Sex differences in neurosteroid and hormonal responses to metyrapone in posttraumatic stress disorder

Rationale

Mechanisms contributing to sex differences in the regulation of acute stress responsivity and their effect on the increased incidence of posttraumatic stress disorder (PTSD) in women are poorly understood. The reproductive hormone, progesterone, through conversion to allopregnanolone (ALLO), suppresses the hypothalamic pituitary adrenal (HPA) axis and has potent anxiolytic effects. The potential that progesterone and allopregnanolone reactivity modulate HPA axis responses and account for sex differences in PTSD has not been previously examined.

Objective

The present study examined the effects of sex and PTSD on adrenocorticotropic hormone (ACTH), progesterone, and allopregnanolone responses to metyrapone and whether progesterone and allopregnanolone reactivity could affect the ACTH response in PTSD.

Methods

Healthy medication-free male and premenopausal follicular phase female participants with chronic PTSD (n?=?43; 49 % female) and controls (n?=?42; 50 % female) completed an overnight metyrapone challenge and ACTH, progesterone, and allopregnanolone were obtained by repeated blood sampling.

Results

The increase in ACTH response to metyrapone was higher in PTSD subjects compared to controls and in women compared to men. Contrary to our initial prediction of an inverse relationship, progesterone and allopregnanolone were positively associated with ACTH. Progesterone and allopregnanolone partially mediated the relationship between PTSD and ACTH.

Conclusions

Our findings of increased ACTH to metyrapone in PTSD and in women may reflect heightened hypothalamic CRF hypersecretion. Progesterone and allopregnanolone partially mediated the ACTH response in PTSD. Further characterizing sex differences in these processes will advance our understanding of the pathophysiology of PTSD, and may ultimately lead to better-targeted, more effective treatment.     

The Ca2+-calmodulin-Ca2+/calmodulin-dependent protein kinase II signaling pathway is involved in oxidative stress-induced mitochondrial permeability transition and apoptosis in isolated rat hepatocytes

Oxidative stress (OS) is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both OS from mitochondrial origin and cell death. Intracellular Ca²⁺ increase plays a permissive role in these events, but the underlying mechanisms are poorly known. We examined in primary cultured rat hepatocytes whether the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidant, model compound. tBOOH (500 μM, 15 min) induced MPTP formation, as assessed by measuring mitochondrial membrane depolarization as a surrogate marker, and increased lipid peroxidation in a cyclosporin A (CsA)-sensitive manner, revealing the involvement of MPTPs in tBOOH-induced radical oxygen species (ROS) formation. Intracellular Ca²⁺ sequestration with BAPTA/AM, CaM blockage with W7 or trifluoperazine, and CaMKII inhibition with KN-62 all fully prevented tBOOH-induced MPTP opening and reduced tBOOH-induced lipid peroxidation to a similar extent to CsA, suggesting that Ca²⁺/CaM/CaMKII signaling pathway fully mediates MPTP-mediated mitochondrial ROS generation. tBOOH-induced apoptosis, as shown by flow cytometry of annexin V/propidium iodide, mitochondrial release of cytochrome c, activation of caspase-3 and increase in the Bax-to-Bcl-xL ratio, and the Ca²⁺/CaM/CaMKII signaling antagonists fully prevented these effects. Intramitochondrial CaM and CaMKII were partially involved in tBOOH-induced MPTP formation, since W7 and KN-62 both attenuated the tBOOH-induced, MPTP-mediated swelling of isolated mitochondria. We concluded that Ca²⁺/CaM/CaMKII signaling pathway is a key mediator of OS-induced MPTP formation and the subsequent exacerbation of OS from mitochondrial origin and apoptotic cell death.     

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Rationale

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABA_A receptor (GABA_AR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABA_ARs.

Objectives

In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine.

Methods

We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABA_ARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion.

Results

We found no evidence that PIP2 depletion affected modulation of GABA_ARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity.

