Anti-hyperalgesic and anti-inflammatory effects of 4R-tobacco cembranoid in a mouse model of inflammatory pain.

4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund's Adjuvant (CFA) into the hind paw of male and female mice. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and von Frey tests, respectively, before and at different time points (2.5 h - 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.

Memantine has a nicotinic neuroprotective pathway in acute hippocampal slices after an NMDA insult.

Memantine is a non-competitive antagonist with a moderate affinity to the N-methyl-d-Aspartate (NMDA) receptor. The present study assessed memantine's neuroprotective activity using electrophysiology of ex-vivo hippocampal slices. Interestingly, a nicotinic component was necessary for memantine's neuroprotection (NP). Memantine demonstrated a bell-shaped dose-response curve of NP against NMDA. Memantine was neuroprotective at concentrations below 3 µM, but the NP declined at higher concentrations (>3 µM) when memantine inhibits the NMDA receptor. Additional evidence that memantine NP is mediated by an alternate mechanism independent of the inhibition of the NMDA receptor is supported by its ability to protect neurons when applied before or after the NMDA insult and in the presence of D(-)-2-Amino-5-phosphonopentanoic acid (APV), the standard NMDA receptor inhibitor. We found several similarities between the memantine NP mechanism and the neuroprotective nicotinic drug, the 4R cembranoid. Memantine's NP requires the release of acetylcholine, the activation of α4ß2, and is independent of MEK/MAPK signaling. Both 4R and memantine require the activation of PI3K/AKT for NP against NMDA-mediated excitotoxicity, although at different concentrations. In conclusion, our studies show memantine is neuroprotective through a nicotinic pathway, similar to the nicotinic drug 4R. This information leads to a better understanding of memantine's mechanisms of action and explains its dose-dependent effectiveness in Alzheimer's and other neurological disorders.

4R-Cembranoid Improves Outcomes after 6-Hydroxydopamine Challenge in Both In vitro and In vivo Models of Parkinson's Disease.

(1S, 2E, 4R, 6R,-7E, 11E)-2, 7, 11-cembratriene-4, 6-diol (4R) is one of the cembranoids found in tobacco leaves. Previous studies have found that 4R protected acute rat hippocampal slices against neurotoxicity induced by N-methyl-D-aspartate (NMDA) and against the toxic organophosphorus compounds paraoxon and diisopropylfluorophosphate (DFP). Furthermore, in vivo, 4R reduced the infarct size in a rodent ischemic stroke model and neurodegeneration caused by DFP. The present study expanded our previous study by focusing on the effect of 4R in Parkinson's disease (PD) and elucidating its underlying mechanisms using 6-hydroxydopamine (6-OHDA)-induced injury models. We found that 4R exhibited significant neuroprotective activity in the rat unilateral 6-OHDA-induced PD model in vivo. The therapeutic effect was evident both at morphological and behavioral levels. 4R (6 and 12 mg/kg) treatments significantly improved outcomes of 6-OHDA-induced PD in vivo as indicated by reducing forelimb asymmetry scores and corner test scores 4 weeks after injection of 6-OHDA (p < 0.05). The therapeutic effect of 4R was also reflected by decreased depletion of tyrosine hydroxylase (TH) in the striatum and substantia nigra (SN) on the side injected with 6-OHDA. TH expression was 70.3 and 62.8% of the contralateral side in striatum and SN, respectively, after 6 mg/kg 4R treatment; furthermore, it was 80.1 and 79.3% after treatment with 12 mg/kg of 4R. In the control group, it was 51.9 and 23.6% of the contralateral striatum and SN (p < 0.05). Moreover, 4R also protected differentiated neuro-2a cells from 6-OHDA-induced cytotoxicity in vitro. The activation of p-AKT and HAX-1, and inhibition of caspase-3 and endothelial inflammation, were involved in 4R-mediated protection against 6-OHDA-induced injury. In conclusion, the present study indicates that 4R shows a therapeutic effect in the rat 6-OHDA-induced PD model in vivo and in 6-OHDA-challenged neuro-2a cells in vitro.

