Salicylic acid in ginseng root alleviates skin hyperpigmentation disorders by inhibiting melanogenesis and melanosome transport.

Abnormal melanogenesis and melanosome transport can cause skin pigmentation disorders that are often treated using ginseng-based formulation. We previously found that phenolic acid compounds in ginseng root could inhibit melanin production and as a skin-whitening agents. However, mechanisms of action underlying effects of ginseng phenolic acid monomers on melanogenesis remain unclear. This study was conducted to investigate effects of salicylic acid, a main ginseng root phenolic acid component, on melanogenesis and melanosome functions in melanocytes of zebrafish and other species. Salicylic acid exhibited no cytotoxicity and reduced melanin levels and tyrosinase activity in B16F10 murine melanoma cells and normal human epidermal melanocytes regardless of prior cell stimulation with α-melanocyte stimulating hormone. Additionally, salicylic acid treatment reduced expression of melanogenic enzymes tyrosinase, tyrosinase-related protein 1 and tyrosinase-related protein 2, while reducing expression of their master transcriptional regulator, microphthalmia-associated transcription factor. Moreover, reduced phosphorylation of cAMP response-element binding protein was observed due to reduced cAMP levels resulting from salicylic acid inhibition of upstream signal regulators (adenylyl cyclase and protein kinase A). Furthermore, salicylic acid treatment suppressed expression of transport complex-associated proteins melanophilin and myosin Va in two UVB-treated melanocytic cell lines, suppressed phagocytosis of fluorescent microspheres by UVB-stimulated human keratinocytes (HaCaT), inhibited protease-activated receptor 2 activation by reducing both Ca2+ release and activation of phosphoinositide 3 kinase/AKT and mitogen-activated protein kinases and induced anti-melanogenic effects in zebrafish. Collectively, these results indicate that salicylic acid within ginseng root can inhibit melanocyte melanogenesis and melanin transport, while also suppressing keratinocyte phagocytic function.

Pulsed electromagnetic fields promote bone formation by activating the sAC-cAMP-PKA-CREB signaling pathway.

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.

Antistress Effects of Rosa rugosa Thunb. on Total Sleep Deprivation-Induced Anxiety-Like Behavior and Cognitive Dysfunction in Rat: Possible Mechanism of Action of 5-HT6 Receptor Antagonist.

Our previous results suggest that the Rosa rugosa Thunb. (family Rosaceae) alleviates endurance exercise-induced stress by decreasing oxidative stress levels. This study aimed to screen and identify the physiological antistress effects of an extract of R. rugosa (RO) on sleep deprivation-induced anxiety-like behavior and cognitive tests (in vivo) and tested for hippocampal CORT and monoamine levels (ex vivo), corticosterone (CORT)-induced injury, N-methyl-d-aspartate (NMDA) receptor, and serotonin 6 (5-hydroxytryptamine 6, 5-HT6) receptor activities (in vitro) in search of active principles and underlying mechanisms of action. We confirmed the antistress effects of RO in a sleep-deprived stress model in rat and explored the underlying mechanisms of its action. In conclusion, an R. rugosa extract showed efficacy and potential for use as an antistress therapy to treat sleep deprivation through its antagonism of the 5-HT6 receptor and resulting inhibition of cAMP activity.

Resveratrol improves hepatic steatosis by inducing autophagy through the cAMP signaling pathway.

SCOPE: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis. METHODS AND RESULTS: Via Oil red O staining, triglyceride, and ß-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid ß-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis. CONCLUSION: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment.

Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate.

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm(-2). Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm(-2) significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm(-2) showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.

Involvements of calcium channel and potassium channel in Danshen and Gegen decoction induced vasodilation in porcine coronary LAD artery.

Danshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been widely used in treating cardiovascular diseases for thousands of years in China. The present study was carried out to evaluate the effects of a Danshen and Gegen decoction (DG) on the vascular reactivity of a porcine isolated coronary artery and the underlying mechanisms involved. Porcine coronary rings were precontracted with 15 nM U46619. The involvement of endothelium-dependent mechanisms was explored by removing the endothelium; the involvement of potassium channels was investigated by the pretreatment of the artery rings with various blockers, and the involvement of the calcium channels was investigated by incubating the artery rings with Ca²âº-free buffer and priming them with high [K⁺] prior to adding CaCl2 to elicit contraction. The involvement of Ca²âº sensitization was explored by evaluating the Rho-activity expression. The results revealed that DG elicited a concentration-dependent relaxation on a U46619-precontracted coronary artery ring. These relaxation responses were not altered by the pretreatment of inhibitors of endothelium-related dilator synthases, cGMP and cAMP pathway inhibitors, potassium channel (BK(Ca), SK(Ca), K(V) and K(ATP)) blockers and endothelium removal. The K(IR) channel blocker BaCl2 only slightly attenuated the DG-induced relaxation. However, the Ca²âº-induced artery contraction was inhibited by DG. Additionally, the expression of the phosphorylated myosin light chain was inhibited by DG whereas the activity of RhoA was not affected. Therefore, DG could be a useful cardioprotective agent for vasodilation in patients who have hypertension.

Hypoglycemic effects of brassinosteroid in diet-induced obese mice.

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50-300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1-15 µM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.

Genistein prevents hyperglycemia-induced monocyte adhesion to human aortic endothelial cells through preservation of the cAMP signaling pathway and ameliorates vascular inflammation in obese diabetic mice.

Hyperglycemia-induced vascular inflammation resulting in the enhanced monocyte-endothelial cell (EC) interaction is the key event in the pathogenesis of atherosclerosis in diabetes. Here, we investigated the effect of isoflavone genistein on hyperglycemia-stimulated vascular inflammation. Human aortic EC (HAEC) were pretreated with genistein before the addition of high glucose (HG; 25 mmol/L) for 48 h. Genistein at a physiological concentration (0.1 µmol/L) significantly inhibited HG-induced adhesion of monocytes to HAEC and suppressed endothelial production of monocyte chemotactic protein-1 (MCP-1) and IL-8. Inhibition of adenylate cyclase or protein kinase A (PKA) significantly attenuated the antiadhesion effect of genistein. Consistently, genistein improved HG-impaired intracellular cAMP production and PKA activity in HAEC. Six-week-old diabetic db/db mice were untreated (db/db) or treated with a diet containing 1 g genistein/kg diet (db/db+G) for 8 wk. Their nondiabetic db/+ mice were used as normal controls. Circulating concentrations of MCP-1/JE and KC were significantly greater, whereas IL-10 concentrations were lower in db/db mice than those in normal mice. Dietary supplementation of genistein did not normalize but significantly suppressed the elevated serum concentrations of MCP-1/JE from 286 ± 30 ng/L to 181 ± 35 ng/L and KC from 321 ± 21 ng/L to 232 ± 20 ng/L while increasing that of IL-10 from 35 ± 4 ng/L to 346 ± 35 ng/L in db/db+G mice. Further, genistein treatment suppressed diabetes-induced adhesion of monocytes to EC by 87% and endothelial secretion of adhesion molecules. We conclude that genistein improves diabetes-caused vascular inflammation, which may be mediated through promoting the cAMP/PKA pathway.

A rapid interference between glucocorticoids and cAMP-activated signalling in hypothalamic neurones prevents binding of phosphorylated cAMP response element binding protein and glucocorticoid receptor at the CRE-Like and composite GRE sites of thyrotrophin-releasing hormone gene promoter.

Glucocorticoids or cAMP increase, within minutes, thyrotrophin-releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at -101/-94 bp and a composite GRE element (cGRE) at -218/-197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br-cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone-bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP- and glucocorticoid-mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP-1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br-cAMP. ChIP assays revealed phospho-CREB, c-Jun, Sp1, c-Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA-polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br-cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE-2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline-stimulated TRH transcription.

Low-energy laser irradiation promotes synovial fibroblast proliferation by modulating p15 subcellular localization.

