Experimental envenoming of mice with venom from the scorpion Centruroides limpidus limpidus: differences in mortality and symptoms with and without antibody therapy relating to differences in age, sex and strain of mouse

C57Bl/6J and BALB/cAnN inbred strains of mice differed significantly in mortality and symptoms when intoxicated subcutaneously with one LD₅₀ of venom from Centruroides limpidus limpidus. Higher mortality was observed in C57Bl/6J than in BALB/cAnN. Also, C57Bl/6J mice more quickly developed muscular and respiratory collapse whilst BALB/cAnN mice were hyperactive before dying. Also, the symptoms in the survivors lasted for 24 h in C57Bl/6J and for 2 h in BALB/cAnN. The age and sex of mice were also related to mortality: younger mice were more resistant than older mice and females were more susceptible than males, especially in the younger groups. Antivenom (horse F(ab′)₂) administration 5–10 min after envenoming of mice with one LD₅₀ rescued 60% of BALB/cAnN and 52% of C57Bl/6J mice, respectively. Results indicate that genetic background, gender and age differences are of consequence in the pathogenesis of C. limpidus scorpion envenomation in mice, and that timely treatment with active antivenom F(ab′)₂ saves a significant fraction of intoxicated mice without statistically significant distinction of strains.     

Equation relating to the Higuchi R-ratio of lubrication

The Higuchi R-ratio of lubrication relates the ratio of upper punch force (F) or pressure (P) to thai sensed by the lower punch (F′ or P′) to the state of lubrication of the mass being compressed. The ratio R = F′/F or P′/P is denoted the lubrication index. It is shown in the article to follow that F' is linear with F, but that the intersect is not zero. The logical approach, in cases where this holds, is therefore to measure the lower punch pressure (P′₁ and P′₂) for two upper punch pressures (P₁ and P₂) and calculate the values of a and b in the relation P′ = a · P + b. The index is then R′ = (P′ − b)/P.     

Neutralization of kinin-releasing enzymes from viperid venoms by antivenom IgG fragments

The activities of kinin-releasing enzymes in the venoms of Vipera xanthina xanthina, V. lebetina obtusa, V. aspis aspis, V. lebetina schweizeri, V. ammodytes ammodytes and V. berus berus were determined using a specific radioimmunoassay for kinin. The kinin-releasing activities of all the viperid venoms measured in vitro were neutralized, to varying extents, by two commercially available monospecific antivenoms in the form of F(ab′)₂ (Zagreb) and Fab (TAb) immunoglobin fragments, indicating a high degree of cross-neutralization of those enzymes.     

Capsaicin potentiates 1,25-dihydoxyvitamin D3- and all-trans retinoic acid-induced differentiation of human promyelocytic leukemia HL-60 cells.

Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5–30 μg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)₂D₃ or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)₂D₃ and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)₂D₃- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)₂D₃ or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)₂D₃- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.     

Stability of thioTEPA and its metabolites, TEPA, monochloroTEPA and thioTEPA-mercapturate, in plasma and urine

The degradation of N,N′,N′′-triethylenethiophosphoramide (thioTEPA) and its metabolites N,N′,N′′-triethylenephosphoramide (TEPA), N,N′-diethylene,N′′-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA-mercapturate in plasma and urine has been investigated. ThioTEPA, TEPA and monochloroTEPA were analyzed using a gas chromatographic (GC) system with selective nitrogen/phosphorous detection; thioTEPA-mercapturate was analyzed on a liquid chromatography-mass spectrometric (LC-MS) system. The influences of pH and temperature on the stability of thioTEPA and its metabolites were studied. An increase in degradation rate was observed with decreasing pH as measured for all studied metabolites. In urine the rate of degradation at 37°C was approximately 2.5±1 times higher than at 22°C. At 37°C thioTEPA and TEPA were more stable in plasma than in urine, with half lives ranging from 9–20 h for urine and 13–34 h for plasma at pH 6. Mono- and dichloro derivatives of thioTEPA were formed in urine and the monochloro derivative was found in plasma. Degradation of TEPA in plasma and urine resulted in the formation of monochloroTEPA. During the degradation of TEPA in plasma also the methoxy derivative of TEPA was formed as a consequence of the applied procedure. The monochloro derivative of thioTEPA-mercapturate was formed in urine, whereas for monochloroTEPA no degradation products could be detected.     

