Attenuation of p53 expression protects against focal ischemic damage in transgenic mice.

Apoptosis or programmed cell death may be involved in neuronal death in the cerebral cortex after a permanent focal ischemic insult. Studies indicate that protein p53 is a major determinant of the cellular mechanism that leads to programmed cell death. Wild-type C57 mice and two groups of transgenic C57 mice, one homozygous and the other heterozygous for a p53 null gene, were subjected to middle cerebral artery occlusion. As expected, the wild-type mice had a large, consistent infarct volume (22.11 +/- 4.59 mm3; n = 10). Both transgenic groups had significantly less ischemic damage than the wild-type control group. However, unexpectedly, the heterozygous group had the least amount of ischemic damage (16.12 +/- 1.71 mm3, n = 11; 27% reduction in infarct size). The ischemic damage in the homozygous group (18.72 +/- 3.48 mm3, n = 9) was significantly less than in the wild-type control (15% reduction in infarct size) but significantly more than in the heterozygous group. Thus, although the absence of p53 expression was protective, greater protection was afforded by reduced expression of p53. These data suggest that attenuated p53 expression may be protective after an ischemic event.

Preclinical neurochemical and electrophysiological profile of 1192U90, a potential antipsychotic.

11192U90 was submitted to receptor binding and monoamine uptake assays. It bound potently at serotonin 5-HT2, dopaminergic D2, serotonin 5-HT1A, and adrenergic alpha 1 and alpha 2 receptors. It also bound to dopaminergic D1, serotonin 5-HT3, serotonin 5-HT4, and sigma sites, albeit with lower affinity. It was essentially inactive at 22 other sites, including those for cholinergic M1 and M2. It weakly inhibited uptake of 3H-norepinephrine, 3H-serotonin and 3H-dopamine. Acute doses of 1192U90 (5 and 20 mg/kg P.O.) increased whole-brain levels of dopamine metabolites but did not affect levels of norepinephrine, dopamine, and serotonin. Subcutaneous injection of 1192U90 (0.8 mg/kg/day) and clozapine (20 mg/kg/day) for 28 days preferentially decreased the number of spontaneously active dopamine cells in the ventral tegmental area (VTA) but not the substantia nigra (SN) of rats, as measured by population sampling. This outcome is characteristics of atypical antipsychotics like clozapine. Acute injections of 1192U90 reversed the rate-inhibiting effects of microiontophoretically applied dopamine and intravenously injected apomorphine and d-amphetamine on dopamine cell firing. Intravenous injection or iontophoretic application of 1192U90 or the 5-HT1A agonist (+/-)8-OH-DPAT inhibited the firing rates of dorsal raphe nucleus (DRN) neurons in rats, and the effects of both compounds were blocked by iontophoretically applied S(-) propranolol, a 5-HT1A antagonist. The results suggest that 1192U90 is a preferential dopamine D2 antagonist as well as a 5-HT1A agonist that may prove to be an atypical antipsychotic.

Interactions between calcium channel compounds and adenosine systems in brain of rat.

A number of organic ligands of calcium channels were investigated for possible actions on several aspects of adenosine systems in the cerebral cortex of rat. The principle findings of the present study were that a number of antagonists of calcium channels and the agonist compound Bay K 8644 inhibited binding to adenosine receptors, binding to nucleoside transporters, and the accumulation of [3H]adenosine with a low microM potency. 2-Nitrophenyl dihydropyridine derivatives were more potent than 3-nitrophenyl dihydropyridine or non-dihydropyridine ligands of calcium channels at inhibiting binding to adenosine receptors. Dihydropyridine ligands of calcium channels were more potent than non-dihydropyridine ligands of calcium channel in inhibiting the binding of [3H]nitrobenzylthioinosine to cortical membranes or inhibiting the accumulation of [3H]adenosine into synaptoneurosomes. However, unlike the case of adenosine receptors, no distinction between 2-nitrophenyl and 3-nitrophenyl dihydropyridine derivatives was observed. In addition, the non-dihydropyridine ligand of calcium channels, diltiazem was a weak inhibitor of the accumulation of [3H]adenosine. These results demonstrate that organic ligands of calcium channels, particularly dihydropyridine compounds, can interact with several aspects of adenosine systems.

