Synthesis, nicotinic acetylcholine receptor binding, antinociceptive and seizure properties of methyllycaconitine analogs

A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl-, and 2-cyclohexylsuccinimidobenzoyl (1b-f) group. The analogs 1b-f were evaluated for their inhibition of [(125)I]iodo-MLA binding at rat brain alpha7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b-f were evaluated for inhibition of binding to rat brain alpha,beta nAChR using [(3)H]epibatidine. At the alpha7 nAChR, MLA showed a K(i) value of 0.87 nM, analogs 1b-e possessed K(i) values of 1.67-2.16 nM, and 1f showed a K(i) value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (K(i)=1.67 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for alpha,beta nAChRs. MLA antagonized nicotine-induced seizures with an AD(50)=2 mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine's antinociceptive effects in the tail-flick assay. Compound 1c (K(i)=1.78 nM at alpha7 nAChR) with an AD(50) value of 1.8 mg/kg was 6.7 times more potent than MLA (AD(50)=12 mg/kg) in antagonizing nicotine's antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against beta-amyloid(1-42), these new analogs which have high alpha7 nAChR affinity and good selectivity relative to alpha,beta nAChRs will be useful biological tools for studying the effects of alpha7 nAChR antagonist and neuroprotection.     

Nicotine-induced phosphorylation of ERK in mouse primary cortical neurons: evidence for involvement of glutamatergic signaling and CaMKII

Extracellular signal-regulated kinase (ERK) is activated in vivo in a number of brain areas by nicotine and other drugs of abuse. Here we show that nicotine stimulation of cultured mouse cortical neurons leads to a robust induction of ERK phosphorylation that is dependent on nicotine concentration and duration of exposure. Calcium/calmodulin-dependent protein kinase II activity is necessary for nicotine-induced ERK phosphorylation and neither cAMP-dependent protein kinase or protein kinase C appear to be involved. Activity of glutamate receptors, L-type voltage-gated calcium channels, and voltage-gated sodium channels are also required for nicotine-induced ERK phosphorylation. Nicotine-induced ERK phosphorylation was inhibited by high concentrations of mecamylamine, however it was not blocked by other broad nicotinic acetylcholine receptor (nAChR) inhibitors (including hexamethonium and chlorisondamine) or nAChR subtype selective inhibitors (such as methyllycaconitine, alpha-bungarotoxin, dihydro-beta-erythroidine, and alpha-conotoxin Au1B). In accord with these pharmacological results, nicotine-induced ERK phosphorylation was normal in primary cultures made from beta2 or alpha7 nAChR subunit knockout mice. The alpha3/beta4 nAChR agonist cytisine did not induce ERK phosphorylation suggesting that alpha3/beta4 nAChRs were not involved in this process. Taken together, these data define a necessary role for glutamatergic signaling and calcium/calmodulin-dependent protein kinase II in nicotine-induced ERK phosphorylation in cortical neurons and do not provide evidence for the involvement of classical nAChRs.     

Expression of functional nicotinic acetylcholine receptors in neuroepithelial bodies of neonatal hamster lung

Pulmonary neuroepithelial bodies (NEB) are presumed airway chemoreceptors involved in respiratory control, especially in the neonate. Nicotine is known to affect both lung development and control of breathing. We report expression of functional nicotinic acetylcholine receptors (nAChR) in NEB cells of neonatal hamster lung using a combination of morphological and electrophysiological techniques. Nonisotopic in situ hybridization method was used to localize mRNA for the beta 2-subunit of nAChR in NEB cells. Double-label immunofluorescence confirmed expression of alpha 4-, alpha 7-, and beta 2-subunits of nAChR in NEB cells. The electrophysiological characteristics of nAChR in NEB cells were studied using the whole cell patch-clamp technique on fresh lung slices. Application of nicotine ( approximately 0.1-100 microM) evoked inward currents that were concentration dependent (EC50 = 3.8 microM; Hill coefficient = 1.1). ACh (100 microM) and nicotine (50 microM) produced two types of currents. In most NEB cells, nicotine-induced currents had a single desensitizing component that was blocked by mecamylamine (50 microM) and dihydro-beta-erythroidine (50 microM). In some NEB cells, nicotine-induced current had two components, with fast- and slow-desensitizing kinetics. The fast component was selectively blocked by methyllcaconitine (MLA, 10 nM), whereas both components were inhibited by mecamylamine. Choline (0.5 mM) also induced an inward current that was abolished by 10 nM MLA. These studies suggest that NEB cells in neonatal hamster lung express functional heteromeric alpha 3 beta 2, alpha 4 beta 2, and alpha 7 nAChR and that cholinergic mechanisms could modulate NEB chemoreceptor function under normal and pathological conditions.     

