Hypoxanthine Research Articles

Nitric Oxide Exerts Different Functions on Porcine Oocytes Cultured in Different Models, Which is Affected by Beta-mercaptoethanol

The present study was conducted to investigate the involvement of nitric oxide (NO) in cumulus expansion, oocyte mortality and meiotic maturation of porcine cumulus enclosed oocytes (CEOs) cultured in two different models when gonadotropins, including follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) were presented or not. And the interaction between NO and β-mercaptoethanol (β-ME), a free radical scavenger was also investigated. Two models refer to spontaneous maturation model and hypoxanthine (HX) medium model. All the 3,433 eligible CEOs were incubated at 39°C and the cumulus expansion, oocyte morphology and nuclear phase were evaluated 44 h after incubation. (1) In spontaneous maturation model, NO stimulates the cumulus expansion and β-ME delayed it. NO doesn't affect the oocyte meiotic resumption but inhibits the oocytes to develop to metaphase II. (2) In HX medium model, NO or β-ME doesn't affect the expansion in the absence of gonadotropins, but in the presence of gonadotropins, NO or β-ME inhibits the expansion. In the presence of gonadotropins, NO inhibits the oocyte meiotic resumption and it especially inhibits the oocyte to develop to metaphase II, and β-ME reverses such inhibitory effects. The cooperation of gonadotropins and β-ME stimulates the meiotic resumption and especially, promotes the CEOs to develop to metaphase II in both models. Moreover, HX might contribute to the fragility of oocyte zona pellucida and gonadotropins, nitric oxide and β-ME could alleviate it separately, and cooperatively. It is concluded that NO exerts different functions in two models and β-ME affected the functions of NO in different models. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 3 : 317-324)

Nitric oxide -- superoxide cooperation in the regulation of renal Na(+),K(+)-ATPase.

The aim of this study was to investigate whether endogenous superoxide anion is involved in the regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities. The study was performed in male Wistar rats. Compounds modulating superoxide anion concentration were infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. We found that infusion of a superoxide anion-generating mixture, xanthine oxidase (1 mU/min per kg) + hypoxanthine (0.2 mumol/min per kg), increased the medullary Na(+),K(+)-ATPase activity by 49.5% but had no effect on cortical Na(+),K(+)-ATPase and either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase. This effect was reproduced by elevating endogenous superoxide anion with a superoxide dismutase inhibitor, diethylthiocarbamate. In contrast, a superoxide dismutase mimetic, TEMPOL, decreased the medullary Na(+),K(+)-ATPase activity. The inhibitory effect of TEMPOL was abolished by inhibitors of nitric oxide synthase (L-NAME), soluble guanylate cyclase (ODQ) and protein kinase G (KT5823). The stimulatory effect of diethylthiocarbamate was not observed in animals pretreated with a synthetic cGMP analogue, 8-bromo-cGMP. An inhibitor of NAD(P)H oxidase, apocynin (1 mumol/min per kg), decreased the Na(+),K(+)-ATPase activity in the renal medulla and its effect was prevented by L-NAME, ODQ or KT5823. In contrast, a xanthine oxidase inhibitor, oxypurinol, administered at the same dose was without effect. These data suggest that NAD(P)H oxidase-derived superoxide anion increases Na(+),K(+)-ATPase activity in the renal medulla by reducing the availability of NO. Excessive intrarenal generation of superoxide anion may upregulate medullary Na(+),K(+)-ATPase leading to sodium retention and blood pressure elevation.

Thiol Oxidation Induced by Oxidative Action of Adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H2O2 (HRP-H2O2) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H2O2 was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H2O2. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H2O2 in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.

Protein kinase C and mitogen-activated protein kinase cascade in mouse cumulus cells: cross talk and effect on meiotic resumption of oocyte.

Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.

Hypoxanthine (HX) inhibition of in vitro meiotic resumption in goat oocytes.

