U. Traversa

Mechanisms of apoptosis induced by purine nucleosides in astrocytes

Astrocytes release adenine‐based and guanine‐based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration‐dependent manner up to 100 μM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 μM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2–3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine‐based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 μM) or propentophylline (100 μM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A1 or A2 adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A3 adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S‐adenosyl‐L‐homocysteine (SAH) and greatly reduced the ratio of S‐adenosyl‐L‐methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A3 adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A3 adenosine receptors. GLIA 38:179–190, 2002.

Specific [3H]‐guanosine binding sites in rat brain membranes

Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine‐based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G‐protein linked cell membrane receptors. Specific binding sites for [3H]‐guanosine were detected on membrane preparations from rat brain. The kinetics of [3H]‐guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122×107 M−1 min−1 and 1.69 min−1, respectively. A single high affinity binding site for [3H]‐guanosine with a KD of 95.4±11.9 nM and Bmax of 0.57±0.03 pmol mg−1 protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [3H]‐guanosine was: guanosine=6‐thio‐guanosine>inosine>6‐thio‐guanine>guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well‐characterized receptors for adenosine and purines. The addition of GTP produced a small concentration‐dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G‐protein. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.

The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase‐3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (∼ 10‐fold over basal apoptotic values) in 18–24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration‐dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 × 10−5 M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi‐proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3‐kinase (PI3K; LY294002, 30 μM) or the MAPK pathway (PD98059, 10 μM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase‐3β (GSK‐3β) and induced an upregulation of Bcl‐2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine‐induced apoptosis by activating multiple pathways, and these are mediated by a Gi‐protein‐coupled putative guanosine receptor.

5′-N-ethylcarboxamido[8-3H]adenosine binds to two different adenosine receptors in membranes from the cerebral cortex of the rat

In the present study it is reported that [3H]NECA binds in a specific and saturable manner to membranes from the cerebral cortex of the rat. Scatchard analysis revealed two binding sites. The high affinity binding site (Kd 10.66 +/- 5 nM, Bmax 0.305 +/- 0.05 pmol/mg prot) was characterized by the following features: maximum binding at 25 degrees C, sensitivity to pretreatment with NEM and regulation by Gpp[NH]p, enhancing of binding in the presence of 1.0 mM MgCl2 and 1.5 mM CaCl2; the rank order of potency of several analogues of adenosine in competing with [3H]NECA for binding, was CHA greater than L-PIA greater than NECA greater than CADO. The low affinity binding site (Kd261.8 +/- 50 nM, Bmax 4.19 +/- 0.33 pmol/mg prot) showed maximum binding at 0 degrees C, insensitivity to pretreatment with NEM up to 1 mM and to regulation by Gpp[NH]p, and inhibition of binding in the presence of MgCl2 and CaCl2. The low affinity site was also present in membranes not pretreated with adenosine deaminase and, even in this condition, an IC50 of 188.5 +/- 36 nM for NECA and an IC50 of 4.35 +/- 0.20 microM for adenosine were found. It is concluded that the high affinity binding site is similar to the A1 adenosine receptors. The low affinity binding site is not classifiable among the A-type adenosine receptors, although it shows peculiar features shared both with the human platelet A2 receptor and the adenosine receptor formerly studied with [3H]adenosine in membranes from the brain of the rat; these results could reflect heterogeneity of adenosine receptors in central nervous system.

