Differentiating the Neuropharmacological Properties of Nicotinic Acetylcholine Receptor-Activating Alkaloids.

Alkaloids that target nicotinic acetylcholine receptors (nAChR) are of great interest because of the critical role they play in mood and anxiety. However, understanding of the neuropharmacological effects of nicotinic alkaloids, such as cotinine and anatabine, is very limited. In this study, we investigated the neuropharmacological effects of three naturally occurring alkaloids-nicotine, cotinine, and anatabine-in vitro and in vivo. A single injection of nicotine induced anxiolytic-like behavioral features in mice by using the SmartCube® behavioral profiling system, while cotinine and anatabine had no detectable effect. The results were corroborated by using the zebrafish novel tank test (NTT), which showed a profound anxiolytic-like effect induced by multiple doses of nicotine after a single 20-min treatment. When the regulation of dopamine and norepinephrine release-the neurotransmitter systems relevant for anxiety-were examined in vitro, we found that nicotine stimulated the release of both norepinephrine and dopamine, while cotinine and anatabine mainly stimulated the dopamine release. The molecular targets of nicotine were confirmed to be nAChRs with its most potent activities against α4ß2 and α6/3ß2ß3 subtypes in vitro. Anatabine was a weaker agonist for these receptors than nicotine. Cotinine was the least potent nAChR compound, only being able to activate α4ß2 and α6/3ß2ß3 subtypes at high doses and no detectable activities against α3ß4 and α7 subtypes at the concentrations tested. The observed effects were unlikely due to the off-target effect, because these alkaloids did not bind or regulate >160 other molecular targets in vitro. Thus, the present results suggest that natural nicotinic alkaloids can induce an anxiolytic-like behavior in nonclinical animal models, potency of which may depend on the activation of various nAChRs and regulation of various neurotransmitter systems. Further investigations would help understand their effects on humans, because non-clinical studies should not be taken as a direct indication for human behavior and nicotine is not risk free.

Synthesis and In Vitro and In Vivo Evaluation of [3H]LRRK2-IN-1 as a Novel Radioligand for LRRK2.

PURPOSE: LRRK2 (leucine-rich repeat kinase 2) has recently been proven to be a promising drug target for Parkinson's disease (PD) due to an apparent enhanced activity caused by mutations associated with familial PD. To date, there have been no reports in which a LRRK2 inhibitor has been radiolabeled and used for in in vitro or in vivo studies of LRRK2. In the present study, we radiolabeled the LRRK2 ligand, LRRK-IN-1, for the purposes of performing in vitro (IC50, K d , B max, autoradiography) and in vivo (biodistribution, and blocking experiments) evaluations in rodents and human striatum tissues. PROCEDURES: [3H]LRRK2-IN-1 was prepared with high radiochemical purity (>99 %) and a specific activity of 41 Ci/mmol via tritium/hydrogen (T/H) exchange using Crabtree's catalyst. For IC50, K d , and B max determination, LRRK2-IN-1 was used as a competing drug for nonspecific binding assessment. The specific binding of the tracer was further evaluated via an in vivo blocking study in mice with a potent LRRK2 inhibitor, Pf-06447475. RESULTS: In vitro binding studies demonstrated a saturable binding site for [3H]LRRK2-IN-1 in rat kidney, rat brain striatum and human brain striatum with K d of 26 ± 3 and 43 ± 8, 48 ± 2 nM, respectively. In rat, the density of LRRK2 binding sites (B max) was higher in kidney (6.4 ± 0.04 pmol/mg) than in brain (2.5 ± 0.03 pmol/mg), however, in human brain striatum, the B max was 0.73 ± 0.01 pmol/mg protein. Autoradiography imaging in striatum of rat and human brain tissues gave results consistent with binding studies. In in vivo biodistribution and blocking studies in mice, co-administration with Pf-06447475 (10 mg/kg) reduced the uptake of [3H]LRRK2-IN-1 (%ID/g) by 50-60% in the kidney or brain. CONCLUSION: The high LRRK2 brain density observed in our study suggests the feasibility for positron emission tomography imaging of LRRK2 (a potential target) with radioligands of higher affinity and specificity.

Arabidopsis AZI1 family proteins mediate signal mobilization for systemic defence priming.

