Identification of Target Genes of Antiarrhythmic Traditional Chinese Medicine Wenxin Keli.

Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an "ingredient-gene-path" network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P < 0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P < 0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P < 0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.

Effects of standardized isopropanolic black cohosh and estrogen on salivary function in ovariectomized rats.

Oral dryness is a common feature in menopausal women. Estrogen therapy can relieve this symptom; however, the underlying mechanism was not clear. Standardized isopropanolic black cohosh (Actaea racemosa; Remifemin) can also relieve menopausal symptoms, such as hot flashes and sweating. Our previous study showed that standardized isopropanolic black cohosh could protect the submandibular gland structure. To investigate the effects and possible mechanisms of action of estrogen and standardized isopropanolic black cohosh on submandibular gland function in ovariectomized (OVX) rats, we measured body weight, daily water consumption, and blood flow in the submandibular glands. Immunohistochemistry and western blotting were used to detect the expression of muscarinic acetylcholine receptors 1 (M1) and 3 (M3), and aquaporin 5 (AQP5) in the submandibular gland. OVX increased daily water consumption and reduced vasodilation in the submandibular gland. It suggested that ovariectomy could damage the salviary function. Moreover, the expression of M1 and M3 receptors decreased, whereas that of AQP5 increased. These changes may explain the dysfunction of saliva secretion in menopause. Estrogen and standardized isopropanolic black cohosh treatment had the same effect on daily water consumption and vasodilation in the submandibular gland. It indicated that estrogen and standardized isopropanolic black cohosh could relieve oral dryness in menopause. However, the mechanism of the two treatments may differ because standardized isopropanolic black cohosh only protected against changes in M1 expression, whereas estrogen protected against variations in M1, M3, and AQP5 expression.

Promoter IV-BDNF deficiency disturbs cholinergic gene expression of CHRNA5, CHRM2, and CHRM5: effects of drug and environmental treatments.

Brain-derived neurotrophic factor (BDNF) promotes maturation of cholinergic neurons. However, how activity-dependent BDNF expression affects specific cholinergic gene expression remains unclear. This study addressed this question by determining mRNA levels of 22 acetylcholine receptor subunits, the choline transporter (CHT), and the choline acetyltransferase (ChAT) in mice deficient in activity-dependent BDNF via promoter IV (KIV) and control wild-type mice. Quantitative RT-PCR revealed significant reductions in nicotinic acetylcholine receptor alpha 5 (CHRNA5) in the frontal cortex and hippocampus and M5 muscarinic acetylcholine receptor (CHRM5) in the hippocampus, but significant increases in M2 muscarinic acetylcholine receptor (CHRM2) in the frontal cortex of KIV mice compared to wild-type mice. Three-week treatments with fluoxetine, phenelzine, duloxetine, imipramine, or an enriched environment treatment (EET) did not affect the altered expression of these genes except that EET increased CHRNA5 levels only in KIV frontal cortex. EET also increased levels of CHRNA7, CHT, and ChAT, again only in the KIV frontal cortex. The imipramine treatment was most prominent among the four antidepressants; it up-regulated hippocampal CHRM2 and frontal cortex CHRM5 in both genotypes, and frontal cortex CHRNA7 only in KIV mice. To the best of our knowledge, this is the first evidence that BDNF deficiency disturbs expression of CHRNA5, CHRM2, and CHRM5. Our results suggest that promoter IV-BDNF deficiency - which occurs under chronic stress - causes cholinergic dysfunctions via these receptors. EET is effective on CHRNA5, while its compensatory induction of other cholinergic genes or drugs targeting CHRNA5, CHRM2, and CHRM5 may become an alternative strategy to reverse these BDNF-linked cholinergic dysfunctions.

ß1/2 or M2/3 Receptors Are Required for Different Gastrointestinal Motility Responses Induced by Acupuncture at Heterotopic or Homotopic Acupoints.

