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The activation of cholinergic pathways by nicotine elicits various physiological and pharmacological effects in mammals. For example, the stimulation of nicotinic acetylcholine receptors (nAChRs) leads to an antinociceptive effect. However, it remains to be elucidated which subtypes of nAChR are involved in the antinociceptive effect of nicotine on nerve injury-induced allodynia and the underlying cascades of the nAChR-mediated antiallodynic effect. In this study, we attempted to characterize the actions of nicotine at the spinal level against mechanical allodynia in an animal model of neuropathic pain, tibial nerve transection (TNT) in rats. It was found that the intrathecal injection of nicotine, RJR-2403, a selective alpha4beta2 nAChR agonist, and choline, a selective alpha7 nAChR agonist, produced an antinociceptive effect on the TNT-induced allodynia. The actions of nicotine were almost completely suppressed by pretreatment with mecamylamine, a non-selective nicotinic antagonist, or dihydro-beta-erythroidine, a selective alpha4beta2 nAChR antagonist, and partially reversed by pretreatment with methyllycaconitine, a selective alpha7 nAChR antagonist. Furthermore, pretreatment with strychnine, a glycine receptor antagonist, blocked the antinociception induced by nicotine, RJR-2403, and choline. On the other hand, the GABAA antagonist bicuculline did not reverse the antiallodynic effect of nicotine. Together, these results indicate that the alpha4beta2 and alpha7 nAChR system, by enhancing the activities of glycinergic neurons at the spinal level, exerts a suppressive effect on the nociceptive transduction in neuropathic pain.

Background Although recent evidence suggests that cyclooxygenase-2 (COX-2) may contribute to the development and management of neuropathic pain, the efficacy of COX-2 inhibitor against neuropathic pain is still unclear. In this study, we investigated the effects of the systemic administration of the selective COX-2 inhibitor meloxicam at an early stage after nerve injury on the development of tactile allodynia in L5 single spinal-nerve injury in rats. Methods Twenty-four young male Sprague-Dawley rats received L5 single spinal-nerve injury. Nerve-injured rats (6 per group) received repeated intraperitoneal administrations of meloxicam (1, 2, or 4 mg/kg) or vehicle 0, 12, 24, and 36 hours after nerve injury. Tactile allodynia was quantified for 4 weeks by use of von Frey filaments. Results In animals given 2 mg/kg and 4 mg/kg, hind-paw withdrawal thresholds 4 weeks after nerve injury were significantly higher compared with those of the vehicle-treated animals. The area under the time-effect curve from preinjury to 4 weeks after nerve injury values were significantly higher in animals treated with 4 mg/kg of meloxicam compared with animals treated with vehicle. Conclusion Systemic administration of 2 mg/kg and 4 mg/kg of meloxicam at an early stage after nerve injury attenuated the development of tactile allodynia. These results suggest that COX-2 may be at least in part involved in the development of tactile allodynia in an L5 single spinal-nerve injury model.

The behavioral and neurochemical effects of high doses of amphetamine administered to BALB/c mice were examined in the presence and absence of co-administered haloperidol (a D2 antagonist), SCH 23390 (a D1 antagonist) and risperidone (a mixed 5-HT2/D2 antagonist). It was observed that mice displayed a dose-dependent increase in stereotypic behavior, oral dyskinesia, and self-injurious behavior (SIB) in response to amphetamine treatment. Furthermore, agents that blocked the SIB reversed the amphetamine-induced release of serotonin. This effect was unrelated to hyperthermia or non-specific sedation (as assessed by measurement of motor activity). These data are interpreted in the context of the underlying basis of murine SIB involving both dopaminergic and serotonergic activation and demonstrate the efficacy of risperidone in treating these behaviors.

To clarify the role of cyclooxygenase in the peripheral nerve on the development of neuropathic pain, we investigated the effects of peri-sciatic administration of indomethacin on the development of allodynia in a model of L5 single spinal nerve injury. Peri-sciatic administration of indomethacin (1 mg/kg) was performed 3, 24, or 72 h after nerve injury (n=6/each). In rats with indomethacin 3 or 24 h after nerve injury, ipsi-lateral paw withdrawal thresholds 7-35 days after nerve injury were significantly higher compared with those in the control group (n=6: without peri-sciatic treatment) (P0.05). However, such efficacy was no longer apparent when indomethacin was administered 72 h after nerve injury. These results suggest that peri-sciatic administration of indomethacin early (less than 24 h) after nerve injury can attenuate the development of allodynia.

To evaluate the relationship between nicotine and immunological inflammation, we investigated the effects of nicotine on plasma extravasation of the passive skin Arthus reaction, elicitated 4 hr after sensitizing skin with antiserum, and serum corticosterone levels in rats. Pretreatment with a single subcutaneous injection of nicotine (0.4 or 0.8 mg/kg) 30 or 60 min. before antigen challenge attenuated the passive skin Arthus reaction immunological inflammation. Serum corticosterone levels were dose-dependently increased 30 and 60 min. after nicotine administration. Both markers co-varied with a similar dose-response and time course after the nicotine-treatment. In addition, we also examined these nicotine-induced responses after bilateral lesions of the hypothalamic paraventricular nucleus; both the nicotine-induced suppression of immunological inflammation and the increased serum corticosterone levels were attenuated in bilateral paraventricular nucleus-lesioned animals. Moreover, the immunological inflammatory decreasing effects of a single subcutaneous injection of nicotine (0.4 mg/kg) were antagonized by intraperitoneal preinjection with mecamylamine (1.0 mg/kg; blocking the brain nicotinic acetylcholine receptors) as well as by subcutaneous preinjection with mifepristone (30 mg/kg; a glucocorticoid receptor antagonist) but not by intraperitoneal preinjection with hexamethonium (2.0 mg/kg; a peripheral nicotinic acetylcholine receptors antagonist). Finally, intraperitoneal preinjection with cycloheximide (2 mg/kg), a protein synthesis inhibitor, abolished both the inhibitory effect of nicotine (0.4 mg/kg) on the dye leakage and the elevation of blood corticosterone levels. These findings indicate that the nicotine-induced decreasing effect on immunological inflammatory response may be related to serum corticosterone levels elevated by an activation of the paraventricular nucleus through the brain nicotinic acetylcholine receptors.