BACKGROUND AND OBJECTIVE: Low-energy laser irradiation (low-level laser therapy) (LELI/LLLT/photobiomodulation) has been found to modulate various biological effects, especially those involved in promoting cell proliferation. Synovial fibroblasts are important in maintaining the homeostasis of articular joints and have strong chondrogenetic capacity. Here, we investigated the effect and molecular basis of LELI on synovial fibroblast proliferation. STUDY DESIGN/MATERIALS AND METHODS: HIG-82 rabbit synovial fibroblasts were cultured, and laser irradiation (660 nm) was applied at the power density of 40 mW/cm(2) for 2 minutes, corresponding to laser fluence of 4.8 J/cm(2). The effect of LELI on cell proliferation, cell cycle progression, and expression of cyclin-dependent kinase inhibitors (CKIs) were investigated. We also examined whether the effects of LELI on HIG-82 cell proliferation were affected by cAMP content, which is known to influence the cell cycle via inducing CKIs. RESULTS: LELI promoted HIG-82 synovial fibroblast proliferation and induced cytoplasmic localization of cyclin-dependent kinase inhibitor p15 (INK4B/CDKN2B). Moreover, the proliferation of HIG-82 synovial fibroblasts was reduced by cAMP, while cAMP inhibitor, SQ22536, induced p15 cytoplasmic localization and as a result, elevated synovial fibroblast proliferation was observed. In addition, the promotive effect of LELI-induced HIG-82 synovial fibroblast proliferation was abolished by cAMP treatment. Our findings suggest that cAMP may be involved in the effect of LELI on synovial fibroblast proliferation. CONCLUSION: We revealed the effect and molecular link involved in synovial fibroblast proliferation induced by 660-nm LELI. Our study provides new insights into the mechanisms by which LELI has biological effects on synovial fibroblast proliferation. These insights may contribute to further investigation on biological effects and application of LELI in regenerative medicine.

Decreased beta-adrenoceptor chronotropic response in selenium-deficient mice: negative crosstalk between iNOS activity and cAMP accumulation.

With the aim to study if selenium (Se) deficiency affects the basal frequency and cardiac response to isoproterenol (ISO), mice were fed a Se-deficient diet (Se-) or the same diet supplemented with 0.2 ppm Se as sodium selenite (Se+) for 4 wk. Atria frequency, cyclic AMP (cAMP) accumulation, nitric oxide synthase (NOS) activity, and beta-adrenoceptor-binding assay were then examined. Results showed that Se-mice have both a reduction in atria frequency as well as in cAMP content but higher NOS activity levels either at basal or after ISO stimulation. These differences were suppressed by feeding Se-mice with a Se-supplemented diet for 1 wk or by inhibition of inducible nitric oxide synthase (iNOS). Alterations observed after ISO stimulation in atria of Se-mice were not related to a beta-adrenoceptor expression modification because specific radioligand-binding parameters in cardiac membranes from Se-mice and Se+ mice were similar. The reduced response on rate and cAMP in atria from Se-mice to direct adenylate cyclase (AC) stimulation by forskolin and the shifted upward levels present in 2-amino-4-methylpyridine-treated Se-mice is in agreement with a negative crosstalk between iNOS activity and AC activity in Se-mice.

Quantification of cAMP antagonist action in vitro and in living cells.

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

8-OH-DPAT attenuates isoproterenol- but not forskolin-stimulated accumulation of cAMP in mediobasal hypothalamus.

Ovariectomized female rats were used to test the possibility that the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT), inhibits cyclic AMP (cAMP) accumulation in the mediobasal hypothalamus. Tissue slices were incubated with forskolin or with the beta-adrenergic receptor agonist, isoproterenol, to stimulate accumulation of cAMP. Both compounds increased accumulation of cAMP. The 5-HT(1A) receptor agonist, 8-OH-DPAT, reduced cAMP accumulation after stimulation by isoproterenol, but not after forskolin stimulation. These findings are discussed in terms of putative differences in the mechanisms whereby 5-HT(1A) receptors are able to inhibit stimulation of adenylate cyclase. The potential significance of these findings to 5-HT(1A) receptor-mediated inhibition of female rat lordosis behavior is also discussed.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons.

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with mu-opioid receptors. Although a direct role for opioids via delta-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the delta-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via delta-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

Curcuma longa extract protects against gastric ulcers by blocking H2 histamine receptors.

