expanded considerably, resulting in a strong imbalance in dNTP pools. NO prodrugs did not cause such an increase in pyrimidine dNTP, suggesting that pyrimidine nucleosides were released by NO-injured macrophages. Altered dNTP levels have been reported to promote mutagenesis and apoptosis. It is suggested that abnormal changes in dNTP pools in tumors might contribute to NO-dependent toxicity.

Animals↗
PubMed2005

HMG-CoA reductase inhibitors upregulate heme oxygenase-1 expression in murine RAW264.7 macrophages via ERK, p38 MAPK and protein kinase G pathways.

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. HMG-CoA reductase inhibitors (statins) possess several anti-inflammatory mechanisms and may be beneficial in the treatment of inflammatory diseases. Our previous study has shown that statins can inhibit iNOS gene expression in murine RAW264.7 macrophages. In this study, we showed that lovastatin, fluvastatin, atorvastatin, simvastatin, mevastatin and pravastatin are able to upregulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), a protein kinase G (PKG) inhibitor (KT5823), a soluble guanylyl cyclase inhibitor (ODQ), a p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of protein kinase C (PKC), protein kinase A (PKA), c-jun N-terminal kinase (JNK) and Rho kinase. Consistent with this notion, our previous study has reported the ability of statins to activate ERK and p38 MAPK in RAW264.7 macrophages. Here we further found the participation of cyclic guanosine monophosphate (cGMP)/PKG pathway for ERK activation in cells stimulated with statin and the ability of statin to induce AP-1 activity, which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, a Ras inhibitor (manumycin A) treatment also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that statins might activate PKG to elicit activations of ERK and p38 MAPK pathways and finally induce HO-1 gene expression, which provides a novel anti-inflammatory mechanism in the therapeutic validity.

Animals↗
PubMed1998

Glutamate neurotoxicity is associated with nitric oxide-mediated mitochondrial dysfunction and glutathione depletion.

The role of mitochondrial energy metabolism in glutamate mediated neurotoxicity was studied in rat neurones in primary culture. A brief (15 min) exposure of the neurones to glutamate caused a dose-dependent (0.01-1 mM) increase in cyclic GMP levels together with delayed (24 h) neurotoxicity and ATP depletion. These effects were prevented by either the nitric oxide (.NO) synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (NAME; 1 mM) or by the N-methyl-D-aspartate (NMDA) glutamate-subtype receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV; 0.1 mM). Glutamate exposure (0.1 mM and 1 mM) followed by 24 h of incubation caused the inhibition of succinate-cytochrome c reductase (20-25%) and cytochrome c oxidase (31%) activities in the surviving neurones, without affecting NADH-coenzyme-Q1 reductase activity. The rate of oxygen consumption was impaired in neurones exposed to 1 mM glutamate, either with glucose (by 26%) or succinate (by 39%) as substrates. These effects on the mitochondrial respiratory chain and neuronal respiration, together with the observed glutathione depletion (20%) by glutamate exposure were completely prevented by NAME or APV. Our results suggest that mitochondrial dysfunction and impairment of antioxidant status may account for glutamate-mediated neurotoxicity via a mechanism involving .NO biosynthesis.

2-Amino-5-phosphonovalerate↗
PubMed1992

Inhibition of nitrovasodilator- and acetylcholine-induced relaxation and cyclic GMP accumulation by the cytochrome P-450 substrate, 7-ethoxyresorufin.