Priming is a major mechanism behind the immunological 'memory' observed during two key plant systemic defences: systemic acquired resistance (SAR) and induced systemic resistance (ISR). Lipid-derived azelaic acid (AZA) is a mobile priming signal. Here, we show that the lipid transfer protein (LTP)-like AZI1 and its closest paralog EARLI1 are necessary for SAR, ISR and the systemic movement and uptake of AZA in Arabidopsis. Imaging and fractionation studies indicate that AZI1 and EARLI1 localize to expected places for lipid exchange/movement to occur. These are the ER/plasmodesmata, chloroplast outer envelopes and membrane contact sites between them. Furthermore, these LTP-like proteins form complexes and act at the site of SAR establishment. The plastid targeting of AZI1 and AZI1 paralogs occurs through a mechanism that may enable/facilitate their roles in signal mobilization.

Agrobacterium†T-DNA-encoded protein Atu6002 interferes with the host auxin response.

Several genes in the Agrobacterium tumefaciens transferred (T)-DNA encode proteins that are involved in developmental alterations, leading to the formation of tumours in infected plants. We investigated the role of the protein encoded by the Atu6002 gene, the function of which is completely unknown. Atu6002 expression occurs in Agrobacterium-induced tumours, and is also activated on activation of plant cell division by growth hormones. Within the expressing plant cells, the Atu6002 protein is targeted to the plasma membrane. Interestingly, constitutive ectopic expression of Atu6002 in transgenic tobacco plants leads to a severe developmental phenotype characterized by stunted growth, shorter internodes, lanceolate leaves, increased branching and modified flower morphology. These Atu6002-expressing plants also display impaired response to auxin. However, auxin cellular uptake and polar transport are not significantly inhibited in these plants, suggesting that Atu6002 interferes with auxin perception or signalling pathways.

A feedback regulatory loop between G3P and lipid transfer proteins DIR1 and AZI1 mediates azelaic-acid-induced systemic immunity.

Systemic acquired resistance (SAR), a highly desirable form of plant defense, provides broad-spectrum immunity against diverse pathogens. The recent identification of seemingly unrelated chemical inducers of SAR warrants an investigation of their mutual interrelationships. We show that SAR induced by the dicarboxylic acid azelaic acid (AA) requires the phosphorylated sugar derivative glycerol-3-phosphate (G3P). Pathogen inoculation induced the release of free unsaturated fatty acids (FAs) and thereby triggered AA accumulation, because these FAs serve as precursors for AA. AA accumulation in turn increased the levels of G3P, which is required for AA-conferred SAR. The lipid transfer proteins DIR1 and AZI1, both of which are required for G3P- and AA-induced SAR, were essential for G3P accumulation. Conversely, reduced G3P resulted in decreased AZI1 and DIR1 transcription. Our results demonstrate that an intricate feedback regulatory loop among G3P, DIR1, and AZI1 regulates SAR and that AA functions upstream of G3P in this pathway.

Polymer-supported organotin reagent for prosthetic group labeling of biological macromolecules with radioiodine.

In this study, we investigated the use of poly-mer-bound precursor for generating a radiolabeled prosthetic group to be used for conjugate labeling of biological macromolecules. For the approach, a trialkyltin chloride in which the tin was bound to a hydrophilic PEG-based resin support via one of the alkyl groups was synthesized. This resin was then used to prepare a resin-bound trialkyltin benzoic acid, which in some cases was further derivatized on-resin by converting it to a succinimidyl ester. Exposure of the resin-bound compounds to electrophilic radioiodine (¹²5I) in either an aqueous or methanol solvent liberated either free radiolabeled [¹²5I]iodobenzoic acid or its succinimidyl ester without co-release of the resin-bound precursors. Radiochemical yield was between 35% and 75%, depending on the solvent system and precursor. As example applications for the released compounds, the amine-reactive N-succinimidyl-[¹²5I]iodobenzoate prosthetic group was used for conjugate radiolabeling of a peptide, tomato plant systemin, and two proteins, albumin and IgG antibody. These results demonstrate that resin-bound organotin precursors in which the compound to be labeled is tethered to the support via the tin group to be substituted can be used to produce radioiodine-labeled aromatic prosthetic groups in good specific activity without the need for HPLC purification. This solid-phase approach is potentially adaptable to kit-formulation for performing conjugate radiolabeling of biological macromolecules.

