Adenosine inhibits human astrocyte proliferation independently of adenosine receptor activation.

Brain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3 days with test drugs. Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase release and by western blot analysis of αII-Spectrin cleavage. 30 µM-Adenosine caused a 40% ± 3% (p < .05, n = 5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100 µM-Homocysteine alone caused 16% ± 3% (p < .05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76% ± 4%, an effect higher (p < .05) than the sum of the effects of adenosine and homocysteine alone (56% ± 5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100 µM) or SAM (50-100 µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.

Adenosine Inhibits Cell Proliferation Differently in Human Astrocytes and in Glioblastoma Cell Lines.

Glioblastoma (GBM) is the most common brain primary tumour. Hypoxic regions in GBM are associated to tumour growth. Adenosine accumulates in hypoxic regions and can affect cell proliferation and survival. However, how proliferating GBM cells respond/adapt to increased adenosine levels compared to human astrocytes (HA) is not clarified and was addressed in the present work. GBM cell lines and HA were treated for 3 days with test drugs. Thirty Adenosine (30 µM) caused a 43% ± 5% (P < 0.05) reduction of cell proliferation/viability in HA, through an adenosine receptor-independent mechanism, but had no effect in GBM cell lines U87MG, U373MG and SNB19. Contrastingly, inhibition of adenosine phosphorylation (using the adenosine kinase (ADK) inhibitor 5-iodotubercidin (ITU) (25 µM)), produced a strong and similar decrease on cell proliferation in both HA and GBM cells. The effect of adenosine on HA proliferation/viability was potentiated by 100 µM-homocysteine. Combined application of 30 µM-adenosine and 100 µM-homocysteine reduced the cell proliferation/viability in all three GBM cell lines, but this reduction was much lower than that observed in HA. Adenosine alone did not induce cell death, assessed by lactate dehydrogenase (LDH) release, both in HA and GBM cells, but potentiated the cytotoxic effect of homocysteine in HA and in U87MG and U373MG cells. Results show a strong attenuation of adenosine anti-proliferative effect in GBM cells compared to HA, probably resulting from increased adenosine elimination by ADK, suggesting a proliferative-prone adaptation of tumour cells to increased adenosine levels.

Serum homocysteine: relationship with circulating levels of cortisol and ascorbate.

BACKGROUND/AIM: High serum levels of homocysteine and cortisol are independent risk factors for several pathologies and their levels can be regulated by some vitamins. Since the relationship between serum concentrations of homocysteine, cortisol and ascorbate has not been assessed in healthy individuals to date, it was the topic of the present work. METHODS: The study group was composed of 20 men and 40 women aged >50 years. Blood samples were collected and serum concentrations of the analytes were quantified. RESULTS: Serum homocysteine levels correlate positively with cortisol (r = 0.36, p <0.01) and age (r = 0.49, p < 0.001), and negatively with ascorbate (r = -0.30, p < 0.05) and folate (r = -0.31, p < 0.05). A negative correlation between serum levels of cortisol and ascorbate (r = -0.30, p < 0.05) was also observed. Multiple linear regression analysis showed that the best independent predictors of serum homocysteine concentration were cortisol (beta = 0.319, p < 0.003), age (beta = 0.529, p < 0.001) and folate (beta = -0.338, p < 0.001). When subjects were divided into tertiles according to their homocysteine concentration, the highest tertile of homocysteine concentration has also higher cortisol (p < 0.005) and lower ascorbate (p < 0.05) concentrations compared with the lowest tertile of homocysteine concentration. CONCLUSION: The association between serum concentrations of homocysteine, cortisol and ascorbate suggests interaction between circulating levels of these molecules.

The Role of cGMP on Adenosine A 1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus.

Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 µM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 µM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway.

Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus.