We examined the effect of the cytochrome P-450 substrate, 7-ethoxyresorufin (7-ER), and its corresponding product, resorufin, on nitrovasodilator- and endothelium-dependent relaxation of isolated rat aorta. The EC50 value for glyceryl trinitrate (GTN) induced relaxation was increased over 100-fold by 7-ER and less than 3-fold by resorufin. The EC50 value for sodium nitroprusside (SNP) induced relaxation was increased approximately 12-fold by 7-ER, acetylcholine (ACh) induced relaxation was abolished, and relaxation induced by isopropylnorepinephrine was not significantly affected. GTN-, SNP-, and ACh-induced increases in cyclic GMP accumulation were inhibited by 7-ER, as were basal cyclic GMP levels in endothelium-intact, but not endothelium-denuded tissues. 7-ER decreased GTN biotransformation in intact aorta and decreased the regioselective formation of glyceryl-1,2-dinitrate. The activation by GTN and SNP of aortic guanylyl cyclase in broken cell preparations was not affected by 7-ER, indicating that the inhibitory effect of 7-ER is probably not due to a direct interaction with guanylyl cyclase. The inhibitory effect of 7-ER on GTN-induced relaxation was not altered by the addition of superoxide dismutase, suggesting that 7-ER does not act by increasing superoxide anion concentration (which would serve to increase the degradation of nitric oxide (NO) formed during vascular GTN biotransformation). Our data provide further evidence for the role of the cytochrome P-450--cytochrome P-450 reductase system in the biotransformation of GTN to an activator (presumably nitric oxide) of guanylyl cyclase. The data are consistent with a mode of action of 7-ER involving either competitive inhibition of vascular cytochrome P-450 or uncoupling of vascular cytochrome P-450 reductase from cytochrome P-450. The data also suggest that the cytochrome P-450 system facilitates NO release from SNP and that 7-ER has an inhibitory effect on endothelial nitric oxide synthase.

Acetylcholine↗
PubMed1980

The preparation and purification of isolated rat corpus-luteum cells and their use in studying the relationship between cholesterol biosynthesis and the lutropin-stimulated formation of progesterone.

Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.

Animals↗
PubMed1993

Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP.

Stimulation of nitric oxide (NO) synthase in endothelial cells by Ca2+ influx leads to increased intracellular levels of cGMP. NO synthase from various sources is known to use tetrahydrobiopterin, flavins, and NADPH as cofactors. We studied the effect of interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide on tetrahydrobiopterin biosynthetic activities in human umbilical vein endothelial cells (HUVEC). These stimuli led to an up to 40-fold increase of GTP cyclohydrolase I (EC 3.5.4.16) activity and to increased accumulation of neopterin and tetrahydrobiopterin in HUVEC. Further enzyme activities of tetrahydrobiopterin biosynthesis, i.e. 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153), remained unchanged. NO synthase activity in protein fractions from homogenates of cells treated with interferon-gamma plus tumor necrosis factor-alpha was not influenced as compared with untreated controls. However, interferon-gamma alone or in combination with tumor necrosis factor-alpha significantly increased intracellular cGMP formation in intact HUVEC by 50 and 80%, respectively. These stimuli increased intracellular tetrahydrobiopterin concentrations up to 14-fold. NO-triggered cGMP formation was similarly increased by incubation of otherwise untreated cells with sepiapterin, leading to elevated intracellular tetrahydrobiopterin levels. Thus, cytokines indirectly stimulate the activity of constitutive NO synthase in HUVEC by upregulating production of the cofactor tetrahydrobiopterin.

Amino Acid Oxidoreductases↗
PubMed1984

Tritrichomonas foetus: partly defined cultivation medium for study of the purine and pyrimidine metabolism.

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose--the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.

Agar↗
PubMed2001

A HMG-CoA reductase inhibitor possesses a potent anti-atherosclerotic effect other than serum lipid lowering effects--the relevance of endothelial nitric oxide synthase and superoxide anion scavenging action.

We have determined whether the anti-atherosclerotic effect of a 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitor (fluvastatin) is mediated through nitric oxide (NO) as well as affecting plasma lipids. NO related vascular responses, endothelial nitric oxide synthase (eNOS) mRNA and superoxide anion (O(2)(-)) release were examined in vascular walls of oophorectomized female rabbits fed 0.5% cholesterol chow for 12 weeks with or without fluvastatin (2 mg/kg per day). Serum lipid profile was not different between two groups. NO dependent responses stimulated by acetylcholine and calcium ionophore A23187 and tone related basal NO response induced by N(G)-monomethyl-L-arginine acetate (L-NMA); nitric oxide synthase inhibitor were all improved by fluvastatin treatment. Endothelium independent vasorelaxation induced by nitroglycerin was not different between the two groups of rabbits' arteries. Fluvastatin treatment increased cyclic GMP concentration in aorta of rabbits. eNOS mRNA expression and O(2)(-) release were measured in aorta using competitive reverse transcription-polymerase chain reaction (RT-PCR) and with lucigenin analogue, 2-methyl-3,7-dihydroimidazol [1,2-a]pyrazine-3-one (MCLA) chemiluminescence methods. eNOS mRNA in the endothelial cells of aorta was significantly up-regulated and O(2)(-) production was significantly reduced in fluvastatin treated rabbit aorta. Anti-macrophage staining area, but not anti-smooth muscle cell derived actin stained area in the aorta was also reduced by fluvastatin treatment. Conclusion, fluvastatin, a HMG-CoA reductase inhibitor, retards the initiation of atherosclerosis formation through the improvement of NO bioavailability by both up-regulation of eNOS mRNA and decrease of O(2)(-) production in vascular endothelial cells, and this means that part of the anti-atherosclerotic effect of fluvastatin may be due to nonlipid factors.

