cAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord.

Using neurokinin 1 receptor (NK1R) internalization to measure of substance P release in rat spinal cord slices, we found that it was induced by the adenylyl cyclase (AC) activator forskolin, by the protein kinase A (PKA) activators 6-Bnz-cAMP and 8-Br-cAMP, and by the activator of exchange protein activated by cAMP (Epac) 8-pCPT-2-O-Me-cAMP (CPTOMe-cAMP). Conversely, AC and PKA inhibitors decreased substance P release induced by electrical stimulation of the dorsal root. Therefore, the cAMP signaling pathway mediates substance P release in the dorsal horn. The effects of forskolin and 6-Bnz-cAMP were not additive with NMDA-induced substance P release and were decreased by the NMDA receptor blocker MK-801. In cultured dorsal horn neurons, forskolin increased NMDA-induced Ca2+ entry and the phosphorylation of the NR1 and NR2B subunits of the NMDA receptor. Therefore, cAMP-induced substance P release is mediated by the activating phosphorylation by PKA of NMDA receptors. Voltage-gated Ca2+ channels, but not by TRPV1 or TRPA1, also contributed to cAMP-induced substance P release. Activation of PKA was required for the effects of forskolin and the three cAMP analogs. Epac2 contributed to the effects of forskolin and CPTOMe-cAMP, signaling through a Raf - mitogen-activated protein kinase pathway to activate Ca2+ channels. Epac1 inhibitors induced NK1R internalization independently of substance P release. In rats with latent sensitization to pain, the effect of 6-Bnz-cAMP was unchanged, whereas the effect of forskolin was decreased due to the loss of the stimulatory effect of Epac2. Hence, substance P release induced by cAMP decreases during pain hypersensitivity.

Synthesis and Biochemical Evaluation of Noncyclic Nucleotide Exchange Proteins Directly Activated by cAMP 1 (EPAC1) Regulators.

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection.

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

In vitro cyst formation of ADPKD cells.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the relentless growth of numerous fluid-filled cysts in the kidneys. Mutations in PKD1 and PKD2, genes that encode polycystin 1 and 2, respectively, are responsible for most cases of ADPKD. Currently, the cellular mechanisms responsible for cyst formation remain poorly understood. In vitro models have been used by researchers to investigate cellular processes for cyst formation in carefully controlled experimental conditions. Madin-Darby canine kidney (MDCK) cells, a distal tubule epithelial cell line, were first used to form 3-dimensional (3-D) cysts within a hydrated collagen gel. This method was applied to epithelial cells cultured from cysts of human ADPKD kidneys, allowing investigators to study cellular mechanisms for cyst growth using cells that harbor the genetic mutations responsible for ADPKD in humans. Studies using ADPKD in vitro cysts have provided insight into cellular processes regulating cell proliferation, fluid secretion, and cell polarity. These assays were used to demonstrate the central role of cAMP agonists, such as arginine vasopressin, on cyst growth; and to test the effectiveness of potential therapeutic agents, including tolvaptan. Results obtained from in vitro cyst experiments demonstrate the translational value of cell model systems for investigating the mechanisms for cyst formation in human ADPKD. In this chapter, we describe protocols for growing ADPKD cells in a 3-D in vitro cyst assay and measuring total cyst volume by microscopy and image analysis.

Physiological cAMP-elevating secretagogues differentially regulate fluid and protein secretions in mouse submandibular and sublingual glands.

The mechanisms underlying the functional differences in sympathetic and parasympathetic regulation of the major salivary glands have received little attention. The acute effects of parasympathetic muscarinic (carbachol)-dependent and combined parasympathetic-dependent plus cAMP-dependent pathways on fluid secretion rates, ion composition, and protein content were assessed using a newly developed ex vivo preparation that allows the simultaneous perfusion of the mouse submandibular (SMGs) and sublingual glands (SLGs). Our results confirm that the muscarinic-dependent pathway accounts for the bulk of salivation in SMGs and SLGs, whereas costimulation with a cAMP-increasing agent (forskolin, isoproterenol, or vasoactive intestinal peptide) did not increase the flow rate. Costimulation with carbachol plus the ß-adrenergic agonist isoproterenol decreased the concentration of NaCl and produced a substantial increase in the protein and Ca2+ content of SMG but not SLG saliva, consistent with a sparse sympathetic innervation of the SLGs. On the other hand, forskolin, which bypasses receptors to increase intracellular cAMP by directly activating the enzyme adenylate cyclase, enhanced the secretion of protein and Ca2+ by both the SMGs and SLGs. In contrast, isoproterenol and vasoactive intestinal peptide specifically stimulated protein secretion in SMG and SLG salivas, respectively. In summary, cAMP-dependent signaling does not play a major role in the stimulation of fluid secretion in SMGs and SLGs, whereas each cAMP-increasing agonist behaves differently in a gland-specific manner suggesting differential expression of G protein-coupled receptors in the epithelial cells of SMGs and SLGs.