Acupuncture at homotopic acupoints or heterotopic acupoints is known to either inhibit or facilitate gastrointestinal motility, depending on the acupoint location. However, little effort has been made to investigate the roles of specific receptors (such as adrenergic and muscarinic acetylcholine receptors) in mediating the effects of acupuncture at heterotopic and homotopic acupoints. Different adrenergic receptor subtypes or cholinergic receptor subtypes are predominantly expressed in various sections of the gut, resulting in variations between the effects of acupuncture at heterotopic or homotopic acupoints on gastrointestinal motility. Here, we investigated the role of ß1/ß2 receptors and M2/M3 receptors in gastrointestinal motility regulated by acupuncture at ST37, a heterotopic acupoint, and ST25, a homotopic acupoint, by simultaneously recording intraluminal pressures in the distal colon and stomach or jejunum and examining fecal phenol red excretion in ß1/2 receptor-knockout mice and M2/3 receptor-knockout mice. We found that knockout of the M2/3 receptor significantly inhibited ST37 acupuncture-induced enhancement of gastric motility, jejunal motility, and colonic motility. Additionally, knocking out of the ß1/2 receptor significantly diminished the ST25 acupuncture-induced inhibition of gastric motility and jejunal motility without significantly altering the enhancement of colonic motility induced by acupuncture at ST25. Acupuncture at ST37 significantly accelerated gastrointestinal transition in ß1/2 receptor-knockout mice and their wild-type littermates. However, this acceleration of gastrointestinal transition was markedly diminished in M2/3 receptor-knockout mice relative to their wild-type littermates. Acupuncture at ST25 significantly increased gastrointestinal transition in ß1/2 receptor-knockout mice and significantly decreased gastrointestinal transition in M2/3 receptor-knockout mice without altering gastrointestinal transition in wild-type littermates of either. Our study revealed that M2/3 receptors are required for the gastrointestinal motility associated with whole gastrointestinal transition enhanced by acupuncture at heterotopic acupoints, whereas ß1/2 receptors are required for the same gastrointestinal motility processes inhibited by acupuncture at homotopic acupoints. Therefore, our findings reveal important biological mechanisms underlying acupuncture treatment of disorders involving gastrointestinal motility dysfunction.

Improving vagal activity ameliorates cardiac fibrosis induced by angiotensin II: in vivo and in vitro.

Cardiac remodeling is characterized by overactivity of the renin-angiotensin system (RAS) and withdrawal of vagal activity. We hypothesized that improving vagal activity could attenuate cardiac fibrosis induced by angiotensin II (Ang II) in vivo and in vitro. Rats were subjected to abdominal aorta constriction (AAC) with or without pyridostigmine (PYR) (31 mg/kg/d). After 8 weeks, PYR significantly decreased Ang II level, AT1 protein expression, and collagen deposition in cardiac tissue and improved heart rate variability, baroreflex sensitivity and cardiac function, which were abolished by atropine. In vitro, treatment of cardiac fibroblasts (CFs) with Ang II (10(-7) M) increased cell proliferation, migration, transformation, and secretory properties, which were significantly diminished by acetylcholine (ACh, 10(-6) M). Subsequently, Ang II significantly increased collagen type I expression as well as metalloproteinase (MMP)-2 expression and activity. Transforming growth factor (TGF)-ß1 expression and Smad3 phosphorylation presented a similar trend. Notably, the knockdown of the acetylcholine M2 receptor by siRNA could abolish ACh anti-fibrotic action. These data implicated cholinesterase inhibitor can increase vagal activity and reduce local Ang II level, and ACh inhibit Ang II pro-fibrotic effects. Our findings suggested that the parasympathetic nervous system can serve as a promising target for cardiac remodeling treatment.

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain.

Repeated stress in combination with pyridostigmine Part II: changes in cerebral gene expression.

