Cyclic AMP Inhibition Research Articles

Mechanism of gabapentinoid potentiation of opioid effects on cyclic AMP signaling in neuropathic pain

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.

Unresolved mystery of cyclic nucleotide second messengers, periplasmic acid phosphatases and bacterial natural competence.

We recently characterized the competitive inhibition of cyclic AMP (cAMP) on three periplasmic acid phosphatases, AphAHi, NadNHi, and eP4 (HelHi), in Haemophilus influenzae Rd KW20. This inhibitory effect is vital for orchestrating the nutritional growth and competence development in KW20. Initially discovered in Escherichia coli, the function of AphA remains however obscure. This study investigates the regulation of E. coli aphA expression under nutrient starvation conditions. Using transcriptional reporters with truncated aphA promoter sequences, we found that starvations of carbon and phosphate, but not amino acid, stimulated aphA expression through distinct promoter regions. Deletions of crp or cyaA abolished aphA expression, confirming their crucial roles. Conversely, CytR deletion increased aphA expression, suggesting CytR's role as a repressor of aphA expression. Additionally, we extended the study of three other second messengers, i.e., cyclic GMP, cyclic UMP, and cyclic CMP, each sharing structural similarities with cAMP. Notably, cGMP competitively inhibits AphAHi's acid phosphatase activity akin to cAMP. In contrast, both cUMP and cCMP stimulate AphAHi's phosphatase activity in a concentration dependent manner. Collectively, these data imply a complicated connection between nucleotide metabolism, AphA, cyclic purine and pyrimidine nucleotides in bacterial nutrient uptake and natural competence.

Targeting Orphan G Protein-Coupled Receptor 17 with T0 Ligand Impairs Glioblastoma Growth.

Simple SummaryGlioblastoma multiforme (GBM), or glioblastoma chemotherapy, has one of the poorest improvements across all types of cancers. Despite the different rationales explored in targeted therapy for taming the GBM aggressiveness, its phenotypic plasticity, drug toxicity, and adaptive resistance mechanisms pose many challenges in finding an effective cure. Our manuscript reports the expression and prognostic role of orphan receptor GPR17 in glioma, the molecular mechanism of action of the novel ligand of GPR17, and provides evidence how the T0 agonist promotes glioblastoma cell death through modulation of the MAPK/ERK, PI3K–Akt, STAT, and NF-κB pathways. The highlights are as follows: GPR17 expression is associated with greater survival for both low-grade glioma (LGG) and GBM; GA-T0, a potent GPR17 receptor agonist, causes significant GBM cell death and apoptosis; GPR17 signaling promotes cell cycle arrest at the G1 phase in GBM cells; key genes are modulated in the signaling pathways that inhibit GBM cell proliferation; and GA-T0 crosses the blood–brain barrier and reduces tumor volume.Glioblastoma, an invasive high-grade brain cancer, exhibits numerous treatment challenges. Amongst the current therapies, targeting functional receptors and active signaling pathways were found to be a potential approach for treating GBM. We exploited the role of endogenous expression of GPR17, a G protein-coupled receptor (GPCR), with agonist GA-T0 in the survival and treatment of GBM. RNA sequencing was performed to understand the association of GPR17 expression with LGG and GBM. RT-PCR and immunoblotting were performed to confirm the endogenous expression of GPR17 mRNA and its encoded protein. Biological functions of GPR17 in the GBM cells was assessed by in vitro analysis. HPLC and histopathology in wild mice and an acute-toxicity analysis in a patient-derived xenograft model were performed to understand the clinical implication of GA-T0 targeting GPR17. We observed the upregulation of GPR17 in association with improved survival of LGG and GBM, confirming it as a predictive biomarker. GA-T0-stimulated GPR17 leads to the inhibition of cyclic AMP and calcium flux. GPR17 signaling activation enhances cytotoxicity against GBM cells and, in patient tissue-derived mesenchymal subtype GBM cells, induces apoptosis and prevents proliferation by stoppage of the cell cycle at the G1 phase. Modulation of the key genes involved in DNA damage, cell cycle arrest, and in several signaling pathways, including MAPK/ERK, PI3K–Akt, STAT, and NF-κB, prevents tumor regression. In vivo activation of GPR17 by GA-T0 reduces the tumor volume, uncovering the potential of GA-T0–GPR17 as a targeted therapy for GBM treatment. Conclusion: Our analysis suggests that GA-T0 targeting the GPR17 receptor presents a novel therapy for treating glioblastoma.

