Effect of disulfiram eye drops on lipid peroxide formation via excessive nitric oxide in lenses of hereditary cataract ICR/f rats

The ICR/f rat is a recessive-type hereditary cataractous strain, and opacity in the lens usually becomes evident at around 75 d of age. We previously found that the instillation of eye drops containing a disulfiram and hydroxypropyl-beta-cyclodextrin inclusion complex (DSF eye drops) delays lens opacification in ICR/f rats. In this study, we attempted to clarify the mechanisms of the delaying effect of DSF eye drops on cataract development in ICR/f rats. The calcium ion (Ca2+) content in the lenses of ICR/f rats increases at 77 d of age, and this elevation is preceded by a decrease in Ca2+-ATPase activity. On the other hand, the levels of nitric oxide (NO) and lipid peroxide (LPO) also increase in the lenses of ICR/f rats at 63 d of age, while the lenses are still transparent. The instillation of DSF eye drops reduces the changes in Ca2+ content, Ca2+-ATPase activity, NO and LPO levels in the lenses of ICR/f rats. The present study demonstrates that excessive NO production induces the increase in LPO, which causes the decrease in Ca2+-ATPase activity, and the increase in Ca2+ content in the lenses of ICR/f rat during cataract development. DSF eye drops have the ability to attenuate the increase in the NO and LPO levels, resulting in a delay in cataract development.     

Crystallin proteins in lenses of hereditary cataractous rat, ICR/f

ICR/f mutation in rat, an inherited disorder, is characterized by the development of cataracts. In this study, we analyzed and compared the crystallins in normal and cataractous rat lenses using gel filtration and two-dimensional gel electrophoresis, and determined the transglutaminase activities and Ca2+ content in the mutant and normal lenses. The Ca2+ content about 10-fold and the activity of transglutaminase was about 1.8-fold higher in the cataractous lenses than in the normal lenses. Analysis of the cataractous lens proteins showed a remarkable decrease in gamma-, betaB1-, betaA3-, and betaA4-crystallin content, accompanied with some increase in alpha-crystallin (or its aggregate). Higher molecular weight proteins were also observed in the cataractous lenses, with molecular masses which correspond to those of cross-linked dimers (43 to 55 kDa) of beta-crystallins. We consider that the mutation accelerates the aggregation of the crystallins, which is associated with their cross-linking by transglutaminase.     

Comparison of the mechanisms of cataract development involving differences in Ca2+ regulation in lenses among three hereditary cataract model rats

We previously found that the increases in Ca2+ content in the lenses of three hereditary cataract model rats, UPL rat (UPLR), Shumiya cataract rat (SCR) and Ihara cataract rat (ICR), are inhibited by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, and that the mechanisms of Ca2+ enhancement in these rat models differ. In this study, we compare the mechanisms for dysfunction in Ca2+ regulation in UPLR, SCR and ICR. Decreases in the activity of Ca2+-ATPase were found in the lenses of SCR and ICR concurrent with cataract development. In contrast, the Ca2+-ATPase activity in UPLR with opaque lenses was higher than in those with transparent lenses. On the other hand, ATP levels were markedly decreased in UPLR with opaque lenses. The expression of cytochrome c oxidase (CCO)-1 mRNA and CCO activity in UPLR lenses was found to decrease during cataract development. The nitric oxide (NO) and lipid peroxide levels were also increased in the lenses of UPLR, SCR and ICR with opaque lenses. In UPLR, excessive NO may cause damage to the mitochondrial genome, resulting in a decrease in ATP production and increase in Ca2+-ATPase activity. The decrease in ATP content may cause the decrease in Ca2+-ATPase function resulting in the elevation in lens Ca2+. In SCR and ICR, excessive NO may cause an enhancement of lipid peroxidation resulting in the oxidative inhibition of Ca2+-ATPase. The decrease in Ca2+-ATPase activity may cause the elevation in the level of lens Ca2+, thus leading to lens opacification. Our findings show that the Ca2+ contents in the cataractous lenses of all three model rats are increased, the mechanisms for this Ca2+ enhancement is different in each rat model.     