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.     

Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells

We have investigated mechanisms of nicotine-induced phosphorylation of extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 microM. The maximal level of nicotine-induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)-induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitors had no significant effect on nicotine-induced ERK phosphorylation. L-Type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. Calcium imaging experiments showed that alpha7-containing nAChR subtypes were functional at 1 microM of nicotine in the nicotine-induced calcium influx, and non-alpha7 nAChRs were prominent in the Ca(2+) influx at 50 microM of nicotine. An expression of dominant inhibitory Ras inhibited nicotine-induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine-induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non-alpha7 nAChRs are involved in nicotine-induced ERK phosphorylation through CaM kinase and the Ras-MAP kinase cascade and most of the nicotine-induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.     

The characterization of central neuromediation in rats fed with magnesium-deprived diet before and after magnesium replenishment

Magnesium (Mg) has been proposed to take part in biochemical dysregulation contributing to psychiatric disorders. The aims of this study was to estimate acute behavioural responses to clonidine (0.1 mg/kg i.p.), d-amphetamine (5 mg/kg, i.p), arecoline (15 mg/kg i.p), nicotine (6 mg/kg i.p.), apomorphine (1.5 mg/kg i.p.) and L-5-hydroxytryptophan (300 mg/kg i.p.) in rats fed with Mg-deprivated diet for 49 days and then treated with organic and inorganic Mg salts (50 mg Mg per kg) ether alone or in combination with pyridoxine (5 mg vitamin B6 per kg). In our study Mg-deficient rats were more sensitive to d-amphetamine-induced motor stereotypes compared with control rats; time of onset of the stereotypies insignificantly decreased by 14.89% and duration of the stereotypies significantly increased by 19.44% (320.36 +/- 19.90 vs. 268.23 +/- 8.17 minutes; p = 0.043). Mg deficiency did not modulate sensitivity to nicotine-induced seizure. The time between nicotine injection and emergence of clonic seizure (seizure latency) in the controls and Mg-deficient rats were 0.80 +/- 0.26 and 0.96 +/- 0.21 minutes respectively. Duration of the seizures in the controls and Mg-deficient rats were 64.93 +/- 7.20 and 79.32 +/- 8.13 minutes. In our study, Mg deficiency did not affect on clonidine- and apomorphine-induced hypothermia. Clonidine produced similar decreases in rectal temperature in controls and Mg-deficient group. In experiments using apomorphine, values of hypothermia were similar to those observed with clonidine. Mg deficiency antagonized 5-hydroxytryptophan-induced head-twitch response. The number of head twitches produced by 5-hydroxytryptophan was significantly (p = 0.49) decreased: twofold in magnesium-deficient rats (1.23 +/- 0.44 per minute) as compared with controls (2.42 +/- 0.52 per minute). Arecoline-induced tremor was comparably less expressed in Mg-deficient rats than in controls. The time between arecoline injection and time of onset of the tremor in the controls and Mg-deficient rats were 92.75 +/- 19.35 and 245.17 +/- 121.86 seconds respectively (p < or = 0.035). Duration of the tremors in the controls and Mg-deficient rats were 1175.58 +/- 127.87 and 703.83 +/- 89.33 seconds (p = 0.015). Magnesium salts (Mg chloride, Mg L-aspartate alone and in combination with B6) were administered through gastric tube during twenty days up to complete compensation oferythrocyte and plasma Mg levels in all experimental groups. In our study administration of Mg salts resulted in normalization of acute behavioural responses in Mg-deficient rats to d-amphetamine, arecoline, and L-5-hydroxytryptophan. Behavioural responses in rats treated with both Mg chloride and Mg L-aspartate in combinations with B6 were comparable with those observed in MagneB6 treatment.     

