BACKGROUND/OBJECTIVES: This study evaluated the effects and molecular mechanisms of the Schisandra chinensis fruit extract (SC) and its major compound gomisin A (GA), on the contractility of rabbit penile corpus cavernosum smooth muscle (PCCSM). MATERIALS/METHODS: PCCSM was exposed to SC or GA after appropriate pretreatment with nitric oxide synthase (NOS) blocker, guanylate cyclase blocker, adenylyl cyclase blocker or protein kinase A blocker. Subsequently, we evaluated the cyclic nucleotide in the perfusate by radioimmunoassay, protein expression level of neuronal NOS (nNOS) and endothelial NOS (eNOS) by western blot, and the interaction of SC or GA with udenafil and rolipram. RESULTS: Both SC and GA induce PCCSM relaxations in a concentration-dependent manner. Pretreatment with NOS blocker, guanylate cyclase blocker, adenylyl cyclase blocker or protein kinase A blocker result in significantly decreased relaxation. SC and GA also induce the levels of cyclic nucleotide in the perfusate in a concentration-dependent manner. Perfusion with GA also showed significantly higher levels of eNOS protein. Furthermore, the udenafil and rolipram induced relaxations of PCCSM were enhanced after exposure to SC and GA. Our results indicate that SC and GA induce the relaxation of PCCSM via the nitric oxide (NO)-cGMP and cAMP signaling pathways. CONCLUSIONS: The SC and GA are potential alternative treatments for men who want to consume natural products to ameliorate erectile function, or who do not respond to the commercially available medicines.
Adenylyl Cyclases ; Biological Products ; Blotting, Western ; Cyclic AMP-Dependent Protein Kinases ; Erectile Dysfunction ; Fruit* ; Guanosine Monophosphate* ; Guanosine* ; Guanylate Cyclase ; Humans ; Lignans ; Male ; Muscle, Smooth* ; Neurons ; Nitric Oxide Synthase ; Nitric Oxide* ; Perfusion ; Phosphodiesterase 5 Inhibitors ; Radioimmunoassay ; Relaxation ; Rolipram ; Schisandra*

PURPOSE: This phase 1 dose-escalation portion of the study evaluated the safety, pharmacokinetics (PK), and antitumor activity of TAK-264 in Asian patients with advanced gastrointestinal (GI) carcinoma or metastatic or recurrent gastric or gastroesophageal junction adenocarcinoma expressing guanylyl cyclase C (GCC). MATERIALS AND METHODS: Adult patients with advanced GI malignancies expressing GCC (H-score ≥ 10) received TAK-264 on day 1 of 3-week cycles as 30-minute intravenous infusions for up to 1 year or until disease progression or unacceptable toxicity. The primary objectives were to evaluate the safety profile including dose-limiting toxicities (DLTs) during cycle 1, determine the maximum tolerated dose (MTD), and characterize the PK profile of TAK-264. RESULTS: Twelve patients were enrolled and treated with 1.2 mg/kg (n=3), 1.5 mg/kg (n=3), or 1.8 mg/kg TAK-264 (n=6). Median number of treatment cycles received was two (range, 1 to 10). None of the patients experienced a DLT and the MTD was not determined. Ten patients (83%) experienced adverse events (AEs). The most common were neutropenia, anorexia, and nausea (each reported by four patients). Five patients (42%) experienced grade ≥ 3 AEs consisting of tumor hemorrhage and hypertension, ascites, adrenal insufficiency, neutropenia and asthenia. Serum exposure to TAK-264 increased proportionally with the dose and the median half-life was approximately 5.5-6.6 days. No patients experienced an objective response. CONCLUSION: TAK-264 demonstrated a manageable safety profile with limited antitumor activity consistent with studies conducted in Western patients with advanced GI malignancies. TAK-264 exposure increased proportionally with the dose.
Adenocarcinoma ; Adrenal Insufficiency ; Adult ; Anorexia ; Ascites ; Asian Continental Ancestry Group* ; Asthenia ; Disease Progression ; Esophagogastric Junction ; Gastrointestinal Neoplasms ; Guanylate Cyclase* ; Half-Life ; Hemorrhage ; Humans ; Hypertension ; Infusions, Intravenous ; Maximum Tolerated Dose ; Nausea ; Neutropenia ; Pharmacokinetics ; Stomach