A comparative study of histamine and K+ effects on (Ca(2+)-Mg2+)-ATPase activity in synaptosomes.

Histamine (10(-4) M) and 60 mM K+, but not 60 mM Na+ or 60 mM choline+, increased the maximal synaptosomal (Ca(2+)-Mg2+)-ATPase activity by 15 and 36% respectively and decreased the extrasynaptosomal Ca2+ concentration necessary to reach it. Histamine and K+ enhanced the synaptosomal (Ca(2+)-Mg2+)-ATPase activity in a concentration-dependent manner. In synaptic plasma membranes histamine (10(-4) M) and 60 mM choline+ were not able to alter the enzymatic activity, however 60 mM K+ and 60 mM Na+ elevated (Ca(2+)-Mg2+)-ATPase activity by 20 and 15%, respectively, without altering the affinity for Ca2+. Histamine effects in synaptosomes were mediated by H2 receptor stimulation. 3-Isobutyl-1-methyl-xanthine (10(-4) M) potentiated (15%) the maximal histamine effect. The slow Ca2+ channel antagonists verapamil and diltiazem, both at 10(-6) M, completely inhibited K+ effects in synaptosomes, however histamine effects were only blocked by verapamil. The data suggest that K+ and histamine effects on synaptosomal (Ca(2+)-Mg2+)-ATPase activity are mediated by increases of intrasynaptosomal Ca2+ levels. Moreover, histamine effects on synaptosomal enzyme activity were mediated by cAMP.     

Variation in glycosidase activity in soluble fractions in ICR/f rat lenses with the progression of cataract formation

The current study reports active glycosidases in the lens of ICR/f rats, which generate a hereditary cataract approximately 90 d after birth, and the variation in enzyme activity with cataract progression. Seven active glycosidases, beta-D-galactosidase, alpha-D-glucosidase, beta-D-glucosidase, beta-D-glucuronidase, beta-D-galactosaminidase, beta-D-glucosaminidase and alpha-D-mannosidase, were detected in ICR/f rat lenses. Of these, beta-D-glucuronidase and beta-D-galactosidase showed a tendency to increase in activity with the cataract progression. Furthermore, beta-D-glucosidase and alpha-D-mannosidase showed a transitory increase in activity at the time of cataract formation. This result suggests that several glycosidases in the lens may be involved in the hereditary cataract formation. The optimal pH and temperature of the seven active glycosidases in rat lenses were also measured in this study.     

Adverse effects of excessive nitric oxide on cytochrome c oxidase in lenses of hereditary cataract UPL rats

The UPL rat is a newly developed hereditary cataract model. We previously found that the ATP content in UPL rat lenses decreases during cataract development, and the decrease in ATP content causes Ca(2+)-ATPase dysfunction resulting in an elevation in Ca(2+) and cataract development. In addition, we reported that the oral administration of disulfiram and aminoguanidine ameliorates the decrease in ATP content and the elevation in Ca(2+) content in UPL rat lenses. In this study, we demonstrate the effect of nitric oxide (NO) on the expression and activity of cytochrome c oxidase (CCO) in normal and UPL rat lenses during cataract development. We also determined the effects of the oral administration of disulfiram and aminoguanidine on the mRNA expression and activity of CCO and NO production in UPL rat lenses. The expression of CCO-1 mRNA in UPL rat lenses, determined by a quantitative real-time RT-PCR method, decreased during cataract development. CCO activity in UPL rat lenses also decreased with aging. On the other hand, the oral administration of disulfiram and aminoguanidine attenuated the decrease in CCO-1 mRNA expression and CCO activity. These results suggest that excessive NO causes the decrease in CCO-1 mRNA expression and CCO activity, and that the decrease in CCO may cause the decrease in ATP production in UPL rat lenses. Disulfiram and aminoguanidine may attenuate the decrease in ATP production, resulting in a delay in cataract development.     

Effect of calpain on hereditary cataractous rat, ICR/f.