Biodistribution of radiolabeled ethanol in rodents.

The biodistribution of [1-(14)C]ethanol in rodents was examined to determine sites of concentration of ethanol or its metabolites that may contribute to its toxicological and pharmacokinetic characteristics. After i.v. administration of [1-(14)C]ethanol in mice, radioactivity showed a widespread distribution among body organs. Determination of the proportion of tissue radioactivity accounted for by volatile [1-(14)C]ethanol versus nonvolatile (14)C metabolites indicated that tissue radioactivity was mostly in the form of the latter, even as early as 5 min after injection, indicating a rapid metabolism of the radiolabeled ethanol to labeled metabolites. In a separate study, radioactivity was imaged using whole-body autoradiography after i.v. administration in rats. High levels of radioactivity were observed in the Harderian gland, preputial gland, and pancreas at 15 and 60 min after injection. High levels of radioactivity were also apparent at the later time point in the intestinal tract, indicating hepatobiliary excretion of radiolabeled metabolites. Moderate levels of radioactivity were present in the liver, lungs, salivary glands, bone marrow, and kidney cortex. In conclusion, after i.v. [(14)C]ethanol administration, radioactivity initially distributes widely among body organs but concentrates in specific tissues at subsequent time points. Especially notable in the current study was the high concentration of radioactivity accumulating in the pancreas. It is thus tempting to speculate that the well documented high incidence of pancreatic disease observed in human chronic alcoholism may be related to a propensity of this organ to accumulate ethanol and/or reactive ethanol metabolites.

Preparation of radioiodinated peptide nucleic acids with high specific activity.

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.

Concentration-dependent conditioned place preference to inhaled toluene vapors in rats.

OBJECTIVES: Toluene is present in many commercial products and is subject to abuse by inhalation. The goal of this study was to extend previous reports indicating that rats will exhibit a positive conditioned place preference to inhaled toluene vapors and to determine the dose-response relationship for inhaled toluene in terms of exposure concentration and number of exposures. For the conditioned place preference experiments rats were exposed to toluene vapors at concentrations of 800, 2000, 3000 or 5000 ppm in one compartment of a three-compartment box. RESULTS: Following six conditioning sessions with toluene, a significant place preference was obtained at 2000 and 3000 ppm, but not at 800 or 5000 ppm. Extending the number of toluene pairings at the 2000 and 3000 ppm concentration to 12 significantly enhanced the place preference compared to that at six pairings. CONCLUSIONS: These experiments extend our previous finding that rats will show a conditioned place preference to inhaled toluene, and indicate that a reinforcing "dose" of toluene depends on both the concentration and number of pairings.

Evaluation of the in vivo receptor occupancy for the behavioral effects of cannabinoids using a radiolabeled cannabinoid receptor agonist, R-[125/131I]AM2233.

G-protein coupled receptors exist in both high and low agonist affinity conformations, with tracer levels of agonist radioligands preferentially binding to the former. The goal of the present study was to characterize the in vivo binding of the aminoalkyindole-based, CB1 receptor agonist, R-[125/131I]AM2233 ((2-[125/131I]iodo-phenyl)-[1-(1-methyl-piperidin-2-yl-methyl)-1H-indol-3-yl]-methanone), and to use this radiotracer to selectively measure the receptor occupancy by the related CB1 receptor agonist, WIN55212-2, to the agonist-preferring affinity state of the receptor. In mouse locomotor assays, both WIN55212-2 and AM2233 (racemic) produced an approximately 60% reduction in activity at 1 mg/kg, (i.v.) and completely inhibited activity at 3 mg/kg, confirming their agonist nature. In ex vivo autoradiography, preferential uptake of R-[131I]AM2233 was apparent in CB1 receptor-rich areas, including globus pallidus, substantia nigra, striatum, cerebellum, and hippocampus. Overall brain uptake of R-[131I]AM2233 was 1.3% injected activity/g at 5 min in mice. Coinjection of 3 mg/kg (i.v.) SR141716A, a CB1 receptor antagonist, with R-[125I]AM2233 inhibited the radiotracer binding almost to nonspecific levels in the striatum, globus pallidus, and substantia nigra, although residual binding to a non-CB1 receptor remained in the hippocampus. In contrast to the effect of SR141716A, coinjection of 10 mg/kg (i.v.) WIN55212-2, a high dose that produced an immediate and profound immobility and catalepsy in the mice, reduced CB1 receptor-specific binding of R-[125I]AM2233 in CB1 receptor-rich areas by only 21-43%. These observations suggest that the behavioral effects of CB1 receptor agonists are manifested with a relatively small fraction of the agonist-preferring affinity state of the receptor occupied.

