Coenzyme Q10 Upregulates Platelet cAMP/PKA Pathway and Attenuates Integrin αIIbß3 Signaling and Thrombus Growth.

SCOPE: Platelet integrin αIIbß3 is the key mediator of atherothrombosis. Supplementation of coenzyme Q10 (CoQ10), a fat-soluble molecule that exists in various foods, exerts protective cardiovascular effects. This study aims to investigate whether and how CoQ10 acts on αIIbß3 signaling and thrombosis, the major cause of cardiovascular diseases. METHODS AND RESULTS: Using a series of platelet functional assays in vitro, it is demonstrated that CoQ10 reduces human platelet aggregation, granule secretion, platelet spreading, and clot retraction. It is further demonstrated that CoQ10 inhibits platelet integrin αIIbß3 outside-in signaling. These inhibitory effects are mainly mediated by upregulating cAMP/PKA pathway, where CoQ10 stimulates the A2A adenosine receptor and decreases phosphodiesterase 3A phosphorylation. Moreover, CoQ10 attenuates murine thrombus growth and vessel occlusion in a ferric chloride (FeCl3 )-induced thrombosis model in vivo. Importantly, the randomized, double-blind, placebo-controlled clinical trial in dyslipidemic patients demonstrates that 24 weeks of CoQ10 supplementation increases platelet CoQ10 concentrations, enhances the cAMP/PKA pathway, and attenuates αIIbß3 outside-in signaling, leading to decreased platelet aggregation and granule release. CONCLUSION: Through upregulating the platelet cAMP/PKA pathway, and attenuating αIIbß3 signaling and thrombus growth, CoQ10 supplementation may play an important protective role in patients with risks of cardiovascular diseases.

Resveratrol Influences Pancreatic Islets by Opposing Effects on Electrical Activity and Insulin Release.

SCOPE: Resveratrol is suggested to improve glycemic control by activation of sirtuin 1 (SIRT1) and has already been tested clinically. Our investigation characterizes the targets of resveratrol in pancreatic beta cells and their contribution to short- and long-term effects on insulin secretion. METHODS AND RESULTS: Islets or beta cells are isolated from C57BL/6N mice. Electrophysiology is performed with microelectrode arrays and patch-clamp technique, insulin secretion and content are determined by radioimmunoassay, cAMP is measured by enzyme-linked immunosorbent assay, and cytosolic Ca2+ concentration by fluorescence methods. Resveratrol (25 µmol L-1 ) elevates [Ca2+ ]c and potentiates glucose-stimulated insulin secretion. These effects are associated with increased intracellular cAMP and are sensitive to the SIRT1 blocker Ex-527. Inhibition of EPAC1 by CE3F4 also abolishes the stimulatory effect of resveratrol. The underlying mechanism does not involve membrane depolarization as resveratrol even reduces electrical activity despite blocking KATP channels. Importantly, after prolonged exposure to resveratrol (14 days), the beneficial influence of the polyphenol on insulin release is lost. CONCLUSION: Resveratrol addresses multiple targets in pancreatic islets. Potentiation of insulin secretion is mediated by SIRT1-dependent activation of cAMP/EPAC1. Considering resveratrol as therapeutic supplement for patients with type 2 diabetes mellitus, the inhibitory influence on electrical excitability attenuates positive effects.

Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

BACKGROUND: The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. METHODS: To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. RESULTS: Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. CONCLUSIONS: Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic disorders.

Glabridin-induced vasorelaxation: Evidence for a role of BKCa channels and cyclic GMP.