Distribution and expression of A1 adenosine receptors, adenosine deaminase and adenosine deaminase-binding protein (CD26) in goldfish brain

The expression patterns of adenosine A(1) receptors (A(1)Rs), adenosine deaminase (ADA) and ADA binding protein (CD26) were studied in goldfish brain using mammalian monoclonal antibody against A(1)R and polyclonal antibodies against ADA and CD26. Western blot analysis revealed the presence of a band of 35 kDa for A(1)R in membrane preparations and a band of 43 kDa for ADA in both cytosol and membranes. Immunohistochemistry on goldfish brain slices showed that A(1) receptors were present in several neuronal cell bodies diffused in the telencephalon, cerebellum, optic tectum. In the rhombencephalon, large and medium sized neurons of the raphe nucleus showed a strong immunopositivity. A(1)R immunoreactivity was also present in the glial cells of the rhombencephalon and optic tectum. An analogous distribution was observed for ADA immunoreactivity. Tests for the presence of CD26 gave positive labelling in several populations of neurons in the rhombencephalon as well as in the radial glia of optic tectum, where immunostaining for ADA and A(1)R was observed. In goldfish astrocyte cultures the immunohistochemical staining of A(1)R, ADA and CD26, performed on the same cell population, displayed a complete overlapping distribution of the three antibodies. The parallel immunopositivity, at least in some discrete neuronal areas, for A(1)Rs, ADA and CD26 led us to hypothesize that a co-localization among A(1)R, ecto-ADA and CD26 also exists in the neurons of goldfish since it has been established to exist in the neurons of mammals. Moreover, we have demonstrated for the first time, that A(1)R, ecto-ADA and CD26 co-localization is present on the astroglial component of the goldfish brain. This raises the possibility that a similar situation is also shown in the glia of the mammalian brain.

Functional adenosine al receptors in goldfish brain: Regional distribution and inhibition of K+-evoked glutamate release from cerebellar slices

In goldfish brain, [3H]cyclohexyladenosine binding sites are ubiquitously distributed with a maximum in the hypothalamus and a minimum in the spinal cord. The binding parameters measured in cerebellar membranes (Kd = 0.88 +/- 0.08 nM; Bmax = 59.65 +/- 2.62 fmol/mg protein) are not significantly different from those of the whole brain. In perfused goldfish cerebellar slices, stimulation of cyclic AMP accumulation by 10(-5) M forskolin was markedly reduced (58.7%) by treatment with 10(-4) M cyclohexyladenosine, an adenosine A1 receptor agonist, and the reduction was reversed in the presence of 10(-4) M 8-cyclopentyltheophylline, a selective A1 receptor antagonist. In the same brain preparation, 30 mM K+ stimulated the release of glutamate, glutamine, glycine and GABA in a Ca(2+)-dependent manner, whereas the aspartate and taurine release was Ca(2+)-independent. Cyclohexyladenosine inhibited the 30 mM K(+)-evoked release of glutamate in a dose-related manner. This effect was reversed by 8-cyclopentyltheophylline. These results support the hypothesis that adenosine A1 receptors present in goldfish cerebellum are involved in the modulation of glutamate transmitter release.

Depressive effects of Chamomilla recutita (L.) Rausch, tubular flowers, on central nervous system in mice*

Summary A lyophilized infusum of tubular flowers of Chamomilla recutita (L) Rausch ( matricaria chamomilla L.), administered i.p. in mice, was investigated in order to verify its action on central nervous system. Basal motility was dose dependently decreased, with 92% of reduction at the dose of 360 mg/kg, without involving motor coordination and muscle relaxation. Exploratory and motor activities registered in the hole-board test, were significantly decreased. A mild hypnogenic effect was displayed at 160 and 320 mg/kg, hexobarbital-induced sleep being significantly potentiated. No toxicity signs were observed at 1440 mg/kg. It was concluded that the camomile effectively displayed a depressive action on the central nervous system.