Ex vivo imaging of fatty acid amide hydrolase activity and its inhibition in the mouse brain.

There is recent behavioral evidence that fatty acid amide hydrolase (FAAH) inhibitors produce a subset of cannabinoid receptor agonist effects, suggesting both anandamide-specific behavioral functions and possible regional differences in FAAH inhibitory effects. Here, we introduce a novel imaging method to quantify regional differences in brain FAAH activity. Upon intravenous [3H]anandamide administration, brain FAAH activity generates [3H]arachidonic acid, which is promptly trapped in membrane phospholipids. As a result, wild-type (WT) brains accumulate tritium in a regionally specific manner that is dependent upon regional FAAH activity, whereas brains from FAAH knockout (KO) mice show a uniform [3H]anandamide distribution. Increasing doses of anandamide + [3H]anandamide fail to alter regional tritium accumulation, suggesting insensitivity toward this process by anandamide-induced changes in regional cerebral blood flow. Regional tritiated metabolite levels in WT brains were highest in the somatosensory and visual cortices and the thalamus. Treatment with methylarachidonyl fluorophosphonate (MAFP) (1 mg/kg i.p.) reduced regional tritium accumulation in the somatosensory and visual cortices (p < 0.01), and at higher doses, the thalamus (p < 0.05). The selective FAAH inhibitor 1-oxazolo[4,5-b]pyridin-2-yl-1-dodecanone (CAY10435), although having similar efficacy as MAFP in reducing tritium in the thalamus and somatosensory and visual cortices, also reduces caudate putamen and cerebellum (p < 0.01) activity. These data indicate FAAH activity generates heterogeneous regional accumulation of [3H]anandamide and metabolites, and they suggest the modulation of endocannabinoid tone by FAAH inhibitors depends upon not only the dose and compound used but also on the degree of FAAH expression in the brain regions examined. This imaging method determines regionally specific FAAH inhibition and can elucidate the in vivo effects of pharmacological agents targeting anandamide inactivation.

Potent cannabinergic indole analogues as radioiodinatable brain imaging agents for the CB1 cannabinoid receptor.

A series of novel aminoalkylindoles was synthesized in an effort to develop compounds that are potent agonists at the CB1 cannabinoid receptor and that are also easily labeled with radioisotopes of iodine for biochemical and imaging studies. 2-Iodophenyl-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]methanone (8, AM2233) had a very high affinity for the rat CB1 receptor, with most of the affinity residing with the (R)-enantiomer. Radioiodinated 8, (R)-8, and (S)-8 were prepared by radioiododestannylation of the tributyltin analogues in high yields, radiochemical purities, and specific radioactivities. In a mouse hippocampal membrane preparation with [131I](R)-8 as radioligand, racemic 8 exhibited a K(i) value of 0.2 nM compared with 1.6 nM for WIN55212-2. In autoradiographic experiments with mouse brain sections, the distribution of radioiodinated 8 was consistent with that of brain CB1 receptors. Again, very little specific binding was seen with the (S)-enantiomer [131I](S)-8 and none occurred with the (R)-enantiomer [131I](R)-8 in sections from CB1 receptor knockout mice. Radioiodinated 8 thus appears to be a suitable radioligand for studies of CB1 cannabinoid receptors.

Toluene inhalation produces a conditioned place preference in rats.