Conclusions

We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABA_ARs by neurosteroids or related compounds.     

Phosphorylation of GABAA receptors influences receptor trafficking and neurosteroid actions

Rationale

Gamma-aminobutyric acid type A receptors (GABA_ARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABA_ARs can be localized at post-synaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABA_ARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABA_ARs are typically activated continuously by ambient GABA concentrations and thus mediate tonic inhibition. The tonic inhibitory currents mediated by extrasynaptic GABA_ARs control neuronal excitability and the strength of synaptic transmission. However, the mechanisms by which neurons control the functional properties of extrasynaptic GABA_ARs had not yet been explored.

Objectives

We review GABA_ARs, how they are assembled and trafficked, and the role phosphorylation has on receptor insertion and membrane stabilization. Finally, we review the modulation of GABA_ARs by neurosteroids and how GABA_AR phosphorylation can influence the actions of neurosteroids.

Conclusions

Trafficking and stability of functional channels to the membrane surface are critical for inhibitory efficacy. Phosphorylation of residues within GABA_AR subunits plays an essential role in the assembly, trafficking, and cell surface stability of GABA_ARs. Neurosteroids are produced in the brain and are highly efficacious allosteric modulators of GABA_AR-mediated current. This allosteric modulation by neurosteroids is influenced by the phosphorylated state of the GABA_AR which is subunit dependent, adding temporal and regional variability to the neurosteroid response. Possible links between neurosteroid actions, phosphorylation, and GABA_AR trafficking remain to be explored, but potential novel therapeutic targets may exist for numerous neurological and psychological disorders which are linked to fluctuations in neurosteroid levels and GABA_A subunit expression.     

Ginsenoside metabolite compound K stimulates glucagon-like peptide-1 secretion in NCI-H716 cells via bile acid receptor activation

Compound K (CK) is a major metabolite of ginsenosides that is absorbed. CK has antidiabetic effects, although the mechanisms underlying the effects of CK have not fully been known. To elucidate the mechanisms underlying the antidiabetic effects of CK, we studied the effects of CK on GLP-1 secretion from NCI-H716 cells, and explored the mechanisms underlying CK-induced GLP-1 secretion. Treatment of NCI-H716 cells with 10, 50, and 100 μM CK significantly increased GLP-1 secretion, and intracellular Ca²⁺ and cAMP levels in a dose-dependent manner. Transfection of NCI-H716 cells with siRNA specific to α-gustducin and siRNA specific to TAS1R3 had no effect on CK-induced GLP-1 secretion and Ca²⁺ increase. However, transfection of NCI-H716 cells with TGR5-specific siRNA significantly inhibited CK-induced GLP-1 secretion and the increase in Ca²⁺ and cAMP levels. Moreover, CK showed human TGR5 agonist activity in CHO-K1 cells transiently transfected with human TGR5. Our data provide a novel mechanism of CK for antidiabetic effects. Moreover, the findings might suggest that CK is a potential agent that has multiple biological functions in the body via GLP-1 secretion and TGR5 activation.     

Ginsenoside compound K inhibits angiogenesis via regulation of sphingosine kinase-1 in human umbilical vein endothelial cells