G Protein-Coupling of Adhesion GPCRs ADGRE2/EMR2 and ADGRE5/CD97, and Activation of G Protein Signalling by an Anti-EMR2 Antibody

The experimental evidence that Adhesion G Protein-Coupled Receptors (aGPCRs) functionally couple to heterotrimeric G proteins has been emerging in incremental steps, but attributing biological significance to their G protein signalling function still presents a major challenge. Here, utilising activated truncated forms of the receptors, we show that ADGRE2/EMR2 and ADGRE5/CD97 are G protein-coupled in a variety of recombinant systems. In a yeast-based assay, where heterologous GPCRs are coupled to chimeric G proteins, EMR2 showed broad G protein-coupling, whereas CD97 coupled more specifically to Gα12, Gα13, Gα14 and Gαz chimeras. Both receptors induced pertussis-toxin (PTX) insensitive inhibition of cyclic AMP (cAMP) levels in mammalian cells, suggesting coupling to Gαz. EMR2 was shown to signal via Gα16, and via a Gα16/Gαz chimera, to stimulate IP1 accumulation. Finally, using an NFAT reporter assay, we identified a polyclonal antibody that activates EMR2 G protein signalling in vitro. Our results highlight the potential for the development of soluble agonists to understand further the biological effects and therapeutic opportunities for ADGRE receptor-mediated G protein signalling.

Dopamine D4 Receptor-Selective Compounds Reveal Structure-Activity Relationships that Engender Agonist Efficacy.

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer’s disease and substance use disorders. To identify new D4R-selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A-412997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide). Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cyclic AMP inhibition assays, and molecular dynamics computational modeling. We identified several novel D4R-selective (Ki ≤ 4.3 nM and >100-fold vs other D2-like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. These compounds highlight receptor–ligand interactions that control efficacy at D2-like receptors and may provide insights into targeted drug discovery, leading to a better understanding of the role of D4Rs in neuropsychiatric disorders.

Comparison of the Pharmacological Profiles of Selective PDE4B and PDE4D Inhibitors in the Central Nervous System

Inhibition of cyclic AMP (cAMP)-specific phosphodiesterase 4 (PDE4) has been proposed as a potential treatment for a series of neuropsychological conditions such as depression, anxiety and memory loss. However, the specific involvement of each of the PDE4 subtypes (PDE4A, 4B and 4C) in different categories of behavior has yet to be elucidated. In the present study, we compared the possible pharmacological effects of PDE4B and PDE4D selective inhibitors, A-33 and D159687, in mediating neurological function in mice. Both compounds were equally potent in stimulating cAMP signaling in the mouse hippocampal cell line HT-22 leading to an increase in CREB phosphorylation. In contrast, A-33 and D159687 displayed distinct neuropharmacological effects in mouse behavioral tests. A-33 has an antidepressant-like profile as indicated by reduced immobility time in the forced swim and tail suspension tasks, as well as reduced latency to feed in the novelty suppressed feeding test. D159687, on the other hand, had a procognitive profile as it improved memory in the novel object recognition test but had no antidepressant or anxiolytic benefit. The present data suggests that inhibitors targeting specific subtypes of PDE4 may exhibit differential pharmacological effects and aid a more efficient pharmacotherapy towards neuropsychological conditions.

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export.

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α2-adrenergic receptors (α2-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRβ. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Adenosine modulates light responses of rat retinal ganglion cell photoreceptors througha cAMP-mediated pathway.

