Nefopam is more potent than carbamazepine for neuroprotection against veratridine in vitro and has anticonvulsant properties against both electrical and chemical stimulation.

Nefopam (NEF) is a known analgesic that has recently been shown to be effective in controlling both neuropathic pain and convulsions in rodents. In this study we compared nefopam to carbamazepine (CBZ), a reference antiepileptic drug (AED), for their ability to protect cerebellar neuronal cultures from neurodegeneration induced by veratridine (VTD). Furthermore, we tested nefopam for protection against both, maximal electroshock-induced seizures (MES), and isoniazid-induced seizures in mice. Both NEF and CBZ were effective in preventing both signs of excitotoxicity and neurodegeneration following exposure of cultures to 5 microM veratridine for 30 min and 24 h, respectively. Concentrations providing full neuroprotection were 500 microM CBZ and 50 microM NEF, while the concentration providing 50% neuroprotection was 200 microM for CBZ and 20 microM for NEF. Neither NEF nor CBZ reduced excitotoxicity following direct exposure of cultures to glutamate, but CBZ failed to reduce increases in intracellular calcium following stimulation of L-type voltage sensitive calcium channels. In vivo, NEF (20 mg/kg i.p.) significantly reduced MES and fully prevented MES-induced terminal clonus (TC). In comparison, NEF was significantly more effective than CBZ in preventing MES, although both drugs were equally effective against MES-induced TC. Furthermore, nefopam provided protection against isoniazid-induced seizures at doses similar to those protecting against MES.

Nefopam inhibits calcium influx, cGMP formation, and NMDA receptor-dependent neurotoxicity following activation of voltage sensitive calcium channels.

Nefopam hydrochloride is a potent non sedative benzoxazocine analgesic that possesses a profile distinct from that of anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanism remains unclear. We have investigated the actions of nefopam on voltage sensitive calcium channels and calcium-mediated pathways. We found that nefopam prevented N-methyl-D-aspartate (NMDA)-mediated excitotoxicity following stimulation of L-type voltage sensitive calcium channels by the specific agonist BayK8644. Nefopam protection was concentration-dependent. 47 muM nefopam provided 50% protection while full neuroprotection was achieved at 100 muM nefopam. Neuroprotection was associated with a 73% reduction in the BayK8644-induced increase in intracellular calcium concentration. Nefopam also inhibited intracellular cGMP formation following BayK8644 in a concentration-dependent manner, 100 muM nefopam providing full inhibition of cGMP synthesis and 58 muM allowing 50% cGMP formation. Nefopam reduced NMDA receptor-mediated cGMP formation resulting from the release of glutamate following activation of channels by BayK8644. Finally, we also showed that nefopam effectively reduced cGMP formation following stimulation of cultures with domoic acid, while not providing neuroprotection against domoic acid. Thus, the novel action of nefopam we report here may be important both for its central analgesic effects and for its potential therapeutic use in neurological and neuropsychiatric disorders involving an excessive glutamate release.

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity.

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 microM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol.

Novel effect of nefopam preventing cGMP increase, oxygen radical formation and neuronal death induced by veratridine.

Nefopam hydrochloride is a potent analgesic compound that possesses a profile distinct from that of opiods or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. Here we have used cultured cerebellar neurons to test the hypothesis that nefopam may modulate voltage sensitive sodium channel (VSSC) activity. Nefopam (100 microM) effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the VSSC activator veratridine. Delayed neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. In contrast, excitotoxicity following direct exposure of neurons to glutamate was not affected. Neuroprotection by nefopam was dose-dependent. 50% protection was obtained at 57 microM while full neuroprotection was achieved at 75 microM nefopam. Veratridine-induced sodium influx was completely abolished in nefopam-treated neurons. Intracellular cGMP and oxygen radical formation following VSSC stimulation by veratridine were also effectively prevented by nefopam. Our data are consistent with an inhibitory action of nefopam on VSSC and suggest that nefopam may modulate the release of endogenous glutamate following activation of these channels. This novel action of nefopam may be of great interest for the treatment of neurodegenerative disorders involving excessive glutamate release and neurotransmission.