Acetylcholine↗
PubMed1988

Enzymic programs of rat bone marrow and the impact of acivicin and tiazofurin.

The in vivo actions of two antimetabolites, acivicin (NSC-163501) and tiazofurin (NSC-286193), were examined on the enzymic programs of rat bone marrow. From the bone marrow of the femurs, 100,000 g supernatant fractions were prepared; enzymic activities were measured by isotopic assays, and cellularity was determined. In the normal bone marrow, the specific activities of pyrimidine de novo synthetic enzymes, CDP reductase, dTMP synthase, CTP synthase, carbamoyl-phosphate synthase II (synthase II), orotidine 5'-phosphate decarboxylase and aspartate carbamoyltransferase, were 1, 2.7, 5, 10, 63 and 601 nmol/hr/mg protein, respectively, whereas those of the salvage enzymes, deoxycytidine, thymidine, cytidine and uridine kinases were 3, 43, 149, and 367 nmol/hr/mg protein, respectively. In purine biosynthesis, the activities of the de novo synthetic enzymes, IMP dehydrogenase, formylglycinamidine ribonucleotide (FGAM) synthase, GMP synthase, amidophosphoribosyl-transferase (AT) and adenylosuccinate synthase were 16, 8, 107, 78 and 124 nmol/hr/mg protein, respectively, and those of the salvage enzymes, adenine, hypoxanthine and guanine phosphoribosyl-transferases, were 340, 407, and 1018 nmol/hr/mg protein, respectively. The sequence of events was elucidated after a single i.p. injection of acivicin (5 mg/kg) or tiazofurin (200 mg/kg). Within 2 hr after acivicin injection, CTP, GMP and FGAM synthases lost 85-90%, while AT and synthase II lost 50 and 80%, respectively, of their activities. The activities rose to near normal range by 72-96 hr. The bone marrow cellularity decreased, reaching a nadir at 24 and 48 hr, and returning to normal range by 72 and 92 hr; thymidine kinase activity followed a similar pattern. Tiazofurin injection depressed IMP dehydrogenase activity to 20% by 2 hr with a rebound to normal range by 48 and 72 hr. The cellularity decreased more slowly, reaching its lowest point at 24 hr and returning to normal range at 72 hr. For acivicin the marked depletion of the activities of the glutamine-utilizing enzymes and for tiazofurin that of IMP dehydrogenase might account, in part at least, for the bone marrow toxicity of these antimetabolites. Because of the presence in the bone marrow of high activities of purine and pyrimidine salvage enzymes, it should be possible to design methods utilizing nucleosides and nucleobases to protect the bone marrow from the action of antimetabolites.

Animals↗
PubMed2002

Reduction of arsenate to arsenite in hepatic cytosol.

After finding that rat liver mitochondria reduce arsenate (AsV) to the more toxic arsenite (AsIII), it was of interest to know if other cell fractions also carried out this process. Postmitochondrial supernatant (PMSN), isolated from rat liver, reduced AsV to AsIII only in the presence of a thiol. Dithiothreitol (DTT) supported the reduction much more effectively than glutathione. Separation of PMSN into microsome and cytosol revealed that the AsV reducing activity resided in the cytosol. AsV-like oxyanions, e.g., phosphate (P(i)) and vanadate, as well as mercurial thiol reagents inhibited the cytosolic AsV reducing activity, indicating the involvement of a P(i)-utilizing SH enzyme. On searching for a reduction partner, it was found unexpectedly that oxidized pyridine nucleotides (NAD(+) or NADP(+)), but not their reduced forms, increased AsIII formation. Some other purine nucleotide derivatives (e.g., AMP, GMP), but not pyrimidine nucleotides, also increased the rate 2-3-fold. Examination of the effect of nucleosides and nucleobases on AsV reduction yielded dramatic results: purine nucleosides (inosine or guanosine) increased the reduction 80-100-fold, whereas purine bases (hypoxanthine or guanine) decreased it 80-90%. Although the retentate obtained by ultrafiltration of cytosol was almost inactive, its AsV reductase activity could be regained by adding the filtrate or inosine or guanosine to the retentate, indicating that endogenous purine nucleosides were essential for AsV reduction by the cytosol. The hepatic cytosol of mice, hamsters, guinea pigs, and rabbits also exhibited AsV reductase activities in the presence of DTT, which were dramatically enhanced by inosine. Thus, the hepatic cytosol of all tested species can reduce AsV to AsIII. The reduction requires the presence of an appropriate thiol as well as a purine nucleoside (inosine or guanosine) and is inhibited by thiol reagents, the AsV analogue phosphate, and purine bases. Characterization of this AsV reductase activity led us to identification of a cytosolic AsV reductase, which is presented in the accompanying paper.

