R. Vertua

Adenosine A1 receptors modulate anxiety in CD1 mice

Abstract The effect of the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) was investigated in CD1 mice by the elevated plus-maze and the light/dark test, two models for measuring anxiety in rodents. CCPA, administered IP, had an anxiolytic effect at 0.3 nmol/kg in the elevated plus-maze and at 1 nmol/kg in the light/dark test. Brain levels of 22 nM were found after administration of 100 nmol/kg CCPA, as measured by ex vivo binding experiments. These values are consistent with the occupancy of adenosine A1 but not A2 receptors by CCPA, and suggest that the anxiolytic-like action of CCPA may be mediated by centrally located adenosine A1 receptors. Both CPT, a selective adenosine A1 receptor antagonist, and IBMX, a non-selective adenosine antagonist, had an anxiogenic effect in the two tests. It is thus possible that purinergic neurons may be involved in the tonic modulation of affective state in mice.

Anti-inflammatory activity of benzopyrones that are inhibitors of cyclo- and lipo-oxygenase.

The anti-inflammatory activity of three benzo-pyrones with prevalent lipooxigenase-inhibitory activity was studied using the Croton oil ear test in mice, in comparison with nordihydroguaieretic acid (NDGA) and indomethacin. Kaempferol, quercetin and NDGA possess a strong and prolonged anti-inflammatory effect, whereas the action of indomethacin appears relevant, but not long-lasting. In contrast the anti-inflammatory activity of esculetin is rather weak, but persistent.

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.

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.

Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex

In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6 - cyclohexyl-[3H]adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks (light on from 7.00 to 19.00 h). Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.

Adenosine receptors in cortical-derived vesicles of the rat: Studies on binding sites and accumulation of cyclic AMP

A vesicular preparation derived from the cerebral cortex of the rat was used to obtain, under the same experimental condition, binding parameters and stimulation data for cyclic AMP. Two analogues of adenosine were employed in the binding studies: [3H]NECA, a mixed A1/A2 agonist and [3H]CHA, a more selective A1 agonist. The [3H]CHA seemed to bind to a single high affinity site (Kd = 1.31 nM, Bmax = 0.327 pmol bound); saturation data for [3H]NECA were resolved for the presence of a high and a low affinity binding site (Kd1 = 3.08 nM, Bmax1 0.115 pmol bound; Kd2 = 204 nM, Bmax2 1.59 pmol bound), but only when calcium ions were omitted from the incubation medium. At 0 degree C, [3H]NECA bound to a single, low affinity site; the presence of calcium ions (1 mM) significantly reduced the affinity of [3H]NECA (Kd 419 nM), with respect to the absence of calcium (Kd 208 nM), without affecting the Bmax value. The influence of calcium ions was also investigated on the binding of [3H]CHA and a reduction of the Bmax value (36%) was found. Regardless of the presence or the absence of calcium ions, NECA stimulated accumulation of cyclic AMP in a dose-dependent way with an EC50 of 2.79 microM; this value did not correlate with the Kd of the low affinity binding site for [3H]NECA. Thus, the purpose of establishing a correlation between binding sites for analogues of adenosine and the site in the cerebral cortex through which the accumulation of cyclic AMP is induced, was not achieved. It is concluded that the stimulatory effect of analogues of adenosine on adenylate cyclase might not be a receptor-mediated effect. The complex influence of calcium ions on affinity and binding capacity of analogues of adenosine is discussed.

Effects of caffeine and chlor-desmethyldiazepam on fighting behavior of mice with different reactivity baselines

The effects of various doses of caffeine and of chlor-desmethyldiazepam on footshock-induced aggressive behavior were examined in mice with different baselines of aggressiveness. Caffeine significantly increased the number of fighting episodes with all the doses tested. This was more evident in mice with low rather than in those with high basal rates of agonistic response. Caffeine caused the appearance of minimal convulsive signs in mice subjected to a threshold electroshock which did not produce any seizure in the controls; it also increased metrazol toxicity. Chlor-desmethyldiazepam enhanced fighting behavior at doses of 0.04 and 0.08 mg/kg, but decreased it at 1.25 mg/kg. The first two doses produced the same effects as caffeine on electroshock test, but did not influence metrazol toxicity.

14C-5-hydroxytryptamine and 3H-d-amphetamine: Uptake and contraction by the rat stomach fundus in vitro

Abstract Binding of radioactively labeled 5-hydroxytryptamine (5-HT) and D -amphetamine (DA) by the isolated rat fundal smooth muscle is independent of contraction. After a short incubation period the two amines bind to extrareceptor sites which are not related to smooth muscle contraction. The uptake of both amines by normal tissues is similar after treatment with neuraminidase plus EDTA (5-HT receptor destruction) and after preincubation with lysergic acid diethylamide (LSD). This may support the hypothesis of a common receptor system for both 5-HT and DA. Neuraminidase also has a relatively aspecific effect on the cell membrane.