Ginsenoside compound K (CK) is a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer (Araliaceae), has long been used to treat against the development of cancer, inflammation, allergies, and diabetes. This study examined the anti-angiogenic properties of CK against sphingosine 1-phosphate (S1P)-induced cell migration via regulation of sphingosine kinase 1 (SPHK1) in human umbilical vein endothelial cells (HUVEC). Studies on S1P-induced cell migration, expression of SPHK1 and MMPs and analysis of sphingolipid metabolites by LC–MS/MS were examined after the treatment of CK (2.5, 5, 10 μg/mL) in HUVEC. S1P produced by SPHK1 is also involved in cell growth, migration, and protection of apoptosis; therefore, we sought to investigate whether ginsenosides are able to regulate SPHK1. For this purpose, we developed an inhibitory assay of SPHK1 activity and an analytical method for detection of S1P and other sphingolipid metabolites in HUVEC. Ginsenoside CK inhibited 100 nM S1P-induced cell migrations in a dose-dependent manner. Among tested ginsenosides, CK exclusively inhibited S1P production, SPHK1 activity and SPHK1 expression in HUVEC, whereas expression of the pro-apoptotic sphingolipids, sphingosine and ceramide, was increased in response to CK. The major subspecies of the increased ceramide was C24:0-ceramide. CK also disrupted the sphingolipid rheostat, which ultimately influences cell fate, and dose-dependently inhibited HUVEC migration by reducing expression of metalloproteinases (MMPs). Ginsenoside CK acts as a unique HUVEC migration inhibitor by regulating MMP expression, as well as the activity of SPHK1 and its related sphingolipid metabolites.     

Zinc Protoporphyrin Polymeric Nanoparticles: Potent Heme Oxygenase Inhibitor for Cancer Therapy

Purpose

Oxidation therapy is an antitumor strategy in which, apoptosis or necrosis is caused by either excess delivery of reactive oxygen species (ROS) as an oxidant or anti-oxidant inhibition. Heme oxygenase (HO) is an anti-oxidant enzyme that plays an important role in cell growth and proliferation. The purpose of this study was to prepare poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with zinc protoporphyrin (ZnPP) to deliver the HO inhibitor into tumor.

Methods

PLGA NPs were prepared using nanoprecipitation technique and their characteristics were optimized by Box-Behnken experimental design. Scanning electron microscopy and in vitro studies consisting of drug release, HO inhibitory effect, cytotoxicity and cellular uptake followed by in vivo biodistribution and blood cytotoxicity were carried out. Internalization of coumerin-6 loaded NPs by PC3 cells was visualized by confocal laser scanning microscopy beside quantitatively analysis.

Results

NPs average size, entrapment efficiency and drug loading were 100.12?±?5.345 nm, 55.6%?±?2.49 and 7.98%?±?0.341 respectively. Equal HO inhibitory effect of NPs compared to free ZnPP was observed. The IC₅₀ value of ZnPP-NPs for PC3 human prostate cancer cells was found to be 2.14?±?0.083 μM.

Conclusion

In conclusion, ZnPP loaded PLGA NPs could exhibit enough HO inhibitory effect against cancer cells to be considered as a promising candidate for cancer treatment investigation.     

The role of ovarian hormone-derived neurosteroids on the regulation of GABAA receptors in affective disorders

Rationale

Neuroactive derivatives of steroid hormones, neurosteroids, can act on GABA_A receptors (GABA_ARs) to potentiate the effects of GABA on these receptors. Neurosteroids become elevated to physiologically relevant levels under conditions characterized by increased steroid hormones. There is considerable evidence for plasticity of GABA_ARs associated with altered levels of neurosteroids which may counteract the fluctuations in the levels of these allosteric modulators.

Objectives

The objective of this review is to summarize the current literature on GABA_AR plasticity under conditions characterized by alterations in neurosteroid levels, such as over the estrous cycle, during puberty, and throughout pregnancy and the postpartum period.

Results

The expression of specific GABA_AR subunits is altered over the estrous cycle, at puberty, and throughout pregnancy and the postpartum period. Inability to regulate δ subunit-containing GABA_ARs throughout pregnancy and the postpartum period is associated with depression-like behavior restricted to the postpartum period.

Conclusions

GABA_AR plasticity associated with alterations in neurosteroid levels represents a homeostatic compensatory mechanism to maintain an ideal level of inhibition to offset the potentiating effects of neurosteroids on GABAergic inhibition. Failure to properly regulate GABA_ARs under conditions of altered neurosteroid levels may increase vulnerability to mood disorders, such as premenstrual syndrome (PMS), premenstrual dysphoric disorder (PMDD), and postpartum depression.     