Treatment with cyclic adenosine monophosphate modulators prior to in vitro maturation alters the lipid composition and transcript profile of bovine cumulus-oocyte complexes and blastocysts.

Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus-oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2h and 16 transcripts after 24h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos.

The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes, increasing ß-adrenoceptor-mediated mitochondrial function and glucose uptake.

Recruitment and activation of brite (or beige) adipocytes has been advocated as a potential avenue for manipulating whole-body energy expenditure. Despite numerous studies illustrating the differences in gene and protein markers between brown, brite and white adipocytes, there is very little information on the adrenergic regulation and function of these brite adipocytes. We have compared the functional (cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, extracellular acidification rates, calcium influx) profiles of mouse adipocytes cultured from three contrasting depots, namely interscapular brown adipose tissue, and inguinal or epididymal white adipose tissues, following chronic treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone. Prototypical brown adipocytes readily express ß3-adrenoceptors, and ß3-adrenoceptor stimulation increases cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, and extracellular acidification rates. Treatment of brown adipocytes with rosiglitazone increases uncoupling protein 1 (UCP1) levels, and increases ß3-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and ß3-adrenoceptors following rosiglitazone treatment, which results in an increase in all ß3-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α1-adrenoceptor mediated increases in calcium influx and glucose uptake (but not mitochondrial function) in inguinal and epididymal white adipocytes. In conclusion, the PPARγ agonist rosiglitazone promotes the induction and function of brite adipocytes cultured from inguinal and epididymal white adipose depots.

PK/PD studies on non-selective PDE inhibitors in rats using cAMP as a marker of pharmacological response.

In recent years, phosphodiesterase (PDE) inhibitors have been frequently tested for the treatment of experimental inflammatory and immune disorders. It is suggested that anti-inflammatory properties of PDE inhibitors are related to their ability to increase cAMP levels. The aim of this study was to verify the hypothesis that cAMP may be a useful marker of pharmacological response following administration of non-selective PDE inhibitors (pentoxifylline and (±)-lisofylline) to endotoxemic rats. Male Wistar rats were administered LPS (1 mg kg-1, i.v.) simultaneously with either compound given at two doses (40 and 80 mg kg-1, i.v.). Levels of cAMP and both compounds in animal plasma were measured by the validated HPLC methods. Pharmacokinetic-pharmacodynamic analysis was performed using basic and modified indirect response (IDR) models II in Phoenix WinNonlin. The results of this study indicate that, in contrast to pentoxifylline, (±)-lisofylline demonstrates a non-linear pharmacokinetics in rats with endotoxemia. In vitro study using human recombinant PDE4B and PDE7A revealed the occurrence of additive interaction between studied compounds. Moreover, (±)-lisofylline is a more potent inhibitor of PDEs compared to pentoxifylline, as evidenced by lower IC50 values. Following administration of both compounds, levels of cAMP in rat plasma increased in a dose-dependent manner. The modified IDR model II better described cAMP levels over time profiles. The validity of the proposed marker was confirmed by measuring plasma TNF-α levels in the studied animals. In conclusion, cAMP may be used in future preclinical and clinical studies of some PDE inhibitors to evaluate the drug concentration-effect relationship.

Butyric acid regulates progesterone and estradiol secretion via cAMP signaling pathway in porcine granulosa cells.

Butyric acid (BA), one of the short chain fatty acids (SCFAs), has positive actions on the metabolism, inflammation, etc. However, whether it influences the reproductive physiology and if so the detail mechanism involved has not yet been determined. In this study, the porcine granulosa cells (PGCs) were treated with gradient concentrations of BA. After 24h culture, 0.05mM BA significantly stimulated the progesterone (P4) secretion (P<0.05), 5mM and 10mM BA significantly inhibited the P4 secretion (P<0.05). Simultaneously, BA up-regulated the estradiol (E2) secretion in a dose dependent manner, 5mM and 10mM BA significantly promoted the E2 level (P<0.05). In addition, 10mM BA significantly promoted the G-protein-coupled receptor 41/43 mRNA (P<0.05). Interestingly, 5mM BA treatment significantly down-regulated cyclic adenosine monophosphate (cAMP) content (P<0.05), steroidogenic acute regulatory (StAR), steroidogenic factor 1 (SF1), P450scc in the mRNA and/or protein level (P<0.05), and these actions were reversed by cAMP activator forskolin (FK). Moreover, the co-treatment of 5mM BA and bupivacaine (BPC, the cAMP inhibitor) significantly accumulated the inhibition action of BPC on cAMP, the secretion of P4, and the abundance of StAR mRNA (P<0.05), inhibited the up-regulation of 5mM BA on the E2 secretion (P<0.05). Further, the Global Proteome and KEGG pathway analysis found that 5mM BA significantly up-regulated the I3LM80 proteins (P<0.05), which is involved in the steroid biosynthesis signaling pathway. 5mM BA significantly decreased the F2Z5G3 protein level (P<0.05), and the cAMP signaling pathway. In conclusion, present findings for the first time demonstrated that BA could regulate the P4 and E2 hormone synthesis in PGCs via the cAMP signaling pathway.