Chorismate synthase(CS, EC:4.2.3.5) catalyses 5-enolpyruvy-shikimate-3-phosphate to form chorismate, which is the essential enzyme for chorismate biosynthesis in organisms. The amino acid sequences of CS from 79 species of higher plants were reported in GenBank at present. 125 amino acid sequences of CS from Baphicacanthus cusia and other 78 species of plants were predicted and analyzed by using various bioinformatics software, including the composition of amino acid sequences, signal peptide, leader peptide, hydrophobic/hydrophilic, transmembrane structure, coiled-coil domain, protein secondary structure, tertiary structure and functional domains. The phylogenetic tree of CS protein family was constructed and divided into eight groups by phylogenetic analysis. The homology comparison indicated that B. cusia shared a high homology with several plants such as Sesamum indicum, Nicotiana tabacum, Solanum tuberosum and so on. The open reading frame(ORF) of all samples is about 1 300 bp, the molecular weight is about 50 kDa, the isoelectric point(pI) is 5.0-8.0 which illustrated that CS protein is slightly basic. The ORF of CS we cloned in B. cusia is 1 326 bp, the amino acid residues are 442, the molecular weight is 47 kDa and pI is 8.11. The CS in B.cusia showed obvious hydrophobicity area and hydrophilicity area, no signal peptide, and may exists transmembrane structure areas. The main secondary structures of CS protein are random coil and Alpha helix, also contain three main structural domains which are an active structural domain, a PLN02754 conserved domain and a FMN binding site. The acquired information in this study would provide certain scientific basis for further study on structure-activity relationship and structure modification of CS in plants in the future.
Acanthaceae ; enzymology ; Amino Acid Sequence ; Computational Biology ; Phosphorus-Oxygen Lyases ; chemistry ; Phylogeny ; Plant Proteins ; chemistry ; Protein Structure, Secondary

Medium-chain fatty acids (MCFAs) are mostly generated from dietary triglycerides and can penetrate the blood-brain barrier. Astrocytes in the brain use MCFAs as an alternative energy source. In addition, MCFAs have various regulatory and signaling functions in astrocytes. However, it is unclear how astrocytes sense and take up MCFAs. This study demonstrates that decanoic acid (DA; C10), a saturated MCFA and a ligand of G(αs) protein-coupled receptors (G(αs)-GPCRs), is a signaling molecule in energy metabolism in primary astrocytes. cAMP synthesis and lactate release were increased via a putative G(αs)-GPCR and transmembrane adenylyl cyclase upon short-term treatment with DA. By contrast, monoamine oxidase B-dependent gamma-aminobutyric acid (GABA) synthesis was increased in primary cortical and hypothalamic astrocytes upon long-term treatment with DA. Thus, astrocytes respond to DA by synthesizing cAMP and releasing lactate upon short-term treatment, and by synthesizing and releasing GABA upon long-term treatment, similar to reactive astrocytes. Our data suggest that astrocytes in the brain play crucial roles in lipid-sensing via GPCRs and modulate neuronal metabolism or activity by releasing lactate via astrocyte-neuron lactate shuttle or GABA to influence neighboring neurons.
Adenylyl Cyclases ; Animals ; Astrocytes* ; Blood-Brain Barrier ; Brain ; Energy Metabolism ; Fatty Acids ; gamma-Aminobutyric Acid* ; Lactic Acid* ; Metabolism ; Mice* ; Monoamine Oxidase ; Neurons ; Triglycerides

Guanylyl cyclase C (GC-C) is a member of a family of enzymes that metabolize GTP to cGMP and was first identified as a receptor for heat-stable enterotoxin. Guanylin (GNY) has since been identified as an endogenous ligand for GC-C in the intestine of several mammalian species. The GNY/GC-C system regulates ion transportation and pH in the mucosa. Recently, it was reported that GC-C and GNY are involved in lipid metabolism in rat mesenteric adipose tissue macrophages. To examine the role of GC-C and GNY in lipid metabolism in cattle, we used a bovine mesenteric adipocyte primary culture system and a coculture system for bovine adipocytes and GNY-/GC-C-expressing macrophages. Fat droplets were observed to accumulate in bovine mesenteric adipocytes cultured alone, whereas few fat droplets accumulated in adipocytes indirectly cocultured with macrophages. We also observed that GC-C was present in bovine mesenteric adipose tissue, and that fat droplet accumulation decreased after in vitro GNY administration. Expressions of mRNAs encoding lipogenic factors decreased significantly in adipocytes after either coculture or GNY administration. These results suggest that the GNY/GC-C system is part of the control system for lipid accumulation in bovine mesenteric adipose tissue.
Adipocytes ; Adipose Tissue* ; Animals ; Cattle* ; Coculture Techniques ; Enterotoxins ; Guanosine Triphosphate ; Guanylate Cyclase* ; Humans ; Hydrogen-Ion Concentration ; In Vitro Techniques ; Intestines ; Ion Transport ; Lipid Metabolism ; Macrophages ; Mucous Membrane ; Rats ; RNA, Messenger