Both adenosine A1 and cannabinoid CB1 receptors trigger similar transduction pathways and protect against neurotoxic insults at the hippocampus, but their combined neuroprotective potential has not been investigated. We set forth to assess the combined action of A1 and CB1 receptors against glutamate NMDA receptor-mediated excitotoxicity at the hippocampus. Cell damage was assessed by measuring propidium iodide (PI) uptake and lactate dehydrogenase (LDH) activity released from cultured hippocampal slices exposed to NMDA. Exposure to NMDA (50 µM) for 1 h increased LDH activity by 92% ± 16%. WIN55212-2 (30 µM), a cannabinoid CB1 receptor agonist, decreased NMDA-induced LDH activity by 53% ± 10% while N6-cyclopentyladenosine (CPA, 100 nM), an adenosine A1 receptor selective agonist, decreased it by 37% ± 11%. The combined inhibitory effect of WIN55212-2 and CPA (88% ± 12%) did not differ from the sum of the individual inhibitory effects of each agonist (90% ± 15%) but was different from the effects of CPA or WIN55212-2 alone. An additive inhibitory effect of co-application of WIN55212-2 (30 µM) and CPA (100 nM) on NMDA (50 µM)-induced PI uptake was also observed in CA3 but not in CA1 area of the hippocampal slice. The results suggest that both CB1 and A1 receptors produce additive cumulative neuroprotection against NMDA-induced excitotoxicity in the hippocampus.

The combined inhibitory effect of the adenosine A1 and cannabinoid CB1 receptors on cAMP accumulation in the hippocampus is additive and independent of A1 receptor desensitization.

Adenosine A1 and cannabinoid CB1 receptors are highly expressed in hippocampus where they trigger similar transduction pathways. We investigated how the combined acute activation of A1 and CB1 receptors modulates cAMP accumulation in rat hippocampal slices. The CB1 agonist WIN55212-2 (0.3-30 µM) decreased forskolin-stimulated cAMP accumulation with an EC50 of 6.6±2.7 µM and an Emax of 31%±2%, whereas for the A1 agonist, N6-cyclopentyladenosine (CPA, 10-150 nM), an EC50 of 35±19 nM, and an Emax of 29%±5 were obtained. The combined inhibitory effect of WIN55212-2 (30 µM) and CPA (100 nM) on cAMP accumulation was 41%±6% (n=4), which did not differ (P>0.7) from the sum of the individual effects of each agonist (43%±8%) but was different (P<0.05) from the effects of CPA or WIN55212-2 alone. Preincubation with CPA (100 nM) for 95 min caused desensitization of adenosine A1 activity, which did not modify the effect of WIN55212-2 (30 µM) on cAMP accumulation. In conclusion, the combined effect of CB1 and A1 receptors on cAMP formation is additive and CB1 receptor activity is not affected by short-term A1 receptor desensitization.

Association of the transcobalamin II gene 776C → G polymorphism with Alzheimer's type dementia: dependence on the 5, 10-methylenetetrahydrofolate reductase 1298A → C polymorphism genotype.

BACKGROUND: Decreased serum concentrations of vitamin B12 are associated with Alzheimer's type dementia. The transcobalamin II gene (TCN2) 776C → G polymorphism affects transcobalamin II function as a carrier of vitamin B12 and might modify its availability. The association of the TCN2 776C → G polymorphism with Alzheimer's type dementia is unclear and was investigated in the present study. METHODS: Case-control study including 27 individuals diagnosed with Alzheimer's type dementia and 28 healthy controls. Serum concentrations of vitamin B12, homocysteine and other analytes were determined and the presence of TCN2 776C → G and 5, 10-methylenetetrahydrofolate reductase 1298A → C polymorphisms genotypes was ascertained by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Serum concentrations of vitamin B12 were lower while those of homocysteine were higher in patients than in controls (P < 0.05). The frequency of individuals carrying at least one 5, 10-methylenetetrahydrofolate reductase 1298C allele was higher (59% versus 32%) while frequency of individuals harbouring at least one TCN2 776G allele was lower (58% versus 86%) in patients than in controls (P < 0.05). Univariate logistic regression showed negative association of TCN2 776CG genotype with Alzheimer's type dementia (OR = 0.17 versus CC genotype, P < 0.02). Multivariate logistic regression identified TCN2 776C → G polymorphism as independent predictor of Alzheimer's type dementia together with higher concentrations of homocysteine, cholesterol and uric acid and lower concentrations of oestradiol. Association of TCN2 776C → G polymorphism with Alzheimer's type dementia was observed for individuals carrying the 5,10-methylenetetrahydrofolate reductase 1298AA genotype but not the AC or CC genotypes, indicating interaction between the two polymorphisms. CONCLUSIONS: The TCN2 776C → G polymorphism is negatively associated with Alzheimer's type dementia, suggesting a protective role against the disease in subjects with the 5, 10-methylenetetrahydrofolate reductase 1298AA genotype.