To improve in vitro maturation and to understand the mechanism for meiotic resumption of oocytes, meiotic progression, and its control by hypoxanthine (HX) were studied in goat oocytes. Ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) and follicular fluid were collected from follicles of different surface diameters (SDs). The meiotic competence and progression of oocytes were observed, and the concentration of HX in the follicular fluid and culture media was measured by high-performance liquid chromatography (HPLC). Full meiotic competence of goat oocytes was acquired in follicles of >/=1.5 mm in SD with 90% of the oocytes developing to metaphase II (MII) stage after 24 hr in culture. The HX concentration in follicular fluid decreased with follicle development, from the highest level of 1.16 mM in </=0.5 mm follicles to the lowest level of 0.45 mM in >/=5 mm follicles. HX inhibited meiotic resumption of goat oocytes in a concentration-related manner but this inhibitory effect declined gradually. When we renewed the medium at 4 hr of HX-199 (TCM-199 supplemented with 4 mM HX) culture, the percentage of oocytes with intact germinal vesicle (GV) did not increase but decreased significantly instead. HPLC measurement of HX in the HX-199 culture drops indicated that the HX concentration declined from 0 hr to 4 hr of culture and after medium renewal at 4 hr of culture. By adding dibutyryl cAMP (db-cAMP) at medium renewal, we found that db-cAMP held up the decline of GV percentages. Together, these results were consistent with the possibility that the decline of HX inhibitory effect was not due to HX depletion but rather due to the negative feedback of the metabolites on its further uptake by oocytes. Goat oocytes were capable of normal nuclear maturation and activation after temporal arrest by HX, but prolonged exposure to HX induced spontaneous activation.

Meiosis-activating sterol protects oocytes from precocious chromosome segregation

Follicular fluid meiosis-activating sterol (FF-MAS) overcomes hypoxanthine (HX)-mediated meiotic arrest in mammalian oocytes. In order to determine whether chromosome segregation was normal in oocytes matured in FF-MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX-arrested mouse oocytes cultured in the absence or presence of FF-MAS. FF-MAS-induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis ( approximately 40 and approximately 90% polar body formation respectively). The majority of oocytes stimulated by FF-MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX-arrested oocytes cultured with or without FF-MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX-arrested oocytes cultured in the presence of FF-MAS but not lanosterol. FF-MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF-MAS-stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF-MAS appears to protect mammalian oocytes from precocious chromatid segregation.

Dual effects of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro

The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24 h in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), N-omega-nitro- l-arginine methyl ester ( l-NAME) or N w-nitro- l-arginine ( l-NNA) (two inhibitors of NO synthase, NOS), and l-arginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10 −3 M l-NAME ( P<0.01) or 10 −3 M l-NNA ( P<0.01) and the inhibition could be reversed by the addition of SNP (10 −5 M). The above mentioned optimal concentration of l-NAME or l-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10 −7, 10 −6, 10 −5 M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1–4 mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1 mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1 mM) could significantly delay GVBD during the first 5 h culture period. The concomitant addition of l-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4 mM, l-arg caused extensive death of both CEOs and DOs. While, administration of 4 mM l-arg and 1 mM l-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with l-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.

Thymidine and hypoxanthine protection patterns of the folic acid-enhanced synergies for combinations of trimetrexate plus a polyglutamylatable inhibitor of purine or thymidylate synthesis against human ileocecal HCT-8 cells

In order to examine the intracellular locus of the folic acid (PteGlu)-enhanced synergies of trimetrexate (TMQ) plus the thymidylate synthase (TS) inhibitor, raltitrexed (RTX), and TMQ plus the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, AG2034, comprehensive protection studies with thymidine (dThd) and hypoxanthine (HX) were conducted in a 96-well plate cell growth inhibition (sulforhodamine B) assay. Current modeling techniques were extended to characterize these protection patterns involving multiple-agent interaction. Wild-type human ileocecal HCT-8 cells and DW2, a subline deficient in folylpoly-gamma-glutamate synthetase (FPGS) were individually treated for 96 h with TMQ, AG2034 and a 1:1 mixture of TMQ:AG2034 or with TMQ, RTX, and a 1:1 mixture of TMQ:RTX in the presence of PteGlu (2.3 or 40 micro M) and the protection agents (10 micro M dThd and/or 100 micro M HX). Drug treatments were randomly assigned to wells. Both isobols and 3-dimensional concentration-effect surfaces were used to assess the nature and the intensity of drug interactions. The structural Hill model was fitted to data with weighted non-linear regression for most cases. A so-called 'double Hill' model was sometimes more appropriate when a plateau in the middle of the concentration-effect curve was found. In HCT-8 and DW2 cells at 2.3 and 40 micro M PteGlu, inhibition of DHFR by TMQ induced antithymidylate and antipurine effects; AG2034 and RTX selectively inhibited de novo purine or thymidine synthesis, respectively. dThd protection increased the PteGlu-enhancement of the TMQ + AG2034 synergy, whereas HX protection increased the PteGlu-enhancement of the TMQ + RTX synergy. The PteGlu-enhanced synergies of TMQ + AG2034 and TMQ + RTX occur primarily through inhibition of purine synthesis and inhibition of thymidylate synthesis, respectively. These results further substantiate the hypothesis that the nonpolyglutamylatable DHFR inhibitor, TMQ, acts as a modulator by decreasing the protection by PteGlu of cells against the polyglutamylatable AG2034 and RTX.