The crystallins in the lenses of ICR/f mutation rat, a known hereditary cataract model, were analyzed during cataractogenesis. Opacification of the mutant lenses was found to be accompanied by changes in crystallin structure and composition, including several deletions of the N-terminals of beta-crystallins and low molecular weight alpha- crystallins. Because similar deletions were observed when the soluble fraction of normal lens protein was incubated with calpain, we considered that calpain could be related to the deletions in mutant lenses. Although measurement of the content of calpain protein by the ELISA method revealed no significant difference between mutant and normal lenses, it was found that the concentrations of Ca2+ and K+ were different between the two lenses and that calpain activity was dependent on both ion concentrations. Endogenous m-calpain in the soluble fraction from normal lenses was activated by addition of 1 mm calcium chloride in the presence of 50 mm KCl (the same concentration as in mutant lenses), and insoluble protein was found in the fraction 1 d after calpain activation. On the other hand, the presence of 120 mm KCl (the concentration in normal lenses) inhibited calpain activity and prevented this insolubilization. These results suggest that calpain in mutant lenses is involved in the proteolysis of crystallins and the progression of cataract formation.     

Transport of trace elements in lenses of normal and hereditary cataract UPL rats

The multitracer technique was applied to the determination of the uptake of trace elements in the lenses of normal and hereditary cataract UPL rats to investigate the transport mechanisms of trace elements during cataract development. Be, Na, Sc, V, Cr, Mn, Fe, Co, Zn, As, Se, Rb, Sr, Y, Zr, Tc, Ru and Rh accumulate in normal and UPL cataract rat lenses. The rates of uptake of trace elements differ among species and also differ between normal and UPL rat lenses. The uptakes of V and Sr are greater in normal rat lenses, while the uptakes of Mn and Co are greater in UPL rat lenses. High concentrations of Zn are transported into normal rat lenses in comparison with other elements. However, the uptake of Se was highest in the lenses of UPL cataract rats. In addition, the difference in Se uptake between the normal and UPL rat lenses was greatest among the tested trace elements. The present study suggests that the transport characteristics of trace elements are different in the lenses of normal and UPL cataract rats. The different transport characteristics of trace elements in the lenses of normal and UPL cataract rats, especially the higher accumulation of Se in UPL rat lenses, may be implicated in cataract development.     

Different effects of endothelin-3 on the Ca2+ discharge induced by agonists and Ca(2+)-ATPase inhibitors in human platelets.

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetic and pharmacodynamic evaluation of the anti-cataract effect of eye drops containing disulfiram and low-substituted methylcellulose using ICR/f rats as a hereditary cataract model

We attempted to develop anti-cataract eye drops using disulfiram (DSF) and low-substituted methylcellulose (MC), and evaluated their anti-cataract effect in terms of the lens opacification vs. age-profile curves using a one-exponential equation. The eye drops were prepared using 0.5% DSF and 2% MC (DSF eye drops), and ICR/f rats, a recessive-type hereditary cataractous strain, were used as the experimental model. Gelation of DSF eye drops containing MC was first observed at about 35°C, close to body temperature. In in vivo transcorneal penetration experiments using rabbit corneas, only diethyldithiocarbamate (DDC) was detected in the aqueous humor, while DSF was not detected. The DDC penetration level of DSF eye drops containing MC was approximately 1.3-fold higher than that of DSF eye drops. The opacification rate constant (k) of ICR/f rat instilled with DSF eye drops with or without MC was lower, and the initial time of opacification (τ) was longer than those of ICR/f rats instilled with saline. Furthermore, the k of ICR/f rats instilled with DSF eye drops with MC was lower than that of ICR/f rats instilled with DSF eye drops without MC. In conclusion, the analysis of kinetic parameters including k and τ using a one-exponential equation provided useful information for clarifying the anti-cataract effect of eye drops. ICR/f rats instilled with DSF eye drops using a low-substituted MC-based drug delivery system demonstrated a delay in cataract development, probably resulting from an increase in the retention of DSF eye drops on the cornea.     

Effects of canatoxin on the Ca(2+)-ATPase of sarcoplasmic reticulum membranes.

