Anticonvulsant action of 2-chloroadenosine injected focally into the perirhinal cortex in amygdaloid kindled rats.

Possible anticonvulsant effects of 2-chloroadenosine injected focally into the perirhinal cortex of amygdala kindled rats were investigated over a 2 h period. Animals were microinfused (1 microl) with 2-chloroadenosine (2-CLA; 5, 10, 15, 25 and 100 nM) or artificial cerebrospinal fluid applied through a cannula located in the perirhinal cortex. At the doses employed, 2-CLA significantly reduced afterdischarge duration and stage 5 seizure duration. The latency to stage 4 seizure was increased only at the highest dose of 2-CLA (100 nM), while even at this dose no significant change in seizure stage could be seen. The maximum effect of 2-CLA was obtained 30 min after microinfusion of the drug. Pre-treatment (intraperirhinal cortex) of animals with the nonselective adenosine antagonist, caffeine (50 microM; 1 microl), blocked the anticonvulsant activity of 2-CLA. These results suggest that adenosine receptors located in the perirhinal cortex may play an important role in the suppression of seizure activity elicited from the amygdala.

Effect of intraperitoneal and intrahippocampal (CA1) 2-chloroadenosine in amygdaloid kindled rats.

Effects of intraperitoneal and intrahippocampal 2-chloroadenosine and caffeine were examined in fully kindled amygdaloid rats. Intraperitoneal administration of 2-chloroadenosine (5 and 10 mg/kg) decreased afterdischarge duration, stage 5 seizure duration and prolonged time taken to reach stage 4 seizure. Only the 10 mg/kg dose induced a significant reduction in seizure stage. Intraperitoneal administration of caffeine (50 mg/kg) increased both afterdischarge duration and stage 5 seizure duration but did not significantly alter other parameters. Intrahippocampal microinfusion of 2-chloroadenosine (1 mM) or caffeine (2 mM) did not alter any of the measured seizure parameters. Intraperitoneal but not intrahippocampal pretreatment of animals with caffeine (50 mg/kg and 2 mM, respectively) blocked the anticonvulsant effects induced by intraperitoneal administration of 2-chloroadenosine. It may therefore be concluded that the adenosine A1 receptors of the CA1 region of the hippocampus do not play a role in mediating the anticonvulsant effects of intraperitoneally administered 2-chloroadenosine in amygdaloid kindled rats.

Adenosine A2A receptors facilitate 45Ca2+ uptake through class A calcium channels in rat hippocampal CA3 but not CA1 synaptosomes.

In the hippocampus, the neuromodulatory role of adenosine depends on a balance between inhibitory A1 responses and facilitatory A2A responses. Since the presynaptic effects of hippocampal inhibitory A1 adenosine receptors are mostly mediated by inhibition of Ca2+ channels, we now investigated whether presynaptic facilitatory A2A adenosine receptors would modulate calcium influx in the hippocampus. The mixed A1/A2 agonist, 2-chloroadenosine (CADO; 1 microM) inhibited veratridine (20 microM)-evoked 45Ca2+ influx into hippocampal synaptosomes of the CA1 or CA3 areas by 24.2 +/- 4.5% and 17.2 +/- 5.8%, respectively. In the presence of the A, antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), the inhibitory effect of CADO (1 microM) on 45Ca2+ influx was prevented in CA1 synaptosomes, but was converted into a facilitatory effect (14.2 +/- 6.7%) in CA3 synaptosomes. The A2A agonist, CGS 21680 (3-30 nM) facilitated 45Ca2+ influx in CA3 synaptosomes, with a maximum increase of 22.9 +/- 3.9% at 10 nM, and was virtually devoid of effect in CA1 synaptosomes. This facilitatory effect of CGS 21680 (10 nM) in CA3 synaptosomes was prevented by the A2A antagonist 8-(3-chlorostyryl)caffeine (CSC; 200 nM), but not by the A1 antagonist, DPCPX (20 or 100 nM). The facilitatory effect of CGS 21680 on 45Ca2+ uptake by CA3 synaptosomes was prevented by the class A calcium channel blocker, omega-agatoxin-IVA (200 nM). These results indicate that presynaptic adenosine A2A receptors facilitate calcium influx in the CA3 but not the CA1 area of the rat hippocampus through activation of class A calcium channels.

Growth-inhibitory effect of 2-CdA on myeloid CFU-GM progenitors in normal human long-term bone marrow cultures and its compensation by G-CSF or IL-3.