Adenosine is an established neuromodulator in the mammalian retina, with A1 adenosine receptors being especially prevalent in the innermost ganglion cell layer. Activation of A1 receptors causes inhibition of adenylate cyclase, decreases in intracellular cyclic AMP (cAMP) levels and inhibition of protein kinase A (PKA). In this work, our aim was to characterize the effects of adenosine on the light responses of intrinsically photosensitive retinal ganglion cells (ipRGCs) and to determine whether these photoreceptors are subject to neuromodulation through intracellular cAMP-related signalling pathways. Using multielectrode array recordings from postnatal and adult rat retinas, we demonstrated that adenosine significantly shortened the duration of ipRGC photoresponses and reduced the number of light-evoked spikes fired by these neurons. The effects were A1 adenosine receptor-mediated, and the expression of this receptor on melanopsin-containing ipRGCs was confirmed by calcium imaging experiments on isolated cells in purified cultures. While inhibition of the cAMP/PKA pathway by adenosine shortened ipRGC light responses, stimulation of this pathway with compounds such as forskolin had the opposite effect and lengthened the duration of ipRGC spiking. Our findings reveal that the modification of ipRGC photoresponses through a cAMP/PKA pathway is a general feature of rat ganglion cell photoreceptors, and this pathway can be inhibited through activation of A1 receptors by adenosine. As adenosine levels in the retina rise at night, adenosinergic modulation of ipRGCs may serve as an internal regulatory mechanism to limit transmission of nocturnal photic signals by ipRGCs to the brain. Targeting retinal A1 adenosine receptors for ipRGC inhibition represents a potential therapeutic target for sleep disorders and migraine-associated photophobia.

Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

The cyclic-nucleotide 3',5'-cyclic AMP (cAMP) is an ancient and widespread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP-cyclic-AMP receptor protein regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein, similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP.

CRP enhances soluble LOX-1 release from macrophages by activating TNF-α converting enzyme

Circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) play an important role in the development and progression of atherosclerosis. We hypothesized that the inflammatory marker C-reactive protein (CRP) might stimulate sLOX-1 release by activating tumor necrosis factor-α converting enzyme (TACE). Macrophages differentiated from THP-1 cells were stimulated with TNF-α and further treated with CRP in the absence or presence of specific inhibitors or small interfering RNA (siRNA). Our results showed that CRP increased sLOX-1 release from activated macrophages in a dose-dependent manner and that these effects were regulated by Fc γ receptor II (FcγRII)-mediated p47(phox) phosphorylation, reactive oxygen species (ROS) production, and TACE activation. CRP also enhanced sLOX-1 release from macrophages derived from peripheral blood mononuclear cells (PBMC) of patients with acute coronary syndrome (ACS). Pretreatment with antibody against FcγRII or with CD32 siRNA, p47(phox) siRNA, apocynin, N-acetylcysteine, tumor necrosis factor-α protease inhibitor 1 (TAPI-1) or TACE siRNA attenuated sLOX-1 release induced by CRP. CRP also elevated serum sLOX-1 levels in a rabbit model of atherosclerosis. Thus, CRP might stimulate sLOX-1 release, and the underlying mechanisms possibly involved FcγRII-mediated p47(phox) phosphorylation, ROS production, and TACE activation.

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Antiplatelet Activity of Phellinus baummii Methanol Extract is Mediated by Cyclic AMP Elevation and Inhibition of Collagen‐activated Integrin‐αIIbβ3 and MAP Kinase

Phellinus baumii is a mushroom that has been used as folk medicine against various diseases and is reported to have antidiabetic, anticancer, antioxidant, antiinflammatory and antihypertensive activities. However, information on the effects of P. baumii extract in platelet function is limited. Therefore, the aim of this study was to examine the impact of a P. baumii methanol extract (PBME) on platelet activation and to investigate the mechanism behind its antiplatelet activity. PBME effects on agonist-induced platelet aggregation, granule secretion, [Ca²⁺](i) mobilization, α(IIb) β₃ activation, cyclic AMP release and mitogen-activated protein kinase (MAPK) phosphorylations were studied using rat platelets. PBME dose-dependently inhibited collagen, thrombin and ADP-induced platelet aggregation with an IC₅₀ of 51.0 ± 2.4, 54.0 ± 2.1 and 53.0 ± 4.3 μg/mL, respectively. Likewise, thrombin-induced [Ca²⁺](i) and collagen-activated ATP secretions were suppressed in PBME treated platelets. Aggregation and ATP secretion were also markedly attenuated by PBME alone or in combination with PP2 (Src inhibitor) and U-73122 (PLC inhibitor) in collagen-stimulated platelets. Besides, PBME treatment elevated basal cyclic AMP levels and inhibited collagen-induced integrin-α(IIb) β₃ activation. Moreover, PBME attenuated extracellular-signal-regulated protein kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1) phosphorylations. Further PD98059 (ERK inhibitor) and SP60025 (JNK inhibitor) reduced collagen-induced platelet aggregation and ATP secretion. In conclusion, the observed PBME antiplatelet activity may be mediated by activation of cyclic AMP and inhibition of ERK2 and JNK1 phosphorylations. Finally, these data suggest that PBME may have therapeutic potential for the treatment of cardiovascular diseases that involve aberrant platelet function.

