Protective effects of autophagy inhibitor 3-methyladenine on ischemia-reperfusion-induced retinal injury.

OBJECTIVE: To investigate the protective effects of autophagy inhibitor 3-methyladenine (3-MA) in a rat model of ischemic-reperfusion injury (IRI). METHODS: Forty Sprague-Dawley male rats (weight 220-250 g) were randomly divided into four groups: a control group (NC, n = 10), a Sham surgery group (n = 10), an IRI group (n = 10), and a 3-MA-treated IRI group [10 µL 3-MA (10 mmol/L) was injected in vitreous after the injury, n = 10]. The retinal IRI was induced by elevating the intraocular pressure to 110 mmHg for 60 min. Hematoxylin and eosin (HE) staining was used to calculate the number of retinal ganglion cells (RGCs). The level of microtubule-associated protein 1A/1B light chain 3 (LC3), Beclin-1, and Caspase-3 in the retina was detected using the immunofluorescence staining method. The LC3, Beclin-1, B-cell lymphoma/leukemia-2 (Bcl-2), and Caspase-3 protein levels were examined by Western blotting. RESULTS: The number of RGCs in IRI group was significantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group was significantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA-treated IRI group was elevated and protein levels of LC3, Beclin-1 were down-regulated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA-treated IRI group presented down-regulated Caspase-3 and up-regulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). CONCLUSIONS: Retina IRI-caused RGCs loss involved activated autophagy pathway and apoptosis, which could be prevented by autophagy inhibitor 3-MA. Autophagy inhibitor 3-MA may act as a potent therapeutic tool in attenuating retina IRI.

Interleukin­17A facilitates osteoclast differentiation and bone resorption via activation of autophagy in mouse bone marrow macrophages.

Interleukin 17A (IL­17A) exerts pleiotropic effects on periodontitis, partially through enhancement of alveolar bone loss. Osteoclasts are the main culprits that absorb alveolar bone. However, studies describing the correlation between IL­17A and osteoclasts are not conclusive. Previously, autophagy was revealed to be involved in osteoclast differentiation and bone resorption. However, the role of autophagy in IL­17A­mediated osteoclast formation is yet to be clarified. In the present study, bone marrow macrophages (BMMs) were treated with or without IL­17A. 3­Methyladenine (3­MA) was applied to inhibit autophagy. Osteoclast formation was detected by tartrate­resistant acid phosphatase (TRAP) staining, immunofluorescence, and scanning electron microscope. The effects of IL­17A on osteoclast­specific genes and autophagy­related genes during osteoclast differentiation were examined by real­time quantitative polymerase chain reaction and western blot analysis. Autophagosomes were observed by transmission electron microscope. Hematoxylin and eosin (H&E), and TRAP staining was adopted to assess alveolar bone destruction and the number of osteoclasts, respectively in a rat periodontitis model. Consequently, IL­17A stimulated osteoclast differentiation and bone resorption of BMMs accompanied by an increase in the mRNA expression of osteoclast­specific genes. Furthermore, IL­17A increased the levels of autophagy­related genes and proteins, and inhibition of autophagy with 3­MA attenuated the IL­17A­mediated osteoclastogenesis. In addition, there was an increase in the number of osteoclasts and alveolar bone resorption with IL­17A treatment in the periodontitis rat model. Collectively, these findings indicated that IL­17A facilitated osteoclast differentiation and bone resorption in vitro and in vivo, which may contribute to the understanding of the molecular basis of IL­17A in alveolar bone destruction and provide insight on the clinical therapeutic targets for periodontitis.

Inhibition of mRNA deadenylation and degradation by ultraviolet light.