The function of the 5-hydroxytryptamine (5-HT) system in the medial vestibular nucleus (MVN) was evaluated using KCl-evoked 5-HT release assessed by in vivo microdialysis. The release of 5-HT in the MVN was increased by the local application of KCl (100 mM) in a calcium-dependent manner. Likewise, perfusion of a 5-HT reuptake blocking agent, clomipramine (1 microM), increased extracellular levels of 5-HT in the MVN. Finally, pretreatment with the selective 5-HT(1A) agonist, 8-hydroxy-2-(di-N-propylamino) tetralin (1 mg/kg, intraperitoneally), attenuated the KCl-evoked 5-HT release. These findings suggest that 5-HT stored in vesicles is released from nerve terminals in the MVN. In addition, its regulation may be mediated by control of somatodendritic 5-HT(1A) receptor activation.

Fiber type distributions, cross-sectional areas (CSAs), and succinate dehydrogenase (SDH) activities of the slow soleus (SOL) muscle and the deep (EDLd) and superficial (EDLs) regions of the fast extensor digitorum longus (EDL) muscle were determined in 10- and 20-week-old male spontaneously hypertensive rats (SHR) and compared with those in age-matched normotensive Wistar-Kyoto rats (WKY). There were no differences in the body weight or the SOL and EDL weight between WKY and SHR at 10 weeks of age, while the body weight and the SOL and EDL weight were lower in SHR than in WKY at 20 weeks of age. A higher percentage of type IIA and type IIC fibers and a lower percentage of type I fibers were observed in SOL of SHR at 10 weeks of age. A higher percentage of type IIB fibers and a lower percentage of type IIA fibers were observed in EDLd and EDLs of SHR at 10 weeks of age. In contrast, there were no differences in the fiber type distribution, except a higher percentage of type IIA fibers and a lower percentage of type I fibers in EDLd of SHR, in SOL, EDLd, or EDLs between WKY and SHR at 20 weeks of age. There were no differences in the CSA, except a smaller CSA of type IIC fibers in SOL of SHR, in SOL, EDLd, or EDLs between WKY and SHR at 10 weeks of age. In contrast, a smaller CSA of type I fibers was observed in SOL of SHR at 20 weeks of age. A smaller CSA of all types of fibers in EDLd and of type IIA fibers in EDLs was observed in SHR at 20 weeks of age. There were no differences in the SDH activity of fibers between WKY and SHR, irrespective of the age and fiber type. It is concluded that age-related differences in the fiber type distribution or CSA are observed in the skeletal muscles between SHR and WKY.

Chronic treatment with dopamine D 2 blockers in schizophrenic patients has been proposed as one of the causes of polydipsia and water intoxication, but this conclusion is still controversial. To investigate the relationship between dopamine D 2 blockers and these syndromes, we designed a behavioral and neurochemical study using hyperosmotic stimulation in the supraoptic nucleus (SON) by microdialysis after chronic treatment with haloperidol in rats. Animals were injected with haloperidol decanoate (20 mg/kg, i.m.) or sesame oil at 2-week intervals for 8 successive weeks. During the 7th week, water-intake was increased 30–60 min after the hyperosmotic stimulation in both groups, but more so in haloperidol-treated animals compared to that in the control group. Moreover, arginine vasopressin (AVP) was released by the hyperosmotic stimulation in SON, but was not significantly different between groups. In addition, striatal dopamine levels 3–4 days after the microdialysis study showed a significant decrease in the haloperidol-treated animals. These results suggest that chronic treatment with haloperidol enhances water-intake produced by hyperosmotic stimulation in the SON but does not increase AVP levels in dialysates following hyperosmotic stimulation. Thus, these symptoms may be mediated by dopaminergic systems in brain.

A relationship between formation of reactive oxygen species (ROS) and energy depletion has been proposed to play an important role in mediating methamphetamine (METH)-induced neurotoxicity. To evaluate this relationship, we examined the effect of the spin-trap agent, α-phenyl-N-tert-butyl nitrone (PBN) on hyperthermia and self-injurious behavior (SIB) and striatal dopamine (DA) depletion produced by METH (4 injections of 4 mg/kg, 2 hr intervals, s.c.) in BALB/c mice. Repeated administration of METH induced hyperthermia, incidence of SIB and striatal DA depletion (84% after 3 days). Pretreatment with PBN (4 injections of 60 or 120 mg/kg, i.p.) reduced METH-induced hyperthermia, but did not significantly attenuate METH-induced SIB or the striatal DA depletion. On the other hand, pretreatment with high doses of PBN (4 injections of 180 or 240 mg/kg, i.p.) protected against METH-induced hyperthermia and SIB, and PBN (180 mg/kg) also completely protected against the acute striatal DA depletion 60 min after the last injection of the drug. However, the long-lasting striatal DA depletion was only attenuated by 52 or 56%, respectively. These results indicate that METH-induced hyperthermia contributes to, but is not solely responsible for METH-induced neurotoxicity, and supports a role for formation of ROS and other mechanisms in the generation of METH-induced striatal dopaminergic neurotoxicity. In addition, the difference in the efficacy of PBN to protect against the acute or long-lasting striatal DA depletion induced by METH may indicate that both ROS formation and other mechanisms are required for METH-induced neurotoxicity to develop.