Proof-of-concept randomized controlled trial of pregnenolone in schizophrenia

Rationale

Preclinical and clinical data suggest that pregnenolone may be a promising therapeutic in schizophrenia. Pregnenolone is neuroprotective and enhances learning and memory, myelination, and microtubule polymerization. Treatment with pregnenolone elevates allopregnanolone (a neurosteroid that enhances GABA_A receptor responses) and pregnenolone sulfate (a positive NMDA receptor modulator). Pregnenolone could thus potentially mitigate GABA dysregulation and/or NMDA receptor hypofunction in schizophrenia via metabolism to other neurosteroids.

Objective

The objective of this study is to conduct a randomized controlled trial of adjunctive pregnenolone in schizophrenia.

Methods

Following a placebo lead-in, 120 participants were randomized to pregnenolone or placebo for 8 weeks (Institute for Mental Health, Singapore). Primary endpoints were changes in MATRICS Consensus Cognitive Battery (MCCB) composite scores (cognitive symptoms), UCSD Performance-based Skills Assessment—Brief (UPSA-B) composite scores (functional capacity), and Scale for Assessment of Negative Symptoms (SANS) total scores (negative symptoms). A modified intent-to-treat analysis approach was utilized.

Results

No significant changes compared to placebo were demonstrated in composite MCCB scores. In contrast, participants randomized to pregnenolone (n?=?56) demonstrated greater improvements in functional capacity (UPSA-B composite changes) compared to placebo (n?=?55), p?=?0.03. Pregnenolone was also superior to placebo in the communication subscale of the UPSA-B (p?r _s?=?0.497, p?n?=?17) but not in males. Mean total SANS scores were very low at baseline and did not improve further post-treatment. Pregnenolone was well-tolerated.

Conclusions

Pregnenolone improved functional capacity in participants with schizophrenia, but did not improve cognitive symptoms over an 8-week treatment period. Neurosteroid changes correlated with functional improvements in female participants. Neurosteroid interventions may exhibit promise as new therapeutic leads for schizophrenia.     

Potential role of nitric oxide synthase isoforms in pathophysiology of neuropathic pain

Neuropathic pain triggers a cascade of events in the sensory neurons. It is the main complication of diabetes after cardiovascular disease. Nitric oxide (NO) produced from nitric oxide synthases (NOS) is an important signaling molecule which is crucial for many physiological processes such as synaptic plasticity, neuronal survival, vasodilation, vascular homeostasis, immune regulation. Overproduction of NO due to changes in NOS isoforms level involves pathological processes such as neurotoxicity, septic shock and neuropathic pain. All three isoforms of NOS as well as their end product, NO have modulatory effect on neuropathic pain. Overactivation of the N-Methyl-d-Aspartate receptor and peroxynitrite formation results in high levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) which suggest that nNOS and eNOS are critical for pain hypersensitivity. Inducible NOS induced in glia by inflammation due to activation of Tumor Necrosis Factor α, Calcitonin Gene Regulating Peptide, Mitogen Activated Protein Kinases, Extracellular signal Regulated Kinase, c-Jun N-terminal kinases can induce neuronal death. This review focuses on different nitric oxide synthases and their role in pathophysiology of neuropathic pain considering NOS as an important therapeutic target.     