Biochemical evidence of functional interaction between mu- and delta-opioid receptors in SK-N-BE neuroblastoma cell line.

Radioligand binding assays and functional experiments revealed that the SK-N-BE neuroblastoma cell line expresses a similar ratio of mu- and delta-opioid receptors, both negatively coupled to adenylyl cyclase through pertussis toxin-sensitive G proteins. Our findings also indicate that some functional interaction occurred between the two opioid subtypes; in fact, long-term exposure to [D-Ala2-N-methyl-Phe4-Gly-ol5]enkephalin (DAMGO), a mu-selective agonist, sensitized the functional response of the delta-selective agonist but not vice versa. It is interesting that in acute interaction experiments, we observed a shift to the right of the concentration-effect curve of either DAMGO or [D-Pen2,5]enkephalin (DPDPE), a delta-selective agonist, as a result of DPDPE or DAMGO administration, respectively. In addition, low doses of naloxone, an antagonist selective for mu receptors, increased the inhibitory effect [D-Ala2-D-Met5]enkephalinamide (DAME), a mixed mu/delta agonist, on adenylyl cyclase activity. Taken overall, these data support the hypothesis of the existence of a cross talk between mu and delta receptors in the SK-N-BE cell line.

Behavioral and biochemical evidence of opioidergic involvement in cocaine sensitization.

Chronic administration of cocaine produces sensitization to its behavioral effects in humans and experimental animals. In the present study, rats treated with cocaine (10 mg/kg, i.p.) once daily for 10 days showed an enhancement in the acute drug stimulation of locomotor activity and stereotypy. Biochemical analysis in the nucleus accumbens of chronic cocaine-treated animals indicated that sensitization of D1 dopamine (DA) receptors had also developed. In fact, stimulation of adenylyl cyclase activity by DA was increased in nucleus accumbens membranes from sensitized rats. Our findings suggest that a novel postsynaptic mechanism, i.e., an increased DA-D1 receptor function, may play a role in the sensitization. A causal relationship between the two events is supported by the observation that neither motor behavioral sensitization nor DA-dependent adenylyl cyclase hyperactivity developed when the opiate antagonist naltrexone (2 mg/kg, s.c.) was given daily for 10 days before cocaine. When given alone, naltrexone was inactive in all respects, which rules out any unspecific action and suggests that its effects may be due to competition at receptors with endogenous opioids mobilized by cocaine. This was indirectly supported by the finding that desensitization of opioid inhibition of adenylyl cyclase developed in nucleus accumbens membranes of cocaine-sensitized rats. Chronic blockade of opioid receptors by naltrexone also counteracted the reinforcing properties of cocaine; conditioned place preference, clearly displayed by cocaine-treated animals, was antagonized in a dose-related manner. Overall, these results confirm that endogenous opioid peptides play an important role in cocaine addiction.

Cholera toxin antagonizes morphine-induced catalepsy through a cyclic AMP-independent mechanism.

We studied the effect of intracerebroventricular pretreatment with pertussis toxin and cholera toxin on morphine catalepsy in rats. Pertussis toxin (1 micrograms/rat, two, three and six days before) did not affect catalepsy evoked by central morphine. Cholera toxin (1 micrograms/rat) did not affect morphine catalepsy after 24 h and 48 h, but significantly reduced it (about 60%) after three and five days. Ten days later the morphine response had totally recovered. This effect was selective, since morphine analgesia was not modified. The reduction of catalepsy appeared unrelated to the ability of cholera toxin to raise cAMP levels, as demonstrated by the different time course of changes in striatal cholera toxin-stimulated adenylate cyclase activity. The effect required an intact cholera toxin molecule and did not occur with a similar dose of cholera toxin-B subunit. These findings demonstrate that catalepsy is an opioid effect not linked to pertussis toxin-sensitive G proteins and suggest that the Gs protein might be involved.

Role of opiates in striatal D-1 dopamine receptor supersensitivity induced by chronic L-dopa treatment.