Curcuma longa has been commonly used as a traditional remedy for a variety of symptoms such as inflammation, gastritis and gastric ulcer. When C. longa extract was administered per os to pylori-ligated rat stomachs, it reduced gastric acid secretion and protected against the formation of gastric mucosal lesions. We therefore tested whether C. longa extract inhibits gastric ulcers by blocking the H(2) histamine receptor. Dimaprit, a H(2) histamine receptor agonist, induced intracellular cAMP production in U937 and HL-60 promyelocytes. Pretreatment with C. longa extract significantly blocked dimaprit-induced cAMP production in a concentration dependent manner, but had no effect on the elevation of cAMP levels triggered by isoproterenol-induced beta(2)-adrenoceptor activation in U937 cells. To identify the active component(s) of C. longa extract, we sequentially fractionated it by extraction with ethyl acetate, n-butanol and water. We found that the ethyl acetate extract showed the most potent H(2)R antagonistic effect against dimaprit-induced cAMP production. However, curcumin, a major component of C. longa extract, showed no H(2)R blocking effect. C. longa ethanol extract and ethylacetate extract also blocked the binding of [(3)H]-tiotidine to membrane receptors on HL-60 cells. These findings suggest that the extract from C. longa specifically inhibits gastric acid secretion by blocking H(2) histamine receptors in a competitive manner.

Evidence for a role of phosphodiesterase 4 in lipopolysaccharide-stimulated prostaglandin E2 production and matrix metalloproteinase-9 activity in human amniochorionic membranes.

Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-alpha and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE(2) production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.

Effect of ethanol on the regulation of corticotropin-releasing factor (CRF) gene expression.

Ethanol stimulates hypothalamic-pituitary-adrenal axis activity in vivo. To determine the cellular and molecular mechanisms through which ethanol regulates corticotropin-releasing factor (CRF) gene expression, we compared the effect of ethanol and forskolin on CRF peptide secretion and messenger RNA levels in hypothalamic primary cell cultures, and on CRF promoter activity in the NG108-15 cell line. CRF secretion, mRNA levels, and gene transcription significantly increased in response to ethanol or forskolin. Mutation of the cAMP-response element (CRE) reduced luciferase activity under basal conditions as well as in response to forskolin or ethanol. On the other hand, plasmid with five CRE repeats yielded dramatically elevated basal luciferase activity and significantly increased upregulation by ethanol. Inclusion of adenosine deaminase reduced the promoter response to ethanol. Finally a PKA inhibitor and a cAMP antagonist both decreased ethanol-induced CRF peptide secretion, gene expression, and transcription. These results suggest that ethanol upregulates CRF expression through cAMP/PKA-dependent pathways.

Effect of the statin atorvastatin on intracellular signalling by the prostacyclin receptor in vitro and in vivo.

Prostacyclin plays a central role within the vasculature. We have previously established that the prostacyclin receptor (IP) undergoes isoprenylation, a lipid modification obligate for its function. The aim of the current study was to investigate the effect of the hydroxy methyl glutaryl co-enzyme A reductase inhibitor atorvastatin on signalling and function of the IP expressed in mammalian whole cells and in platelets isolated from patients undergoing therapeutic intervention with atorvastatin. Initially, the effect of atorvastatin on signalling by the human (h) and mouse (m) IP overexpressed in human embryonic kidney 293 cells and the hIP endogenously expressed in human erythroleukaemic 92.1.7 cells was investigated. Atorvastatin significantly reduced IP-mediated cAMP generation (IC(50) 6.6-11.1 microm) and [Ca(2+)](i) mobilization (IC(50) 7.2-16.4 microm) in a concentration-dependent manner, but had no effect on signalling by the nonisoprenylated beta(2) adrenergic receptor or the alpha or beta isoforms of the human thromboxane A(2) receptor (TP). Moreover, atorvastatin significantly reduced IP-mediated crossdesensitization of signalling by TP alpha (IC(50) 10.4 microm), but not by TP beta. In contrast to the whole-cell data, atorvastatin therapy did not interfere with IP-mediated cAMP generation or IP-induced inhibition of TP-mediated aggregation of platelets isolated from human volunteers undergoing therapeutic intervention with atorvastatin (10-80 mg per daily dose). In conclusion, while data generated in whole cells indicated that atorvastatin significantly impairs signalling by both the hIP and mP, the in vivo clinical data indicated that, at the administered therapeutic dose, atorvastatin does not significantly compromise IP signalling and function in humans.