BACKGROUND AND PURPOSE: Glabridin is a major flavonoid in Glycyrrhiza glabra (licorice) root, a traditional Asian medicine. Glabridin is reported to have anti-atherogenic, anti-inflammatory and anti-nephritic properties; however its effects on vascular tone remain unexplored. EXPERIMENTAL APPROACH: We examined the effect of glabridin on rat main mesenteric artery using isometric myography and also ELISA to measure cGMP levels. KEY RESULTS: Glabridin (30µM) relaxed arteries pre-constricted with the thromboxane A2 analog U46619 (0.2µM) by ~60% in an endothelium-independent manner. Relaxation to 30µM glabridin was abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1µM) and by the BKCa channel blocker tetraethyammonium (1mM) but was unaffected by the estrogen receptor antagonist ICI182780. The concentration-response curve to glabridin (0.1 to 30µM) was downshifted by the KATP channel blocker glibenclamide (10µM), the KV channel blocker 4-aminopyridine (300µM), and the KIR blocker BaCl2 (30µM). In U46619-contracted arteries partially relaxed by 0.1µM sodium nitroprusside, application of 10 and 30nM glabridin caused additional vasorelaxation. Glabridin (30µM) approximately doubled tissue [cyclic GMP]. Application of the phosphodiesterase inhibitor isobutylmethylxanthine caused a much larger rise in [cyclic GMP], and glabridin failed to cause vasorelaxation or a further rise in [cGMP] when co-applied with IBMX. CONCLUSIONS AND IMPLICATIONS: Vasorelaxation to glabridin is dependent on the opening of K+ channels, particularly BKCa, probably caused by a rise in cellular [cyclic GMP] owing to phosphodiesterase inhibition. In the presence of sodium nitroprusside an effect of glabridin is observed at nM concentrations, similar those measured in plasma following human ingestion of licorice flavonoid oil.

Study on the mechanisms of the bronchodilator effects of Folium Eriobotryae and the selected active ingredient on isolated guinea pig tracheal strips.

CONTEXT: Folium Eriobotryae (FE), the dry leaf of Eriobotrya japonica (Thunb.) Lindl. (Rosaceae), has been widely used to treat respiratory disorders. OBJECTIVE: To examine the bronchodilatory activity of FE and the potential mechanisms involved. MATERIALS AND METHODS: The effects of ethyl acetate fraction of FE (EFE) (0.05-0.3 mg/mL) on the isolated tracheal strips, and ursolic acid (UA) (5-30 µg/mL) that was the main constituent of EFE, were tested in vitro. Meanwhile, acetylcholine (Ach) and histamine (His)-induced bronchospasm were conducted in vivo in guinea pig. Furthermore, mechanisms of relaxant effects of EFE and UA were evaluated in the absence and presence of specific inhibitors. RESULTS: With in vitro studies, the contractile response evoked by Ach or His (EC50 = 0.21 and 0.16 mg/mL) was decreased by EFE, and UA caused a concentration-dependent relaxation precontracted by His (EC50 = 23.2 µg/mL). With in vivo studies, EFE strongly prolonged preconvulsive time similar to isoprenalin. The bronchodilator effects of EFE could be blocked by propranolol (1 µM), NG-nitro-l-arginine methyl ester (l-NAME) (100 µM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) (1 µM). EFE also inhibited the contraction in Ca2+-free medium and produced rightward parallel displacement of CaCl2 curves. In addition, the relaxant effects of UA could only be blocked by l-NAME and ODQ. DISCUSSION AND CONCLUSION: These results suggest that bronchodilator activities of EFE were related to activation of ß-adrenoceptor and NO/cGMP pathway. Blockage of Ca2+ channels and inhibition of IP3R-mediated internal Ca2+ release were also involved. Additionally, UA produced relaxant effects by the NO/cGMP pathway.

Inhibition of phosphodiesterase 2 reverses impaired cognition and neuronal remodeling caused by chronic stress.

Chronic stress and neuronal vulnerability have recently been recognized as factors contributing to cognitive disorders. One way to modify neuronal vulnerability is through mediation of phosphodiesterase 2 (PDE2), an enzyme that exerts its action on cognitive processes via the control of intracellular second messengers, cGMP and, to a lesser extent, cAMP. This study explored the effects of a PDE2 inhibitor, Bay 60-7550, on stress-induced learning and memory dysfunction in terms of its ramification on behavioral, morphologic, and molecular changes. Bay 60-7550 reversed stress-induced cognitive impairment in the Morris water maze, novel object recognition, and location tasks (object recognition test and/or object location test), effects prevented by treatment with 7-NI, a selective inhibitor of neuronal nitric oxide synthase; MK801, a glutamate receptor (NMDAR) inhibitor; myr-AIP, a CaMKII inhibitor; and KT5823, a protein kinase G inhibitor. Bay 60-7550 also ameliorated stress-induced structural remodeling in the CA1 of the hippocampus, leading to increases in dendritic branching, length, and spine density. However, the neuroplasticity initiated by Bay 60-7550 was not seen in the presence of 7-NI, MK801, myr-AIP, or KT5823. PDE2 inhibition reduced stress-induced extracellular-regulated protein kinase activation and attenuated stress-induced decreases in transcription factors (e.g., Elk-1, TORC1, and CREB phosphorylation) and plasticity-related proteins (e.g., Egr-1 and brain-derived neurotrophic factor). Pretreatment with inhibitors of NMDA, CaMKII, neuronal nitric oxide synthase, and protein kinase G (or protein kinase A) blocked the effects of Bay 60-7550 on cGMP or cAMP signaling. These findings indicate that the effect of PDE2 inhibition on stress-induced memory impairment is potentially mediated via modulation of neuroplasticity-related NMDAR-CaMKII-cGMP/cAMP signaling.