The behaviors associated with the neurotoxic effects of methamphetamine were evaluated in BALB/c mice. Hyperthermia and behavioral observations were measured 60 min after each subcutaneous injection of methamphetamine (4x4 or 8 mg/kg) or saline, each given 2 h apart. The behavioral observations included stereotyped behaviors, incidence of hemorrhage in breast, salivation and self-injurious behavior (SIB). Repeated administration of methamphetamine produced these behavioral changes and hyperthermia, but resulted in hypothermia by the final injection (8 mg/kg). In addition, the methamphetamine treatment induced a long-lasting dopamine depletion of similar magnitude in the 4 and 8 mg/kg-treated animals. In a time course study striatal monoamine levels were measured 60 min after each injection of these doses. The first and second injections of methamphetamine (8 mg/kg) produced a drastic increase in striatal 3-methoxytyramine; this failed to occur after the third or fourth injection of the same dose. In contrast, 4 mg/kg of methamphetamine also produced an increase in 3-methoxytyramine after the second and third injections of the drug and, in this case, these were maintained for the duration of the treatment. Striatal 3, 4-dihydroxyphenylacetic acid levels also drastically decreased following both doses of methamphetamine, suggesting inhibition of monoamine oxidase in striatum. Moreover, a single injection of methamphetamine increased striatal 2,3-dihydroxybenzoic acid formation. These results suggest that the incidence of hyperthermia, SIB and striatal dopamine neurotoxicity are closely linked to striatal dopamine release and inhibition of monoamine oxidase produced by methamphetamine in BALB/c mice.

The expression of cyclooxygenase-2 (COX-2) and striatal dopamine (DA) depletion in BALB/cAnNcrj (BALB/c) mice following a neurotoxic dose of methamphetamine (METH) was investigated. METH-treatment (4 mg/kg x 4, 2 h intervals, s.c.) induced a significant hyperthermia and a persistent depletion of striatal DA levels 72 h after the treatment. COX-2, a marker of the cytotoxic effect of inflammation and oxidative stress and thiobarbituric acid (TBA) were significantly induced in the striatum 72 h after the METH-treatment, but not in the hippocampus. These results suggest that COX-2 may participate in METH-induced neurotoxicity in striatum.

The cross-sectional areas and succinate dehydrogenase activities of different types of fibers in different regions of the plantaris and tibialis anterior muscles in 10-week-old male rats were determined using quantitative histochemistry. The muscle fibers were classified as type I, type IIA, or type IIB according to their adenosine triphosphatase activities. There were no regional differences in either the mean cross-sectional area or the mean succinate dehydrogenase activity of type IIA fibers in both muscles. In contrast, type IIB fibers in the deep region of both muscles had smaller cross-sectional areas and higher succinate dehydrogenase activities than those in the superficial and middle regions. These data suggest the presence of regional differences in the cross-sectional area and succinate dehydrogenase activity of type IIB fibers in the muscle.

We demonstrated previously that endothelin-1 (10(-14) to 10(-8) M) promotes lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E(2) production through endothelin ET(B) receptors effects which are up-regulated by lipopolysaccharide. In the present study, we confirmed these findings and showed that prostaglandin E(2) (10(-6) to 10(-5) M) inhibited the lipopolysaccharide plus endothelin-1-induced cyclooxygenase 2 expression more profoundly as compared to its inhibition of the lipopolysaccharide-induced cyclooxygenase 2 expression. The endothelin ET(B) receptor selective antagonist, N-cis-2, 6-dimethylpiperidino-carbonyl-L-gamma-methyl-leucyl-D-L-methoxy carbon yl-tryptophanyl-D-norleucine (BQ788), partly inhibited this suppression. Interestingly, the expression of endothelin ET(B) receptors in macrophages was increased by lipopolysaccharide plus prostaglandin E(2) (10(-8) to 10(-5) M) about 1.6-fold compared with that evoked by lipopolysaccharide stimulation alone. We also showed that treatment with endothelin-1 at 10(-14) M (15 min) elevated an intracellular cyclic AMP concentration in macrophages stimulated by lipopolysaccharide or lipopolysaccharide plus prostaglandin E(2) (10(-6) M) for 6 h, and the elevation in the latter cells was more pronounced. These results suggested that endothelin-1 shows an opposite modulation of lipopolysaccharide-induced cyclooxygenase 2 expression in macrophages through endothelin ET(B) receptors, depending on the level of extracellular prostaglandin E(2), and the changes of intracellular cyclic AMP by endothelin-1 may be involved in this mechanism.

Administration of neurotoxic doses of methamphetamine (8 mg/kg, intraperitoneally x 4 times, at 2 hr intervals) caused a significant decrease in dopamine and 3,4-dihydroxyphenylacetic acid and an increase in 3-methoxytyramine levels in the striatum along with a decrease in serotonin and 5-hydroxyindoleacetic acid levels in the striatum and hippocampus. In addition, the methamphetamine treatment caused an increase in rat rectal temperature. Intraventricular injection of salicylate 105 min. after the last injection of methamphetamine produced an increase in 2,3- and 2,5-dihydroxybenzoic acid in the striatum and hippocampus. Moreover, the ratio of 2,3-dihydroxybenzoic acid to salicylate was significantly increased in the striatum, but not in the hippocampus. These results indicate that the hydroxyl radical may play an important role in methamphetamine-induced neurotoxicity in rat striatum and that its formation may be the result of methamphetamine-induced release of dopamine.

We investigated the modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E 2 production by mouse peritoneal macrophages. Our previous report showed that endothelin-1 at concentrations above 10 −11 M induced cyclooxygenase 2 expression through mainly endothelin ET B receptors and that an endothelin ET B receptor-mediated process was not involved in cyclooxygenase 2 activation in macrophages stimulated by lipopolysaccharide for 4 h. In the present study, when macrophages were stimulated by lipopolysaccharide for 12 h in the presence of endothelin-1 (10 −15 to 10 −8 M), cyclooxygenase 2 expression and prostaglandin E 2 production were enhanced by 1.2- to 1.6-fold. The endothelin ET B receptor selective antagonist, BQ788 ( N - cis -2,6-dimethylpiperidino-carbonyl- l -γ-methyl-leucyl- d - l -methoxycarbonyl-tryptophanyl- d -norleucine), significantly inhibited this synergistic effect of endothelin-1. In addition, the cyclooxygenase 2-selective inhibitor, NS398 ( N -[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide), also suppressed this effect. Western blot analysis showed that the endothelin ET B receptor was up-regulated by lipopolysaccharide in a time- and concentration-dependent manner, and that this up-regulation was inhibited by NS398. From these results, we conclude that endothelin-1 promotes lipopolysaccharide-induced cyclooxygenase 2 activation in the delayed phase through endothelin ET B receptors up-regulated by lipopolysaccharide.