Pharmacokinetics and Metabolism of 4R-Cembranoid.

4R-cembranoid (4R) is a natural cyclic diterpenoid found in tobacco leaves that displays neuroprotective activity. 4R protects against NMDA, paraoxon (POX), and diisopropylfluorophosphate (DFP) damage in rat hippocampal slices and against DFP in rats in vivo. The purpose of this study was to examine the metabolism and pharmacokinetics of 4R as part of its preclinical development as a neuroprotective drug. 10 µM 4R was found to be very stable in plasma for up to 1 hr incubation. 4R metabolism in human microsomes was faster than in the rat. Ten metabolites of 4R were detected in the microsomal samples; 6 dihydroxy and 4 monohydroxy forms of 4R. Male rats received a single dose of 4R at 6 mg/kg i.v., i.m., or s.c. The i.v. group had the highest plasma concentration of 1017 ng/mL. The t1/2 was 36 min and reached the brain within 10 min. The brain peak concentration was 6516 ng/g. The peak plasma concentration in the i.m. group was 163 ng/mL compared to 138 ng/mL in the s.c. group. The t1/2 of 4R after i.m. and s.c. administration was approximately 1.5 hr. The brain peak concentration was 329 ng/g in the i.m. group and 323 ng/g for the s.c. group. The brain to plasma ratio in the i.v. group was 6.4, reached 10 min after dose, whereas in the i.m. and s.c. groups was 2.49 and 2.48, respectively, at 90 min after dose. Our data show that 4R crosses the BBB and concentrates in the brain where it exerts its neuroprotective effect.

Actions of octocoral and tobacco cembranoids on nicotinic receptors.

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies.

Tobacco cembranoids protect the function of acute hippocampal slices against NMDA by a mechanism mediated by alpha4beta2 nicotinic receptors.

Nicotine has been reported to be neuroprotective in experimental and epidemiological studies. In addition to nicotine, tobacco and cigarette smoke contain cembranoids, which are antagonists of neuronal nicotinic receptors (nAChR). Exposure of hippocampal slices to N-methyl-D-aspartate (NMDA) decreases the population spikes (PS). This parameter has been used as a measure of excitotoxicity. Surprisingly, both nicotine and tobacco cembranoids protected against NMDA and this neuroprotection was not blocked by methyllycaconitine (MLA), an antagonist of alpha7 nAChR. On the contrary, MLA had a neuroprotective effect of its own. We examined the effect of the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) on the neuroprotection against NMDA. DHbetaE, a selective antagonist of alpha4beta2 nAChR, inhibited the neuroprotection by nicotine, 4R, and MLA, suggesting the involvement of alpha4beta2 nAChRs in the neuroprotection. The cell-signaling pathways underlying the neuroprotection by 4R and by nicotine are different. The activity of phosphatidylinositol-3 kinase (PI3K) was required in both cases; however, 4R required the activity of L-type calcium channels and CAM kinase, whereas nicotine required the extracellular signal regulated kinase-1,2 (ERK) and protein kinase C (PKC). In addition, 4R did not enhance total phospho-ERK-1/2 but increased the amount of total Akt/PKB phosphorylated on the activation site and of glycogen synthase kinase 3-beta phosphorylated on the inhibitory site. Total levels of phosphoenzymes are presented instead of the ratio of phospho- over total enzyme because in preliminary experiments total ERK-1/2 levels were slightly increased by 4R. In conclusion, these findings demonstrate that there are two different nicotinic neuroprotective mechanisms mediated by alpha4beta2.

Nicotinic receptors differentially regulate N-methyl-D-aspartate damage in acute hippocampal slices.