Glutamine and the maintenance of meiotic arrest in mouse oocytes: influence of culture medium, glucose, and cumulus cells.

The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.

Nitric oxide produced by cumulus cells stimulates maturation of mouse oocytes

The effect of sodium nitroprusside (SNP), a donor of nitric oxide, on meiotic maturation of mouse oocytes was studied by injecting Nw-nitro-L-arginine methyl ester (L-NAME) intra-peritoneal (ip), a nitric oxide synthase inhibitor, or culturing oocytes in the medium supplemented with L-NAME or hypoxanthine (HX) to arrest the spontaneous oocyte maturationin vitro. The results showed that the inhibitory effect of L-NAME by injecting 10 mg/kg ip on extrusion of the first polar body only could be reversed by injecting 2.5 mg/kg SNP with L-NAME simultaneously (P < 0.05). Half an hour later ten mice died when given 10 mg/kg SNP ip. The treatment of some concentrations of SNP (10−7, 10−6, 10−5 mol/L) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes in the presence of HX. However, other concentrations of SNP (10−8, 10−4, 10−3 mol/L) had no effect on HX-arrested oocyte meiotic maturation. The optimal concentration of SNP on CEOs had no effect on DOs. The dose of 10−3 mol/LL-NAME demonstrated a significant suppression in formation of PB1, but not in GVBD. This inhibition was reversed by the addition of SNP. These results indicated that the physiological levels of NO produced by cumulus cells could stimulate meiotic maturation of mouse oocytes bothin vivo andin vitro.

In vitro activation of dibromoacetonitrile to cyanide: role of xanthine oxidase.

Dibromoacetonitrile (DBAN) is a disinfection byproduct of chlorination of drinking water. Epidemiological studies indicate that it might present a potential hazard to human health. The present work provides evidence for DBAN activation to cyanide (CN(-)) by the hypoxanthine (HX)/xanthine oxidase (XO)/iron (Fe) system in vitro. Optimum conditions for the oxidation of DBAN to CN(-)were characterized. Addition of the sulfhydryl compounds glutathione, N-acetyl- L-cysteine or dithiothreitol significantly enhanced the rate of CN(-)release. A high positive correlation existed between hydroxyl free radical ((*)OH) generation and CN(-) formation. Addition of the (*)OH scavengers mannitol or dimethylthiourea to the reaction mixtures resulted in a significant decrease in the rate of DBAN oxidation. Addition of the antioxidant enzymes catalase or superoxide dismutase resulted in a significant decrease in the rate of DBAN oxidation. The iron chelator desferrioxamine significantly decreased CN(-) formation. The maximum velocity (V(max)) and Michaelis-Menten constant (K(m)) of the reaction were assessed. Allopurinol competitively inhibited the reaction, while folic acid uncompetitively inhibited the reaction. In conclusion, (*)OH generated by the HX/XO/Fe system are implicated in DBAN oxidation. The present results represent a novel pathway for DBAN activation and might be important in explaining DBAN-induced toxicity.

Pyruvate utilization by mouse oocytes is influenced by meiotic status and the cumulus oophorus.