The aim of this study was to investigate the effect and action mechanism of quercetin on penile corpus cavernosum smooth muscle (PCCSM). PCCSM precontracted with phenylephrine (Phe) was treated with four different concentrations of quercetin (10−7, 10−6, 10−5 and 10−4 M). PCCSM were preincubated with N-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) to block nitric oxide synthase and guanylate cyclase, respectively. The changes in PCCSM tension were recorded, and cyclic nucleotides in the perfusate were measured by radioimmunoassay. The interactions of quercetin with phosphodiesterase type 5 inhibitors (PDE5-Is) such as sildenafil, udenafil and mirodenafil, were also evaluated. PCCSM relaxation induced by quercetin occurred in a concentrationdependent manner. The application of quercetin to PCCSM pre-treated with L-NAME and ODQ significantly inhibited the relaxation. Quercetin significantly increased cGMP in the perfusate. Furthermore, quercetin enhanced PDE5-Is-induced relaxation of PCCSM. Quercetin relaxed the PCCSM by activating the NO-cGMP signaling pathway, and it may be a therapeutic candidate or an alternative treatment for patients with erectile dysfunction who do not completely respond to PDE5-Is.
Erectile Dysfunction ; Guanylate Cyclase ; Humans ; Male ; Muscle, Smooth* ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase ; Nucleotides, Cyclic ; Phenylephrine ; Phosphodiesterase 5 Inhibitors ; Quercetin* ; Radioimmunoassay ; Relaxation ; Sildenafil Citrate

OBJECTIVETo study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD).

METHODSA total of 40 SHR rats were randomly divided into five groups: ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA.

RESULTSCompared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (P<0.05). Compared with the ADHD model group, the methylphenidate hydrochloride group and the medium- and high-dose baicalin groups had a significant increase in the ATPase activity (P<0.05), a significant reduction in the LDH activity (P<0.05), and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the methylphenidate hydrochloride group, the high-dose baicalin group had significantly greater changes in these indices (P<0.05). Compared with the low-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05); the medium- and high-dose baicalin groups had a significant reduction in the LDH activity (P<0.05) and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the medium-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05).

CONCLUSIONSBoth methylphenidate hydrochloride and baicalin can improve synaptosomal ATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

Adenosine Triphosphatases ; metabolism ; Adenylyl Cyclases ; physiology ; Animals ; Attention Deficit Disorder with Hyperactivity ; drug therapy ; physiopathology ; Cyclic AMP ; physiology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; Flavonoids ; pharmacology ; therapeutic use ; L-Lactate Dehydrogenase ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Signal Transduction ; drug effects ; Synaptosomes ; chemistry ; ultrastructure

MDL-12330A is a widely used adenylyl cyclase (AC) inhibitor that blocks AC/cAMP signaling. In this study, we demonstrated a novel antitumor activity of this drug in gastric carcinoma (GC) cell lines. In these GC cells, MDL-12330A reduced cell viability and induced cell death in a concentration-dependent manner. At a moderate concentration (~20 µM), MDL-12330A mainly induced apoptotic death whereas at concentrations greater than 20 µM, it increased non-apoptotic cell death. The induction of apoptosis was at least partially regulated by CHOP-mediated DR5 upregulation, as detected by immunoblotting and gene interference assays. More importantly, low concentrations of MDL-12330A effectively enhanced recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis and clonogenicity in these gastric cancer cells. This study demonstrates a possible role of MDL-12330A as a potential sensitizer to TRAIL, and suggests a novel therapeutic strategy targeting gastric cancer cells.
Adenylyl Cyclases ; Apoptosis* ; Cell Death ; Cell Line ; Cell Survival ; Humans ; Immunoblotting ; Stomach Neoplasms* ; Tumor Necrosis Factor-alpha ; Up-Regulation*

OBJECTIVETo explore possible mechanism of electroacupuncture (EA) for regulating immune function in anxiety disorder (AD) rats by observing the effect of acupuncture on the histology of thymus and expressions of atrial natriuretic peptide (ANP) and natriuretic peptide receptor type A (NPR- A) in thymus.