Inhibition of stimulated Jurkat cell adenosine 3',5'-cyclic monophosphate synthesis by the immunomodulatory compound HR325.

HR325 (2-cyano-3-cyclopropyl-3-hydroxy-N-[3'-methyl-4'(trifluoromethyl)-phenyl]-propenamide) is an immunomodulatory compound through pyrimidine biosynthesis inhibition with antiproliferative properties which was derived from the isoxazol compound A77 1726 [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide]. During studies of the effects on early signal transduction events of this type of compound, it was found that HR325 dose-dependently inhibited adenosine 3',5'-cyclic monophosphate (cAMP) synthesis by Jurkat cells stimulated with prostaglandin E(2), (PGE(2)), cholera toxin (CTX), or forskolin (FKN). The potency of inhibition by HR325 of FKN-stimulated cells (IC(50) 30.4 microM) was approximately 3-fold higher than that of the other agonists (11.6 and 11.7 microM) and was independent of time of preincubation for both PGE(2) and FKN. Interestingly, A77 1726, an analogue of HR325, displayed a markedly different profile of stimulus-dependent potencies. The inhibition of cAMP synthesis by HR325 when stimulated by both PGE(2) and FKN was unaffected by glucose supplementation, in contrast to HR325-inhibited ATP levels, which were restored under such conditions. Further studies revealed that HR325 reduced intracellular ATP levels by uncoupling oxidative phosphorylation, albeit with a 1000-fold lower potency than the antihelmintic drug niclosamide. In addition, glucose supplementation experiments showed that, in contrast to HR325, the niclosamide-mediated reduction of ATP levels was wholly responsible for its inhibition of PGE(2)- and FKN-stimulated cAMP synthesis.

Role of activation of calcium-sensitive K+ channels and cAMP in opioid-induced pial artery dilation.

The present study was designed to investigate the role of activation of Kca+2 channels and cAMP in opioid-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Methionine enkephalin, an endogenous mu agonist, elicited dilation that was modestly attenuated by the Kca+2 channel antagonist, iberiotoxin (10(-7) M) (7 +/- 1, 11 +/- 1 and 16 +/- 1 vs. 4 +/- 1, 7 +/- 1, and 11 +/- 1% for methionine enkephalin 10(-10), 10(-8), 10(-6) M in the absence and presence of iberiotoxin, respectively). Dilator responses to leucine enkephalin and dynorphin, endogenous delta and kappa agonists, as well as the synthetic analogues DAMGO, DPDPE, deltorphin and U50488H all were similarly attenuated by iberiotoxin. Dilation in response to methionine enkephalin was accompanied by increased CSF cAMP concentration (1170 +/- 21, 1358 +/- 22, 1473 +/- 26, and 1575 +/- 24 fmol/ml for control, 10(-10), 10(-8), 10(-6) M methionine enkephalin, respectively). Methionine enkephalin-induced dilation was attenuated by Rp 8-bromo cAMPs (10(-5) M), a cAMP antagonist (7 +/- 1, 11 +/- 1 and 17 +/- 1 vs. 2 +/- 1, 4 +/- 1, and 7 +/- 1% for methionine enkephalin 10(-10), 10(-8), and 10(-6) M in the absence and presence of Rp 8-bromo cAMPs, respectively). Dilation by the other endogenous and synthetic opioid analogues was also accompanied by elevated CSF cAMP and attenuated by Rp 8-bromo cAMPs. Additionally, dilation produced by the cAMP analogue, 8-bromo cAMP, was blunted by iberiotoxin. These data show that both cAMP and activation of Kca+2 channels contribute to opioid-induced pial artery dilation. Further, these data suggest that opioids elicit dilation, at least in part, via the sequential release of cAMP and subsequent activation of Kca+2 channels by this second messenger.