Although in neuronal cultures nicotine was reported to prevent early and delayed excitotoxic death, no studies with nicotinic drugs have been done with acute hippocampal slices. We investigated the effect of nicotine and methyllycaconitine (MLA) on the toxicity of N-methyl-d-aspartate (NMDA) in the CA1 area of hippocampal slices. The excitotoxic effect of NMDA was assessed as decreased recovery of the capability to produce synaptically evoked population spikes (PSs). Application of nicotine or MLA before NMDA application increased the recovery of PSs. This electrophysiological recovery was used as a measure of the early neuroprotective events. The neuroprotection conferred by both nicotine and MLA was inhibited by dihydro-beta-erythroidine, showing mediation of neuroprotection by alpha 4 beta 2 neuronal nicotinic receptors (nAChRs). Because nicotine activates alpha 4 beta 2 and other nAChR subtypes, whereas 10 nM MLA inhibits the alpha 7 subtype, we propose the involvement of a neuronal circuitry-dependent mechanism for nicotinic neuroprotection. The effect of nicotine downstream from the receptors was investigated using inhibitors of cell signaling. The results suggest that the effect of nicotine is mediated by tyrosine receptor kinases, 1,2-phosphatidylinositol-3 kinase, and the mitogen-activated extracellular signal-regulated kinases. Although nicotine neuroprotection is Ca2+-dependent, neither L-type Ca2+ channels nor calmodulin-dependent protein kinase is involved in the effect of nicotine. In summary, these results suggest that in acute slices nicotinic protection is initiated either by direct activation of alpha 4 beta 2 or indirectly by inhibition of alpha 7 followed by signal transduction involving tyrosine kinases, phospholipid-dependent kinases, and mitogen-activated kinases.

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.

Spermine does not compete with omega-conotoxin GVIA in the striatum radiatum of the hippocampal slice.

The effect of spermine (Spm) and of omega-conotoxin GVIA (CTX) on the population excitatory postsynaptic potentials (pEPSP) in stratum radiatum of the CA1 area were compared. CTX decreased irreversibly the initial slope of pEPSP by 57%. Spm produced a maximum inhibition of 85% with an apparent dissociation constant of 0.85 mM and a maximum Hill coefficient larger than 3. The effect of Spm was mostly reversible. Preincubation with Spm did not protect the slice from the irreversible effect of CTX suggesting that they interact with different sites. Since CTX and Spm inhibited pEPSPs with very different affinities and reversibilities a kinetic model was developed to compare their effects. This model relates the inhibitors' binding to presynaptic voltage-activated Ca2+ channels (VACC) with inhibition of pEPSP. The model suggest that: all CTX and Spm effects can be explained by inhibition of VACC. Spm and CTX do not compete for the same site. CTX inhibits 20% (N-type) and Spm 40% of channels (probably the Q-type). More than three Spm molecules bind per one channel molecule, while one CTX is sufficient to inhibit channel function. The model also illustrates that the inhibitor concentration-pEPSP inhibition curves display a Hill coefficient similar to that for inhibitor binding.

Interaction with objects by group-living Saimiri sciureus

Se ha estudiado la conducta de un grupo de Saimiri sciureus en presencia de objetos novedosos, no comestibles. El grupo de 21 animales, de ambos sexos y varias edades, vivía en un corral cubierto ubicado al aire libre. Diez objetos de diversas formas, colores, materiales y tamaños fueron introducidos en el corral y el número de monos que interactuaban con cada objeto se registró usando una técnica de muestreo. También se hicieron observaciones cualitativas sobre el tipo de interacción. Los mismos objetos se volvieron a presentar al día siguiente, repetiendo las determinaciones. Este experimento, con algunas variantes se repitió tres veces durante un período de seis meses. Durante el primer día, la interacción con los objetos fue máxima inmediatamente después de la presentación de los mismos y caía abruptamente por el resto del día. Al segundo día, la habitación era menos notoria y el puntaje total fue similar en ambos días. Las características que influyeron en la cantidad de interacción fueron el material de los objetos y probablemente la complejidad estructural de los mismos. Ni el color ni el peso tuvieron influencia estadísticamente significativa. Aquellos objetos que fueron tocados más frecuentemente despertaban una intensa actividad investigativa y vigoroso juego social. Estos resultados difieren de algunos estudios anteriores que reportaron falta de interês por objetos no comestibles en Saimiris