Modulation of cGMP accumulation by adenosine A1 receptors at the hippocampus: influence of cGMP levels and gender.

Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100 µM) and Bay 60-7550 (10 µM), and cAMP accumulation, induced by Forskolin (20 µM) and Rolipram (50 µM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100 nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2 U/ml) unmasked a CPA (0.03-300 nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4 nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100 µM) abolished the CPA (100 nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP.

Kinetics of Angiotensin I alteration of conformation on different hydrophobic interaction chromatographic surfaces.

In the present study, Angiotensin I (Ang I) will be used as model peptide to assess on-column alteration of conformation phenomena. Adsorptive behavior of Ang I on various commercial hydrophobic interaction surfaces (Butyl, Octyl and Phenyl - Sepharose), under different conditions, was investigated. In order to calculate the cis-trans isomerization rate constants of Ang I on the stationary phase's surface, the first and second moments of the proline peptide elution profiles were determined. The activation energies for the isomerization process on Phenyl and Butyl Sepharose were also calculated. Results suggest that the stationary phase catalyzes Ang I isomerization and that catalysis is dependent on hydrophobic interaction ligand nature.

Serum homocysteine: interplay with other circulating and genetic factors in association to Alzheimer's type dementia.

OBJECTIVES: To study the interplay between serum concentrations of homocysteine, steroid hormones and vitamins B and mutations in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, in association to Alzheimer's type dementia (ATD). DESIGN AND METHODS: Case-control study including 19 individuals diagnosed with ATD and 36 healthy controls. Serum concentrations of the analytes were determined and MTHFR 1298A-->C mutation was screened by PCR-RFLP. RESULTS: Multivariable logistic regression analysis identified homocysteine (OR=1.92, P<0.01), cholesterol (OR=1.14, P<0.001), estradiol (OR=0.728, P<0.001), uric acid (OR=2.42, P<0.02), vitamin B(12) (OR=0.984, P<0.004) and MTHFR 1298A-->C mutation (OR=6.01, P<0.04) as independent predictors of ATD. Positive interaction between homocysteine and uric acid, creatinine, urea or cortisol (P<0.02) and negative interaction between homocysteine and vitamin B(12) or MTHFR 1298A-->C mutation (P<0.03) were observed. CONCLUSIONS: High serum concentrations of homocysteine, cholesterol and uric acid, and low concentrations of estradiol and vitamin B(12), as well as the MTHFR 1298A-->C mutation are simultaneously associated to ATD.

Pertussis toxin-sensitive G proteins mediate the inhibition of basal phosphoinositide metabolism caused by adenosine A1 receptors in rat hippocampal slices.

The adenosine A1 receptor selective agonist, N6-cyclopentyladenosine (CPA, 300 nM) inhibited basal accumulation of [3H]inositol phosphates ([3H]InsPs), but not the total levels of membrane [3H]-phosphoinositides, in rat hippocampal slices. This action of CPA was not significantly modified when synaptic transmission was blocked with tetrodotoxin (TTX, 200 nM) but was prevented in slices pre-incubated with pertussis toxin (PTX, 5 microg/mL) for 12-16 hr. Neither PTX nor TTX, when applied in the absence of CPA, influenced basal [3H]InsPs accumulation. It is concluded that the inhibition of the basal phosphatidylinositol metabolism by adenosine A1 receptor activation is independent of neurotransmission and involves a PTX-sensitive G protein, probably of the Gi/Go family.