Toluene is a widely abused solvent with demonstrated addictive potential in humans. Here we explore if conditioned place preference can be used to study the abuse-related effects of inhaled toluene in rats. Animals were confined to a distinctive compartment of a three-compartment chamber while exposed to toluene vapor and later tested for preference for that compartment compared to appropriate control subjects. In this study, a flame ionization detector was used for on-line monitoring of toluene vapor concentrations inside the conditioning apparatus coupled with computerized recording of the time spent by the animals on the test day in each of the chambers. Sprague-Dawley rats were exposed to 810, 1895 or 4950 ppm of toluene vapors in either the black or white compartment during 30-min pairing sessions given every other day alternating with air exposure for the total of six pairings for each treatment. Rats that received air in both sides (control group) did not show any preference for either side with approximately equal time spent in each compartment on the test day (241 +/- 33 and 234 +/- 34 s, for white and black box, respectively). However, the 1895- and 4950-ppm test groups, but not the 810-ppm group, demonstrated a significant preference for the side paired with toluene exposure. When a subsequent test session was performed during toluene exposures, no conditioned place preference was observed. Thus, toluene produced a clear conditioned place preference that appears to be most evident when animals are not intoxicated. This procedure should be useful for further studies of the abuse-related effects of abused inhalants.

In vivo imaging of the brain cannabinoid receptor.

The CB1 cannabinoid receptor is expressed in the brain at levels sufficient to serve as potential target for in vivo imaging using positron emission tomography (PET) or single photon emission computed tomography methodology. To date, the most promising radioligands for the in vivo imaging of this receptor have structures based on that of the cannabinoid antagonist, SR141716A. Rodent data obtained using these in vivo radiotracers has demonstrated that both the behavioral and neurochemical effects of cannabinoids occur at very low levels of receptor occupancy. More recently, an agonist radiotracer based on the structure of aminoalkylindole cannabinoids has also been examined for in vivo labeling of CB1 receptors. Although rodent studies have indicated that in vivo imaging of CB1 receptors is feasible, at the present time this receptor has still to be successful imaged in a human PET study.

Effect of SA4503 on the electrically evoked release of (3)H-acetylcholine from striatal and hippocampal rat brain slices.

Recent microdialysis data has shown that the systemic administration of the selective sigma(1) receptor agonist SA4503 increases the extracellular levels of acetylcholine (ACh) in the hippocampus, but not the striatum, of freely moving rats. In the present study, we examined the effect of SA4503 on the electrically evoked release of (3)H-ACh in rat brain slices isolated from the hippocampus and striatum. At 100 and 300 nM concentrations of SA4503, the electrically evoked release of (3)H-ACh was increased in hippocampal but not striatal slices. Concentrations below 100 nM did not alter the electrically evoked release of (3)H-ACh in either brain area. These results tentatively suggest that the increase in extracellular ACh levels observed in the hippocampus after the systemic administration of SA4503 could in part be related to its interaction with sigma(1) receptors in the hippocampus.

Conditioned place preference to morphine in cannabinoid CB1 receptor knockout mice.

Recent reports have suggested an involvement of the brain cannabinoid system in the morphine-reward pathway. To address this question we evaluated whether CB1 receptor knockout mice would show a conditioned place preference to morphine. CB1 receptor knockout mice developed a strong place preference to 4 and 8 mg/kg morphine, similar to that in wild-type Swiss-Webster mice. This data thus does not support a contribution of the brain cannabinoid system to morphine reward.

"Nonhedonic" food motivation in humans involves dopamine in the dorsal striatum and methylphenidate amplifies this effect.

The drive for food is one of the most powerful of human and animal behaviors. Dopamine, a neurotransmitter involved with motivation and reward, its believed to regulate food intake in laboratory animals by modulating its rewarding effects through the nucleus accumbens (NA). Here we assess the involvement of dopamine in "nonhedonic" food motivation in humans. Changes in extracellular dopamine in striatum in response to nonhedonic food stimulation (display of food without consumption) were evaluated in 10 food-deprived subjects (16-20 h) using positron emission tomography (PET) and [11C]raclopride (a D2 receptor radioligand that competes with endogenous dopamine for binding to the receptor). To amplify the dopamine changes we pretreated subjects with methylphenidate (20 mg p.o.), a drug that blocks dopamine transporters (mechanism for removal of extracellular dopamine). Although the food stimulation when preceded by placebo did not increase dopamine or the desire for food, the food stimulation when preceded by methylphenidate (20 mg p.o.) did. The increases in extracellular dopamine were significant in dorsal (P < 0.005) but not in ventral striatum (area that included NA) and were significantly correlated with the increases in self-reports of hunger and desire for food (P < 0.01). These results provide the first evidence that dopamine in the dorsal striatum is involved in food motivation in humans that is distinct from its role in regulating reward through the NA. In addition it demonstrates the ability of methylphenidate to amplify weak dopamine signals.