Serotonergic and dopaminergic involvement in the mechanism of action of R-(−)-2, 5-dimethoxy-4-bromoamphetamine (DOB) in cats

Intraperitoneally administered R-(?)- and S-(+)- enantiomers of 2,5-dimethoxy-4-bromoamphetamine were evaluated for their ability to induce head-body shake, limb flick and abortive grooming behaviors in cats. The R-(?)-enantiomer was consistently more effective than the S-(+)-isomer in all three behavioral measures. Dose-response relationships were evident for head-body shakes and limb flicks for both enantiomers, but reliable abortive grooming responses appeared only after the higher doses of R-(?)-DOB. Cinanserin and methysergide pretreatments effectively antagonized the induction of head-body shakes and limb flicks by 0.1 mg/kg R-(?)-DOB. In addition, haloperidol pretreatment significantly antagonized the appearance of these behaviors suggesting that dopaminergic as well as serotonergic stimulation is involved in the elicitation of these cat behaviors by R-(?)-DOB.     

Involvement of DDAH/ADMA/NOS pathway in nicotine-induced endothelial dysfunction

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is a key contributor for endothelial dysfunction. Decrease in activity of dimethylarginine dimethylaminohydrolase (DDAH), a major hydrolase of ADMA, causes accumulation of ADMA under cardiovascular abnormalities. The study was to determine whether nicotine-induced endothelial dysfunction is related to modulating DDAH/ADMA/NOS pathway. Four-week oral nicotine treatment (5 mg/kg/day) significantly increased the plasma level of ADMA and decreased aortic DDAH expression as well as impaired endothelial function in Sprague-Dawley rats. Similarly, the medium levels of both ADMA and lactate dehydrogenase were markedly elevated in umbilical vein endothelial cells (HUVECs) treated with nicotine (10 microM) for 48 h. Nicotine-induced endothelial damages were markedly attenuated by L-arginine or overexpression of DDAH-II. Nicotine greatly downregulated both mRNA and protein levels of DDAH-II, and decreased DDAH activity in HUVECs. HUVECs express alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), whose antagonists could block these effects of nicotine mentioned above. Intracellular Ca2+ chelator did not affect nicotine-induced decrease in DDAH-II mRNA level. In conclusion, nicotine modulates DDAH/ADMA/NOS pathway of endothelial cell via activation of alpha7 nAChR, which may be involved in endothelial dysfunction associated to smoking.     

alpha7 nicotinic receptor gene promoter polymorphisms in inbred mice affect expression in a cell type-specific fashion

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.     

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.     

+]-Huperzine A treatment protects against N-methyl-D-aspartate-induced seizure/status epilepticus in rats

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood-brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [-]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [-]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20mug/kg NMDA (intracerebroventricular) 20-30min later. For post-exposure, rats were treated with [+]-Hup A (3mg/kg, intramuscularly) 1min after NMDA (20mug/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [-]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.     

Effect of adrenergic antagonists and cyclooxygenase inhibitors on the nicotine-induced hypothalamic-pituitary-adrenocortical activity.

Nicotine is a potent stimulus for the hypothalamic-pituitary-adrenal (HPA) axis. Systemic nicotine acts via central mechanisms to stimulate by multiple pathways the release of ACTH from the anterior pituitary corticotrops and corticosterone from the adrenal cortex. Nicotine may stimulate indirectly the hypothalamic paraventricular nucleus, the site of the corticotropin-releasing hormone (CRH) neurons which activates ACTH release. In the present studies an involvement of adrenergic system and prostaglandins synthesized by constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) in the nicotine-induced HPA response in rats was investigated. Nicotine (2.5-5 mg/kg i.p.) significantly increased plasma ACTH and corticosterone levels measured 1 hr after administration. Adrenergic receptor antagonists or COX inhibitors were injected i.p. 15 min prior to nicotine and the rats were decapitated 1 hr after the last injection. Prazosin (0.01-0.1 mg/kg), an alpha1-adrenergic antagonist, significantly decreased the nicotine-evoked ACTH and corticosterone secretion. Yohimbine (0.1-1.0 mg/kg), an alpha2-adrenergic antagonist, moderately diminished ACTH response, and propranolol (0.1-10 mg/kg), a beta-adrenergic antagonist, did not significantly alter the nicotine-induced hormones secretion. Pretreatment with piroxicam (0.2-2.0 mg/kg), a COX-1 inhibitor, considerably impaired the nicotine-induced ACTH and corticosterone secretion. Compound NS-398 (0.2-5.0 mg/kg), a selective COX-2 blocker did not markedly alter these hormones secretion, and indomethacin (2 mg/kg), a non-selective COX inhibitor significantly diminished ACTH response. These results indicate that systemic nicotine stimulates the HPA axis indirectly, and both adrenergic system and prostaglandins are significantly involved in this stimulation. Noradrenaline, stimulating postsynaptic alpha1-adrenergic receptors, and prostaglandins, synthesized by COX-1 isoenzyme, are of crucial significance in the nicotine-induced ACTH and corticosterone secretion.     