Structure-activity studies on the potentiation of benzodiazepine receptor binding by ethylenediamine analogues and derivatives.

The effect of ethylenediamine analogues on in vitro binding of [3H]-diazepam to crude cerebral cortical synaptosomal membranes in the rat was studied. Ethylenediamine significantly increased [3H]-diazepam binding to a maximum potentiation of 154% control (EC50 = 1.8 X 10(-4) M) and was the most active compound studied in terms of both potency and the maximum potentiation observed. Potentiation of [3H]-diazepam binding by ethylenediamine analogues is dependent on carbon-chain length, appears to require two terminal amino groups, and is not observed in the rigid analogues studied. Potentiation of [3H]-diazepam binding by ethylenediamine analogues is mediated largely by a change in receptor number and not receptor affinity. Results are discussed in terms of the possible nature of the ethylenediamine binding site.

Anticonvulsant actions of the putative gamma-aminobutyric acid (GABA)-mimetic, ethylenediamine.

1 Ethylenediamine, 31.6-1000 mg/kg intraperitoneally, inhibited the convulsive effects of pentylenetetrazol, 100 mg/kg (i.p.) in mice. 2 Ethylenediamine, 100-1000 mg/kg (i.p.) increased the convulsion threshold to the intravenous infusion of three convulsants in the order pentylenetetrazol greater than bicuculline greater than strychnine. 3 The benzodiazepine antagonist R0 15-1788, 10 mg/kg (i.p.), significantly inhibited the anticonvulsant action of diazepam, 50 micrograms/kg, but not ethylenediamine, 1000 mg/kg. 4 These results clearly indicate that ethylenediamine has anticonvulsant properties and are consistent with the hypothesis that ethylenediamine is a gamma-aminobutyric acid (GABA)-mimetic.

Inhibitory action of gamma-aminobutyric acid on the excitatory but not inhibitory innervation of the rat anococcygeus muscle.

1 The effects of gamma-aminobutyric acid (GABA), ethylenediamine, 3-aminopropane sulphonic acid and (+/-)-baclofen have been examined on the responses to stimulation of the adrenergic excitatory and non-adrenergic non-cholinergic inhibitory innervation of the rat anococcygeus muscle in vitro. 2 GABA produced a dose-related depression of the contractile responses to field stimulation. Ethylenediamine and baclofen also depressed the contractile responses, though they were less potent than GABA. 3-Aminopropane sulphonic acid was almost inactive. The inhibitory action of GABA was not modified by phentolamine, propranolol or bicuculline methylbromide. 3 GABA did not affect the contractile responses of the anococcygeus muscle to noradrenaline, phenylephrine or carbachol in untreated muscles or those treated with 6-hydroxydopamine in vitro. 4 In preparations in which tone was raised by continuous perfusion with carbachol in the presence of phentolamine, field stimulation relaxed the muscle. GABA had no effect on this inhibitory response, and did not itself produce any relaxation. 5 It is concluded that GABA exerts a presynaptic inhibitory action on the excitatory adrenergic but not on the inhibitory innervation of the anococcygeus muscle, and that the GABA receptor involved exhibits properties of the previously described GABAB site.

Covariation of alpha 2-adrenoceptor density and function following irreversible antagonism with EEDQ.

1. Administration of the irreversible antagonist, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, (EEDQ, 2 mg kg-1, i.p.) to mice reduced binding of [3H]-RX 821002 (2-methoxy-idazoxan) to alpha 2-adrenoceptors in whole mouse brain by 75% 24 h later. The receptor binding returned over time only being reduced by 25% by 16 days post administration; the time taken for binding to return to 50% of control levels was estimated to be 5.25 days. 2. EEDQ administration also resulted in the loss of the sedative effect of the alpha 2-adrenoceptor agonist, medetomidine, measured by the holeboard test of directed exploration and locomotor activity. Agonist-induced sedation returned to control values by 8 days post EEDQ administration. 3. EEDQ administration also resulted in the loss of the hypothermic response to medetomidine (0.1 mg kg-1, i.p.). Medetomidine-induced hypothermia returned to control values by 12 days post EEDQ administration. 4. Pretreatment with the selective alpha 2-adrenoceptor antagonist, RX 821002 (0.1-3.0 mg kg-1, i.p.) 45 min before EEDQ prevented the loss of alpha 2-adrenoceptors as well as the blockade of medetomide-induced sedation and hypothermia by EEDQ. 5. The results of these experiments indicate that there is significant receptor reserve for alpha 2-adrenoceptor-mediated behavioural and physiological responses.