Succinate dehydrogenase (SDH) activities and cross-sectional areas (CSAs) of different types of fibers in the superficial (EDLs) and deep (EDLd) regions of the extensor digitorum longus and soleus (SOL) muscles and the left ventricular muscle of the heart (HEART) of 10-week-old male rats were determined using quantitative histochemistry and a computer-assisted image processing system. The fibers were classified as type I, type IIA, type IIB, or type IIC according to their histochemically assessed adenosine triphosphatase activities. The mean SDH activity was higher and the mean CSA was smaller in type IIA fibers than in type IIB fibers in both the EDLs and EDLd. The mean SDH activity of type IIA fibers in the SOL was higher than that of type I fibers. Fibers in the HEART showed the highest mean SDH activity and the smallest mean CSA among all fiber types in the muscles examined. There was an inverse correlation between CSA and SDH activity for the different fiber types in different muscles. These data suggest that the SDH activity of fibers in muscle is fiber type- and size-specific, and that the highest SDH activity of fibers in the left ventricular muscle of the heart contributes to their functional properties, i.e., high fatigue resistance.

The effects of an endothelin ET a -receptor selective antagonist, S-0139, were examined using dogs given endothelin-1 (ET-1) into the subarachnoid space. ET-1 at 40 pmol apparently constricted the basilar artery in anesthetized dogs and caused various grades of ataxia, facial clonus, nystagmus and other features in conscious dogs, partially mimicking those which have been reported for conscious rats. S-0139 could completely inhibit both the vasoconstriction and behavioral changes. It could also alleviate the behavioral changes caused by ET-1 in conscious dogs when given after the severe ataxia. We concluded that ET-1 in the subarachnoid space produces behavioral changes via endothelin ET a -receptor mediation similar to its cerebral vasoconstricting action, at least, in dogs.

Psychological stress without any physical stimuli caused a rapid and marked increase in the level of heat shock protein (HSP) 70 mRNA in rat aorta, but had little effect on the other tested tissues. The maximum increase in HSP70 mRNA level in the aorta was observed at 0.5-1 hr after the stress, and then it declined. Moreover, this stress also increased the level of HSP70 protein in the aorta, but had little effect on the other tested tissues. These results indicate that exposure of rats to mild psychological stress results in the induction of HSP70, especially in the blood vessels.

Localization of nicotinic acetylcholine receptor (nAChR) alpha 4 subunits was investigated in rat cerebral cortex using a monoclonal antibody against alpha 4 subunits. The antibody depleted more than 70% of the [3H]methylcarbamylcholine choline binding activity of the solubilized membrane fraction. By light microscopy alpha 4-like immunoreactivity (alpha 4-LI) was found through layers II to VI. The immunostaining was the most prominent in cell bodies and apical dendrites of pyramidal cells in layer V. By electron microscopy most immunoreaction products were observed in the rough endoplasmic reticulum, cytoplasmic matrix and synaptic membranes. Alpha 4-LI was detected in the postsynaptic membranes of neuronal cell bodies and apical dendrites. These findings suggest that alpha 4-containing subtypes serve as one possibly the postsynaptic nAChR in rat cerebral cortex.

1. Comparison between the chronotropic and inotropic effects induced by nicotine in the right and left atrial muscles of rat was investigated. 2. Nicotine (300 μM and 1 mM) significantly produced negative chronotropic and inotropic effects in the right atrium. The effects were concentration-dependent, and reached a maximum about 30 sec after nicotine administration (1 mM). The responses were attenuated with time even during exposure to nicotine (desensitization). 3. In the left atrium, nicotine (300 μM and 1 mM) caused a negative inotropic effect. The depression was decreased with an increase in the frequencies of stimulation (1–3 Hz). The negative inotropic effect reached a maximum at approx. 60 sec later. 4. Nicotine (1 mM) caused a greater negative inotropic response in the right atrium than in the left atrium by 23% (stimulated at 3 Hz). The effects were not modified by atropine (1 μM) and hexamethonium (1 mM). 5. There was no difference between single and cumulative administrations of nicotine in both right and left atrial muscles. 6. These results suggest that the negative chronotropic and inotropic effects of nicotine may be due mainly to a direct action on the cell membrane of rat atria, accompanied with down regulation of nicotinic ACh receptors. The difference between the right and left atria could result from a different innervation of the autonomic nervous system.

Acute toxicity of nicotine was examined in old (24 months) and young (6 weeks) Wistar rats. There were no significant age differences in the mortality and convulsive responses induced by an intraperitoneal injection of nicotine (24.5 mg/kg). The lethal nicotine levels in blood and cortex and the latent period to death in old rats were larger than those in young rats. Cortical and blood nicotine levels 15 min. after the nicotine injection in old rats were also higher than those in young rats. Nicotine significantly increased dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in striatum and hippocampus in young rats, but not those in old rats. Moreover, nicotine-induced elevations in blood levels of corticosterone in old rats were also less than those in young rats. On the other hand, the cytochrome P-450 content, the nicotine oxidase activity and the flavin-containing monooxygenase activity in liver showed age-related decreases in the young, the middle-aged (12 months) and the old rats. These results indicate that the acute toxicity of nicotine in old rats reflects the decreases in hepatic nicotine metabolism and in brain sensitivity to nicotine.