Diverse effects of variant doses of dexamethasone in lithium-pilocarpine induced seizures in rats

Corticosteroids are used in the management of several epileptic aliments; however, their effectiveness in combating seizures remains controversial, with pro- and anti-convulsive effects ascribed. The current study aimed to address the modulatory effect of dexamethasone (DEX) utilizing 3 dose levels (5, 10, and 20 mg/kg body mass of male Wistar rat) in the rat lithium-pilocarpine (Li-PIL) epilepsy model. Li-PIL induced seizures that were associated with neuronal cell loss in the CA3 region, and increased prostaglandin (PG)E(2), tumor necrosis factor (TNF)-α, interleukin (IL)-10, nitric oxide, and neutrophil infiltration in the hippocampus. However, Li-PIL compromised the oxidant-antioxidant balance of the hippocampus. Effective anticonvulsant activity was only observed with 10 mg DEX/kg body mass, which reduced seizure production and incidence, as well as neuronal cell loss in the CA3 region. At this anticonvulsant dose, enhancements in the antioxidant system and IL-10, as well as suppression of altered inflammatory markers were observed. Conversely, doubling the dose showed a tendency to shorten seizure latency, and neither affected seizure incidence nor CA3 neuronal cell loss. These effects were associated with an increase in levels of PGE(2) and TNF-α. The present study found a lack of protection at 5 mg DEX/kg body mass, an anticonvulsant effect at 10 mg/kg, and a loss of protection at 20 mg/kg in the Li-PIL epilepsy model, which indicates that there is an optimal dose of DEX for preventing the induction of seizures.     

Ontogeny of susceptibility to the convulsant, Ro 5-4864, and its relationship to audiogenic seizure susceptibility in inbred mice

The postnatal development of susceptibility to the convulsant effects of Ro5-4864 (4'-chlorodiazepam) was characterized in two inbred mouse strains (DBA/2J and BALB/c ByJ) which as adults differ markedly in their response to this convulsant. Onset of susceptibility to a dose of Ro5-4864 which caused a high frequency of clonic seizures in adults was observed at 10 days of age in DBA/2 mice, but not until 35 days in BALB/c By mice. At 14 days of age an abrupt increase in susceptibility to Ro5-4864-induced tonic seizures was found in DBA/2 but not BALB/c By mice. Both the peak of tonic seizure susceptibility (21 days) and the time course of its subsequent age-dependent decline closely paralleled the change in audiogenic seizure susceptibility in the DBA/2 strain. PK11195 (40 mg/kg) blocked Ro5-4864 (25 mg/kg)-induced, age-dependent tonic seizures but had no effect on clonic seizure induction in the same mice. These observations establish that both the susceptibility to Ro5-4864 in adult mice and the postnatal time course for development of susceptibility to this convulsant are inherently different in these two strains of mice. The lack of coincidence between the developmental onset of susceptibility to Ro5-4864-induced seizures and the onset of supersensitivity to Ro5-4864-induced tonic seizures during the period of peak audiogenic seizure susceptibility in DBA/2 mice implies that more than one neurochemical mechanism is involved in the ability of Ro5-4864 to induce seizures in this strain. However, the blockade of Ro5-4864-induced tonic seizures by PK11195 suggests that peripheral type benzodiazepine receptors may mediate this effect.     

Nicotine increases the expression of high affinity nerve growth factor receptors in both in vitro and in vivo

Impairment in nerve growth factor (NGF)-mediated support to basal forebrain cholinergic neurons may represent an initial insult to certain neural cells in Alzheimer's disease (AD). High affinity NGF receptor (TrkA) levels are decreased in AD brains as compared to age-matched control brains. One of the approaches suggested for the treatment of AD exploits the ability of small molecular substances to enhance the expression of endogenous growth factors and/or their receptors. The purpose of this study was to determine whether treatment with nicotine in both in vitro and in vivo settings would increase the neural expression of TrkA receptors. Using a differentiated PC12 neuronal-like system, chronic nicotine treatment increased cell surface TrkA receptor expression. Nicotine's action was blocked by co-treatment with either the non-competitive nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine or with the alpha7 nAChR-selective antagonist methyllycaconitine. Surprisingly, certain low doses of mecamylamine alone also increased TrkA receptor levels. Rats prepared with chronic indwelling intravenous catheters were continuously infused with nicotine to deliver a total dose of 12 mg/kg over 24 hr. This treatment resulted in a significant 44% increase in TrkA receptor expression in the hippocampus. As in the cell experiments, mecamylamine also increased hippocampal TrkA receptor expression. In fact, the ratio of the maximal mecamylamine response to the maximal nicotine response that was measured in vitro, i.e., 0.43 was remarkably similar to that for the in vivo experiment, i.e., 0.47. Since in our previous studies the increase in TrkA expression produced by nicotine was shown to be related to its cytoprotective actions, these results suggest that nicotine's neuroprotective actions might also be mediated through the drug's interaction with central alpha7 nAChRs and subsequent increase in TrkA receptor expression.     