Characterization of [3H]Ro 5-4864 binding to calmodulin using a rapid filtration technique.

The benzodiazepine [3H]Ro 5-4864 bound specifically and saturably to an apparently homogenous, univalent species of binding site on the calmodulin molecule with an associated equilibrium dissociation rate constant (Kd) of 644 +/- 121 nM. The rates of association (K1) and dissociation (K-1) governing this interaction were estimated to be 7.66 x 10(3) M-1 sec-1 and 2.9 x 10(-3) sec-1, respectively, yielding a non-equilibrium determination of the Kd to be 379 nM. Such binding of [3H]Ro 5-4864 was protein-, pH-, and temperature-dependent and demonstrated pharmacological selectivity. Only benzodiazepine compounds (chlordiazepoxide, diazepam and Ro 5-4864) inhibited [3H]Ro 5-4864 binding to calmodulin with inhibitory equilibrium dissociation constants (Ki) less than 10 microM. The benzodiazepine compounds Ro 15-1788 and flunitrazepam did not displace [3H]Ro 5-4864 binding to calmodulin nor did a number of pharmacologically active non-benzodiazepine compounds (Ki values greater than 10 microM). Consideration of the stoichiometry yielded an approximate mole ratio of 0.90:1.0 (Ro 5-4864: calmodulin), suggesting that there is one binding site for Ro 5-4864 per molecule of calmodulin. The data reveal that the binding of [3H]Ro 5-4864 to calmodulin fulfills the major criteria of a ligand binding to a receptor. Such an interaction may underlie some of the pharmacological actions of Ro 5-4864-like compounds.

Comparative aspects of nitrobenzylthioinosine and dipyridamole inhibition of adenosine accumulation in rat and guinea pig synaptoneurosomes.

Dipyridamole (DPR) and nitrobenzylthioinosine (NBI) inhibition of adenosine accumulation in synaptoneurosomes derived from rat cerebral cortex, rat cerebellum, guinea pig cerebral cortex and guinea pig cerebellum was investigated. The inhibition of adenosine accumulation by NBI was observed to be distinctly biphasic in both guinea pig and rat synaptoneurosomes. Such biphasic inhibition consisted of a nM potency component to inhibition, accounting for 20-30% of the maximum inhibition, and a ?M potency component, accounting for the remaining 70-80% maximum inhibition. Such an inhibitory profile contrasts sharply with that of DPR which appears monophasic, with a mean IC(50) of between 10(?7) M and 10(?6) M, in all rat and guinea pig synaptoneurosomes preparations studied. Further differences between the potency of NBI and DPR in inhibiting [(3)H]adenosine accumulation were also noted. DPR was more potent in inhibiting [(3)H]adenosine accumulation in guinea pig cerebellar synaptoneurosomes than in cerebral cortex synaptoneurosomes. In rat synaptoneurosomes, the reverse selectivity was observed. DPR was also 2-6 fold (depending on brain region of comparison) more potent in inhibiting adenosine accumulation in guinea pig synaptoneurosomes than in inhibiting such accumulation in rat synaptoneurosomes. In contrast, NBI was approximately equipotent in inhibiting adenosine accumulation in rat and guinea pig synaptoneurosomes. Additional binding studies using [(3)H]NBI are also reported. The data presented are entirely consistent with the hypotheses that (1) NBI and DPR bind to functionally relevant sites and (2) there are different populations of nucleoside transporters in mammalian brain.

Regional induction of c-fos mRNA by NMDA: a quantitative in-situ hybridization study.