Targeting EGFR mutants with non-cognate kinase inhibitors in non-small cell lung cancer

Considering that the human protein kinase family members share high conservation in both primary sequence and advanced structure, and a large number of small-molecule inhibitors have already been developed to target these different members, we herein are interested in whether certain existing inhibitors that were originally designed for other (cognate) kinases can also bind efficiently to (non-cognate) epidermal growth factor receptor (EGFR) mutants and simultaneously lie low affinity to wild-type EGFR. To explore this notion, a structure-based quantitative structure–activity relationship model was derived from 77 crystal structure-solved, affinity-known kinase–inhibitor complexes. We employed this model to profile a systematic interaction map of 2 cognate and 11 non-cognate kinase inhibitors with wild-type EGFR and its several important NSCLC-related mutants. As might be expected, the cognate inhibitors exhibited generally high affinity to both wild-type and mutant EGFR, while most non-cognate inhibitors have low binding potency for EGFR. However, few combinations of non-cognate inhibitors and EGFR mutants, such as TAE684 and T790M, SKI606 and L858R, and R406 and T790M, show a favorable interaction as compared to the same inhibitors with wild-type EGFR. A further kinase assay was performed to determine the inhibitory activities of compound TAE684, originally developed as a ALK kinase inhibitor, against wild-type and T790M mutant EGFR; it is revealed that the TAE684 inhibited the mutant with ~tenfold higher potency than the wild-type enzyme. This work would help to establish a new strategy for the new use of an old drug.     

MDMA administration during adolescence exacerbates MPTP-induced cognitive impairment and neuroinflammation in the hippocampus and prefrontal cortex

Rationale

We have recently shown that chronic exposure to 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) of adolescent mice exacerbates dopamine neurotoxicity and neuroinflammatory effects elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the substantia nigra and striatum at adulthood.

Objectives

The present study investigated whether the amplification of MPTP effects by previous treatment with MDMA extends to the limbic and cortical regions and consequently affects cognitive performance.

Methods

Mice received MDMA (10 mg/kg, twice a day/twice a week) for 9 weeks, followed by MPTP (20 mg/kg?×?4 administrations), starting 2 weeks after MDMA discontinuation. Complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) were evaluated by immunohistochemistry in both the hippocampus and the medial prefrontal cortex (mPFC) to measure microglia and astroglia activation. These neurochemical evaluations were paired with an assessment of cognitive performance by means of the novel object recognition (NOR) and spontaneous alternation tasks.

Results

MPTP administration to MDMA-pretreated mice elicited a stronger activation of CD11b and GFAP in both the hippocampus and the mPFC compared with either substance administered alone. Furthermore, NOR performance was lower in MDMA-pretreated mice administered MPTP compared with mice that received either substance alone.

Conclusions

These results demonstrate that MDMA–MPTP negative interactions extend to the limbic and cortical regions and may result in cognitive impairment, providing further evidence that exposure to MDMA may amplify the effects of later neurotoxic insults.     

MDMA effects consistent across laboratories

Rationale

Several laboratories have conducted placebo-controlled drug challenge studies with 3,4-methylenedioxymethamphetamine (MDMA), providing a unique source of data to examine the reliability of the acute effects of the drug across subject samples and settings. We examined the subjective and physiological responses to the drug across three different laboratories and investigated the influence of prior MDMA use.

Methods

Overall, 220 healthy volunteers with varying levels of previous MDMA experience participated in laboratory-based studies in which they received placebo or MDMA orally (1.5 mg/kg or 125-mg fixed dose) under double-blind conditions. Cardiovascular and subjective effects were assessed before and repeatedly after drug administration. The studies were conducted independently by investigators in Basel, San Francisco, and Chicago.

Results

Despite methodological differences between the studies and differences in the subjects’ drug use histories, MDMA produced very similar cardiovascular and subjective effects across the sites. The participants’ prior use of MDMA was inversely related to feeling “Any Drug Effect” only at sites testing more experienced users.

Conclusions

These data indicate that the pharmacological effects of MDMA are robust and highly reproducible across settings. There was also modest evidence for tolerance to the effects of MDMA in regular users.     

Acylation of Exenatide by Glycolic Acid and its Anti-Diabetic Activities in db/db Mice

Purpose

To prepare acylated exenatide analogues and investigate their biological properties for guiding the development of PLGA formulations of exenatide.