Repeated administration of L-dihydroxyphenylalanine (L-dopa) to rats lesioned with monolateral intranigral injections of 6-hydroxydopamine counteracted the increased density of striatal [3H]spiroperidol binding sites induced by the lesion. On the contrary, the treatment with L-DOPA further enhanced the hypersensitivity of adenylate cyclase to dopamine stimulation that follows striatal denervation. In addition, the apomorphine-induced rotations were strongly potentiated. The latter effect was antagonized by morphine given acutely shortly before the dopamine agonist. On the other hand, the efficacy of [D-Ala2]-methionine enkephalinamide to inhibit striatal adenylate cyclase was decreased in 6-hydroxydopamine-lesioned rats chronically treated with L-dopa. Moreover, in these animals, when naltrexone was given chronically together with L-dopa, the supersensitivity of the enzyme to dopamine stimulation did not develop. Finally, in 6-hydroxydopamine-lesioned rats, chronic morphine, similarly to L-dopa, further enhanced the responses of adenylate cyclase to dopamine stimulation. These data suggest that prolonged indirect activation of striatal opiate receptors and their consequent desensitization could be among the causes of the hyperactivity of D-1 dopamine receptors that follows chronic L-dopa treatment.

Pertussis toxin inhibits morphine analgesia and prevents opiate dependence.

Six days after intracerebroventricular pretreatment of rats with pertussis toxin (PTX 0.5 microgram/rat) there was a marked decrease in the antinociceptive effect of morphine, regardless of the route of opioid administration (into the periaqueductal gray matter, intrathecally or intraperitoneally) or the analgesic test used (tail flick and jaw opening reflex). PTX pretreatment also partially attenuated the naloxone-precipitated withdrawal syndrome in morphine-dependent rats, significantly reducing teeth chattering, rearing and grooming. These in vivo findings indicate that G-protein-dependent mechanisms are involved in morphine analgesia and dependence. The biochemical mechanism could be related to ADP ribosylation of Gi coupled to the adenylate cyclase system, but an interaction of PTX with other G-proteins linked to different second messengers or directly to ionic channels cannot be excluded.

Cross-talk between different intracellular signalling pathways in the rat hippocampus.

The activation of alpha 1-adrenergic receptors in rat hippocampal slices enhances polyphosphoinositide (PPI) breakdown and cyclicAMP (cAMP) accumulation. The latter effect is antagonized by different protein kinase C (PKC) inhibitors and mimicked by a diacylglycerol (DAG) analogue, 1,2-diolein, which activates PKC, suggesting that cAMP synthesis is indirectly affected by alpha 1-adrenoceptors through the stimulated generation of DAG upon PPI hydrolysis. Furthermore the elevation of hippocampal cAMP decreases the ability of alpha 1-receptor agonists to enhance PPI breakdown. It is proposed that the observed effects are part of a complex cross-talk between PPI and AC signalling pathways operating in hippocampal neurons.

Role of dopamine in manganese neurotoxicity.

Manganese chloride increased cell mortality when added to human fibroblast cultures. The toxicity of the metal was greatly enhanced by dopamine; this effect was antagonized by the presence in the culture medium of catalase and superoxide dismutase enzymes. Manganese chloride also caused a marked decrease of striatal dopamine concentrations when infused into rat substantia nigra. Manganese neurotoxicity was lowered by pretreating the animals with drugs that reduced striatal dopamine turnover rate. Administration of an antioxidant, such as vitamin E, also partially prevented striatal dopamine decline induced by intranigral manganese infusion. Therefore, the decreased availability or autoxidation of dopamine attenuated manganese neurotoxicity. These findings are in agreement with previous observations suggesting that manganese increases toxic products originating from dopamine catabolism.

Effect of aspirin on serotonin and met-enkephalin in brain: correlation with the antinociceptive activity of the drug.

Intravenous administration of acetyl salicylate of lysine, a soluble salt of aspirin, reduced in rats the firing discharge of thalamic neurones, evoked by noxious stimuli. Concomitantly, concentrations of 5-hydroxyindole acetic acid increased, while those of met-enkephalin-like immuno-reactive derivatives were decreased in several areas of the brain. Similar electrophysiological and biochemical responses were obtained by administering tryptophan or 5-hydroxytryptophan plus carbidopa. The effect of aspirin on the evoked firing of the thalamic neurones was counteracted by pretreating the animals with metergoline. On the other hand, naloxone did not antagonize the inhibitory effect of aspirin and 5-hydroxytryptophan on pain-induced neuronal excitation. These data indicate that a serotonin-, but not a naloxone-sensitive opiate mechanism, may be relevant for aspirin-mediated antinociception.