The regional expression of c-fos mRNA following acute NMDA administration has been mapped by quantitative in-situ hybridization using 35S-c-fos DNA probe. NMDA-induced expression of c-fos mRNA is discrete, largely restricted to the dentate gyrus of the hippocampus and piriform cortex. This distribution is different from the much more widespread distribution of NMDA receptors detected by ligand autoradiography. Expression of c-fos mRNA induced by 225 mg kg-1 NMDA was stereospecifically blocked by pretreatment with the NMDA receptor/ionophore complex blocker, MK-801. Larger doses of NMDA (greater than 175 mg kg-1) were needed for increased expression of c-fos mRNA. Animals which had seizures after acute NMDA administration always had high levels of c-fos mRNA, but equally high levels of c-fos mRNA expression were found in some seizure-free animals. Thus overt seizure activity may not be necessary for c-fos mRNA expression.

Multidrug resistance in MCF-7 human breast cancer cells is associated with increased expression of nucleoside transporters and altered uptake of adenosine.

The rate of adenosine uptake and the corresponding expression of nucleoside transporters were studied in several MCF-7 human breast-cancer cell lines that express different levels of multidrug resistance (MDR). Kinetic studies of adenosine transport in these cell lines revealed that the mean apparent Km and Vmax values for the nucleoside transporters increased with increasing MDR. The apparent Km and the apparent Vmax of Adriamycin-resistant (ADR10) cell lines were respectively 3.2- and 1.8- fold those of Adriamycin-sensitive wild-type (WT) cells (P less than 0.001). A partially revertant cell line (ADR10rev) that was derived from the ADR10 line and was partially sensitive to Adriamycin exhibited apparent Km and Vmax parameters that lay between those of the ADR10 and WT cells (P less than 0.001 vs ADR10 cells; P less than 0.05 vs WT cells). ADR10 cell membranes bound greater than 4 times more of the nucleoside transporter blockers [3H]-nitrobenzylthioinosine [( 3H]-NBI) and [3H]-dipyridamole [( 3H]-DPR) than did WT cell membranes per unit protein (P less than 0.0001). Scatchard analysis revealed a 2-3 times greater density for nucleoside transporters in ADR10 membranes as compared with those in WT membranes. ADR10rev membranes bound less [3H]-NBI and [3H]-DPR than did ADR10 membranes (P less than 0.001), but they bound more of the blockers than did WT membranes (P less than 0.05). A 2.5-h exposure to 200 nM phorbol-12,13-dibutyrate (PDBu), which activates protein kinase C (PKC) and induces WT cells to exhibit a 4-fold increased transient MDR phenotype, increased the apparent Km of WT cells for adenosine transport by greater than 2 times (P less than 0.001) to a value close to that found for the ADR10 cells. An identical exposure of ADR10 cells to PDBu produced no significant effect. The apparent Km of ADR10rev cells was increased 1.4 times by a 2.5-h PDBu exposure. None of the cell lines were affected by a 2.5-h exposure to 200 nM phorbol-13,10-diacetate (PDA), a much less active phorbol, or vehicle. These results suggest that MDR in MCF-7 cells is associated with changes in nucleoside transport, including both the number of transporters and their rate of transport, and that such changes can be partially mimicked by stimulation of PKC.

Ontogenetic appearance of the adenosine receptor precedes N-protein coupling in rat forebrain.

The ontogenetic development of rat forebrain adenosine receptors labelled by [3H]cyclohexyladenosine [( 3H]CHA) and to the corresponding susceptibility of such [3H]CHA binding to the non-hydrolysable GTP analogue, GppNHp is reported. The present studies reveal that: (1) in neonatal forebrain, [3H]CHA binding can be detected but there is little or no GppNHp-induced reduction in such binding; (2) susceptibility of [3H]CHA binding to GppNHp develops slowly with maximum (adult) levels of inhibition not being observed until approximately 16 days postpartum; (3) changing susceptibility of adenosine receptors to GppNHp is due to the maximal effect of GppNHp increasing with time. The potency of GppNHp remains constant at around 6.3 X 10(-7)M over this period of change; (4) Scatchard analysis of [3H]CHA binding to 30-day forebrain membranes reveals the presence of two binding sites--a high-affinity, low-capacity site and a low-affinity, high-capacity site. In the presence of 10(-4)M GppNHp, only a low affinity, high capacity site is detected; (5) Scatchard analysis of [3H]CHA binding to 6-day forebrain membranes (where GppNHp has no effect) reveals the presence of only a single low-affinity, high-capacity binding site. The data strongly suggests that in very young neonates adenosine receptors can be detected but that many detected binding sites are not functionally linked to associated N-proteins.