Post-transcriptional mechanisms contribute to the changes in gene expression induced by cell stress. The effect of UV-B light on mRNA degradation in HeLa cells was investigated using a transcriptional chase system to determine the decay kinetics of tet-off vector-derived mRNAs containing or lacking a destabilizing AU-rich element. Degradation of both mRNAs was strongly inhibited in cells exposed to UV-B light. Removal of the poly(A)-tail, considered a crucial step in mRNA degradation, was strikingly impaired. UV light also inhibited deadenylation and degradation of endogenous mRNA of the chemoattractant cytokine interleukin (IL)-8. Both effects occurred rapidly and independently of newly induced genes. Importantly, stabilization of IL-8 mRNA was accompanied by a strong increase in the duration of IL-8 protein formation. Furthermore, general inhibition of protein synthesis, a hallmark of the response to cell stress, required far higher doses of UV-B than inhibition of mRNA deadenylation and degradation. The difference in sensitivity of cells to these effects of UV-B light establishes a dose range in which mRNA stabilization can lead to dramatically enhanced expression of proteins derived from normally unstable mRNAs, such as those of inflammatory cytokines, growth factors and proto-oncogenes, and thereby have a major impact on the response to UV light.

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway.

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.

Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4 and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral cortex and hippocampus.

Stimulation of N-methyl-D-aspartate (NMDA) receptors on neurons activates both cAMP and cGMP signaling pathways. Experiments were carried out to determine which phosphodiesterase (PDE) families are involved in the hydrolysis of the cyclic nucleotides formed via this mechanism, using primary neuronal cultures prepared from rat cerebral cortex and hippocampus. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) potentiated the ability of NMDA to increase cAMP and cGMP. However, among the family-selective inhibitors, only the PDE4 inhibitor rolipram enhanced the ability of NMDA to increase cAMP in the neurons. In contrast, only the PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) enhanced the ability of NMDA to increase cGMP. Neither adenosine nor an adenosine deaminase inhibitor mimicked the effect of EHNA; this suggests that EHNA's inhibition of PDE2, not its effects on adenosine metabolism, mediates its effects on NMDA-stimulated cGMP concentrations. The PDE inhibitor-augmented effects of NMDA on cAMP and cGMP formation were antagonized by 5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate (MK-801), verifying NMDA receptor mediation. In contrast, only NMDA-mediated cGMP formation was affected by altering either nitric oxide signaling or guanylyl cyclase; this suggests that NMDA-induced changes in cAMP are not secondary to altered cGMP concentrations. Overall, the present findings indicate that cAMP and cGMP formed in neurons as a result of NMDA receptor stimulation are hydrolyzed by PDE4 and PDE2, respectively. Selective inhibitors of the two PDE families will differentially affect the functional consequences of activation of these two signaling pathways by NMDA receptor stimulation.

Inhibition of 6-methyladenine formation decreases the translation efficiency of dihydrofolate reductase transcripts.

Cycloleucine was used to inhibit the formation of internal N6-methyladenosine residues in the messenger ribonucleic acid transcripts from cultured methotrexate resistant mouse sarcoma cells. Cells cultured in cycloleucine produced transcripts deficient in N6-methyladenosine residues and the 2'-O-methylated nucleosides of the cap structure; however, the formation of the 7-methylguanine nucleoside of the cap was not effected. Cytoplasmic polyadenylated transcripts were isolated from cells which had been pretreated with media containing cycloleucine and translated in an in vitro translation assay. The levels of translated dihydrofolate reductase were then analyzed by polyacrylamide gel electrophoresis. The amount of dihydrofolate reductase protein produced from the transcripts of the cycloleucine treated cells was 20% less than untreated transcripts. Ribonuclease protection assays demonstrated little difference in the cytoplasmic levels of dihydrofolate reductase transcripts between treated and untreated cells suggesting that the decrease in translation efficiency was not caused solely by an alteration in the processing or cytoplasmic transport of the transcripts. Translation of in vitro transcribed transcripts showed the presence of 2'-O-methylated nucleosides in the cap structure had a negative effect on translation efficiency, demonstrating that the results observed from cycloleucine treatment could not be due to the inhibition of 2'-O-methylation in the cap. These experiments therefore suggest that an inhibition of N6-methyladenosine residues in dihydrofolate reductase transcripts significantly alters their rate of translation.

Inhibition of intestinal metabolism of the antiviral ester prodrug bis(POC)-PMPA by nature-identical fruit extracts as a strategy to enhance its oral absorption: an in vitro study.