METHODSTotally 34 SD healthy rats were randomly divided into the blank control group (n = 10), the model group (n = 12), the EA group (n = 12). Anxiety model was established in rats of the model group and the EA group by using chronic unpredictable stress (CUS) stimulation. EA (15/25 Hz) at Neiguan (PC6) and Shenmen (HT7) was performed in the EA group, with 15-min needle retaining, once every other day, 15 days in total. Needle was fixed at same acupoints for 15 min without electric stimulus in the other two groups. Anxiety-like behavior was measured by elevated plus-maze (EPM) test. Pathological changes of thymus tissue were observed by optical microscope. Expressions of ANP and NPR-A in thymus were measured by immunohistochemical assay.

RESULTSThe thymus tissue in the model group was severely atrophied, with unclear structure of thymic lobules, unclear margin of thymic medulla, loosely arranged lymphocytes ,and obviously enlarged volume of thymic corpuscle. The thymus tissue in the EA group was mildly atrophied, with existent structure of thymic lobules, clear margin of thymic medulla, densely arranged lymphocytes in cortical region, and widened medullary area. Com- pared with the blank control group, the percentage of open-arms entries (OE%) in the total QE times ob- viously decreased in the model group (P < 0.05), ANP expression obviously increased (P < 0.05), and NPR-A expression obviously decreased (P < 0.01). Compared with the model group, OE% was obviously elevated (P < 0.05), ANP expression obviously decreased (P < 0.05), and NPR-A expression obviously increased (P < 0.01) in the EA group.

CONCLUSIONEA not only could reduce anxiety of rats, but also could improve chronic stress induced thymus injury through intervening synthesis and secretion of ANP, as well as the expression of NPR-A (a specific receptor of ANP).

Acupuncture Points ; Animals ; Anxiety Disorders ; therapy ; Atrial Natriuretic Factor ; metabolism ; Electroacupuncture ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Atrial Natriuretic Factor ; metabolism ; Thymus Gland ; pathology

OBJECTIVETo compare the effect of Shen-Fu Injection (SFI) and epinephrine on the expression of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) in a pig model with post-resuscitation myocardial dysfunction.

METHODSVentricular fibrillation (VF) was electrically induced in Wu-zhi-shan miniature pigs. After 8 min of untreated VF and 2 min of cardiopulmonary resuscitation (CPR), all animals were randomly administered a bolus injection of saline placebo (SA group, n=10), SFI (0.8 mg/kg, SFI group, n=10) or epinephrine (20 μg/kg, EPI group, n=10). After 4 min of CPR, a 100-J shock was delivered. If the defibrillation attempt failed to attain restoration of spontaneous circulation (ROSC), manual chest compressions were rapidly resumed for a further 2 min followed by a second defibrillation attempt. Hemodynamic variables were recorded, and plasma concentrations of catecholamines were measured. Adenylate cyclase (AC), cyclic adenosine monophosphate (cAMP) and the expressions of β1-adrenoceptor (AR) and SERCA 2a were determined.

RESULTSCardiac output, left ventricular dp/dtmax and negative dp/dtmax were significantly higher in the SFI group than in the SA and EPI groups at 4 and 6 h after ROSC. The expression of β1-AR and SERCA2a at 24 h after ROSC were significantly higher in the SFI group than in the SA and EPI groups (P<0.05 or P<0.01).

CONCLUSIONSThe administration of epinephrine during CPR decreased the expression of SERCA2a and aggravated postresuscitation myocardial function (P<0.01). SFI attenuated post-resuscitation myocardial dysfunction, and the mechanism might be related to the up-regulation of SERCA2a expression.

Adenylyl Cyclases ; metabolism ; Animals ; Blotting, Western ; Cardiac Output ; drug effects ; Cardiopulmonary Resuscitation ; Cyclic AMP ; metabolism ; Dopamine ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Epinephrine ; blood ; Heart Ventricles ; drug effects ; metabolism ; physiopathology ; Hemodynamics ; drug effects ; Injections ; Male ; Myocardium ; enzymology ; pathology ; Norepinephrine ; blood ; Receptors, Adrenergic, beta-1 ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Swine ; Swine, Miniature ; Up-Regulation ; drug effects