Ontogeny of adenosine uptake sites in guinea pig brain: differential profile of [3H]nitrobenzylthioinosine and [3H]dipyridamole binding sites.

The ontogenetic profile of adenosine uptake sites was investigated in guinea pig cerebral cortex and cerebellum using as ligand probes the uptake inhibitors, [3H]nitrobenzylthioinosine ([3H]NBI) and [3H]dipyridamole ([3H]DPR). In cerebral cortex [3H]NBI binding was highest at E50 and decreased subsequently until P28 while in cerebellum after a first peak at E50 and a subsequent decline it increased again until P28. [3H]DPR binding increased by 25% from E40 to P28 in cerebral cortex while in cerebellum hardly any binding could be detected before E50 and it afterwards increased by more than 250% until P28. Scatchard analysis demonstrated that [3H]NBI labeled approximately as many sites as [3H]DPR in cerebral cortex at E44 while at P28 [3H]DPR labeled more than double as many sites. Accordingly, NBI was more potent in displacing [3H]DPR binding at E44 than at P28. These findings suggest that part of the [3H]DPR binding sites, i.e. the NBI-insensitive one develops later than [3H]NBI binding sites during ontogeny in guinea pig cerebral cortex and cerebellum.

Adenosinergic modulation of caffeine-induced c-fos mRNA expression in mouse brain.

The adenosine receptor antagonist, caffeine, transiently induced proto-oncogene c-fos mRNA in mouse brain in a dose-dependent fashion. In situ hybridization revealed that caffeine-induced c-fos expression was high in caudate-putamen and olfactory tubercle at both subconvulsive and convulsive doses. The pattern of c-fos mRNA distribution following caffeine administration differs from that reported after seizures induced by electroconvulsive shock (ECS) or other chemical convulsants, and closely parallels the distribution of adenosine A2 receptors. Furthermore, the potent adenosine A2 receptor agonist, 5'-N-ethylcarboxamide adenosine (NECA) blocked caffeine-induced c-fos expression whereas the adenosine A1 receptor ligand, N6-cyclohexyladenosine (CHA), had no effect. This study suggests that the caffeine-induced expression of c-fos mRNA may be mediated by the adenosine A2 receptor in mouse brain.

Evidence for adenosine A2 receptor involvement in the hypomobility effects of adenosine analogues in mice.

The hypomobility induced by a series of adenosine analogues was investigated using a holeboard test and their behavioral potencies correlated to their reported adenosine A1 and A2 receptor affinities. All of the adenosine analogues dose dependently inhibited both exploratory behavior (head dipping) and locomotor activity. The rank order of potency 5'-ethylcarboxamido adenosine (NECA) greater than 5'-methylcarboxamido adenosine (MECA) greater than N6-[(R)-1-methyl-2-phenylethyl]adenosine (R-PIA) greater than N6-cyclohexyladenosine (CHA) = N6-cyclopentyladenosine (CPA) greater than N6-[(S)-1-methyl-2-phenylethylladenosine (S-PIA) greater than N6-(2-hydroxyethyl)adenosine (2-OH-ethyl) was observed for inhibiting both activities. The behavioral potency of these adenosine analogues correlates extremely well with their reported A2 receptor affinity (r2 = 0.93, P less than 0.01 and r2 = 0.86, P less than 0.05 for locomotor and head dipping activity respectively), but very poorly with their reported A1 receptor affinity (r2 less than 0.02, P greater than 0.50 for both activities). These results suggest that adenosine A2 receptors, but not A1 receptors, may be involved in the hypomobility induced by adenosine analogues.

Hypothermic effects of alkylxanthines: evidence for a calcium-independent phosphodiesterase action.