Stereochemistry of the metabolism of MDMA to MDA

The chiral derivatizing reagent N-trifluoroacetyl-L-prolyl chloride (LTPC) was used to form diastereomers of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) which were resolved on an achiral gas chromatographic column using a mass spectrometer as a detector. Rats were subcutaneously dosed with 40 mg/kg of (+/-) MDMA.HCl and blood was obtained by decapitation four hours after dosing. Plasma was separated and extracted. The extract was derivatized on-column with LTPC. In addition to the two MDMA isomers, the demethylated metabolites, S(+) and R(-)-MDA were identified. In all experimental groups (male rats, food deprived male rats, female rats, post partum female rats, and mice) dosed with racemic MDMA, higher levels of the S(+) isomer of MDA relative to the R(-) MDA isomer were observed. This may be significant since it has been shown that the S(+) isomer of MDMA is the more neurotoxic isomer of the racemic drug of abuse MDMA.     

Nicotine-induced sensitization to ambulatory stimulant effect produced by daily administration into the ventral tegmental area and the nucleus accumbens in rats.

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.     

Effects of L-arginine on prevention and treatment of lithium-pilocarpine-induced status epilepticus

The effects of various doses of L-arginine, a nitric oxide substrate, on lithium-pilocarpine-induced seizures were studied in rats. Rats were implanted with chronic, stainless steel screw electrodes epidurally for electrocortical recordings. A control group received 3 mEq/kg LiCl (i.p.) and 24 h later 45 mg/kg pilocarpine HCl (i.p.). Two different experimental procedures were followed: (1) L-arginine was applied in doses of 100 mg/kg, 300 mg/kg or 500 mg/kg (i.p.), 30 min before pilocarpine injection; (2) 300 mg/kg, 500 mg/kg or 1000 mg/kg (i.p.) L-arginine was injected either 5 min or 30 min after the onset of status epilepticus (SE). L-arginine (300 mg/kg) injected 30 min before pilocarpine significantly reduced the percentage of SE, but did not change the latency to SE or 24-hour survival. These parameters were not significantly affected by the 100 mg/kg or 500 mg/kg dose of L-arginine. On the other hand, no dose of L-arginine that was applied after SE had begun, had any significant influence on the seizures. We concluded that L-arginine may prevent seizure activity in some but not all doses, and does not have any effect on the ongoing seizure activity.     

Mechanisms mediating canine renal vasoconstriction induced by nicotine infusion

We investigated the respective contributions of the renin-angiotensin and alpha-adrenergic systems to nicotine-induced, canine, renal vasoconstriction by using saralasin (4 micrograms/kg/min) and phentolamine (25 micrograms/kg/min) blockade respectively. Nicotine infusion (0.024 mg/kg/min) increased mean arterial blood pressure (MABP) (114 +/- 3.0 to 219 +/- 8.0 mmHg) and decreased total renal blood flow (TRBF) (3.12 +/- 0.34 to 1.60 +/- 0.37 ml/min/g). Nicotine infusion produced a significantly lesser blood flow in outer cortex (OC), inner cortex (IC), and outer medulla (OM) compared to control dogs. The intrarenal-artery infusion of saralasin or phentolamine had no effect on the nicotine-induced MABP changes. Phentolamine infusion prior to nicotine resulted in a significantly greater TRBF (P less than 0.01), OC (p less than 0.001), IC (p less than 0.001) and OM (p less than 0.01) flow than in the group that received nicotine only. Saralasin pretreatment prior to nicotine resulted only in a significantly (p less than 0.01) greater OC flow than nicotine only. Our data suggest that while angiotensin II mediates a portion of the action of nicotine on the OC renal vasculature, the alpha adrenergic system predominates as the mediator of nicotine-induced renal vasoconstriction in the first 7 minutes of nicotine infusion.