PURPOSE: To explore the usefulness of fruit extracts as enhancers of the oral absorption of esterase-sensitive prodrugs. METHODS: Inhibition of esterase-mediated degradation by nature-identical fruit extracts was evaluated using 1) p-nitrophenylacetate (model substrate for esterase-activity) in rat intestinal homogenates and 2) bis(isopropyloxycarbonyloxymethyl)-(R)-9-[(2-phosphonomethoxy++ +)propyl]adenine [bis(POC)-PMPA] (esterase-sensitive prodrug of the antiviral agent PMPA) in Caco-2 cell homogenates and in intestinal homogenates from rat, pig and man. Subsequently, transport of the ester prodrug was studied across Caco-2 monolayers in the presence or absence of fruit extracts. RESULTS: In homogenates from rat ileum, the esterase activity could be reduced significantly by the inclusion of fruit extracts (1%): the initial enzymatic degradation of p-nitrophenylacetate was inhibited by 77% (strawberry), 16% (passion fruit) and 57% (banana). A similar inhibition of bis(POC)-PMPA metabolism by fruit extracts was observed in intestinal homogenates from several species and in homogenates from Caco-2 cells. Transport of total PMPA across Caco-2 monolayers was enhanced 3-fold by co-incubation with strawberry extract (1%). The fraction of intact prodrug appearing in the acceptor compartment increased from virtually zero to 67%. CONCLUSIONS: The results suggest that co-incubation with nature-identical fruit extracts might be useful as a strategy to enhance the transepithelial transport of esterase-sensitive prodrugs through inhibition of intracellular metabolism of the prodrug.

Intravascular adenosine: the endothelial mediators of its negative dromotropic effects.

Intravascular adenosine may exert its negative dromotropic effect via activation of luminal coronary endothelial receptors, which suggests the presence of transcellular dromotropic mediators of endothelial origin, perhaps nitric oxide (NO) and prostaglandins. We decided to test this hypothesis in isolated guinea pig hearts retrogradely perfused with Krebs-Henseleit solution. A pair of stimulating electrodes were placed in the right atria and the auricular-ventricular (A-V) delay recorded by means of a recording electrode placed on the left atria and an electrode placed on the tip of the ventricle. Hearts were paced at a rate of 3.8 +/- 0.2 Hz and perfused at a coronary flow of 9 +/- 0.25 ml/min. To obtain dose-response curves, single doses (as boluses) of different concentrations of adenosine were infused and the maximal increase in A-V delay induced by each dose was determined. Agents that inhibit NO accumulation, such as N(G)-nitro-L-arginine methyl ester (L-NAME) and oxyhemoglobin, diminished the effect of adenosine while NO-sparing agents, such as superoxide dismutase and dithiotreitol, enhanced the adenosine effect. Infusion of NO and the NO donor morpholinosydnonimine increased the A-V delay in a dose-dependent manner. In addition, the dose-response curve for adenosine was displaced downward and to the right by indomethacin, indicating also the involvement of prostaglandins. Infusion of L-NAME in addition to indomethacin further diminished the effects of adenosine, indicating that NO and prostaglandins acted simultaneously. To selectively activate intravascular endothelial adenosine receptors, adenosine amino congener (ADAC), an adenosine A1 receptor agonist, was covalently coupled to 2 X 10(6) Da dextran. When intracoronarily infused, the dextran-ADAC complex remains in the blood vessel lumen because it is too large to diffuse to the interstitium. On intracoronary administration, the dextran-ADAC complex caused a negative dromotropic effect which was diminished by L-NAME and indomethacin. Our data indicate that the dromotropic effect caused by intracoronarily administered adenosine is the result solely of activation of intravascular endothelial adenosine receptors, possibly type A , and that NO and prostaglandins are synergistic endothelial mediators of this effect.

Effects of adenosine deaminase inhibition on blood pressure in old spontaneously hypertensive rats.