Caffeine induces a dose-dependent decrease in core body temperature in mice and the hypothermia induced by a 100 mg/kg dose of caffeine was seen to persist for greater than 160 min. Other alkylxanthines including theophylline, enprophylline, isbutylmethylxanthine and 1,3-dipropyl-7-methylxanthine also showed dose-dependent reductions in body temperature. The dose of these drugs required to reduce body temperature by 2 degrees C was calculated and correlated with the affinities for the compounds at adenosine A1 and A2 receptors and their activities in inhibiting calcium dependent and independent phosphodiesterases. Significant relationships were found between the 2 degrees C hypothermic dose (HD2) and soluble and membrane calcium-independent phosphodiesterase inhibiting activity (r2s = 0.950 and 0.940, respectively). No significant relationship was seen between HD2 and soluble calcium-dependent phosphodiesterase inhibiting activity or with A2 adenosine receptor affinity. The relationship between HD2 and A1 adenosine receptor affinity (r2 = 0.739) did however almost reach statistical significance. These results would suggest that phosphodiesterase inhibition, instead of or in addition to adenosine receptor blockade, may play an important role in the effects of alkylxanthines on body temperature.

Intracerebroventricular pertussis toxin enhances sensitivity to N-methyl-D-aspartate-induced seizures in mice.

The effect of pretreatment with pertussis toxin on N-methyl-D-aspartate (NMDA)-induced seizures was investigated in mice. In animals treated with pertussis toxin (0.5 micrograms/animal i.c.v) five days prior to testing the convulsant ED50 of NMDA was calculated to be 18 mg/kg whereas it was calculated to be 107 mg/kg in sham-treated animals. These results suggest the pertussis toxin enhances sensitivity to NMDA, possibly via its actions on inhibitory G-proteins.

Opioid receptor mediation of the hypothermic response to caffeine.

Caffeine and other methylxanthines induce a dose-dependent reduction in core body temperature in mice. These experiments investigated the effects of neurotransmitter and neuromodulator antagonists on caffeine-induced hypothermia. Pretreatment with the alpha 2-adrenoceptor antagonist, atipamezole; the beta-adrenoceptor antagonist, propranolol; the dopamine antagonist, haloperidol; or the benzodiazepine receptor antagonist, flumazenil had no intrinsic effects on core body temperature nor did they interact significantly with the hypothermic effects of caffeine. The alpha 1-adrenoceptor antagonist, prazosin and the 5-HT receptor antagonist, metergoline significantly enhanced the hypothermic effects of caffeine, probably involving a combined effect with their intrinsic hypothermic actions. Pretreatment with the opiate receptor antagonist, naloxone (3 mg/kg i.p.), had no intrinsic effect on core body temperature but attenuated the hypothermic effect of caffeine reflected in a parallel shift to the right in the caffeine dose-effect curve. The naloxone-induced attenuation of the hypothermic effects of caffeine was also seen to be dose-dependent. The results reveal that opiate receptors (but not adrenoceptors, 5-HT, dopamine or benzodiazepine receptors) may play a role in modulating the hypothermic action of caffeine and possibly other methylxanthines.

High expression of nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue.

The presence of both nitrobenzylthioinosine-sensitive and nitrobenzylthioinosine-insensitive dipyridamole binding sites in postmortem human ependymal tissue is reported. Displacement studies using 15 nM [3H]dipyridamole revealed 50-60% of the sites were sensitive to nitrobenzylthioinosine. Non-linear analysis of binding isotherms to estimate the density of nitrobenzylthioinosine-sensitive and -insensitive sites revealed a maximum number of nitrobenzylthioinosine-sensitive binding sites (Bmax) of 395 +/- 19 fmol/mg protein and a nitrobenzylthioinosine-insensitive Bmax of 3910 +/- 700 fmol/mg protein (corresponding Kd values of 0.1 nM and 114 nM respectively). Thus there are approximately 10 times as many nitrobenzylthioinosine-insensitive sites as nitrobenzylthioinosine-sensitive [3H]dipyridamole binding sites in human ependymal membranes.

Mapping rat brain structures activated during ethanol withdrawal: role of glutamate and NMDA receptors.

Brain structures activated during ethanol withdrawal have been mapped by visualizing c-fos mRNA expression. The regional distribution of c-fos mRNA in brain during ethanol withdrawal can be mimicked by acute injection of N-methyl-D-aspartic acid (NMDA) and is stereospecifically blocked by the NMDA receptor antagonist, MK-801. The findings reveal that the dentate gyrus and piriform cortex are selectively activated during ethanol withdrawal and suggest that this may be mediated by glutamate activation of NMDA receptors.