Effects of the desensitization by morphine of the opiate-dependent adenylate cyclase system in the rat striatum on the activity of the inhibitory regulatory G protein.

Opiates act through a specific receptor to inhibit the striatal adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4,6.1.1] and stimulate a high-affinity GTPase (EC 3.6.1). The present study analyzes the functions of the striatal adenylate cyclase complex following chronic morphine treatment in the rat. The inhibitory effects of GTP on basal adenylate cyclase activity, between 10(-6) and 10(-4) M, were reduced. Moreover, the half-maximal inhibitory concentration of the opiate receptor agonist (D-Ala2-Met5)-enkephalinamide (DAME) on striatal adenylate cyclase activity was increased by about four times, whereas the maximal effect was reduced in membranes from treated rats. In parallel, the half-maximal stimulatory concentration of DAME on GTPase was increased by two times, and the maximal stimulation was reduced from 60 to 25%. Binding studies performed with [3,5-3H]DAME (saturation curves) and with [3H]naloxone (competition curves) did not show any change in opiate receptor numbers and affinity. Moreover, the kinetics of the activation of the inhibitory GTP binding protein (Gi) which transduces the opiate receptor effect on adenylate cyclase showed a small but significant delay. Therefore, hypofunction of Gi can be, at least in part, responsible for the observed desensitization by morphine of the opiate-dependent GTPase and adenylate cyclase.

Manganese neurotoxicity: effects of L-DOPA and pargyline treatments.

Single, monolateral injection into rat substantia nigra of manganese chloride produced within two weeks from its administration a loss of dopamine in the striatum ipsilateral to the injected side. The effect was dose-dependent and was not extended to serotoninergic terminals present in this brain area, whose content in serotonin and 5-hydroxyindoleacetic acid was not affected. When L-DOPA + carbidopa or pargyline were given to these animals the decrease of striatal dopamine was more marked. Moreover, rats treated two weeks before with a dose of manganese chloride that produced a 70-80% drop in striatal dopamine concentrations, rotated ipsilaterally to the dopamine-depleted striatum when injected with apomorphine, suggesting that in these animals the stimulatory effects of apomorphine were more relevant in striatum where presynaptic dopaminergic neurons were not affected by manganese chloride. These data indicate that the alterations of dopaminergic postsynaptic receptors may be different in parkinsonian and in manganese-intoxicated patients and that current therapy used for Parkinson's disease could be a hazard in treating manganese poisoning.

Differential effect of repeated treatment with L-dopa on dopamine-D1 or -D2 receptors.

Unilateral degeneration of the nigro-striatal dopaminergic pathway with 6-hydroxydopamine induced contralateral rotations to apomorphine injection, increased [3H]-spiroperidol binding and enhanced sensitivity of adenylate cyclase to dopamine stimulation in lesioned striata. Prolonged L-DOPA administration counteracted the increased density of [3H]-spiroperidol binding sites but further enhanced the hypersensitivity of adenylate cyclase to dopamine. Also apomorphine-induced contralateral rotations were potentiated. This effect was antagonized by SCH-23390. These results suggest that dopaminergic D1 and D2 receptors are differently affected by prolonged L-DOPA treatment.

Dopamine receptor changes in response to prolonged treatment with L-dopa.

Unilateral lesions of the nigro-striatal dopamine (DA) pathway induced contralateral rotations to apomorphine, increased (3H)-spiroperidol binding and enhanced the sensitivity of striatal adenylate cyclase to DA stimulation. Prolonged L-dopa administration counteracted the increased density of (3H)-spiroperidol binding sites but further enhanced the hypersensitivity of adenylate cyclase to DA and decreased the inhibitory effect of opiates on this enzyme. The apomorphine-induced contralateral rotations were also strongly potentiated. On the contrary the binding of (3H)-SCH-23390 was affected neither by DA nerve degeneration nor by chronic L-dopa treatment. These results suggest that DA-D1 and DA-D2 receptors are differently affected by prolonged L-dopa treatment. The biochemical changes of DA-D1 receptors associated with adenylate cyclase seem to be correlated with the enhanced behavioural responses to apomorphine and could be a consequence of a decreased opiate inhibitory tone on the enzyme. The increased supersensitivity of the DA-D1 receptors may play a role in the clinical changes seen in parkinsonian patients following chronic use of L-dopa.