Inhibition of phosphodiesterase 2 augments cGMP and cAMP signaling to ameliorate pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin contributes to PH pathogenesis, and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP, which underpin the bioactivity of NO and prostacyclin. PDE5 inhibitors (eg, sildenafil) are licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH. METHODS AND RESULTS: The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide- and treprostinil-evoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), trepostinil, inorganic nitrate (NO donor), or a PDE5 inhibitor. Proliferation of pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension was reduced by BAY 60-7550, an effect further enhanced in the presence of atrial natriuretic peptide, NO, and treprostinil. CONCLUSIONS: PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity and is additive with prostacyclin analogues, PDE5 inhibitor, and NO. PDE2 inhibition represents a viable, orally active therapy for PH.

New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate.

Green tea is rich in polyphenol flavonoids including catechins. Epigallocatechin 3-gallate (EGCG) is the most abundant and potent green tea catechin. EGCG has been extensively studied for its beneficial health effects as a nutriceutical agent. Based upon its chemical structure, EGCG is often classified as an antioxidant. However, treatment of cells with EGCG results in production of hydrogen peroxide and hydroxyl radicals in the presence of Fe (III). Thus, EGCG functions as a pro-oxidant in some cellular contexts. Recent investigations have revealed many other direct actions of EGCG that are independent from anti-oxidative mechanisms. In this review, we discuss these novel molecular mechanisms of action for EGCG. In particular, EGCG directly interacts with proteins and phospholipids in the plasma membrane and regulates signal transduction pathways, transcription factors, DNA methylation, mitochondrial function, and autophagy to exert many of its beneficial biological actions.

Treatment of erectile dysfunction: new targets and strategies from recent research.

In recent years, research on penile erection has increasingly been centered on the molecular mechanisms involved. Major progress has been made in the field and at present a whole number of neurotransmitters, chemical effectors, growth factors, second-messenger molecules, ions, intercellular proteins, and hormones have been characterized as components of the complex process of erection. This knowledge has led to the discovery of several new therapeutic targets and multiple medical approaches for the treatment of erectile dysfunction (ED). This review focuses on the progress made in this field within the last few years.

Cyclic adenosine monophosphate and brain-derived neurotrophic factor decreased oxidative stress and apoptosis in developing hypothalamic neuronal cells: role of microglia.

BACKGROUND: We have previously shown that ethanol (EtOH) increases cellular apoptosis to developing neurons via the effects on oxidative stress of neurons directly and via increasing production of microglia-derived factors. To study further the mechanism of EtOH action on neuronal apoptosis, we determined the effects of 2 well-known PKA activators, dibutyryl cAMP (dbcAMP) and brain-derived neurotrophic factor (BDNF), on EtOH-activated oxidative stress and apoptotic processes in the hypothalamic neurons in the presence and absence of microglial cells' influence. METHODS: In enriched neuronal cells from fetal rat hypothalami treated with EtOH or with conditioned medium from EtOH-treated microglia, we measured cellular apoptosis by the free nucleosome assay and the levels of cAMP, BDNF, O²â», reactive oxygen species (ROS), nitrite, glutathione (GSH), and catalase following treatment with EtOH or EtOH-treated microglial culture conditioned medium. Additionally, we tested the effectiveness of dbcAMP and BDNF in preventing EtOH or EtOH-treated microglial conditioned medium on cellular apoptosis and oxidative stress in enriched hypothalamic neuronal cell in primary cultures. RESULTS: Neuronal cell cultures following treatment with EtOH or EtOH-activated microglial conditioned medium showed decreased production levels of cAMP and BDNF. EtOH also increased apoptotic death as well as oxidative status, as demonstrated by higher cellular levels of oxidants but lower levels of antioxidants, in neuronal cells. These effects of EtOH on oxidative stress and cell death were enhanced by the presence of microglia. Treatment with BDNF or dbcAMP decreased EtOH or EtOH-activated microglial conditioned medium-induced changes in the levels of intracellular free radicals, ROS and O²â», nitrite, GSH, and catalase. CONCLUSIONS: These data support the possibility that EtOH by acting directly and via increasing the production of microglial-derived factors reduces cellular levels of cAMP and BDNF to increase cellular oxidative status and apoptosis in hypothalamic neuronal cells in primary cultures.