Neuropeptide Y Y5 Receptor Promotes Cell Growth through Extracellular Signal-Regulated Kinase Signaling and Cyclic AMP Inhibition in a Human Breast Cancer Cell Line

Overexpression of neuropeptide Y (NPY) and its receptor system has been reported in various types of cancers. NPY Y5 receptor (Y5R) has been implicated in cell growth and angiogenesis. However, the role of Y5R in breast cancer is unknown. To identify the role of Y5R in breast cancer, we screened several breast cancer cell lines to examine the expression of Y5R and its function in breast cancer. All screened cell lines express both Y1 receptor and Y5R except BT-549, which expresses mainly Y5R. Binding studies showed that NPY, Y5R-selective agonist peptide, and Y5R-selective antagonist (CGP71683A) displaced (125)I-PYY binding in BT-549 cell membranes in a dose-dependent manner. The displacement studies revealed the presence of two binding sites in Y5R with IC(50) values of 29 pmol/L and 531 nmol/L. NPY inhibited forskolin-stimulated cyclic AMP accumulation with an IC(50) value of 52 pmol/L. NPY treatment of BT-549 cells induced extracellular signal-regulated kinase phosphorylation but did not alter intracellular calcium. Y5R activation stimulates BT-549 cell growth, which is inhibited by CGP71683A, pertussis toxin, and extracellular signal-regulated kinase blockade. CGP71683A alone induced cell death in a time- and dose-dependent manner in Y5R-expressing cells. The stimulation of MDA MB-231 cell migration by NPY is inhibited by CGP71683A. Together, our results suggest that Y5R plays an important role in cancer cell growth and migration and could be a novel therapeutic target for breast cancer.

Elevated cyclic AMP and PDE4 inhibition induce chemokine expression in human monocyte-derived macrophages

Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE(2)). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.

γ-Aminobutryic Acid

γ-Aminobutryic Acid

Inhibition of cyclic AMP production is not reduced in HEK293 cells expressing the alpha‐2C adrenergic receptor Del322–325 polymorphism

The human alpha‐2C adrenergic receptor inhibits norepinephrine release in the sympathetic nervous system. A four residue deletion in the third intracellular loop of this receptor, Del322–325, has been reported to exhibit reduced signaling in vitro (Small, et al., 2000) and be a potential risk factor for heart failure, presumably due to excess norepinephrine release and action. We attempted to replicate the reduced signaling, with the hypothesis that decreased signaling is due to decreased coupling of Del322–325 to Gi proteins. Our objective was to compare cyclic AMP inhibition of alpha‐2C wild type and Del322–325 receptors stably expressed in HEK293 cells. Cyclic AMP production was stimulated by 30 μM forskolin, and alpha‐2 agonists were used to inhibit this production in clones with high or low receptor expression levels. WT and Del322–325 clones with high expression levels (Bmax = 1500 and 2300 fmol/mg) did not differ in their percent inhibition (94 and 98%) or in their potencies (0.5 and 0.4 nM) for UK‐14,304. Clonidine also showed no difference between WT and Del322–325 clones with high Bmax (93 and 97%, 2.2 and 1.7 nM EC50s). Similar results were obtained in clones with low Bmax (20 and 20 fmol/mg). Therefore, in HEK293 cells, Del322–325 does not have a reduced ability to inhibit cyclic AMP production in comparison to WT.Supported by a donation from Procter and Gamble.