Canatoxin, a toxic protein isolated from the seeds of Canavalia ensiformis, was studied for its effects on the sarcoplasmic reticulum vesicles obtained from rabbit skeletal muscle. Canatoxin inhibited Ca2+ accumulation catalysed by the Ca(2+)-ATPase, without affecting the hydrolytic activity of this enzyme or membrane permeability to Ca2+. The effects of canatoxin were dose dependent, but not time dependent. It is concluded that canatoxin interacts with the Ca2+ pump and uncouples Ca2+ uptake from Ca(2+)-dependent ATP hydrolysis.     

Ca2+ increase and Ca(2+)-influx in human tracheal smooth muscle cells: role of Ca2+ pools controlled by sarco-endoplasmic reticulum Ca(2+)-ATPase 2 isoform.

1. The contribution of sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA)-regulated Ca2+ stores to the increase in intracellular free calcium ([Ca2+]i) induced by bradykinin (BK) was investigated in fura-2 loaded human tracheal smooth muscle cells (TSMC). For this purpose, we used thapsigargin, a selective inhibitor of Ca(2+)-ATPases of intracellular organelles. 2. Thapsigargin (10(-9) to 10(-6) M) induced a dose-dependent increase in [Ca2+]i in the presence of external Ca2+ with an EC50 value of 7.33 +/- 1.26 nM. In Ca(2+)-free conditions, the addition of Ca2+ (1.25 mM) caused an increase in [Ca2+]i which was directly proportional to the pre-incubation time of the cells with thapsigargin. Net increases of 60 +/- 9, 150 +/- 22 and 210 +/- 27 nM were obtained after 1, 3 and 5 min, respectively. 3. In the presence of extracellular Ca2+, BK induced a typical biphasic increase in [Ca2+]i with a fast transient phase and a sustained phase. The sustained component was reversed by addition of a bradykinin B2-receptor antagonist (Hoe 140, 10(-6) M) to the buffer as well as by deprivation of Ca2+. The transient phase induced by BK, histamine and carbachol was inhibited in a time-dependent way by preincubation of the cells with thapsigargin. 4. Comparative western blotting of human TSMC membranes using anti-SERCA2 isoform-specific antibodies clearly showed the greater expression of the 100-kDa SERCA2-b isoform compared with the SERCA2-a isoform.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of enhanced lipid peroxidation in the liver of Long-Evans cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rat is a mutant strain of rats that accumulate copper (Cu) in the liver in much the same way as individuals who suffer from Wilson's disease (WD) and has been suggested as a model for this disease. Lipid peroxidation (LPO) is considered to be involved in the toxic action of Cu in the livers of LEC rats. We investigated the mechanism of LPO in the livers of LEC rats showing apparent signs of hepatitis. Several-fold higher LPO levels were observed in post-mitochondrial supernatant (S-9) fraction of livers from hepatitic LEC rats than in those from Wistar rats. To mimic living cells, we introduced NADPH-generating system (NADPH-gs) into the S-9 incubation system. Thus was ensured a constant supply of NADPH to vital enzymes that may be directly or indirectly involved in the generation and/or elimination of reactive oxygen species (ROSs), such as glutathione reductase (GSSG-R), which require NADPH for their reactions. The levels of LPO in liver S-9 from hepatitic LEC rats were further increased by incubating liver S-9 at 37 °C in the presence of NADPH-gs. This increase was inhibited by EDTA, butylated hydroxytoluene (BHT), and catalase (CAT), suggesting that some metal, most likely the accumulated Cu, and ROSs derived from hydrogen peroxide (H₂O₂) are involved in the increased levels of LPO in the livers of hepatitic LEC rats. The requirement of NADPH-gs for enhanced LPO in the livers of hepatitic LEC rats indicates the consumption of NADPH during reactions leading to LPO. It is known that H₂O₂, and consequently hydroxyl radical are generated during Cu–catalyzed glutathione (GSH) oxidation. The cyclic regeneration of GSH from GSSG by NADPH-dependent GSSG-R in the presence of NADPH-gs may cause sustained generation of hydroxyl radical in the presence of excess free Cu. The generation of H₂O₂ in S-9 fraction of livers from hepatitic LEC rats was observed to be significantly higher than that in S-9 fraction of livers from non-hepatitic LEC rats and Wistar rats. Moreover, in addition to the reported decrease in glutathione peroxidase (GPX) activity, we found that CAT activity was markedly decreased in LEC rats with hepatitis. The increased generation of H₂O₂ with reduced activities of GPX and CAT may result in cellular accumulation of H₂O₂ in the liver of hepatitic LEC rats. Taken altogether, it is suggested that the accumulated H₂O₂ undergoes the Fenton-type reaction with also accumulated free Cu, thus generating hydroxyl radical in the livers of hepatitic LEC rats and increasing LPO levels in these animals. Received: 20 April 1999 / Accepted: 2 September 1999     