Direct regulation of the proglucagon gene by insulin, leptin, and cAMP in embryonic versus adult hypothalamic neurons.

The proglucagon gene is expressed not only in the pancreas and intestine but also in the hypothalamus. Proglucagon-derived peptides have emerged as potential regulators of energy homeostasis. Whether leptin, insulin, or cAMP activation controls proglucagon gene expression in the hypothalamus is not known. A key reason for this has been the inaccessibility of hypothalamic proglucagon-expressing neurons and the lack of suitable neuronal cell lines. Herein we describe the mechanisms involved in the direct regulation of the proglucagon gene by insulin, leptin, and cAMP in hypothalamic cell models. Insulin, through an Akt-dependent manner, significantly induced proglucagon mRNA expression by 70% in adult-derived mHypoA-2/10 neurons and significantly suppressed it by 45% in embryonic-derived mHypoE-39 neurons. Leptin, via the Janus kinase-2/ signal transducer and activator of transcription-3 pathway, caused an initial increase by 66 and 43% at 1 h followed by a decrease by 45 and 34% at 12 h in mHypoA-2/10 and mHypoE-39 cells, respectively. Furthermore, cAMP activation by forskolin up-regulated proglucagon expression by 87% in mHypoE-39 neurons and increased proglucagon mRNA, through Epac activation, in the mHypoE-20/2 neurons. Specific regions of the proglucagon promoter were regulated by cAMP signaling, as determined by transient transfections, whereas mRNA stability assays demonstrate that insulin and leptin increase proglucagon mRNA stability in the adult cells. These findings suggest that insulin, leptin, and cAMP act directly, but differentially, on specific hypothalamic neurons to regulate proglucagon gene expression. Because proglucagon-derived peptides are potential regulators of energy homeostasis, an understanding of hypothalamic proglucagon neurons is important to further expand our knowledge of alternative feeding circuits.

Analgesic effect of iridoid glycosides from Paederia scandens (LOUR.) MERRILL (Rubiaceae) on spared nerve injury rat model of neuropathic pain.

Iridoid glycosides of Paederia scandens (IGPS) is a major active component isolated from traditional Chinese herb P. scandens (LOUR.) MERRILL (Rubiaceae). The aim of the present study was to investigate the analgesic effect of IGPS on spared nerve injury (SNI) model of neuropathic pain. The SNI model in rats was established by complete transection of the common peroneal and tibial distal branches of the sciatic nerve, leaving the sural branch intact. The mechanical withdrawal threshold (MWT) in response to mechanical stimulation was measured by electronic von Frey filaments on day 1 before operation and on days 1, 3, 5, 7, 10, and 14 after operation, respectively. Nitric oxide synthase (NOS) activity and nitric oxide (NO) production of spinal cord were measured by spectrophotometry and its cyclic guanosine monophosphate (cGMP) content by radioimmunoassay, mRNA expression of inducible NOS (iNOS) and protein kinase G type I (PKG-I, including PKG Ια and PKG Iß) of spinal cord were analyzed by RT-PCR. There was a marked mechanical hypersensitivity response observed on day 1 after operation in SNI model, which accompanied with decreased MWT. Treatment with IGPS (70, 140, 280 mg/kg) significantly alleviated SNI-induced mechanical hypersensitivity response evidenced by increased MWT; as well as markedly decreased NOS activity, NO and cGMP levels. At the same time, IGPS (70, 140, 280 mg/kg) could also inhibit mRNA expression of iNOS, PKG Ια and PKG Iß in the spinal cord. The results suggested that IGPS possesses antinociceptive effect, which may be partly related to the inhibition of NO/cGMP/PKG signaling pathway in the rat SNI model of neuropathic pain.

Exendin-4 directly improves endothelial dysfunction in isolated aortas from obese rats through the cAMP or AMPK-eNOS pathways.