Mechanisms of inhibitory effects of zinc and cadmium ions on agonist binding to adenosine A1 receptors in rat brain

The dose-dependent inhibition of zinc and cadmium ions of agonist binding to A1 adenosine receptors in rat brain is prevented by histidine and cysteine, respectively. In the present study, the possible different mechanisms of Zn2+ and Cd2+ inhibitions were examined. The effects of Zn2+ and Cd2+ on equilibrium binding parameters of the agonists N6-cyclohexyl-[2,8-3H]-adenosine ([3H]CHA) or chloro-N6-cyclopentyl-adenosine ([3H]CCPA) and the antagonist cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were compared with those effects of reagents or binding conditions which altered histidyl or cysteinyl residues of the A1 receptor. Zn2+ pretreatment did not change A1 agonist or antagonist affinity, but did reduce the Bmax. The inhibitory effects of Zn2+ pretreatments were also maintained after several membrane washings. Diethylpyrocarbonate, a histidine-specific alkylating reagent, behaved like zinc ions: pretreatment with A1 agonist protected the histidyl residues of the [3H]CHA binding site against modification by Zn2+, while the modification of the protonation state of the nitrogen of the imidazole group of histidines by changing pH indicated that the interactions of Zn2+ with the histidyl residues were feasible with their unprotonated form. These findings suggest the formation of coordination bonds between Zn2+ and histidines critical for [3H]CHA or [3H]DPCPX binding, which may prevent the ligand interaction with the specific sites without modifying the binding kinetics of radioligand to the non-chelated recognition sites. Cd2+ pretreatment reduced the [3H]CCPA affinity, but did not modify the affinity of the antagonist [3H]DPCPX, the Bmax remaining unaffected. As with cadmium effects, the oxidation of the thiol group of cysteine by dithionitrobenzoic acid (DTNB) reduced [3H]CCPA affinity without changing the number of binding sites. The reducing reagent dithiothreitol, which alone was unable to modify [3H]CCPA binding, overcame the inhibiting effects of both Cd2+ and DTNB. These findings suggest that cadmium ions may oxidize SH groups of cysteines localized on the A1 receptor molecule or a cysteine localized in the region of G(i)alpha subunit involved in the coupling with receptors. This mechanism can justify potential conformational modifications of the receptor molecule producing the decrease in affinity.

Inhibitory and excitatory effects of adenosine antagonists on spontaneous locomotor activity in mice.

The behavioral effect of the adenosine antagonists CPT, PACPX, DPCPX and PD 115,199 on spontaneous locomotor activity was investigated in mice after parenteral administration. CPT, PACPX and PD 115,199 affected locomotor activity in a biphasic way. Doses in the nanomolar/kg range significantly reduced locomotion (PACPX> or =PD 115,199>>CPT). Higher doses were progressively less active until they became ineffective or slightly stimulated locomotion. NECA, a mixed A1/A2 agonist, and CCPA, a highly selective A1 agonist, also induced a biphasic behavior, with low doses stimulating and high doses inhibiting locomotion. The stimulant effect of 1 nmol/kg NECA was antagonized by depressant doses of antagonists, whereas antagonists-induced hypomotility was potentiated by a depressant dose of NECA (20 nmol/kg). It is suggested that the blockade of A1 receptors by antagonists is probably responsible for reducing locomotor activity, whereas the activation of A2 receptors by agonists is likely responsible for reducing locomotion in mice.

Characterization of A1 adenosine receptors in membranes from whole goldfish brain

Abstract 1. 1. A1 adenosine receptor binding was investigated using the selective agonist [3H]-cyclohexl-adenosine ([3H]CHA) on membranes prepared from adult (12–14 cm length) goldfish whole brain. 2. 2. The A1 receptor agonist [3H]-cyclohexyladenosine bound saturably, reversibly and with high affinity (Kd = 1.20 ± 0.08 nM; Bmax = 6.8 ± 3.02 fmol/mg protein). 3. 3. The specific binding was increased by Mg2+ and inhibited by guanosine 5′-triphosphate (Ki = 5.81 ± 1.54 μM). 4. 4. In equilibrium competition experiments, the adenosine analogs R-phenylisopropyladenosine, cyclohexyladenosine, N-ethylcarboxamidoadenosine, 2-chloroadenosine, S-phenylisopropyladenosine and the antagonists 8-cyclopentyl-1,3-dipropylxanthine and theophylline all displaced [3H]-cyclohexyladenosine from high-affinity binding sites with the rank order of potency in displacing characteristics of an A1 adenosine receptor.