Analysis of various nucleosides in plasma using solid phase extraction and high-performance liquid chromatography with UV detection

The National Cancer Institute (NCI) has screened many nucleosides for antiviral activity to the HIV-1 virus. Drugs demonstrating antiviral activity are tested in animal models to evaluate their toxicity and pharmacokinetic characteristics. These drugs are subsequently evaluated for efficacy in human clinical trials. Sensitive analytical methodology is needed to quantify nucleosides in plasma and other biological matrices in support of these studies. Battelle has modified and validated a reversed phase high-performance liquid chromatography (HPLC) method for several of these nucleosides that could be easily adapted for similar compounds. Methods have been validated for 6-chloro-2′,3′-dideoxyguanosine (6ClddG), 6-chloro-2′,3′-dideoxyinosine (6ClddI) and their primary metabolites 2′,3′-dideoxyguanosine (ddG) and 2′,3′-dideoxyinosine (ddI) in both rat and dog plasma containing EDTA. The method has also been validated for 2′-fluoro-2′,3′-dideoxyara-adenosine (βFlddA) and its primary metabolite 2′-β-fluorodideoxyinosine (βFddI) in rat plasma containing heparin. Calibration plasma standards were prepared over ranges of 0.1–10 μg ml⁻¹ for βFlddA and βFddI, 0.1–50 μg ml⁻¹ for 6ClddG and ddG, and 0.25–50 μg ml⁻¹ for 6ClddI and ddI in plasma containing 4 μg ml⁻¹ pentostatin. The addition of pentostatin to the plasma samples inhibits in-vitro deamination of the drug after collection. Quality control (QC) standards were prepared containing the appropriate anticoagulant and 4 μg ml⁻¹ pentostatin at concentrations within each of the bracketed calibration ranges in plasma. These methods have been successfully applied to plasma samples generated during various animal studies.     

Efficacy of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and diphenyl diselenide on cadmium induced testicular damage in mice

The deleterious effect of acute cadmium-intoxication in mice testes was evaluated. Animals received a single dose of CdCl₂ (2.5 or 5 mg/kg, intraperitoneally) and a number of toxicological parameters in mice testes were examined, such as δ-aminolevulinic acid dehydratase (δ-ALA-D) activity, lipid peroxidation, hemoglobin and ascorbic acid contents. Furthermore, the parameters that indicate tissue damage such as plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were also determined. Thus, a possible protective effect of 2,3-dimercapto-1-propane-sulfonic acid (DMPS) and diphenyl diselenide (PhSe)₂ were studied. The results demonstrated an inhibition of δ-ALA-D activity, a reduction of ascorbic acid and an increase of lipid peroxidation induced by cadmium, indicating testes damage. Furthermore, we observed an increase of plasma LDH, AST and ALT activities. DMPS (400 mol/kg) and (PhSe)₂ (100 μmol/kg) partially protected from the inhibitory effect of 2.5 mg/kg CdCl₂ on δ-ALA-D and from the increase of TBARS (thiobarbituric acid reactive species) levels. (PhSe)₂ therapy was effective in ameliorate ascorbic acid content when the cadmium dose was 2.5 mg/kg. Treatment with DMPS and (PhSe)₂, individually or combined, was inefficient in reducing cadmium-induced plasma LDH and ALT activity increase. The use of combined therapy (DMPS plus (PhSe)₂) proved to be efficient in decreasing cadmium levels in testes and in ameliorating plasma AST activity from animals that received the highest dose of cadmium.     