Opiate and dopamine stimulate different GTPase in striatum: evidence for distinct modulatory mechanisms of adenylate cyclase.

Previous studies demonstrated that opiate inhibition of adenylate cyclase (AC) in striatal membranes is related to an opiate-stimulated GTPase with a low Km. Dopamine (DA) also dose-dependently activates a high affinity GTPase, with a pattern of stimulation and a receptor selectivity (D1 type) similar to those observed in DA activation of striatal AC. Moreover, the DA- and the opiate-sensitive GTPase activities have different sensitivities to agents that affect the inhibition of AC, such as Na+ and N-ethylmaleimide (NEM), or the stimulation, such as cholera toxin (CTX). Thus, the impairment of opiate-dependent inhibition of AC in the absence of Na+ ions or after NEM pretreatment of the membranes is parallel with preferential impairment of the opiate-dependent GTPase. On the contrary, selective blocking by CTX of the DA-dependent GTPase leads to the enhancement of AC stimulation by DA. These results suggest that DA activation of striatal AC is related to a GTPase that is specifically stimulated by DA and is associated with the Ns protein. A distinct Ni protein seems to be responsible for the opiate effect on AC and GTPase.

Possible involvement of endogenous opiates in the tolerance to the anorectic effect of fenfluramine.

Repeated administration of fenfluramine leads to a rapid and progressive loss of its effectiveness in reducing food intake. The animals tolerant to the anorectic effect of fenfluramine had markedly low basal hypothalamic serotonin (5-HT) levels. In this brain area the levels of [Met5]enkephalin-like immunoreactive material were, on the contrary, significantly higher in fenfluramine-tolerant animals than in controls. In tolerant animals the drug failed to further decrease 5-HT concentrations unless it was given at doses also reducing food intake. On the other hand, in acute experiments, morphine pretreatment potentiated and naloxone antagonized fenfluramine-induced depletion of striatal and hypothalamic 5-HT stores. In addition, when given to fenfluramine-tolerant rats, morphine restored the efficacy of the anorectic agent. After morphine pretreatment, fenfluramine depleted 5-HT and reduced food intake in tolerant animals. These findings, while further substantiating the importance of 5-HT in mediating fenfluramine anorexia, also suggest that endogenous opiates may play an important role in the processes through which tolerance to this drug develops. Fenfluramine reduces food intake by releasing 5-HT and tolerance to its anorectic effect would be a consequence of an inability to further release 5-HT. However, because release of 5-HT by fenfluramine seems to be modulated by opiates, repeated administration of fenfluramine might alter such modulatory mechanisms and tolerance to the effects of the drug would develop.

Presence of opiate receptors on striatal serotoninergic nerve terminals.

After degeneration of serotoninergic neurons induced by either transection of the ascending neuronal pathways originating from the nucleus raphe dorsalis or intraventricular 5,6-dihydroxytryptamine administration, the number of binding sites for [3H]D-Ala2, Met5-enkephalinamide was significantly reduced. This decrease in binding sites does not seem to be related to the opiate receptors present on dopaminergic terminals, nor is it due to a simple decrease in serotoninergic neuronal tone, since after p-chlorophenylalanine (100 mg/kg X 4 days) the number of striatal binding sites for the opiate ligand was not diminished. On the other hand, shortly after mechanical interruption of the raphe-striatal serotoninergic fibers, at a time when the metabolic processes are still functioning in the lesioned neurons, morphine still increased the striatal content of 5-hydroxyindoleacetic acid. These results suggest the presence of opiate receptors on striatal serotoninergic terminals, where they may modulate the presynaptic activity of these neurons.