Dynamic changes in the expression pattern of ecto-5'-nucleotidase in the rat model of cortical stab injury.

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate-limiting enzyme in the ectonucleotidase pathway is ecto-5'-nucleotidase (e-5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e-5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e-5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double-immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e-5NT-positive astrocytes, accounting for up-regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e-5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI.

The effect of MK-801 on motor activity and c-Fos protein expression in the brain of adolescent Wistar rats.

The changes that occur during adolescence have a profound impact on the brain and behavior later in life. In this work we examined changes in motor activity during habituation to a novel environment and after treatment with MK-801 (0.025, 0.05, 0.1mg/kg) in peripubertal, pubertal and adult Wistar rats. The involvement of the motor cortex and striatum in motor activity was assessed by analyzing changes in c-Fos protein levels that served as an indicator of neuronal activity. During the habituation period, locomotor activity in peripubertal rats was higher during the first 10 min than in other groups. The same amount of stereotypy-like movements was detected in all three groups. MK-801 induced dose- and age-dependent changes in motor activity. Peripubertal rats were the most sensitive to treatment with MK-801. We also report a surprising finding that systemic application of MK-801 induced a similar age-related profile of changes in motor activity and c-Fos protein expression in the motor cortex but no c-Fos induction in the striatum. Our results demonstrate that, depending on the phase of adolescence the same dose of MK-801 affected behavioral functions in a different manner and that activity of the motor cortex rather than striatal activity was linked to drug-motor activity interactions.

Purine analogs sensitize the multidrug resistant cell line (NCI-H460/R) to doxorubicin and stimulate the cell growth inhibitory effect of verapamil.

The resistant cell line NCI-H460/R and its counterpart NCI-H460 were used to investigate the ability of purine analogs to overcome multidrug resistance (MDR) that seriously limit the efficacy of lung cancer regimens with chemotherapeutic agents. Two purine analogs, sulfinosine (SF) and 8-Cl-cAMP, exerted dose-dependent effects on cell growth in both parental and resistant cell lines. They significantly decreased mdr1 expression in NCI-H460/R cells. Low concentrations (1 microM) of SF and 8-Cl-cAMP in combination with doxorubicin (DOX) exerted synergistic growth inhibition in both cell lines. Pretreatment with SF and 8-Cl-cAMP improved the sensitivity to DOX more than verapamil (VER), the standard modulator of MDR. The increased accumulation of DOX observed after the treatment with SF and 8-Cl-cAMP was consistent with the results obtained with VER. VER stimulated the effect of 8-Cl-cAMP on DOX cytotoxicity and mdr1 expression. Combinations of either SF or 8-Cl-cAMP with VER at clinically acceptable concentrations exhibited synergistic effects on cell growth inhibition in the resistant cell line. SF and 8-Cl-cAMP modulated MDR in NCI-H460/R cells, especially when applied before DOX administration. This feature, together with their ability to reverse MDR, renders the purine analogs (in combination with VER) as potential candidates for improving the clinical activity of existing lung cancer therapeutics.

Sulfinosine enhances doxorubicin efficacy through synergism and by reversing multidrug resistance in the human non-small cell lung carcinoma cell line (NCI-H460/R).

A resistant non-small cell lung carcinoma cell line-NSCLC (NCI-H460/R) was established in order to investigate the potential of sulfinosine (SF) to overcome multidrug resistance (MDR). The cytotoxicity of doxorubicin (DOX) in NCI-H460/R cells was enhanced by interaction with SF. SF improved the sensitivity of resistant cells to DOX when NCI-H460/R cells were pretreated with SF. Synergism was accompanied by the accumulation of cells in S and G(2)/M phases. Pretreatment with SF was more potent in improving the sensitivity to DOX than verapamil (VER). The decrease of mdr1 and topo II alpha expression (assessed by RT-PCR), was consistent with the DOX accumulation assay and cell cycle analysis. Also, SF significantly decreased intracellular glutathione (GSH) concentration. These results point to SF as a potential agent of MDR reversal and a valuable drug for improving chemotherapy of NSCLC.

Amyloid precursor protein 96-110 and beta-amyloid 1-42 elicit developmental anomalies in sea urchin embryos and larvae that are alleviated by neurotransmitter analogs for acetylcholine, serotonin and cannabinoids.