Methods

The acylated exenatide analogues were prepared by reaction with glycolic acid (GA), one constitutional unit of PLGA, and characterized by HPLC-MS/MS and Circular Dichroism (CD). The pharmacokinetic properties and anti-diabetic activities were studied in SD rats and db/db mice, respectively.

Results

Structural characterizations of the acylated products showed that one to four glycolic acids (GAs) were connected to the primary amine groups of exenatide, and there was a conversion of α-helix to β-sheet to some extent. Pharmacokinetic studies in SD rats revealed that acylated exenatides had a similar Tmax with that of the prototype drug, whereas the Cmax and the AUC values of the adducts were significantly decreased. Biological activity tests demonstrated that exenatide and acylated exenatide analogues had similar in vivo antidiabetic activities in terms of controlling blood glucose concentration, HbA1c level, body weight and food intake.

Conclusions

These findings suggest that GA conjugated exenatide had no influence on the peptide efficacy, therefore it’s not necessary to inhibit exenatide acylation in PLGA formulations during the peptide release process.     

Differential responses of two related neurosteroids to methylphenidate based on ADHD subtype and the presence of depressive symptomatology

Rationale

Attention deficit with hyperactivity disorder is a neurodevelopmental disorder associated with alterations in the prefrontal cortex via dopaminergic and noradrenergic neurotransmission. Neurosteroids (e.g. allopregnanolone and dehydroepiandrosterone) modulate the release of multiple neurotransmitters.

Objective

This study aims to determine the baseline concentrations and daily variations in allopregnanolone and dehydroepiandrosterone in children with attention deficit hyperactivity disorder (ADHD) and to determine the effect of chronic administration of methylphenidate on clinical symptoms and on the concentrations of these two neurosteroids.

Methods

We included 148 children aged 5 to 14 years, subdivided into two groups: ADHD group (n?=?107, with a diagnosis of ADHD (DSM-IV-TR criteria), further classified in subtypes by an “attention deficit and hyperactivity scale” and subgroups by the “Children’s Depression Inventory”) and a control group (n?=?41). The clinical workup included blood samples that were drawn at 20:00 and 09:00 hours, at inclusion in both groups, and after 4.61?±?2.29 months of treatment only in the ADHD group, for measurements for allopregnanolone and dehydroepiandrosterone. Factorial analysis, adjusted for age and gender, was performed by using Stata 12.0.

Results

Methylphenidate induced the doubling of allopregnanolone levels in the predominantly inattentive ADHD patients without depressive symptoms (27.26?±?12.90 vs. 12.67?±?6.22 ng/ml, morning values). Although without statistical differences, baseline dehydroepiandrosterone levels were higher and slightly increased after methylphenidate in the ADHD subtype with depressive symptoms (7.74?±?11.46 vs. 6.18?±?5.99 ng/ml, in the morning), opposite to the lower baseline levels, and further decrease after methylphenidate in the inattentive subtype with depressive symptoms.

Conclusions

Different neurosteroids may have different baseline concentrations and differential responses to methylphenidate treatment as a function of ADHD subtype and subgroup. These differential responses may be a clinical marker of ADHD subtype and/or co-morbidities.     

Analysis of the SMAD4 gene in asthma

Considering the importance of the TGF-β signaling pathway for normal lung function and especially its roles in inflammation and tissue remodeling, key features of asthma pathology, it can be assumed that these molecules may harbor mutations in asthmatics. The aim of this study was to analyze the SMAD4 gene in patients with asthma. Analysis has encompassed exons 10, 11, 12 and 13 encoding the carboxy-terminal (MH2) domain of the SMAD4 protein, where mutations most frequently occur. The study included 50 patients (20 men and 30 women) with asthma aged between 17 and 73 years (average age 45.2±15.6 years). Polymerase chain reaction (PCR) was used to amplify exons 10, 11, 12 and 13 of the SMAD4 gene and the obtained PCR products were subjected to direct DNA sequencing. No nucleotide changes were found in any of the analyzed exons in either of the subjects. Based on the results of this study, it seems that mutations in the carboxy-terminal (MH2) domain of the SMAD4 are not present in asthmatic patients. Future research should be directed at the analysis of the complete gene, including regulatory elements, in order to resolve the exact role of SMAD4 in asthma.     