To date, cDNA studies and purification of nicotinic acetylcholine receptors (nAChRs) have shown that there are many different subtypes of the receptors in the central and peripheral nervous systems. This review focuses on the animal and avian brain nAChRs, their diversity, pharmacological properties, distribution and regulation. nAChR subtypes are classified as alpha beta types and alpha-bungarotoxin binding proteins. Each subtype contains two or more subunits. Each subunit combination leads to a distinct pattern of sensitivity to nicotinic agonists in reconstituted systems of Xenopus oocytes. Nicotine has been shown to increase the levels of intracellular free Ca through both nAChR types. The mRNAs for each subunit are uniquely expressed in animal and avian brains, although beta 2 is expressed in most regions of the brains. This shows somewhat overlapping patterns of gene expression. Monoclonal antibodies against each subunit make it possible to investigate the distribution of the subunits in brains and neurons. nAChRs have been shown to be regulated by nicotine administration and phosphorylation of the receptors. The regulatory mechanisms have been extensively studied, but still remain obscure. The structural and functional diversity of neuronal nAChRs is probably important in nicotine addiction, tolerance and reverse tolerance.

The convulsive movements, blood levels of corticosterone and pharmacokinetics of nicotine after an acute intraperitoneal injection of nicotine (5 mg/kg) were examined in young (6-week-old) and old (2-year-old) rats. In pharmacokinetic study, blood nicotine levels during the elimination phase were significantly higher in old rats than in young rats. However, the duration of convulsions and the elevation of corticosterone levels after the nicotine injection showed significant decreases in old rats compared with those in young rats. These differences of nicotine-induced responses between young and old rats may be involved in the decrease in nicotine sensitivity.

The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 microM) did not cause large changes in catecholamine secretion evoked by Ca2+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabilized cells with [gamma-32P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca(2+)-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 microM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 microM TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Ca(2+)-induced catecholamine secretion, and cAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.

Using chromatography on DE-52 and acetylcholine-Affi-Gel columns, nicotinic acetylcholine receptor was purified to approximately 10,000 fold from Lubrol extract of rat brain with a recovery of 15%. The purified preparation contained no cholinesterase activity. α-Bungarotoxin did not inhibit [ 3 H]acetylcholine binding to the purified preparation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 4 major protein bands with apparent molecular weights of 53,000, 67,000, 80,000 and 108,000. When nicotinic acetylcholine receptor was eluted with either carbachol or nicotine from the affinity column, these major bands were found on SDS-PAGE gels. Immunoblot analysis showed that the M r 80,000 protein was an acetylcholine-binding subunit and that the M r 48,000 protein, a minor band on SDS-PAGE gel, was a structural subunit.

The present study was conducted to evaluate the contribution of endothelin (ET) to the pharmacodynamic response to chronic cigarette smoke in spontaneously hypertensive rats (SHR). The contribution of ET was studied consequent to the hemodynamic response following 8 weeks of cigarette smoke by determining the changes in tissue ET-1 content and ET receptors. The blood pressure (BP) at the early phase of smoking and the heart rate (HR) 24 h later were apparently reduced in SHR, while the HR at the early phase was transiently elevated in normotensive Wistar Kyoto (WKY) rats. Tissue ET-1 levels in the hypothalamus, striatum, and cortex of SHR were higher than those in WKY rats, and these higher levels in SHR were reduced by exposure to chronic cigarette smoke. The ET-1 contents in the medulla oblongata and midbrain of both strains were clearly increased by smoke exposure, although the levels of SHR and WKY rats were not different. In addition, the immunoreactivity of the ET type A receptor in the adrenal glands and type B receptor in the kidneys of SHR showed a different response to smoke exposure as compared to WKY rats. Our present findings suggest that the changes of ETs may relate to the pharmacodynamic effects of chronic cigarette smoke. Keywords:: endothelin, spontaneously hypertensive rat, cigarette smoking, hypertension, endothelin receptor

Recent evidence suggested that cyclooxygenase-2 (COX-2) expression in the peripheral nerve early after nerve injury might be involved in the development of neuropathic pain. Although previous investigators have demonstrated that COX-2 is expressed in peripheral nerve at a late phase (2-4 weeks) after nerve injury, COX-2 up-regulation at an early phase after nerve injury has not been determined. Using immunohistochemistry, we observed biphasic increases of COX-2 expression after L5 single spinal nerve injury. The first increment of COX-2 positive cells was noted 1 day after nerve injury and these cells co-expressed the Schwann cell marker S-100. A second increment was noted after 7-14 days and these cells co-expressed the macrophage marker ED-1. These results suggested that the cellular sources of COX-2 expression might be different between the early and late phases after nerve injury.

1. The fibre type distributions, cross-sectional areas (CSA) and succinate dehydrogenase (SDH) activities in the deep (EDLd) and superficial (EDLs) regions of the extensor digitorum longus (EDL) muscle in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were determined after exposure to cigarette smoke at three different dose levels using a smoking machine. 2. Rats were exposed to cigarette smoke at a rate of 15 puffs/min for 20 min/day with 23 cigarettes (low-dose level), 26 cigarettes (medium-dose level) or 30 cigarettes (high-dose level) for 8 weeks. 3. No changes in fibre type distribution, CSA or SDH activity were observed after exposure to cigarette smoke at the low- and medium-dose levels, irrespective of the muscle region or strain. 4. No change in fibre type distribution was observed after exposure to cigarette smoke at the high-dose level, irrespective of the muscle region or strain. A decreased CSA of type IIA and type IIB fibres in EDLd and increased SDH activity of all types of fibres in EDLd and EDLs were observed in WKY rats after exposure to cigarette smoke at the high-dose level. In addition, a decreased CSA of type IIB fibres in EDLd and type IIA and type IIB fibres in EDLs and increased SDH activity of type IIB fibres in EDLd were observed in SHR after exposure to cigarette smoke at the high-dose level. 5. The smaller CSA and higher SDH activity of fibres in EDL observed in WKY rats and SHR after exposure to cigarette smoke at the high-dose level, but not at the low- and medium-dose levels, are adaptive responses, indicating that heavy cigarette smoke affects the morphological and metabolic properties of fibres in skeletal muscle.