In this study, the effects of meiotic status on the energy substrate dynamics of mouse oocyte-cumulus cell complexes (OCCs) and denuded oocytes (DOs) have been examined. In the first series of experiments, OCCs from PMSG-primed, immature mice were cultured in minimum essential medium in 8-microl microdrops under a variety of conditions, and the medium and oocytes were sampled for pyruvate and glucose concentration and for meiotic status. Oocytes in control medium underwent germinal vesicle breakdown within 3 hr and the OCCs displayed a time-dependent increase in pyruvate consumption, but the glucose concentration changed very little. Treatment with IBMX or dbcAMP, which maintained complete meiotic arrest, suppressed pyruvate consumption, but slightly more glucose was consumed than in controls. Hypoxanthine (HX) allowed up to 10% of the oocytes to resume maturation, and pyruvate and glucose consumption resembled that of control OCCs. FSH added to HX-containing medium stimulated significant glucose consumption and pyruvate production. In general, a reciprocal relationship was observed between glucose and pyruvate consumption. When the energy substrate dynamics were compared with meiotic status of the oocytes, pyruvate consumption was associated with the maturation process. Although HX maintained oocytes in the germinal vesicle stage, the meiotic arrest was "leaky," allowing increased pyruvate consumption. Additional experiments showed that DOs at either the prophase I or metaphase II stages consumed less pyruvate than oocytes actively engaged in meiotic maturation. DOs oxidized significantly more pyruvate than OCCs, and glycolytic metabolism of glucose lowered the oxidation rate in OCCs. Furthermore, while 5-6.2 times more pyruvate was consumed by OCCs than by DOs in the absence of glucose, oxidation did not mediate the meiosis-inducing effect of pyruvate, since less of this substrate was oxidized by OCCs than by DOs. We conclude that meiotically active oocytes have a greater requirement for pyruvate than prophase I- or metaphase II-arrested oocytes and that meiotic status can influence the metabolism not only of oocytes, but also of the OCCs.

Effects of reactive oxygen species on actin filament polymerisation and amylase secretion in mouse pancreatic acinar cells

The present study investigates the effect of reactive oxygen species (ROS) on actin filament reorganisation and its relevance to exocytosis in pancreatic acinar cells. Treatment of pancreatic acini with cholecystokinin (CCK-8) induced spatial and temporal changes in actin filament reorganisation with an initial depolymerisation of the apical actin barrier followed by an increase in the actin filament content in the subapical area leading to amylase release. Hydrogen peroxide (H 2O 2) increased actin filament content and potentiated the polymerising effects of CCK-8 in these cells but abolished the disruption of the apical actin layer and amylase release induced by CCK-8. Similar to CCK-8, ROS generated by the oxidation of hypoxanthine (HX) with xanthine oxidase (XOD) induced an initial decrease in actin filaments located under the apical membrane followed by a smaller increase in the content of actin filaments in the subapical area. XOD-generated ROS are able to increase amylase release in pancreatic acini although combination with CCK-8 leads to abnormal exocytosis. We provide evidence that indicates that CCK-8- and ROS-induced actin reorganisation is entirely dependent on Ca 2+ mobilisation and independent of PKC activation. The regulation of the actin cytoskeleton by ROS might be involved in radical-induced cell injury in pancreatic acinar cells.

Separation of six purine bases by capillary electrophoresis with electrochemical detection

Capillary zone electrophoresis with electrochemical detection (ED) has been employed for the separation and determination of adenine (A), guanine (G), theophylline (Thp), hypoxanthine (HX), xanthine (Xan) and uric acid (UA). Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300 μm carbon disc electrode at a working potential of +0.95 V (versus saturated calomel electrode (SCE)). The six purine bases can be well separated within 14 min in a 40 cm length fused-silica capillary at a separation voltage of 10 kV in a 100 mmol/l borate buffer (BB, pH 10.0). The current response was linear over about three orders of magnitude with detection limits (S/N=3) ranging from 0.157×10 −6 to 0.767×10 −6 mol/l for all compounds. The proposed method was successfully applied to determine Thp in tea and aminophylline tablets, UA in human urine, and two purine bases in DNA.

Detection of oxidant-induced slight chromosomal damage in cells by subsequent exposure to X-rays.