Reversed-phase high-performance liquid chromatography for evaluating the distribution of pharmaceutical substances in suppository base-phosphate buffer system

The aim of this study was to assess the potency of the reversed-phase high-performance liquid chromatography (RP-HPLC) for in vitro evaluation of the distribution behavior of common drugs between one of the generally used suppository bases Witepsol H₁₅ and the rectal liquid which is imitated by a phosphate buffer, pH 7.2. The distribution coefficients (log K) of nine compounds — paracetamol, caffeine, diclofenac, propyphenazone, indomethacin, codeine base, codeine phosphate, phenobarbital acid and phenobarbital sodium salt were determined by the classical ‘shake-flask’ method followed by RP-HPLC quantitative assay. The capacity factors log k′ of the compounds were determined on reversed-phase C₁₈ column at a number of methanol–5 mM phosphate buffer, pH 7.2 mobile phases containing different percentages of methanol (φ_MeOH). The apparent capacity factors log k′_w^app were derived by extrapolation of the methanol concentration to zero and using the correction for ionization, the real capacity factors log k′_w were calculated. The lipophilicity of the compounds was assessed by the partition coefficients CLOGP and the distribution coefficients CLOGD_7.2, calculated for the n-octanol–water system. Correlations between log k′_w and CLOGP, log k′_w^app and CLOGD_7.2, log k′_w^app and log K were found. The last correlation indicated that the parameter log k′_w^app was suitable for evaluating the distribution behavior of the studied drugs in the examined Witepsol H₁₅-rectal liquid system. The predictive power of this correlation was tested by a set of nine non-congeners. It was shown that the classical ‘shake-flask’ method for determination of the distribution behavior of the studied drugs between the suppository base Witepsol H₁₅ and the phosphate buffer, pH 7.2 might be replaced by the RP-HPLC technique due to its priorities of rapid, stable and reproducible experiments.     

Teratogenic effects of diphenyl diselenide in Wistar rats

Diphenyl diselenide is an organoselenium compound with potential therapeutic use. The present study evaluates the effects of single maternal subcutaneous injection of 50 and 100 mg/kg diphenyl diselenide [(PhSe)₂] at gestational days (GD) 6, 10 or 17 in Wistar rats. The highest dose of (PhSe)₂ was also administered at GD 7–12. External and internal fetal soft-tissue examination was performed at GD 20. No mortality was observed in fetuses or dams at any (PhSe)₂ treatment group. Neither did exposure to (PhSe)₂ cause significant changes to fetal body weight, organ weight, or fetal size when administered at GD 6–8, 10–12 or 17. Exposure to 100 mg/kg (PhSe)₂ at GD 9 produced significant changes in fetal biometry (crown-rump (CR) length) and body weight. No significant increase in the proportion of fetuses with external visible abnormalities was observed in groups exposed to (PhSe)₂. Skeletal anomalies were observed in fetuses in the GD 9–11 treatment groups and included incomplete ossification of cranial bones, misshapen and incomplete ossification of sternebrae, reduced sternebrae number, wavy and extra ribs, incomplete ossification of fore and hindpaw bones and incomplete ossification of sacral and caudal bones. We conclude that maternal administration of (PhSe)₂ during GD 7–12 led to increased incidences of these skeletal variations or anomalies, but did not cause externally visible malformations in rat fetuses.     

The effects of cyclopentane and cyclopentene analogues of GABA at recombinant GABA(C) receptors.