To evaluate the relationship between nicotine and immunological inflammation, we investigated the effects of nicotine on plasma extravasation of the passive skin Arthus reaction, elicited 4 hr after sensitizing skin with antiserum, and serum corticosterone levels in rats. Pretreatment with a single subcutaneous injection of nicotine (0.4 or 0.8 mg/kg) 30 or 60 min. before antigen challenge attenuated the passive skin Arthus reaction immunological inflammation. Serum corticosterone levels were dose-dependently increased 30 and 60 min. after nicotine administration. Both markers co-varied with a similar dose-response and time course after the nicotine-treatment. In addition, we also examined these nicotine-induced responses after bilateral lesions of the hypothalamic paraventricular nucleus; both the nicotine-induced suppression of immunological inflammation and the increased serum corticosterone levels were attenuated in bilateral paraventricular nucleus-lesioned animals. Moreover, the immunological inflammatory decreasing effects of a single subcutaneous injection of nicotine (0.4 mg/kg) were antagonized by intraperitoneal preinjection with mecamylamine (1.0 mg/kg; blocking the brain nicotinic acetylcholine receptors) as well as by subcutaneous preinjection with mifepristone (30 mg/kg; a glucocorticoid receptor antagonist) but not by intraperitoneal preinjection with hexamethonium (2.0 mg/kg; a peripheral nicotinic acetylcholine receptors antagonist). Finally, intraperitoneal preinjection with cycloheximide (2 mg/kg), a protein synthesis inhibitor, abolished both the inhibitory effect of nicotine (0.4 mg/kg) on the dye leakage and the elevation of blood corticosterone levels. These findings indicate that the nicotine-induced decreasing effect on immunological inflammatory response may be related to serum corticosterone levels elevated by an activation of the paraventricular nucleus through the brain nicotinic acetylcholine receptors.

The effects of pretreatment with MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydropyridine) on the acute and long-term effects of methamphetamine on striatal dopamine were evaluated in BALB/c mice. Four subcutaneous injections of a non-toxic dose of MPTP (8 mg/kg, at 2 hr intervals) were followed three days later by a toxic regimen of methamphetamine (four injections of 4 mg/kg, at 2 hr intervals) and mice were sacrificed immediately or three days later. Control mice received saline in place of the MPTP or methamphetamine and mice were observed for acute changes in body temperature, self-injurious behaviour, and striatal dopamine metabolites, or long-term changes in striatal dopamine levels, tyrosine hydroxylase immunoreactivity and glial fibrillary acidic protein. It was observed that pretreatment with MPTP protected mice against the acute increase in body temperature caused by the methamphetamine but, at the same time, delayed the occurrence of self-injurious behaviour following the repeated injections of methamphetamine. Likewise, pretreatment with MPTP attenuated the long-term depletion of striatal dopamine induced by the methamphetamine as well as the large increase in glial fibrillary acidic protein and the reduction in tyrosine hydroxylase immunoreactivity. The MPTP-treatment itself did not alter any of these neurotoxic markers. Finally, the acute decrease in 3,4-dihydroxyphenyacetic acid levels and increased ratio of 3-methoxytyramine/dopamine observed 60 min. after a single injection of methamphetamine (4 mg/kg) were also attenuated in MPTP-treated mice. These results are discussed in the context of the hypothesis that the low-dose treatment with MPTP may modify exchange diffusion across the striatal cell membrane thereby altering the acute and long-lasting effects of methamphetamine.

The effects of intracisternal administration of endothelin-1 on blood-brain barrier permeability were examined in dogs and rats. Single doses of endothelin-1 elevated blood-brain barrier permeability about two times compared with control groups in both species. However, repeated dosing with endothelin-1 at a 24 hr intervals caused a highly enhanced disruption of blood-brain barrier permeability in dogs, but not in rats, whereas the repeated administration with a 48 hr interval markedly increased the blood-brain barrier permeability in both species of animals (dogs: 923%, rats: more than 661%). Moreover, this abnormally enhanced permeability of blood-brain barrier in dogs was completely blocked by pretreatment with the endothelin ET-A receptor selective antagonist, S-0139, administered prior to either the first or second dosing with endothelin-1. From these results, we conclude that a repeated attack of endothelin-1 from the adventitial site of brain blood vessels produces a severe disruption of blood-brain barrier permeability through an endothelin ET-A receptor-mediated process.

Fiber type distributions, cross-sectional areas (CSAs) and succinate dehydrogenase (SDH) activities in the soleus (SOL) muscle in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were determined after exposure to cigarette smoke at three different dosage levels using a smoking machine. The rats were exposed to cigarette smoke at a rate of 15 puffs/min, for 20 min/day with 23 cigarettes (low-dosage), 26 cigarettes (medium-dosage) or 30 cigarettes (high-dosage) for 8 weeks. There were no effects on the body weight, SOL weight, fiber type distribution, CSA or SDH activity of WKY after exposure to cigarette smoke, irrespective of the dosage level. In contrast, the body weights of SHR were lower after exposure to cigarette smoke, irrespective of the dosage level. The SOL weights of SHR were lower after exposure to cigarette smoke at the medium- and high-dosage levels. A lower percentage of type I fibers, a higher percentage of type IIA fibers and a smaller CSA of both type I and type IIA fibers were observed in SHR after exposure to cigarette smoke at the high-dosage level, but not at the low- and medium-dosage levels. These results indicate that heavy cigarette smoke causes changes in the enzyme histochemical properties with a reduced CSA of the SOL in SHR, but not in WKY.

The neurotoxic damage caused by methamphetamine (METH) is characterized by nerve terminal destruction and/or degeneration of the dopaminergic and serotonergic systems in striatum and hippocampus. It has been hypothesized that intraneural dopamine (DA) redistribution from synaptic vesicles to cytoplasmic compartments produced by METH is an important factor for its neurotoxicity. The METH-induced redistribution of DA is thought to occur after an increased production of DA-based reactive oxygen species (ROS) (e.g., oxygen radicals and hydroxyl radicals) by auto-oxidation or enzymatic degradation, and METH-induced ROS produces an oxidative stress and depletion of energy stores. Furthermore, the glutamatergic system and nitric oxide (NO) may also contribute to METH-induced neurotoxicity. Recently, studies using several knockout strains of mice lacking the DA transporter, the monoamine vesicle transporter-2, c-fos, or neuronal NO synthase confirm a possible role of these factors in METH-induced neurotoxicity. Moreover, it has been proposed that METH causes the apoptosis and activation of cell-death-related genes. For example, METH-induced neurotoxicity is reduced in bcl-2-over expressing neural cell and p53 knockout mice and also induces the activation of caspase 3. Therefore in this review, we discuss the relationship between ROS formation, oxidative stress, and apoptosis in METH-induced neurotoxicity.