We examined whether slight oxidative stress and/or damage in cells could be amplified by subsequent ionizing irradiation and thus become detectable as obvious chromosomal damage. WIL2-NS cells, a human B lymphoblastoid cell line, were pretreated with an oxidant and then exposed to X-rays at 0.25 or 0.5 Gy. The chromosomal damage in the cells was evaluated by cytokinesis-block micronucleus (CBMN) assay. Pretreatment with a superoxide-generating system (hypoxanthine (HX)/xanthine oxidase (XO), 1 and 2 mU/ml of XO), tert-butyl hydroperoxide (t-BuOOH, 10 and 100 microM) or H2O2 (5 microM) alone did not induce significant chromosomal damage, but the oxidant-induced damage increased significantly with subsequent irradiation. The tested dose of these oxidants did not induce significant changes in cell viability, the nuclear division index, and the concentration of antioxidants, indicating that only weak oxidative stress was introduced into the cells. These results suggest that low-dose oxidant-induced chromosomal damage becomes detectable as obvious chromosomal damage with subsequent ionizing irradiation in vitro.

XANTHINE OXIDASE ACTIVATION IN MILD GESTATIONAL HYPERTENSION

Objective: We hypothesized that activation of the xanthine oxidase (XO) enzyme system is a potential source of free radicals in pregnancy-induced hypertension (PIH). Methods: A prospective observational study was carried out on 16 pregnant women who met the criteria of gestational hypertension [rise in blood pressure (BP) of 30 mm Hg systolic or 15 mm Hg diastolic after 20 weeks gestation or BP>140/90 mm Hg if earlier pressure is unknown] without proteinuria or any signs of renal impairment. Fourteen women with a clinically normal pregnancy matched for maternal age, parity, and gestational age acted as pregnant controls. Nonpregnant control women were members of the laboratory staff (n=15). Main Outcome Measures: Concentrations of free sulfhydryl (SH) groups, purine catabolites, lipid peroxidation products in plasma, and blood carboxyhemoglobin levels were used to follow oxidative stress and potential hemolysis. A noninvasive measurement of functional XO activity was carried out (i.e., the urinary ratio of the two metabolites of caffeine was estimated). Results: A pronounced oxidative stress was demonstrated in plasma samples of patients with hypertension by the elevated concentrations of uric acid and lipid peroxidation products. A reduced level of free sulfhydryl groups and an increased concentration of hypoxanthine (HX) were shown in normotensive pregnant individuals. The XO activity index was substantially higher in overweight pregnant subjects with mild hypertension [0.849±0.096 (p<0.01)] than in normotensive pregnant women or in age-matched nonpregnant subjects [0.596±0.105, 0.542±0.049 (means±SD), respectively]. Conclusions: Our study of mildly hypertensive pregnant subjects provides additional evidence of the putative role of XO activation as a source of free radicals in the early stage of endothelial dysfunction.

Preliminary study of the vasorelaxant effects of (+)-nantenine, an alkaloid isolated from Platycapnos spicata, in rat aorta.

In this work, the potential vasorelaxant activity of (+)-nantenine, an alkaloid isolated from Platycapnos spicata, was studied for the first time in rat aorta. (+)-Nantenine (3 - 30 microM) totally relaxed, in a concentration-dependent manner and with almost equal effectiveness, the contractions induced by noradrenaline (NA) or by a high KCl concentration (60 mM) in intact rat aortic rings. Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (10 microM) or tetraethylammonium (TEA, 2 mM) did not significantly modify the vasorelaxant effects of this aporphine alkaloid. In the experiments in Ca(2+)-free medium, (+)-nantenine (10 microM) had no effect on caffeine-induced contractions. Furthermore, in the studies with radiolabelled Ca(2+), (+)-nantenine (3 - 30 microM) did not modify the basal uptake of (45)Ca(2+) but decreased, in a concentration-dependent fashion, the influx of (45)Ca(2+) induced by NA and KCl in endothelium-containing and endothelium-denuded rat aortic rings. In addition, (+)-nantenine (3 - 30 microM) was ineffective to scavenge superoxide anion (O(2) (-)) radicals generated by the hypoxanthine (HX)-xanthine oxidase (XO) system and/or to inhibit XO activity. These results indicate that: a) the vasorelaxant effects of (+)-nantenine in rat aorta are due, at least in part, to a blockage of Ca(2+) influx through transmembrane calcium channels, b) the activation of ATP-sensitive K(+) channels (K(ATP)) and large conductance Ca(2+)-activated K(+) channels (K(Ca)) present in smooth muscle cells, the presence (integrity) of endothelial system, an inhibitory action on XO enzymatic activity and/or O(2)(-) radicals scavenging properties are not involved in the vascular effects of (+)-nantenine in rat aorta described above.