AIMS: To determine whether exendin-4 might directly improve endothelial dysfunction in aorta isolated from high-fat diet-induced obese rats. METHODS: Wistar rats were randomly divided into control and obesity (OB) groups and fed. Vascular segments of obese rats were incubated in organ bath in the presence or absence of exendin-4. Nitric oxide (NO) production and nuclear transcription factor kappa B expression in vascular rings were measured. The aortic rings of the obese rats were then incubated in an organ bath with exendin-4 in the presence or absence of the following inhibitors: the AMPK, the adenylate cyclase and the NO synthase inhibitor. RESULTS: The maximum endothelium-dependent vasodilatation (EDV) value was severely reduced in the OB group. Exendin-4 treatment significantly increased the NO level, improved endothelium-dependent vasodilatation and reduced expression of NF-κB in the obese group. The beneficial effect of exendin-4 on EDV in obese rats was partly attenuated in the presence of the specific inhibitors. CONCLUSION: Exendin-4 directly improves impaired EDV of aortae from obese rats. The beneficial effect of exendin-4 appears to be mediated in part via stimulation of cAMP/AMPK-related signalling pathways and enhancement of endothelial nitric oxide synthase activity.

[Survey of clinical and experimental researches on mechanisms of acupuncture treatment of bronchial asthma].

In the present paper, the authors review the development of experimental and clinical studies on acupuncture treatment of bronchial asthma in recent 10 years. Regarding clinical studies, results showed that acupuncture could (1) regulate cardiac-pulmonary function; and (2) adjust immune state and relieve inflammatory reactions in bronchial asthma patients. Animal experiments showed that acupuncture could function in (1) improving pulmonary function; (2) reducing accumulation of the peripheral eosinophile granulocytes (EOS), relieving the infiltration of inflammatory cells in the air-passage mucosa and promoting the apoptosis of EOS in the lung and air-passages; (3) down-regulating the expression of air-passage remodeling-related protein insulin growth factor-1; (4) suppressing the secretion of tumor necrosis factor and endothelin; (5) attenuating allergic reaction; (6) regulating neuroendocrine activity; and (7) modulating intracellular second messenger activities. However, rigorous clinical study design is not enough, so that the reliability of the results is limited. In spite of many indicators of animal experiments have been selected, but their correlations are not in close association, resulting in poor complementation and mutual identification of the acquired findings. For this reason, its clinical efficacies need to be researched further according to principles of evidence-based medicine.

8-Phenylthio-adenines stimulate the expression of steroid hydroxylases, Cav3.2 Ca²âº channels, and cortisol synthesis by a cAMP-independent mechanism.

The regulation of cortisol synthesis and the expression of genes coding for steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10-50 µM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2'-OMe-cAMP, but not 8HPT-2'-OMe-cAMP or 8MeOPT-2'-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 steroid hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10-200 µM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca(2+) antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Ca(v)3.2 Ca(2+) current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Ca(v)3.2 Ca(2+) channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.

Induction of differentiation by panaxydol in human hepatocarcinoma SMMC-7721 cells via cAMP and MAP kinase dependent mechanism.

Panaxydol (PND) is one of the main non-peptidyl small molecules isolated from the lipophilic fractions of Panax notoginseng. The present study was carried out to demonstrate the potential effects of panaxydol on the induction of differentiation of human liver carcinoma cell lines SMMC-7721. Cell viability was evaluated by MTT method and Trypan blue exclusion assay respectively. The changes of morphology were detected by transmission electron microscope. Inhibitors were applied to detect the signaling pathway of differentiation. The level of intracellular cyclic AMP was determined by radioimmunoassay. The expression of p-ERK, Id1, and p21 were determined by Western blot. We found that panaxydol inhibit the proliferation of SMMC-7721 cells and caused the morphology and ultrastructure changes of SMMC-7721. Moreover, panaxydol dose-dependently increased the secretion of albumin and alkaline phosphatase activity, and decreased the secretion of AFP correspondingly. These changes of differentiation markers in SMMC-7721 can be reversed by the protein kinase A inhibitor RpcAMPS and by MAP kinase kinase 1/2 inhibitor U0126 or sorafenib. Intracellular cAMP was elevated by panaxydol in SMMC-7721 cells. Panaxydol dose-dependently decreased the expression of regulatory factors Id1 and increased the protein levels of p21 and p-ERK1/2 correspondingly. It suggested panaxydol might be of value for further exploration as a potential anti-cancer agent via cAMP and MAP kinase-dependent mechanism.

A fission yeast-based platform for phosphodiesterase inhibitor HTSs and analyses of phosphodiesterase activity.