We examined the effect of intracisternal application of endothelin-1 (ET-1) on the permeability of fluorescein into the cerebrospinal fluid (CSF) in beagle dogs in order to evaluate its role in disruption of blood-brain barrier (BBB) permeability seen in the subarachnoid hemorrhage animal model. Intracisternal application of their autologous blood for producing a canine two-hemorrhage model revealed an enhanced fluorescein permeability into the CSF together with the development of cerebral vasospasm. A single dose of ET-1 (40 pmol/animal) significantly increased penetration of fluorescein compared with that in normal dogs. Although its magnitude was much less than that in the two-hemorrhage model after the first administration of ET-1, the second challenge of the same dose of ET-1 with a 48-h interval produced marked disruption of BBB permeability similar to those in the animal model. Moreover, the ET-1-induced enhancement of fluorescein permeability into the CSF was completely prevented by intracisternal pretreatment with an endothelin ET(A)-receptor selective antagonist, S-0139 (0.03 mg/kg), as were the ET-1-induced cerebral vasoconstriction and behavioral changes as previously reported. Thus, we conclude that ET-1 acting on the adventitial site of brain in dogs contributes to the disruption of BBB permeability via endothelin ET(A)-receptor mediation.

1. 1. The authors investigated the influence of psychological stress (PSY-stress) on sensitization to nicotine (0.5 mg/Kg S.c.)-induced ambulatory stimulation. 2. 2. Rats were exposed to the emotional responses of animals which received foot-shock (FS), which they, themselves, did not receive. Ten daily exposures to PSY-stress for 20 min enhanced sensitization to the nicotine-induced ambulatory stimulation compared to that in non-stress rats. 3. 3. However, the increased serum corticosterone levels following nicotine (0.5 mg/Kg S.c.) administered 24 hr after the tenth injection of nicotine in the behavioral study was almost the same in the rats exposed to PSY-stress as compared to non-stress rats. 4. 4. These results suggest that PSY-stress may promote sensitization to nicotine-induced ambulatory stimulation and that the combined effect of PSY-stress and nicotine would facilitate the development of sensitization to nicotine.

Repeated administration of methamphetamine (METH; 2 and 4 mg/kg, s.c. four times every 2 h) caused hyperthermia and a dose-dependent depletion of striatal dopamine levels 3 days after the METH-treatment in both BALB/cAnNCrj (BALB) and DBA/2NCrj (DBA) mice, but these responses were lower in C57BL/6NCrj (C57BL) mice. An acute decrease of striatal dopamine levels 30 min after the last injection of METH (4 mg/kg) was observed in both BALB and DBA mice, while an increase in dopamine was observed in C57BL mice. Striatal 3-methoxytyramine levels were drastically increased in both DBA and C57BL mice after this same treatment. Moreover, pretreatment with the superoxide dismutase inhibitor, diethyldithiocarbamate (200 mg/kg, i.p.) exacerbated the METH (4 mg/kg)-induced striatal dopamine-depletion in BALB mice. In addition, pretreatment with an inhibitor of poly(ADP-ribose) polymerase, benzamide (160 mg/kg, s.c.), significantly attenuated the METH (4 mg/kg)-induced striatal dopamine depletion in both BALB and DBA mice. These results suggest that both BALB and DBA mice possess a higher sensitivity to the METH-induced striatal dopaminergic neurotoxicity compared to C57BL mice. In addition, the striatal dopaminergic neurons of BALB mice may be more vulnerable to METH-induced oxidative stress as compared to that in C57BL mice.

1. The authors investigated the ambulatory activity and water intake of rats during each 12 hr light and dark cycle for one week following four s.c. injections of 4 or 8 mg/kg of methamphetamine (METH). 2. Administration of the higher METH dose caused an increase in activity during the dark cycle on days 1 through 6 with the maximal increase on day 3 while the increase in activity during the light cycle was observed only on day 1. 3. Water intake increased the first day after administration of both METH doses, but returned to baseline by day 3. 4. Administration of both METH doses induced hyperthermia and the 8 mg/kg dose produced depletions of striatal dopamine and striatal, hippocampal and hypothalamic serotonin on day 3 but only in hippocampal serotonin by day 7. 5. These results demonstrate that high doses of METH produce a long-lasting increase in activity during the dark cycle and a transient increase in water intake. The behavioral changes which occurred during the dark cycle appear to be related to the depletion of central dopamine and/or serotonin.

We examined the prophylactic effect of a novel nonpeptide endothelin (ET) A-receptor selective antagonist, S-0139, using a canine two-hemorrhage model and an ET-1-induced cerebral vasospasm model. The agent markedly prevented cerebral vasospasm in the canine two-hemorrhage model when given intracisternally or intravenously by continuous daily dosing. An efficacious intravenous method was to apply a relatively high initial dose followed by daily sustaining administration at a much lower dose, which alone would have been ineffective. The need for sustaining dosing may imply daily successive attacks of ETs in the cerebral vessel compartment after the introduction of autologous blood into the subarachnoid space. A small amount of S-0139 was detected from the cerebrospinal fluid (CSF) with an apparent lag time after its disappearance from the plasma following intravenous dosing of 0.83 mg/kg/min for 12 min, however, cerebral vasoconstriction induced by ET-1 dosing from the adventitial side was clearly inhibited during such a lag period. Moreover, its movement into the CSF was greatly enhanced after the application of autologous blood to the animals. From these results, we conclude that ET-1 play a major role in producing delayed cerebral vasospasm in this canine two-hemorrhage model, and S-0139 effectively antagonizes the action of ET-1 even by intravenous treatment because it moves easily into the cerebral vessel compartment from plasma.