The pharmacological effects of the enantiomers of cis-3-aminocyclopentanecarboxylic acids ((+)- and (−)-CACP), the enantiomers of trans-3-aminocyclopentanecarboxylic acids ((+)- and (−)-TACP), and the enantiomers of 4-aminocyclopent-1-ene-1-carboxylic acids ((+)- and (−)-4-ACPCA) were studied on human homomeric ρ₁ and ρ₂ GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp methods. These compounds are conformationally restricted analogues of γ-aminobutyric acid (GABA) held in a five-membered ring. (+)-TACP (EC₅₀ (ρ₁)=2.7±0.2 μM; EC₅₀ (ρ₂)=1.45±0.22 μM), (+)-CACP (EC₅₀ (ρ₁)=26.1±1.1 μM; EC₅₀ (ρ₂)=20.1±2.1 μM) and (−)-CACP (EC₅₀ (ρ₁)=78.5±3.5 μM; EC₅₀ (ρ₂)=63.8±23.3 μM) were moderately potent partial agonists at ρ₁ and ρ₂ GABA_C receptors, while (−)-TACP (100 μM inhibited 56% and 62% of the current produced by 1 μM GABA at ρ₁ and ρ₂ receptors, respectively) was a weak partial agonist with low intrinsic activity at these receptors. In contrast, (+)-4-ACPCA (K_i (ρ₁)=6.0±0.1 μM; K_i (ρ₂)=4.7±0.3 μM) did not activate GABA_C ρ₁ and ρ₂ receptors but potently inhibited the action of GABA at these receptors, while (−)-4-ACPCA had little effect as either an agonist or an antagonist. The affinity order at both GABA_C ρ₁ and ρ₂ receptors was (+)-TACP>(+)-4-ACPCA(+)-CACP>(−)-CACP(−)-TACP(−)-4-ACPCA. This study shows that the cyclopentane and cyclopentene analogues of GABA affect GABA_C receptors in a unique manner, defining a preferred stereochemical orientation of the amine and carboxylic acid groups when binding to GABA_C receptors. This is exemplified by the partial agonist, (+)-TACP, and the antagonist, (+)-4-ACPCA.     

Intrinsic sympathomimetic activity of beta-adrenoceptor antagonists: down-regulation of cardiac beta 1- and beta 2-adrenoceptors

Prolonged treatment of cultured rat heart muscle cells containing β₁- and non-muscle cells containing β₂-adrenoceptors with β-adrenoceptor antagonists devoid of intrinsic sympathomimetic activity had no effect on β-adrenoceptor density. In contrast, antagonists with intrinsic sympathomimetic activity decreased β-adrenoceptor density and response (adenylate cyclase stimulation) in both heart muscle (β₁) and non-muscle cells (β₂) by a maximum of about 50%. An even larger down-regulation of β-adrenoceptors and loss of receptor-stimulated adenylate cyclase activity was induced by the full endogenous agonist, noradrenaline, with the β-adrenoceptors of heart muscle cells (β₁) being much more sensitive to the β₁-selective noradrenaline than the heart non-muscle cell β₂-adrenoceptors. When combined with noradrenaline, the antagonists with intrinsic sympathomimetic activity prevented the action of noradrenaline at both β₁- and β₂-adrenoceptors, thereby leading to an apparent up-regulation of receptor density and response. This apparent reversal from an agonist to an antagonist action was observed at much lower concentrations of noradrenaline at β₁- than at β₂-adrenoceptors. The data presented indicate that the β-adrenoceptor antagonists with intrinsic sympathomimetic activity, but not those without, upon prolonged treatment decrease the density and responsiveness of both β₁- and β₂-adrenoceptors in cultured rat heart cells. This suggests that the intrinsic sympathomimetic activity of these agents is not a subtype-selective component. Furthermore, the agonist and antagonist activity of these agents apparently depends on the concomitant presence of an endogenous full agonist and an its own affinity and that of the partial agonist for the β-adrenoceptor subtype.     

Effect of maturation and aging on beta-adrenergic signal transduction in rat kidney and liver