As neutropenia is a common side effect of treatment with 2-chlorodeoxyadenosine (2-CdA), we investigated the myelosuppressive action of 2-CdA in Dexter-type human long-term bone marrow cultures (LTBMCs). LTBMCs were incubated with varying doses of 2-CdA (5 to 20 nM/L) during the first week. At 20 and 10 nM/L 2-CdA, we found a marked reduction in colony-forming unit-granulocyte/macrophage (CFU-GM) production throughout the culture period of 7 weeks (maximum reduction to 3.5% of untreated control cultures with 20 nM/L and to 27.2% with 10 nM/L, respectively). Even the lowest 2-CdA dose tested (5 nM/L) strongly reduced the number of CFU-GM progenitors during the first 3 weeks (maximum reduction to 52.4% of untreated controls), but this effect was transient, and values had recovered to normal within in 5 weeks. 2-CdA was also shown to cause a dose-dependent decrease in long-term culture-initiating cell (LTCIC) detections after 5 weeks in culture (49.6% of control cultures with 10 nM/L 2-CdA and 14% with 20 nM/L 2-CdA, respectively). When 2-CdA was added to LTBMCs initiated on preformed irradiated stromal feeder layers, similar results on CFU-GM production were obtained, indicating that the effects observed were not secondary to effects on the formation of a supportive layer. In addition, IL-6-concentrations in the supernatant of LTBMCs measured at various intervals after the addition of fresh medium with or without 2-CdA showed no significant decrease in cultures treated with 2-CdA. As neutropenia has been shown to be associated with a small but significant risk of fatal infection, we subsequently investigated the reversal potential of the 2-CdA effect by addition of recombinant human granulocyte colony-stimulating factor (rhG-CSF) or rh interleukin-3 (rhIL-3). The weekly addition of 100 ng/mL rhG-CSF counteracted the 2-CdA-mediated decrease in CFU-GM numbers during the entire period of 7 weeks, reaching statistical significance from weeks 3 to 7 (p < 0.05). Addition of rhIL-3 (100 ng/mL) showed an enhancement of CFU-GM output in 2-CdA-treated cultures that resulted in their numbers exceeding those in control cultures (without 2-CdA) from weeks 1 to 5 (p < 0.05) with a maximum increase of 5.1-fold over the parallel control value at week 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Trophic actions of 2-chloroadenosine and bFGF on cultured myenteric neurones.

The effects of the stable adenosine analogue, 2-chloroadenosine (2-CA) and basic fibroblast growth factor (bFGF) on myenteric neurones in dissociated cell culture were examined. 2-CA had no effect on neuronal numbers, but increased neurite length, in a dose-dependent manner. bFGF increased both the number of myenteric neurones and neurite length. When 2-CA was applied together with bFGF, an enhanced increase in neurite outgrowth, but no additional increase in neuronal numbers was observed. 2-CA-induced effects were blocked by the adenosine antagonist 8-(p-sulphophenyl)theophylline. These results show, for the first time, that both purines and bFGF may have trophic actions on myenteric neurones and also indicate that purines enhance some effects of bFGF, in a synergistic manner.

Cyclosporin A inhibits 2-chloroadenosine-induced DNA cleavage in mouse thymocytes.

Incubation of mouse thymocytes with the adenosine analogue 2-chloroadenosine resulted in enhanced internucleosomal DNA fragmentation which could be inhibited by the immunosuppressive drug cyclosporin A. In order to be effective, cyclosporin A had to be added to thymocyte preparations at the same time as 2-chloroadenosine. Since cyclosporin A is a selective inhibitor of calcineurin, our data suggest a possible role for calcineurin as a signaling intermediate in the apoptotic pathway activated in thymocytes through adenosine receptors. However, at the present time we cannot exclude the possibility that the inhibitory effect of cyclosporin A on 2-chloroadenosine-induced apoptosis may be mediated through a calcineurin-independent process.

The influence of naloxone on the effects of adenosine receptor agonists in analgesic tests and binding studies.

Naloxone (1 mg/kg ip) reduced analgesic effect of R-phenylisopropyladenosine (R-PIA-0.2 mg/kg sc) in hot plate and tail-immersion tests in mice and in tail-immersion test in rats. Also the effect of 2-chloroadenosine (2-CADO-2 mg/kg sc) was significantly reduced by naloxone in mice in both nociceptive tests. Naloxone induced partial reduction of analgesic effects of 5'-N-ethylcarboxamideadenosine (NECA-0.02-0.05 mg/kg sc) in mice and rats. Binding studies revealed that the affinity of adenosine agonists (R-PIA and NECA) to opioid receptors was about 5000 times weaker than the corresponding affinity of naloxone.

Neutrophils from asthmatics exhibit diminished responsiveness to 2-chloroadenosine which is reversed by theophylline. Evidence for a cyclic-AMP-independent pathway on human neutrophils.