Amyloid precursor protein (APP) is overexpressed in the developing brain and portions of its extracellular domain, especially amino acid residues 96-110, play an important role in neurite outgrowth and neural cell differentiation. In the current study, we evaluated the developmental abnormalities caused by administration of exogenous APP(96-110) in sea urchin embryos and larvae, which, like the developing mammalian brain, utilize acetylcholine and other neurotransmitters as morphogens; effects were compared to those of beta-amyloid 1-42 (Abeta42), the neurotoxic APP fragment contained within neurodegenerative plaques in Alzheimer's Disease. Although both peptides elicited dysmorphogenesis, Abeta42 was far more potent; in addition, whereas Abeta42 produced abnormalities at developmental stages ranging from early cleavage divisions to the late pluteus, APP(96-110) effects were restricted to the intermediate, mid-blastula stage. For both agents, anomalies were prevented or reduced by addition of lipid-permeable analogs of acetylcholine, serotonin or cannabinoids; physostigmine, a carbamate-derived cholinesterase inhibitor, was also effective. In contrast, agents that act on NMDA receptors (memantine) or alpha-adrenergic receptors (nicergoline), and that are therapeutic in Alzheimer's Disease, were themselves embryotoxic, as was tacrine, a cholinesterase inhibitor from a different chemical class than physostigmine. Protection was also provided by agents acting downstream from receptor-mediated events: increasing cyclic AMP with caffeine or isobutylmethylxanthine, or administering the antioxidant, a-tocopherol, were all partially effective. Our findings reinforce a role for APP in development and point to specific interactions with neurotransmitter systems that act as morphogens in developing sea urchins as well as in the mammalian brain.

Sea urchin embryonic development provides a model for evaluating therapies against beta-amyloid toxicity.

Accumulation of beta-amyloid protein is an Alzheimer's disease hallmark but also may be mechanistically involved in neurodegeneration. One of its cleavage peptides, Abeta42, has been used to evaluate the mechanisms underlying amyloid-induced cytotoxicity and targeting of acetylcholine systems. We studied Sphaerechinus granularis sea urchin embryos which utilize acetylcholine and other neurotransmitters as morphogens. At a threshold concentration of 0.1 microM Abeta42, there was damage to the larval skeleton, accumulation of ectodermal cells in the blastocoele and underdevelopment of larval arms. Raising the Abeta42 concentration to 0.2-0.4 microM produced anomalies depending on the stage at which Abeta42 was introduced: at the first cleavage divisions, abnormalities appeared within 1-2 cell cycles; at the mid-blastula stage, the peak period of sensitivity to Abeta42, gastrulation was blocked; at later stages, there was progressive damage to the larval skeleton, digestive tract and larval spicules, as well as regression of larval arms. Each of these anomalies could be offset by the addition of lipid-permeable analogs of acetylcholine (arachidonoyl dimethylaminoethanol), serotonin (arachidonoyl serotonin) and cannabinoids (arachidonoyl vanillylamine), with the greatest activity exhibited by the acetylcholine analog. These results indicate that sea urchin embryos provide a model suitable to characterize the mechanisms underlying the cytotoxicity of Abeta42, as well as providing a system that enables the rapid screening of potential therapeutic interventions. The protection provided by neurotransmitter analogs, especially that for acetylcholine, points to unsuspected advantages of existing therapies that enhance cholinergic function, as well as indicating novel approaches that may prove protective in Alzheimer's disease.

The sea urchin embryo, an invertebrate model for mammalian developmental neurotoxicity, reveals multiple neurotransmitter mechanisms for effects of chlorpyrifos: therapeutic interventions and a comparison with the monoamine depleter, reserpine.

Lower organisms show promise for the screening of neurotoxicants that might target mammalian brain development. Sea urchins use neurotransmitters as embryonic growth regulatory signals, so that adverse effects on neural substrates for mammalian brain development can be studied in this simple organism. We compared the effects of the organophosphate insecticide, chlorpyrifos in sea urchin embryos with those of the monoamine depleter, reserpine, so as to investigate multiple neurotransmitter mechanisms involved in developmental toxicity and to evaluate different therapeutic interventions corresponding to each neurotransmitter system. Whereas reserpine interfered with all stages of embryonic development, the effects of chlorpyrifos did not emerge until the mid-blastula stage. After that point, the effects of the two agents were similar. Treatment with membrane permeable analogs of the monoamine neurotransmitters, serotonin and dopamine, prevented the adverse effects of either chlorpyrifos or reserpine, despite the fact that chlorpyrifos works simultaneously through actions on acetylcholine, monoamines and other neurotransmitter pathways. This suggests that different neurotransmitters, converging on the same downstream signaling events, could work together or in parallel to offset the developmental disruption caused by exposure to disparate agents. We tested this hypothesis by evaluating membrane permeable analogs of acetylcholine and cannabinoids, both of which proved effective against chlorpyrifos- or reserpine-induced teratogenesis. Invertebrate test systems can provide both a screening procedure for mammalian neuroteratogenesis and may uncover novel mechanisms underlying developmental vulnerability as well as possible therapeutic approaches to prevent teratogenesis.