The characteristics of the β-adrenergic signal transduction system were analyzed in kidney and liver membrane preparations from neonatal (2–3 days), mature (2 months), and old (2 years) rats. When comparing kidneys from adult to neonatal rats, we found a higher β-receptor density and a higher percentage of β₁-receptor subtype, lower immunoreactive G_s-protein, a lower ratio between the high and low molecular weight splice variant of G_s, lower immunoreactive G_i-protein, and lower basal adenylate cyclase activity. When comparing livers from adult to neonatal rats, we found lower β-receptor density and basal adenylate cyclase activity. Very few differences could be detected when comparing mature to old kidneys or livers. Stimulated adenosine 3′,5′-cyclic monophosphate (cAMP) synthesis was tissue- and age-dependent. In liver, G-protein- and β-receptor-stimulated cAMP synthesis mirrored basal adenylate cyclase activity and was highest in liver from neonatal animals. In contrast, cAMP synthesis was significantly more stimulated in kidneys from mature animals than from neonatal and senescent rats. We conclude that: (i) the stoichiometry of the components within the β-receptor/G-protein/adenylate cyclase complex is not fixed but is both tissue- and age-dependent; (ii) adenylate cyclase enzyme activity is possibly but not necessarily the rate-limiting step in the β-receptor-mediated synthesis of cAMP; and (iii) there is in vivo evidence for a preferential co-expression of the large splice variant of the G_s-protein and β₂-receptor subtype. It is speculated that this could have important physiological consequences for the development of the kidney.     

Peripheral adenosine 5′-triphosphate enhances nociception in the formalin test via activation of a purinergic p2X receptor

The pronociceptive effects of adenosine 5′-triphosphate (ATP) were examined in the low concentration formalin model (0.5%) by coadministration of ATP, ATP analogs (,β-methylene-ATP and 2-methylthio-ATP) and antagonists (suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) with formalin and determining effects on the expression of flinching behaviours. Coadministration of ATP (5–500 nmol) with formalin enhanced phase 2 (12–60 min after injection) but not phase 1 (0–10 min after injection) responses. ,β-methylene-ATP (0.5–50 nmol) but not 2-methylthio-ATP (50–500 nmol) produced a similar enhancement of activity, generating an order of potency of ,β-methylene-ATP, ATP2-methylthio-ATP. This enhancement was primarily expressed in the latter part of phase 2, 30–60 min after injection. Coadministration of suramin 50–500 nmol, a non-selective P_2X and P_2Y purinoceptor antagonist and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid 5–500 nmol, a selective P_2X purinoceptor antagonist, dose-dependently inhibited the augmentation of the formalin response by ATP 50 nmol, but did not reduce the response to formalin itself. Pretreatment for 30 min with higher doses of suramin inhibited the response to formalin (0.5%, 1.5%) and this appeared to be by a systemically mediated action as it was seen following administration into the contralateral paw. The results of this study provide evidence in support of a P_2X purinoceptor mediated augmentation of the pain signal by ATP. The delayed time-course of the effect suggests that it may occur in concert with other mediators that are recruited by the inflammatory process, rather than reflecting a direct depolarization of sensory nerves. Other behavioural paradigms may be required to examine the fast onset, direct effect. Suramin appears to exert both local and systemic effects on the expression of pain behaviours in response to formalin.     

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABA_A receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABA_A (₁β₂γ_2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABA_A receptors. The GABA enhancing action of E-6375 on recombinant GABA_A receptors was unaffected by the subtype of the isoform (i.e. _xβ₂γ_2L; x=1–3) within the receptor, but was increased by the omission of the γ_2L subunit. Receptors incorporating β₂, or β₃, subunits were more sensitive to modulation by E-6375 than those containing the β₁ subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the β-subunit. The various in vivo actions of general anaesthetics may be mediated by GABA_A receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABA_A receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.     