Organophosphates (OP) represent a potential threat in terrorism or during military conflicts. Due to its faculty to protect cholinesterase (ChE) activity against irreversible inactivation by OP, pyridostigmine bromide (PB) was used as a prophylaxis treatment during the first Persian Gulf War. To explain dysfunctions reported by Gulf War Veterans (GWV), it was suggested a potentiation of the operational stress effects by PB given to soldiers. Our companion paper (see part 1 in the same journal issue) describes that PB treatment administered in repeated stress conditions results in long-term perturbations of learning and social behaviour. The present paper examines, in adult male Wistar rats, consequences of the association of repeated stress and PB treatment on gene expression in hypothalamus and hippocampus. PB treatment (1.5 mg/kg/day) was orally administered 30 min before each stress session to inhibit 40% of blood ChE as recommended by NATO. 10 days of stress alone induce a decrease in hypothalamic Il-1alpha expression. Treatment with PB alone increases mineralocorticoid receptor expression in hypothalamus which means that PB may thus modify stress perception by animals. Stressed-PB animals showed increase in hippocampal expression of BDNF, TrkB and CamKIIalpha, three genes implicated in memory development. As a supplement to previous studies showing behavioural and biochemical effects of the association of stress with PB, our data reveal that behavioural effects of this association may be linked with genomic changes in hippocampus. Mechanisms underlying these modifications and their link with memory disturbances reported by GWV remain to be further determined.

Coupling ion channels to receptors for biomolecule sensing.

Nanoscale electrical biosensors are promising tools for diagnostics and high-throughput screening systems. The electrical signal allows label-free assays with a high signal-to-noise ratio and fast real-time measurements. The challenge in developing such biosensors lies in functionally connecting a molecule detector to an electrical switch. Advances in this field have relied on synthetic ion-conducting pores and modified ion channels that are not yet suitable for biomolecule screening. Here we report the design and characterization of a novel bioelectric-sensing platform engineered by coupling an ion channel, which serves as the electrical probe, to G-protein-coupled receptors (GPCRs), a family of receptors that detect molecules outside the cell. These ion-channel-coupled receptors may potentially detect a wide range of ligands recognized by natural or altered GPCRs, which are known to be major pharmaceutical targets. This could form a unique platform for label-free drug screening.

The effects of five alkaloids from Bulbus Fritillariae on the concentration of cAMP in HEK cells transfected with muscarinic M(2) receptor plasmid.

The aim of this study was to investigate the effects of five alkaloids, namely verticine, verticinone, imperialine, imperialine-3beta-D-glucoside, and puqietinone, purified from Bulbus Fritillariae and used as an antitussive drug in traditional Chinese medicine, on their antimuscarinic M(2) function and the cAMP level of HEK cells transfected with muscarinic M(2) receptor plasmid. By transfecting the HEK cells with the method of calcium phosphate co-precipitation and screening with G418, the cells stably expressing M(2) receptor were identified. The expression of M(2) receptor in HEK cells was confirmed by both RT-PCR and western blot. The cAMP level in the treated cells was analyzed with RIA method ((125)I-cAMP KIT). And the results suggested that the five alkaloids could significantly elevate the cAMP concentration in the HEK cells transfected with muscarinic M(2) receptor plasmid (p < 0.01).

Retinoic acid reverses the airway hyperresponsiveness but not the parenchymal defect that is associated with vitamin A deficiency.

Airway hyperresponsiveness (AHR) is influenced by structural components of the bronchial wall, including the smooth muscle and connective tissue elements and the neuromuscular function. AHR is also influenced by parenchymally derived tethering forces on the bronchial wall, which maintain airway caliber by producing outward radial traction. Our previous work has shown that vitamin A-deficient (VAD) rats exhibit cholinergic hyperresponsiveness and a decrease in the expression and function of the muscarinic-2 receptors (M2R). We hypothesized that if decreases in radial traction from airway or parenchymal structures contributed to the VAD-related increase in AHR, then the radial traction would normalize more slowly than VAD-related alterations in neurotransmitter signaling. Rats remained vitamin A sufficient (VAS) or were rendered VAD and then maintained on the VAD diet in the presence or absence of supplementation with all-trans retinoic acid (RA). VAD was associated with an approximately twofold increase in respiratory resistance and elastance compared with VAS rats. Exposure to RA for 12 days but not 4 days restored resistance and elastance to control (VAS) levels. In VAD rats, AHR was accompanied by decreases in bronchial M2R gene expression and function, which were restored after 12 days of RA supplementation. Subepithelial bronchial elastic fibers were decreased by approximately 50% in VAD rats and were significantly restored by RA. The increase in AHR that is associated with VAD is accompanied by decreases in M2R expression and function that can be restored by RA and a reduction in airway elastic fibers that can be partially restored by RA.