In vitro effects of metal ions (Fe2+, Mn2+, Pb2+) on sperm motility and lipid peroxidation in human semen

The effects of divalent manganese ion (Mn2+), ferrous iron (Fe2+), and lead ion (Pb2+) on human sperm motility and lipid peroxidation were examined. Human semen from healthy male volunteers was incubated with 0, 5, 50, or 500 ppm divalent metal ions, and the sperm motility was determined at 0, 2, 4, 6, or 8 h by microscopy. Malondialdehyde (MDA) levels in seminal plasma was measured by high-performance liquid chromatography after 8 h of exposure. The results showed that 500 ppm Mn2+ or Pb2+ significantly inhibited sperm motility without an accompanying change in seminal MDA levels. Incubation with Fe2+ significantly inhibited sperm motility at 5 ppm, associated with a marked rise in MDA levels. Our results suggested that Fe2+ may induce lipid peroxidation to inhibit sperm motility. In the case of Mn2+ and Pb2+ there is an absence of seminal lipid peroxidation and the observed inhibition of sperm motility at high concentrations is not biologically or environmentally relevant.     

Effects of calcium antagonists on (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities of rat cortical synaptosomes

1. The effects of 11 calcium antagonists on (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities of rat cortical synaptosomes were studied. 2. All the calcium antagonists studied had inhibitory effects on ouabain-sensitive (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities in synaptosomes at high concentrations (10 or 100 microM). 3. Calcium antagonists such as trifluoperazine, flunarizine and cinnarizine had inhibitory effects on Ca2+-ATPase activity at low concentrations (1-10 microM). 4. Trifluoperazine and La3+ had inhibitory effects on Mg2+-ATPase activity at low concentration (1 microM). 5. Our results suggest that most of the calcium antagonists studied have little effects on neuronal (Na+ + K+)-ATPase, Mg2+-ATPase and Ca2+-ATPase activities at therapeutic dose ranges (1 microM or lower).     

绞股蓝在大鼠肝的抗脂质过氧化作用

目的：观察绞股蓝（GP）对四氯化碳（CCl4)所致肝脏脂质过氧化的干预作用。方法：将Wistar大鼠随机分为4组：A（对照组）;B（CCL4）;C（GP);D(CCL4＋CP）。结果：CCL4组的脂质过氧化物（LPO）含量明显高于对照组,谷胱甘肽过氧化物酶（GSH－Px）活性低于对照组;而同时给予GP的D组可减弱CCL4诱发的上述作用。结论：GP能减弱CCL4对肝脏的损害。     

红花对实验性大鼠肝纤维化的抑制作用

目的：观察红花对实验性大鼠肝纤维化的影响,并探讨其作用机制。方法：在CCl4诱导实验性大鼠肝纤维化3周后．给予红花5.0g/kg,每天1次腹腔注射．同时设正常对照及CCl4对照组。分别用放免法检测血清透明质酸(HA)、层粘连蛋白(LN)、3型前肢原(PCⅢ)。HE及VG法染色观察组织的病理变化。免疫组织化学法检测肝组织Desmin的表达。结果：红花治疗组大鼠肝组织纤维化程度计分、血清HA、LN、PCⅢ浓度、肝组织中Desmin染色阳性细胞面积低于CCl4对照组,差异显著。结论：红花抑制肝星状细胞的激活和转化．具有一定的抗肝纤维化作用。