Calphostin C, a protein kinase C inhibitor, reduced phorbol ester-induced enhancement of catecholamine secretion but not primary Ca2+-induced secretion from digitonin-permeabilized bovine adrenal medullary cells, indicating that this compound selectively inhibited protein kinase C-dependent secretion.

Whether or not corticotropin-releasing factor (CRF) containing synaptic vesicles are located in axon terminals with nicotinic acetylcholine receptor (nAChR) in the median eminence (ME) of the rat was examined by electron microscopic double-labeling immunocytochemistry combining the pre-embedding avidin-biotin-peroxidase complex (ABC) method for nAChR with the post-embedding immunogold staining method for CRF. nAChR-like immunoreactivity (nAChR-LI) was found in the cell membranes of the axon terminals in the ME. CRF-like immunoreactivity (CRF-LI) was found in dense granular vesicles (about 100 nm in diameter) in the axon terminals. Double-labeling method revealed that some of nAChR-LI axon terminals were found to contain CRF-LI dense granular vesicles. The results indicate that nicotine may act on nAChR in axon terminals to release CRF.

Immunocytochemical localization of neuronal nicotinic acetylcholine receptor (nAChR) was examined in rat hypothalamus. Monoclonal antibody against alpha 4 ACh-binding subunits of nAChR was used in the avidin-biotin-peroxidase complex (ABC) immunocytochemical method at both the light and electron microscopic levels. By light microscopy nAChR-like immunoreactivity was found in many neuronal cell bodies and their fibers in the paraventricular nucleus (PVN) and in many axons and axon terminals in the median eminence (ME). The immunoreactivity of nAChR was the most intense in the ME. By electron microscopy immunoreaction products occurred on the rough endoplasmic reticulum, nuclear envelope, cytoplasmic matrices and postsynaptic densities of synaptic junctions in some neurons in the parvocellular part of the PVN. In the external layer of the ME, nAChR-like immunoreactivity was found over the entire plasma membranes of many axon terminals. Involvement of nAChRs in the release of neurotransmitters and neuropeptides both in the PVN and the ME is discussed.

The participation of protein kinases in phosphorylation of nicotinic acetylcholine receptor (nAChR) in electric organ and muscle has been precisely investigated in vitro and in vivo whereas phosphorylation of neuronal nAChR is not yet fully characterized. Here, we first report the in vitro phosphorylation of brain nAChR. nAChR purified from rat brains was phosphorylated in vitro by cAMP-dependent protein kinase (PKA), immunoprecipitated with monoclonal antibody against the receptor, and subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. PKA specifically phosphorylated nAChR on the α4 subunits, and H8, an inhibitor of PKA, inhibited completely the phosphorylation. Under the conditions used, a maximal stoichiometry of the phosphorylation by PKA was near to 1 mol of phosphate/mol of the α4 subunits. The 32 P-labeled subunits were digested with S. aureas V8 protease followed by SDS-PAGE autoradiography and the resultant phosphopeptide maps revealed three distinct phosphopeptide bands, one major band and two minor bands. Phosphoamino acid analysis of the 32 P-labeled α4 subunits showed that serine residues were exclusively phosphorylated. Based on these results, participation of PKA in the regulation of neuronal nAChR is discussed.

1. The effects of phorbol esters on canine Purkinje fibers were examined using conventional microelectrode techniques. 2. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (PDB), which are specific activators of protein kinase C (PKC), decreased the action potential amplitude and the maximum rate of depolarization (Vmax) at 3 x 10(-7) M or higher. These phorbol esters had little effect on the resting potential. 3. PDB (1-3 x 10(-7) M) also reduced the contractile force, accompanied with initial increase (in 5 out of 8 experiments), whereas TPA did not decrease it to any significant extent. 4. An inactive analog of phorbol esters, 4-alpha-phorbol-12,13-didecanoate (PDD), decreased the action potential amplitude and Vmax, and slightly increased the action potential duration. However, PDD failed to produce any inotropic effect. 5. Post-rest potentiation of the contractile force after a rest from stimulation for 30 sec was inhibited in the presence of 3-10 x 10(-7) M TPA or 3 x 10(-7) M PDB. 6. Isoproterenol 10(-7) M augmented the action of PDB 3 x 10(-7) M. 7. These results suggest that activation of PKC may modulate myocardial Ca2+ homeostasis and influence the excitation-contraction process.

Bilateral injections of nicotine (30 micrograms/side) into the ventral tegmental area (VTA) and the nucleus accumbens (NACC) increased the ambulatory activity in rats. Moreover, daily injections of nicotine (10, 20 and 30 micrograms/side) into the VTA and the NACC for 6 successive days produced sensitization to the ambulatory stimulant effect of nicotine. Sensitization produced by daily injections of nicotine (20 micrograms/side) into both the sites was maintained for withdrawal periods of 10 days. Mecamylamine (2 mg/kg, i.p.), SCH23390 (0.05 mg/kg, i.p.) and spiperone (0.1 mg/kg, i.p.) antagonized nicotine-induced sensitization to the ambulatory stimulant nicotine-induced sensitization to the ambulatory stimulant effect produced by daily injections into the VTA. These results suggest that nicotine-induced sensitization to the ambulatory stimulant effect involves the stimulation of the mesolimbic dopaminergic pathway through the nicotinic acetylcholine receptor (nAChR) in the VTA and the NACC.

Nicotinic acetylcholine receptor (nAChR) purified from rat brains by cholinergic ligand affinity chromatography was characterized. Monoclonal antibody 299, which binds an acetylcholine (ACh) binding subunit termed alpha 4, depleted more than 85% of [3H]ACh binding activity of the purified preparation. A number of cholinergic agonists strongly inhibited [3H]ACh binding to the purified nAChR, whereas potential antagonists were less effective than the agonists. These results show that most of the purified nAChR contains alpha 4 subunit and the pharmacological properties are preserved upon purification.