The resolution of acute inflammation induced by cyclic AMP is dependent on annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.

Specific visible radiation facilitates lipolysis in mature 3T3-L1 adipocytes via rhodopsin-dependent ß3-adrenergic signaling.

The regulation of fat metabolism is important for maintaining functional and structural tissue homeostasis in biological systems. Reducing excessive lipids has been an important concern due to the concomitant health risks caused by metabolic disorders such as obesity, adiposity and dyslipidemia. A recent study revealed that unlike conventional care regimens (e.g., diet or medicine), low-energy visible radiation (VR) regulates lipid levels via autophagy-dependent hormone-sensitive lipase (HSL) phosphorylation in differentiated human adipose-derived stem cells. To clarify the underlying cellular and molecular mechanisms, we first verified the photoreceptor and photoreceptor-dependent signal cascade in nonvisual 3T3-L1 adipocytes. For a better understanding of the concomitant phenomena that result from VR exposure, mature 3T3-L1 adipocytes were exposed to four different wavelengths of VR (410, 505, 590 and 660nm) in this study. The results confirmed that specific VR wavelengths, especially 505nm than 590nm, increase intracellular cyclic adenosine monophosphate (cAMP) levels and decrease lipid droplets. Interestingly, the mRNA and protein levels of the Opn2 (rhodopsin) photoreceptor increased after VR exposure in mature 3T3-L1 adipocytes. Subsequent treatment of mature 3T3-L1 adipocytes at a specific VR wavelength induced rhodopsin- and ß3-adrenergic receptor (AR)-dependent lipolytic responses that consequently led to increases in intracellular cAMP and phosphorylated HSL protein levels. Our study indicates that photoreceptors are expressed and exert individual functions in nonvisual cells, such as adipocytes. We suggest that the VR-induced photoreceptor system could be a potential therapeutic target for the regulation of lipid homeostasis in a non-invasive manner.

ß-Adrenergic induction of lipolysis in hepatocytes is inhibited by ethanol exposure.

In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the ß-adrenergic (ß-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to ß-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the ß-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). ß-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this ß-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in ß-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that ß-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.

Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules.

Endothelial exocytosis of Weibel-Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis.

Exenatide induces an increase in vasodilatory and a decrease in vasoconstrictive mediators.

In view of the known vasodilatory effects of glucagon-like peptide-1 and exenatide, we investigated the effects of exenatide on vasoactive factors. We analysed blood samples and mononuclear cells (MNCs) from a previous study, collected after a single dose and 12 weeks of exenatide or placebo treatment in a series of 24 patients with type 2 diabetes mellitus. After exenatide treatment, plasma concentrations of atrial natriuretic peptide, cyclic guanyl monophosphate (cGMP) and cyclic adenyl monophosphate increased significantly at 12 weeks. Plasma cGMP and adenylate cyclase expression in MNCs increased significantly after a single dose. Angiotensinogen concentration fell significantly 2 hours after a single dose and at 12 weeks, while renin and angiotensin II levels fell significantly only after a single dose and not after 12 weeks of treatment. Exenatide also suppressed the plasma concentration of transforming growth factor-ß and the expression of P311 in MNCs at 12 weeks. Thus, exenatide induces an increase in a series of vasodilators, while suppressing the renin-angiotensin system. These changes may contribute to the overall vasodilatory effect of exenatide.

Essential Role of CFTR in PKA-Dependent Phosphorylation, Alkalinization, and Hyperpolarization During Human Sperm Capacitation.

Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm, and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work, we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3- -entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility, and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH, and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. J. Cell. Physiol. 232: 1404-1414, 2017. © 2016 Wiley Periodicals, Inc.

Effects of AMPK activation on lipolysis in primary rat adipocytes: studies at different glucose concentrations.

Adipose tissue plays a key role in energy homeostasis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important intracellular energy sensor. Effects of activation of AMPK by aminomidazole-4-carboxamide ribonucleotide (AICAR) on lipolysis in the rat adipocytes were determined in the presence of 3 or 12 mM glucose. Response to epinephrine or dibutyryl-cAMP was higher in the presence of 12 mM glucose. AICAR decreased lipolysis, also when glucose was replaced by alanine or succinate and without decrease in cAMP levels. AICAR attenuated epinephrine-induced decrease in adenosine triphosphate (ATP) levels, reduced glucose uptake and lactate release. These results indicate that short-term activation of AMPK by AICAR in the rat adipocytes inhibits lipolysis, due to changes in the final, followed by protein kinase A (PKA), steps of the lipolytic cascade and improves intracellular energy status. Similar effects of AICAR were observed in the presence of 3 and 12 mM glucose, which indicates that the AMPK system is operative at high glucose concentrations.