Animals↗
PubMed2005

Nitric oxide stimulates PC12 cell proliferation via cGMP and inhibits at higher concentrations mainly via energy depletion.

We investigated the mechanisms by which nitric oxide (NO) from an NO donor (DETA/NO) regulates proliferation of pheochromocytoma PC12 cells. The NO donor stimulated proliferation at low concentrations, but reversibly and completely inhibited proliferation at higher concentrations. The stimulation (but not the inhibition) of proliferation was apparently due to NO stimulation of soluble guanylate cyclase to produce cGMP, as it was prevented by a specific cyclase inhibitor (ODQ), and replicated by a cell-permeable form of cGMP. The NO-induced cytostasis was not reversed by inhibitors of MEK kinase or poly(ADP-ribose)polymerase, or by treatments that bypass inhibition of ribonucleotide reductase or ornithine decarboxylase. Cytostatic concentrations of DETA/NO strongly inhibited respiration of PC12 cells, and specific respiratory inhibitors (rotenone, myxothiazol, or azide) caused complete cytostasis. Uridine and pyruvate reversed the cytostasis induced by the specific respiratory inhibitors, but not that induced by DETA/NO. However, the combination of uridine, pyruvate, and N-acetyl-cysteine did reverse DETA/NO-induced cytostasis. DETA/NO strongly and progressively inhibited glycolysis measured by glucose consumption, lactate production, and ATP level, and a specific glycolytic inhibitor (5 mM 2-deoxy-d-glucose) caused complete cytostasis. Our results indicate that NO at low concentrations increases cell proliferation via cGMP, while high concentrations of NO block proliferation via inhibition of both glycolysis and respiration, causing energy depletion.

Acetylcysteine↗
PubMed2003

17beta-estradiol activates ICI 182,780-sensitive estrogen receptors and cyclic GMP-dependent thioredoxin expression for neuroprotection.

Clinical studies suggest that estrogen may improve cognition in Alzheimer's patients. Basic experiments demonstrate that 17beta-estradiol protects against neurodegeneration in both cell and animal models. In the present study, a human SH-SY5Y cell model was used to investigate molecular mechanisms underlying the receptor-mediated neuroprotection of physiological concentrations of 17beta-estradiol. 17beta-estradiol (<10 nM) concomitantly increased neuronal nitric oxide synthase (NOS1) expression and cell viability. 17beta-estradiol-induced neuroprotection was blocked by the receptor antagonist ICI 182,780, also prevented by inhibitors of NOS1 (7-nitroindazole), guanylyl cyclase (LY 83,583), and cGMP-dependent protein kinase (PKG) (Rp-8-pCPT-cGMPs). In addition to the expression of NOS1 and MnSOD, 17beta-estradiol increased the expression of the redox protein thioredoxin (Trx), which was blocked by the inhibition of either cGMP formation or PKG activity. The expression of heme oxygenase 2 and brain-derived neurotrophic factor was not altered. Estrogen receptor-enhanced cell viability against oxidative stress may be linked to Trx expression because the Trx reductase inhibitor, 5,5'-dithio-bis(2-nitrobenzoic acid) significantly reduced the cytoprotective effect of 17beta-estradiol. Furthermore, Trx (1 microM) inhibited lipid peroxidation, proapoptotic caspase-3, and cell death during oxidative stress caused by serum deprivation. We conclude that cGMP-dependent expression of Trx--the redox protein with potent antioxidative and antiapoptotic properties--may play a pivotal role in estrogen-induced neuroprotection.

Cell Survival↗