We have shown previously that neutrophils (PMNs) from patients with asthma have a more potent stimulated respiratory burst than normals and that their respiratory burst is significantly less suppressed with exposure to 2-chloroadenosine (2-CADO). The present studies investigated the basis of this defect in responsiveness to 2-CADO. PMNs obtained from asthmatics either not on theophylline (minus theophylline) or taking theophylline (plus theophylline) generated significantly more superoxide in response to 2 x 10(-8) M FMLP (2.08 +/- 0.36 nmol/5 x 10(5) PMNs (minus theophylline) (P less than 0.01 compared to controls) vs. 2.16 +/- 0.44 (plus theophylline) (P less than 0.01) as compared to controls (1.05 +/- 0.17 nmol). In the presence of FMLP (2 x 10(-8) M), PMNs from the minus theophylline cohort had less 2-CADO (10(-6) M)-mediated suppression of superoxide generation as compared to controls (38.3 +/- 3.8% vs. 67.1 +/- 3.8%; (P less than 0.001). The plus theophylline group exhibited suppression values similar to controls (64.5 +/- 7.2%). Theophylline, in the presence of a physiological concentration of 2-CADO (0.1 microM) accentuated the suppression of the respiratory burst in normals (74.1 +/- 5.9%, 80.1 +/- 4.9% (P less than 0.02) and 84.7 +/- 3.8% (P less than 0.02) at 0, 10, and 100 microM, respectively). PMNs from asthmatics not taking theophylline demonstrated suppression values of 46.2 +/- 6%, 53.8 +/- 6.6% (P = NS), and 63.2 +/- 7.1% (P less than 0.01), respectively. Resting PMNs from normal controls generated 0.97 +/- 0.20 pmol cAMP/10(7) cells compared to 2.83 +/- 0.75 pmol in the presence of 0.1 microM 2-CADO. The combination of 2-CADO and theophylline (10-100 microM) produced cAMP concentrations not significantly different from that observed with 2-CADO alone. These findings support the existence of a novel cAMP-independent adenosine receptor in PMNs. The specific binding of 10(-8)M 3H-labeled 2-CADO (in delta cpm) was 10,358 +/- 1502 (P less than 0.001 compared to controls), 5468 +/- 843 (NS compared to controls), and 3751 +/- 477 in the plus theophylline group, minus theophylline group, and controls, respectively. Such up-regulation of specific binding may represent the effects of theophylline as shown by the specific binding of [3H]2-CADO in PMNs from normal controls exposed to 10 microM theophylline for 30 min (6013 +/- 969) compared to unexposed PMNs (3768 +/- 656; P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Anticonvulsant action of 2-chloroadenosine injected focally into the inferior colliculus and substantia nigra.

Focal injection of 2-chloroadenosine into the substantia nigra protects Sprague-Dawley rats against electroshock seizures (50 mA. 60 Hz, 0.2 s) and genetically epilepsy prone rats against audiogenic seizures. 2-Chloroadenosine infusion into the substantia nigra pars reticulata provided significant protection against electroshock seizures (1.66, 8.33 and 25 nmol) and audiogenic seizures (66, 331 pmol and 1.66 nmol). High doses of 2-chloroadenosine injected into the substantia nigra pars compacta resulted in a similar suppression of electroshock seizure activity (8.33 nmol) and audiogenic seizures (1.66 nmol). No anticonvulsant activity was observed when 2-chloroadenosine was infused into the entopeduncolar nucleus. Lower doses of 2-chloroadenosine provided significant protection against audiogenic seizures (66 and 331 pmol) when injected into the inferior colliculus. Aminophylline antagonised these effects, indicating that purinergic mechanisms are involved in both audiogenic and electroshock seizures. In addition, the similarity of these effects to those elicited by excitatory amino acid antagonists in the inferior colliculus and substantia nigra pars reticulata is consistent with 2-chloroadenosine acting by reducing excitatory transmission.

2-Chloroadenosine decreases long-term potentiation in the hippocampal CA1 area of the rat.

The effect of the adenosine (ADO) analogue 2-chloroadenosine (CADO) on frequency-induced long-term potentiation (LTP) of the responses evoked by stimulation of the Schaffer fibres and recorded in CA1 area was studied in hippocampal slices of the rat. CADP significantly decreased LTP of the population spikes (PS) (EC50 = 0.28 microM), and LTP of the field excitatory postsynaptic potentials (f.e.p.s.p) (EC50 = 0.33 microM). These effects were reversed by the ADO receptor antagonist 8-phenyltheophylline (8-PT) (2.5 microM). It is concluded that CADO decreases LTP through activation of a xanthine-sensitive ADO receptor.

Inhibition by chlordiazepoxide of adenosine-stimulated glucagon secretion.

The action of a water soluble benzodiazepine, chlordiazepoxide (CDZ) on the stimulatory effect of adenosine on glucagon secretion from the isolated pancreas of the rat perfused in presence of 2.8 mM glucose was studied. CDZ 10(-7) and 10(-6) M had no effect per se on glucagon secretion under our experimental conditions. In contrast, CDZ 10(-6) M (but not 10(-7) M) markedly reduced the peak of glucagon secretion provoked by adenosine, 2-chloroadenosine (1.65 C 10(-6) M) and by a stable analogue, 5'-N-ethylcarboxamidoadenosine or NECA (1.65 X 10(-8) M). This peripheral interaction between CDZ and adenosine seemed to be specific, since CDZ did not modify the peak of glucagon secretion induced by (-)isoproterenol (10(-8) M). Our results demonstrate an inhibitory effect of CDZ on adenosine-stimulated glucagon secretion.