Enhanced alcohol-seeking behavior by nicotine in the posterior ventral tegmental area of female alcohol-preferring (P) rats: modulation by serotonin-3 and nicotinic cholinergic receptors

Rationale

Alcohol and nicotine co-use can reciprocally promote self-administration and drug-craving/drug-seeking behaviors. To date, the neurocircuitry in which nicotine influences ethanol (EtOH) seeking has not been elucidated. Clinical and preclinical research has suggested that the activation of the mesolimbic dopamine system is involved in the promotion of drug seeking. Alcohol, nicotine, and serotonin-3 (5-HT₃) receptors interact within the posterior ventral tegmental area (pVTA) to regulate drug reward. Recently, our laboratory has reported that systemic administration of nicotine can promote context-induced EtOH seeking.

Objectives

The goals of the current study were to (1) determine if microinjections of pharmacologically relevant levels of nicotine into the pVTA would enhance EtOH seeking, (2) determine if coadministration of nicotinic cholinergic receptor antagonist (nACh) or 5-HT₃ receptor antagonists would block the ability of nicotine microinjected into the pVTA to promote EtOH seeking, and (3) determine if 5-HT₃ receptors in the pVTA can modulate EtOH seeking.

Results

Nicotine (100 and 200 μM) microinjected into the pVTA enhanced EtOH seeking. Coinfusion with 200 μM mecamylamine (nACh antagonist) or 100 and 200 μM zacopride (5-HT₃ receptor antagonist) blocked the observed nicotine enhancement of EtOH seeking. The data also indicated that microinjection of 1 μM CPBG (5-HT₃ receptor agonist) promotes context-induced EtOH seeking; conversely, microinjection of 100 and 200 μM zacopride alone reduced context-induced EtOH seeking.

Conclusions

Overall, the results show that nicotine-enhanced EtOH-seeking behavior is modulated by 5-HT₃ and nACh receptors within the pVTA and that the 5-HT₃ receptor system within pVTA may be a potential pharmacological target to inhibit EtOH-seeking behaviors.     

Quantitative and pattern recognition analyses of magnoflorine,spinosin, 6′′′-feruloyl spinosin and jujuboside A by HPLC in Zizyphi Semen

Two rapid and simple HPLC methods with UV detector to determine three main compounds (magnoflorine, spinosin and 6′′′-feruloyl spinosin) and evaporative light scattering detector (ELSD) to determine jujuboside A were developed for the chemical analyses of Zizyphi Semen. Magnoflorine, spinosin, and 6′′′-feruloyl spinosin were separated with an YMC J’sphere ODS-H80 column (250 mm × 4.6 mm, 4 μm) by the gradient elution followed by the isocratic elution using methanol with 0.1 % formic acid and water with 0.1 % formic acid as the mobile phase. The flow rate was 1.0 mL/min. Jujuboside A was separated by HPLC–ELSD with YoungJinBioChrom Aegispak C18-L column (250 mm × 4.6 mm, 5 μm) column in a gradient elution using methanol with 0.1 % formic acid (A) and water with 0.1 % formic acid as the mobile phase. These two methods were fully validated with respect to linearity, precision, accuracy, stability, and robustness. These HPLC methods were applied successfully to quantify four compounds in a Zizyphi Semen extract. The HPLC analytical methods were validated for pattern recognition analysis by repeated analysis of 91 seed samples corresponding to 48 Zizyphus jujuba var. spinosa (J01–J48) and 43 Zizyphus mauritiana (M01–M43). The results indicate that these methods are suitable for a quality evaluation of Zizyphi Semen.