Hypoxanthine transport in human tumour cell lines. Relationship to the inhibition of hypoxanthine rescue by dipyridamole

Hypoxanthine (HPX) uptake was investigated in four human tumour cell lines previously characterised as being sensitive ( ds: A549 and MCF7) or insensitive ( di: COR-L23 and T-47D) to dipyridamole (DP)-induced inhibition of HPX rescue from antipurine antifolate-induced growth inhibition. The aim of the study was to determine the mechanism underlying the differential sensitivity of HPX rescue to DP. The time–course of HPX uptake in the two ds cell lines was different in comparison to the two di cell lines. The initial rate of HPX uptake in the di cell lines was more rapid than in the ds cell lines such that at 60 sec the amount of HPX taken up by the former was 2–6 times higher than that taken up by the later. The K t and T max for HPX transport in di COR-L23 cells were 870 μM and 4.75 μM/10 6 cells/min and 1390 μM and 1.78 μM/10 6 cells/min in ds A549 cells. HPX transport was not sodium-dependent in these cells. Equilibrative nucleoside transporter 2 (ENT2)-mediated thymidine transport was also higher in di cells. DP inhibited HPX uptake into ds cell lines by ≥48% and by ≤20% in the di cell lines. Competition studies with HPX and thymidine transport via ENT2 indicated an overlap between nucleoside and nucleobase transport transporters in the breast cancer cell lines (MCF7 and T-47D). These studies showed that more rapid and extensive HPX uptake, as well as reduced sensitivity to DP inhibition, is associated with the inability of DP to prevent HPX rescue from antipurine antifolate-induced growth inhibition in certain human tumour cell lines.

A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes

This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a positive stimulus in the absence of functional gap junctional communication. No effect of GA was seen in dbcAMP-arrested oocytes. A kinetics experiment showed that when CEO were cultured in dbcAMP +/- FSH, meiotic induction was initiated after 3 h and germinal vesicle breakdown reached 60% by 6 h. When GA was added to the cultures at different times after the initiation of culture (0, 2, 3, 4 and 5 h), meiotic induction was immediately blocked. In addition, measurement of OCC coupling revealed that no reduction in coupling occurred during this induction period in the absence of GA. It is concluded that cumulus cells can secrete a positive factor, but that this is normally overridden by inhibitory influences transmitted through the gap junction pathway in intact complexes. Furthermore, upon exposure of complexes to a meiosis-inducing stimulus, a positive gap-junction-mediated signal now predominates to trigger germinal vesicle breakdown, and this signal is utilised throughout the induction period.

Protein kinase C and meiotic regulation in isolated mouse oocytes.

In this study, the possible role of protein kinase C (PKC) in mediating both positive and negative actions on meiotic maturation in isolated mouse oocytes has been examined. When cumulus cell-enclosed oocytes (CEO) were cultured for 17-18 hr in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, each of the five different activators and five different antagonists of PKC stimulated germinal vesicle breakdown (GVB) in a dose-dependent fashion. One of the activators, phorbol-12-myristate 13-acetate (PMA), also triggered GVB in CEO arrested with isobutylmethylxanthine or guanosine, but not in those arrested with dibutyryl cyclic AMP. When denuded oocytes (DO) were cultured for 3hr in inhibitor-free medium, all PKC activators suppressed maturation (<10% GVB compared to 94% in controls), while the effect of PKC antagonists was negligible. Four of the five antagonists reversed the meiosis-arresting action of HX in DO. PMA transiently arrested the spontaneous maturation of both CEO and DO, with greater potency in DO. The stimulatory action of PMA in HX-arrested oocytes was dependent on cumulus cells, because meiotic induction occurred in CEO but not DO. PKC activators also preferentially stimulated cumulus expansion when compared to antagonists. A cell-cell coupling assay determined that the action of PMA on oocyte maturation was not due to a loss of metabolic coupling between the oocyte and cumulus oophorus. Finally, Western analysis demonstrated the presence of PKCs alpha, beta1, delta, and eta in both cumulus cells and oocytes, but only PKC epsilon was detected in the cumulus cells. It is concluded that direct activation of PKC in the oocyte suppresses maturation, while stimulation within cumulus cells generates a positive trigger that leads to meiotic resumption.