Fission yeast strains have been engineered so that their growth behavior reflects the activity of heterologous cyclic nucleotide phosphodiesterases (PDEs). These strains can be used in High-Throughput Screens (HTSs) for PDE inhibitors that possess "drug-like" characteristics, displaying activity in a growth stimulation assay over a 48-h period. Through three generations of development, a collection of strains expressing 10 of the 11 mammalian PDE families that is appropriate for small molecule inhibitor screening has been generated in our laboratory. Strains unable to synthesize cyclic nucleotides allow characterization of PDE activity in that the enzyme's potency is reflected in the amount of either cAMP or cGMP that must be added to the growth medium to stimulate cell growth. In the future, this system could be used to screen cDNA libraries for biological regulators of target PDEs and for the construction of strains that co-express PDEs and associated regulatory proteins to facilitate molecular and genetic studies of their functions and, in particular, to identify whether different PDE-partner protein complexes show distinct patterns of inhibitor sensitivity.

Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats.

Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.

Intracellular leptin-signaling pathways in hypothalamic neurons: the emerging role of phosphatidylinositol-3 kinase-phosphodiesterase-3B-cAMP pathway.

Leptin is secreted primarily by fat cells and acts centrally, particularly in the hypothalamus, to reduce food intake and body weight. Besides the classical JAK2 (Janus kinase-2)-STAT3 (signal transducer and activator of transcription-3) pathway, several non-STAT3 pathways play an important role in mediating leptin signaling in the hypothalamus. We have demonstrated that leptin action in the hypothalamus is mediated by an insulin-like signaling pathway involving stimulation of PI3K (phosphatidylinositol-3 kinase) and PDE3B (phosphodiesterase-3B), and reduction in cAMP levels, and that a PI3K-PDE3B-cAMP pathway interacting with the JAK2-STAT3 pathway constitutes a critical component of leptin signaling in the hypothalamus. It appears that defective regulation of multiple signaling pathways in the hypothalamus causes central leptin resistance, a major cause of obesity. In this regard, we have shown that leptin resistance in hypothalamic neurons following chronic central infusion of this hormone is associated with a defect in the PI3K-PDE3B-cAMP, and not due to compromised signaling in the JAK2-STAT3 pathway. Similarly, the PI3K, but not the STAT3, pathway is impaired in the hypothalamus during the development of diet-induced obesity. Additionally, our recent work suggests that suppressor of cytokine signaling-3 negatively regulates the PI3K pathway of leptin signaling in the hypothalamus, a mechanism expected to play a significant role in diet-induced obesity. Together, the PI3K-PDE3B-cAMP pathway appears to emerge as a major mechanism of leptin signaling in the hypothalamus in regulating energy balance.

Intracellular signaling cascades induced by relaxin in the stimulation of capacitation and acrosome reaction in fresh and frozen-thawed bovine spermatozoa.

Relaxin is one of the 6-kDa peptide hormones, which acts as a pleiotropic endocrine and paracrine factor. Our previous studies revealed that sperm capacitating medium containing relaxin induced capacitation and acrosome reaction (AR) in fresh and frozen-thawed porcine or bovine spermatozoa. However, the intracellular signaling cascades involved with capacitation or AR induced by relaxin was unknown. Therefore, the present study was designed to investigate the intracellular signaling cascades involved with capacitation and AR induced by relaxin in fresh and frozen-thawed bovine spermatozoa. Spermatozoa were incubated in sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium supplemented with (40 ng ml(-1)) or without relaxin, and subjected to evaluation of chlortetracycline staining pattern, cholesterol efflux, Ca(2+)-influx, intracellular cyclic adenosine monophosphate (cAMP) and protein tyrosine phosphorylation. Capacitation and AR were increased (P<0.05) in both fresh and frozen-thawed spermatozoa incubated with relaxin. Cholesterol effluxes were greater in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa incubated with relaxin than the spermatozoa incubated without relaxin. Ca(2+)-influxes were also significantly stimulated by relaxin in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa. The Sp-TALP medium containing relaxin influenced the generation of intracellular cAMP in the fresh (P<0.01) and frozen-thawed (P<0.05) spermatozoa, and exhibited higher exposure of protein tyrosine phosphorylation in both sperm types than the medium devoid of relaxin. Therefore, the results postulate that relaxin exerts the intracellular signaling cascades involved with capacitation and AR through accelerating the cholesterol efflux, Ca(2+)-influx, intracellular cAMP and protein tyrosine phosphorylation in fresh and frozen-thawed bovine spermatozoa.