The effects of tiazofurin on basal and amphetamine-induced motor activity in rats.

The effects of tiazofurin (TR; 2-beta-d-ribofuranosylthiazole-4-carboxamide), a purine nucleoside analogue on basal and amphetamine (AMPH)-induced locomotor and stereotypic activity of adult Wistar rat males were studied. The animals were injected with low (3.75, 7.5, and 15 mg/kg ip) and high (62.5, 125, and 250 mg/kg ip) TR doses. Neither low nor high TR doses influenced basal locomotor and stereotypic activity in comparison with the corresponding controls treated with saline only. However, pretreatment with TR at any dose applied, except for the lowest one, significantly decreased AMPH-induced (1.5 mg/kg ip) locomotor activity, while AMPH-induced stereotypic activity was inhibited with the two highest TR doses. In addition, TR was detected in the brain by HPLC already 15 min after the injection (125 mg/kg ip) to reach a maximum 2 h after the administration and was detectable in this tissue during the next 4 h. Our results indicate that TR modifies central regulation of the motor activity, possibly by influencing dopaminergic (DA-ergic) transmission.

Sulfinosine-induced cell growth inhibition and apoptosis in human lung carcinomas in vitro.

In spite of tremendous effort for improved therapy, lung cancer remains the leading cause of cancer-related deaths worldwide. In the present study, we used the novel purine ribunocleoside sulfinosine and evaluated its antiproliferative and apoptotic outcome on the non-small cell lung carcinoma cell line (NSCLC) and the small cell lung carcinoma cell line (SCLC). Using a BrdU incorporation-test sulfinosine inhibited cell growth in a dose dependent-manner. ID50 values were 4.65 +/- 0.17 microM in the case of NSCLC cells, and 3.59 +/- 0.81 microM in the case of SCLC cells. MTT testing revealed that IC50 values were 6.24 +/- 0.77 microM for NSCLC and 5.68 +/- 0.58 microM for SCLC. Inhibitory concentrations (IC50 and ID50) for sulfinosine were nonsignificantly lower in SCLC cells compared to NSCLC cells, indicating similar susceptibility of the cells. Flow-cytometric analysis, TUNEL staining, DNA laddering and cell death ELISA test were used to investigate apoptotic cell death. Our results demonstrated that high concentrations of sulfinosine can cause typical DNA laddering, a hallmark for apoptosis. Evidence of free nucleosomes and enzymatic labeling of fragmented DNA confirmed apoptosis involvement in sulfinosine cytotoxicity. In addition, flow-cytometric analysis showed that 25 microM sulfinosine arrested cell cycle progression at the G2M phase and induction of apoptosis in both cell lines. From these results, we concluded that sulfinosine may act as an anticancer agent and further studies may prove its efficacy in lung cancer cells. Thus the biological effects of sulfinosine may be due to modulation of cell growth, cell death, and cell cycle regulatory molecules.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep.

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63+/-3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.

The linkage of glucose to tiazofurin decreases in vitro uptake into rat glioma C6 cells.

The aim of this study was to analyse the uptake of the synthetic nucleoside tiazofurin and glucoso-linker-tiazofurin conjugate (GLTC) into rat C6 glioma cells in vitro. Results indicated that C6 cells accumulated [3H] tiazofurin slowly with time and that accumulation was reduced by the presence of unlabelled GLTC in the medium which implies that GLTC competes with tiazofurin for transport sites. Uptake of [14C] 2 deoxy-glucose into these cells was very rapid and was not affected by the presence of unlabelled GLTC. To prove the true rate of uptake, the HPLC analysis of cellular extract was performed. After the 360 min of incubation in medium that contained 0.15 mM of tiazofurin, the sum of the concentration of tiazofurin and it's metabolite thiazole-adenine dinucleotide (TAD) in the cells was a total of approximately 4.8% of the amount added to each flask. After the same period of incubation in medium which contained 0.15 mM of GLTC, the sum of concentrations of conjugate, free tiazofurin and TAD represented less than 1/3 of the total concentration measured after the incubation with free tiazofurin and was further reduced in the presence of dipyridamole. Therefore, it can be concluded that GLTC shows some affinity for the nucleoside transporter, but the actual rate of uptake is low.