Sensitivity of GBM cells to cAMP agonist-mediated apoptosis correlates with CD44 expression and agonist resistance with MAPK signaling.

In some cell types, activation of the second messenger cAMP leads to increased expression of proapoptotic Bim and subsequent cell death. We demonstrate that suppression of the cAMP pathway is a common event across many cancers and that pharmacological activation of cAMP in glioblastoma (GBM) cells leads to enhanced BIM expression and apoptosis in specific GBM cell types. We identified the MAPK signaling axis as the determinant of cAMP agonist sensitivity in GBM cells, with high MAPK activity corresponding to cAMP resistance and low activity corresponding to sensitization to cAMP-induced apoptosis. Sensitive cells were efficiently killed by cAMP agonists alone, while targeting both the cAMP and MAPK pathways in resistant GBM cells resulted in efficient apoptosis. We also show that CD44 is differentially expressed in cAMP agonist-sensitive and -resistant cells. We thus propose that CD44 may be a useful biomarker for distinguishing tumors that may be sensitive to cAMP agonists alone or cAMP agonists in combination with other pathway inhibitors. This suggests that using existing chemotherapeutic compounds in combination with existing FDA-approved cAMP agonists may fast track trials toward improved therapies for difficult-to-treat cancers, such as GBM.

Cyclic AMP Effectors Regulate Myometrial Oxytocin Receptor Expression.

The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1ß increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1ß had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation.

Internalized Receptor for Glucose-dependent Insulinotropic Peptide stimulates adenylyl cyclase on early endosomes.

Until very recently, G-protein dependent signal of GPCRs was thought to originate exclusively from the plasma membrane and internalized GPCRs were considered silent. Here, we demonstrated that, once internalized and located in the membrane of early endosomes, glucose-dependent Insulinotropic receptor (GIPR) continues to trigger production of cAMP and PKA activation. Direct evidence is based on identification of the active form of Gαs in early endosomes containing GIPR using a genetically encoded GFP tagged nanobody, and on detection of a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, compared to endosomes without GIP. Furthermore, decrease of the sustained phase of cAMP production and PKA activation kinetics as well as reversibility of cAMP production and PKA activity following GIP washout in cells treated with a pharmacological inhibitor of GIPR internalization, and continuous increase of cAMP level over time in the presence of dominant-negative Rab7, which causes accumulation of early endosomes in cells, were noticed. Hence the GIPR joins the few GPCRs which signal through G-proteins both at plasma membrane and on endosomes.

α-MSH modulates cell adhesion and inflammatory responses of synovial fibroblasts from osteoarthritis patients.

INTRODUCTION: The synovium is a target for neuropeptides. Melanocortins have attained particular attention as they elicit antiinflammatory effects. Although synovial fluid from patients with rheumatic diseases contains α-melanocyte-stimulating hormone (α-MSH) it is unknown whether synovial fibroblasts generate α-MSH and respond to melanocortins. METHODS: Synovial tissue was obtained from osteoarthritis (OA) patients. Cells were isolated and prepared either as primary mixed synoviocytes or propagated as synovial fibroblasts (OASFs). Melanocortin receptor (MC) and proopiomelanocortin (POMC) expression were investigated by endpoint RT-PCR, immunofluorescence and Western immunoblotting. Functional coupling of MC1 was assessed by cAMP and Ca(2+) assays. Cell adhesion was monitored by the xCELLigence system. Secretion of α-MSH, tumour necrosis factor (TNF), interleukin (IL)-6 and IL-8 was determined by ELISA. RESULTS: OASFs in vitro expressed MC1. MC1 transcripts were present in synovial tissue and appropriate immunoreactivity was detected in synovial fibroblasts in situ. OASFs contained truncated POMC transcripts but neither full-length POMC mRNA, POMC protein nor α-MSH were detectable. In accordance with this only truncated POMC transcripts were present in synovial tissue. α-MSH increased cAMP dose-dependently but did not alter calcium in OASFs. α-MSH also enhanced adhesion of OASFs to fibronectin and reduced TNF, IL-6 and IL-8 secretion in primary mixed synoviocyte cultures. In OASFs, α-MSH modulated basal and TNF/IL-1ß-mediated secretion of IL-6 and IL-8. CONCLUSION: Synovial fibroblasts express MC1in vitro and in situ. α-MSH elicits biological effects in these cells suggesting an endogenous immunomodulatory role of melanocortins within the synovium. Our results encourage in vivo studies with melanocortins in OA models.