Adenosine is an ubiquitously occurring endogenous nucleoside that via cell surface receptors exerts multiple antihypertensive actions, and mediates a number of biological responses that may reduce cardiovascular disease risk. Therefore modulation of endogenous levels of adenosine may offer beneficial effects in hypertension. The objective of this study was to determine whether inhibition of adenosine deaminase lowers blood pressure in spontaneously hypertensive rats (SHR). We investigated the effects of erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, on hemodynamic and renal parameters in 16-week-old and 36-week-old SHR and normotensive Wistar Kyoto rats (WKY) and in 36-week-old SHR and WKY pretreated with 1,3-dipropyl-8-p-sulfopheznylxanthine (DPSPX, an adenosine antagonist that does not enter the brain and is restricted to the extracellular space). Adenosine deaminase inhibition with EHNA (10 mg/kg, iv.) produced a marked fall in arterial blood pressure in older (MABP 162.0+/-7.6 mmHg and 120.7+/-11.7 mmHg for baseline and EHNA period, respectively; p<0.01), but not younger, SHR, whereas no effects on blood pressure were observed in age-matched normotensive WKY rats. EHNA did not affect renal hemodynamic and excretory function in any of six groups of animals. DPSPX blocked the antihypertensive effects of EHNA, suggesting that the effects of EHNA on blood pressure are mediated via peripheral adenosine receptors. Further studies are required to elucidate why inhibition of adenosine deaminase lowers blood pressure only in older SHR. The present data suggest that inhibition of adenosine deaminase may provide beneficial effects in older hypertensives and lead us to propose that design and use of extracellular adenosine deaminase inhibitors may offer cardiovascular protection in hypertension.

Nephrotoxicity induced by a single dose of adenine: effects of 4-aminopyrazolo[3,4-d]pyrimidine and allopurinol.

The effects of allopurinol and 4-aminopyrazolo[3,4-d]pyrimidine (4APP) on adenine-induced renal injury in mice were examined. Plasma urea nitrogen (UN) and creatinine levels increased after the oral administration of adenine to mice. However, plasma UN and creatinine levels decreased inversely with the dose of 4APP when a different dosage of 4APP was administered together with adenine. Yet 4APP did not have any effect on the UN or creatinine levels when 4APP was administered after adenine administration. Plasma UN and creatinine levels increased in the allopurinol-administered group as in the adenine-administered group. Moreover, from light microscopic observation by hematoxylin-eosin staining, microvacuolic changes in the proximal tubuli were detected in the mouse kidney in the adenine-administered group, and epithelial cell loss, degeneration and microvacuolic changes in the proximal tubuli were observed in the mouse kidney in the adenine-and-allopurinol-administered group. However, there were no changes in the proximal tubuli in the mouse kidney in the adenine-and-4APP-administered group. These findings suggested that 4APP inhibits the action of adenine in the mouse kidney.

Purine base transport in wild-type and mycophenolic acid-resistant Tritrichomonas foetus.

The purine base transport systems of wild-type and mycophenolic acid-resistant (MPAR) Tritrichomonas foetus have been characterized. Wild-type T. foetus has two carriers, one for hypoxanthine (Km = 0.7 +/- 0.3 mM, Vm = 80 +/- 20 pmol microliters-1min-1) and guanine (Km = 0.09 +/- 0.02 mM, Vm = 17 +/- 3 pmol microliters-1min-1), and a second for xanthine (Km = 0.6 +/- 0.2 mM, Vm = 25 +/- 5 pmol microliters-1min-1). Adenine transport was not saturable (k = 0.16 +/- 0.01 min-1) and therefore appears to enter the parasite by passive diffusion through the membrane. T. foetus MPAR has lost the hypoxanthine/guanine transporter. Xanthine and adenine transport are similar in wild-type and MPAR T. foetus. No purine nucleoside transporter could be identified.

[Antagonism in the action of kinetin and actinomycin D on the cytogenetic effect of radiation]. / Ob antagonizme v deistvii kinetina i aktinomitsina D na tsitogeneticheskii éffekt oblucheniia.

A study was made of the effect of kinetin when administered separately or in combination with actinomycin D on the chromosomal aberrations in Crepis capillaris L. irradiated by X-rays. Dry seeds were treated after irradiation or seeds soaked, prior to irradiation. Significant decrease in the frequency of chromosomal aberrations was shown when actinomycin D was administered separately and prior to kinetin. The latter used separately and prior to actinomycin D slightly increased the level of the aberrations, as compared with the control. Noticeable radioprotective effect was observed after treatment of seeds with kinetin, prior to irradiation.