Puerarin protects rat pancreatic islets from damage by hydrogen peroxide

Periodate-oxidized 2′,3′-dialdehyde ATP (oxidized ATP) has been used extensively as a selective antagonist at P2X₇ receptors, although P2X₇-independent actions on pro-inflammatory cytokine release have also been reported. Because P2X₇ receptors in astrocytes have been suggested as potential targets of anti-inflammatory drug therapy, we examined the effect of oxidized ATP on β-actin expression and superoxide production of RBA-2 type-2 astrocytes known to possess P2X₇ receptors. Oxidized ATP per se decreased β-actin expression time and dose dependently. Treatment with oxidized ATP for 8 h caused an approximately 50% decrease in β-actin expression whereas other P2 receptor antagonists, brilliant blue G (BBG), suramin and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), were not effective. In addition, oxidized ATP per se decreased the intracellular superoxide concentration, whereas ATP and the P2X₇ receptor-selective agonist 3′-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) stimulated intracellular superoxide production, an effect inhibited by oxidized ATP. In addition, oxidized ATP neither affected cellular viability nor affected interleukin-1β, converting enzyme (ICE)-like protease activity in these astrocytes. To further elucidate the mechanism, the effects of oxidized ATP on intracellular superoxide concentration and β-actin expression were examined in a P2X₇ receptor-negative astrocyte cell line, IA-1g1. Oxidized ATP-induced a time-dependent decrease in intracellular superoxide concentration whereas oxidized ATP had no effect on β-actin expression. Nevertheless, oxidized ATP altered f-actin cytoskeleton arrangement in IA-1g1 astrocytes. Taken together, these results indicate that oxidized ATP per se caused a cell specific decrease in β-actin expression in RBA-2 type-2 astrocytes. In addition, oxidized ATP decreased intracellular superoxide concentrations and altered f-actin cytoskeleton arrangement in both P2X₇ receptor-positive and -negative astrocytes. Thus, we conclude from these results that the effects of oxidized ATP on actin and superoxide are mediated through mechanisms that are at least in part, independent of P2X₇ receptors.     

Evaluation of nicotinic receptors agonists and antagonists against paraoxon exposed PC12 cells

Chronic and acute exposure to organophosphate pesticides may lead to persistent neurological and neurobehavioral effects, which cannot be explained by acetylcholinesterase (AChE) inhibition alone. In an attempt to elucidate the mechanism by which paraoxon affects the nicotinic receptors gene expression, the effects of exposure of PC12 cells to 100 μM concentrations of paraoxon for 48 h in the presence and the absence of nicotinic acetylcholine receptors (nAChRs) agonists and antagonists were characterized. Paraoxon at 100 μM significantly inhibited AChE activity. On the mRNA level, the ₄ and β₂ subunits of nAChR mRNA were significantly decreased in the cells exposed to paraoxon. On the protein level, ₄ and β₂ subunits of nAChR protein were also significantly reduced. Mecamylamine (10 μM), dihydro-β-erythroidine (DHβE) (5 μM) and nicotine (10 μM) efficiently prevented the decrease of ₄ and β₂ nAChR mRNA and protein in PC12 cells, but carbamaylcholine a weak agonist of nAChR was not efficient. These observations suggest that ₄β₂ nAChRs are involved in paraoxon related toxicity and nicotinic receptors antagonists could play some protective role against organophosphate related damages.     

Evaluation of the role of nicotinic acetylcholine receptor subtypes and cannabinoid system in the discriminative stimulus effects of nicotine in rats

Male Wistar rats were trained to discriminate (−)-nicotine (0.4 mg/kg) from saline under a two-lever, fixed-ratio 10 schedule of water reinforcement. During test sessions the following drugs were coadministered with saline (substitution studies) or nicotine (0.025–0.4 mg/kg; combination studies): the ₄β₂ nicotinic acetylcholine receptor subtype antagonist dihydro-β-erythroidine (DHβE), the non-selective nicotinic acetylcholine receptor subtype antagonist mecamylamine, the ₇ nicotinic acetylcholine receptor subtype antagonist methyllycaconitine (MLA), the ₄β₂ nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA), the cannabinoid CB₁ receptor antagonist/partial agonist rimonabant, the cannabinoid CB₂ receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methybenzyl)pyrazole-3-carboxamide (SR 144528), the cannabinoid CB_1/2 receptor agonists (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)-methanone mesylate (WIN 55,212-2), the endogenous cannabinoid agonist and non-competitive ₇ nicotinic acetylcholine receptor subtype antagonist anandamide, the anandamide uptake and fatty acid amide hydrolase inhibitor N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM-404), the fatty acid amide hydrolase inhibitor cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB 597), AM-404 + anandamide or URB 597 + anandamide. 5-IA (0.01 mg/kg) fully substituted for nicotine, while other drugs were inactive. In combination studies, DHβE and mecamylamine dose-dependently attenuated the discriminative stimulus effects of nicotine and the full substitution of 5-IA, while MLA, rimonabant, SR 144528, CP 55,940, WIN 55,212-2, and URB 597 did not alter the nicotine cue. Pretreatment with AM-404 + anandamide or URB 597 + anandamide weakly enhanced nicotine-lever responding. Our pharmacological analyses demonstrates that the expression of nicotine discrimination is under the control of nicotinic acetylcholine receptor subtypes composed of ₄β₂ (but not of ₇) subunits. Furthermore, we excluded the involvement of either cannabinoid CB₁ and CB₂ receptors or increases in the endocannabinoid tone in the nicotine discrimination.     

Effects of adenosine A2 receptor agonists on the excitation of capsaicin-sensitive afferent sensory nerves in airway tissues

We examined the effects of adenosine analogues on the asthmatic reactions induced by the stimulation of capsaicin-sensitive afferent sensory nerves. Intravenous (i.v.) injection of adenosine A₂ receptor agonists, 5′-(N-ethylcarboxamido)-adenosine (NECA) and 2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamido-adenosine (CGS 21680), dose dependently inhibited capsaicin-induced guinea-pig bronchoconstriction (1–1000 nmol kg⁻¹), whereas i.v. administration of the adenosine A₁ receptor agonist, N⁶-cyclo-hexyladenosine (CHA), did not affect it (1000 nmol kg⁻¹). Intratracheal injection of NECA (0.05–5 nmol site⁻¹) and CGA 21 680 (0.05−5 nmol site⁻¹) also reduced capsaicin-induced constriction in a dose-dependent manner. However, NECA (1000 nmol kg⁻¹) failed to inhibit substance P-induced guinea-pig bronchoconstriction. NECA (1–1000 nmol kg⁻¹) dose-dependently inhibited cigarette smoke-induced rat tracheal plasma extravasation, but not substance P-induced reaction. NECA (0.1–10 μM) and CGS 21 680 (10 μM) significantly blocked the capsaicin-induced release of substance P-like immunoreactivity from guinea-pig lung, whereas CHA (10 μM) had no effect. This evidence suggests that adenosine A₂ receptors modulate negatively the excitation of capsaicin-sensitive afferent sensory nerves and substance P release from their endings in airway tissues.     

Beneficial effects of the sigma1 receptor agonists igmesine and dehydroepiandrosterone against learning impairments in rats prenatally exposed to cocaine

In utero cocaine (IUC) exposure results in offspring rats in complex neurochemical and behavioral alterations, particularly affecting learning and memory processes. We examined here the impact of IUC exposure on memory functions in male and female offspring rats and report that selective sigma₁ (σ₁) receptor agonists are effective in reversing the deficits. Dams received a daily cocaine, 20 mg/kg ip, injection between gestational days E17 to E20. Learning was examined in offspring between day P30 and P41 using delayed alternation in the T-maze, water-maze learning and passive avoidance. Both male and female rats prenatally exposed to cocaine showed delayed alternation deficits and impairments of acquisition of a fixed platform position in the water maze, as shown by higher acquisition latencies and diminutions of time spent in the training quadrant during the probe test. The acquisition of a daily changing platform position also demonstrated impaired working memory. Finally, passive avoidance deficits were observed. Pretreatment with the synthetic σ₁ agonist igmesine (0.1–1 mg/kg ip) or the neuroactive steroid dehydroepiandrosterone (DHEA 10–40 mg/kg ip) reversed the prenatal cocaine-induced learning deficits in offspring rats for each test. The σ₁ antagonist BD1063 (1 mg/kg ip) failed to affect performances alone but blocked the igmesine and DHEA effects, confirming the involvement of the σ₁ receptor. IUC exposure thus results in marked memory deficits, affecting spatial and nonspatial short- and long-term memories in juvenile male and female offspring rats. The activation of the σ₁ neuromodulatory receptor allows a complete behavioral recovery of the memory functions in prenatally cocaine-exposed rats.