Inhibition of Basal and Stimulated Release of Endothelin-1 from Guinea Pig Tracheal Epithelial Cells in Culture by Beta 2-adrenoceptor Agonists and Cyclic AMP Enhancers

The effects of cyclic AMP-related compounds and beta adrenoceptor agonists on the basal and lipopolysaccharide (LPS)-stimulated release of endothelin-1 (ET-1) from guinea-pig tracheal epithelial cells (GPTEpCs) in culture were studied. Forskolin (a potent activator of adenylyl cyclase), 8-bromo-cyclic AMP (a cyclic AMP analogue), salbutamol and salmeterol (two beta 2-adrenoceptor agonists), were used to increase cyclic AMP levels. Cultured GPTEpCs released ET-1 continuously over a 24 h incubation period. The values reached 1,938 +/- 122 pg/mg of total cell proteins after 24 h. LPS (10 microg/ml) significantly stimulated the release of ET-1 by 1.6- to 1.8-fold, up to 1,262 +/- 56 pg/mg total cell proteins after an 8 h incubation period. Compound 8-bromo-cyclic AMP (10(-5), 10(-4) and 10(-3) M) reduced the basal release of ET-1 from GPTEpCs by up to 31% (P < 0.01) and the LPS stimulated release by up to 42% (P < 0.05), after an 8 h incubation period. Forskolin (10(-6), 10(-5) and 10(-4) M) also inhibited the basal release of ET-1 by up to 28% (P < 0.05) and LPS-stimulated release of ET-1 by up to 50% (P < 0.05), after an 8 h incubation period. At the concentration of 10(-5) M, forskolin increased cyclic AMP levels in GPTEpCs by 17-fold (P < 0.001) in the medium, 15 min after the beginning of the incubation. Salbutamol (10(-8) to 10(-6) M) had no effect on the basal production and release of ET-1 after 8 h. Conversely, this short acting beta 2-adrenoceptor agonist significantly reduced LPS-mediated increase of ET-1 production by up to 55% (P < 0.05) after an 8 h incubation period. Salmeterol (10(-9) M to 10(-5) M) inhibited basal and LPS-stimulated production and release of ET-1 after an 8 h incubation period (between 44 and 51%, P < 0.01). Both salbutamol and salmeterol (10(-6) M) increase cyclic AMP levels by five- and twofold, respectively (P < 0.05). In summary, these observations indicate that beta 2-adrenoceptor agonists or cyclic AMP enhancers can modulate both basal and more markedly, the enhanced production of ET-1 from LPS-activated guinea pig airway EpCs. In addition, these compounds increase cyclic AMP levels in the cells. It is suggested that there is a correlation between cyclic AMP increase and inhibition of ET-1 release by guinea pig airway EpCs. Since ET-1 production was shown to be elevated in asthmatic subjects and in patients suffering from other inflammatory lung disorders, the inhibition of its production by beta adrenoceptor agonists, such as salbutamol and salmeterol, could be added to their therapeutical benefits.

Differential Expression of CXCL12 and CXCR4 During Human Fetal Neural Progenitor Cell Differentiation

Stromal cell-derived factor 1 alpha (SDF-1alpha, CXCL12) and its receptor CXCR4 play an important role in the central nervous system (CNS) development and adulthood by mediating cell migration, enhancing precursor cell proliferation, assisting in neuronal circuit formation, and possibly regulating migration during repair. The expression pattern of CXCR4 and CXCL12 during neurogenesis has not been thoroughly elucidated. In this study, we investigated the expression of CXCL12 and CXCR4 during neural progenitor cells (NPC) differentiation by microarray analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) using human fetal NPC as a model system. The production of CXCL12 was measured by enzyme-linked immunosorbent assay (ELISA). CXCR4 expression was determined by florescence-activated cell sorting (FACS) analysis, immunocytochemical staining, and CXCR4-mediated inhibition of cyclic AMP (cAMP) accumulation. Our data demonstrated that CXCR4 expression is significantly upregulated when NPC are differentiated into neuronal precursors, whereas CXCL12 is upregulated when differentiated into astrocytes. We also provide evidence that CXCR4 localization changes as neurons mature. In neuronal precursors, CXCR4 is localized in both neuronal processes and the cell body, whereas in mature neurons, it is primarily expressed on axons and dendrites. This differential expression of CXCR4 and CXCL12 may be important for the temporal regulation of neuronal migration and circuit formation during development and possibly in adult neurogenesis and repair.

Blocking CXCR4-Mediated Cyclic AMP Suppression Inhibits Brain Tumor GrowthIn vivo

The chemokine CXCL12 and its cognate receptor CXCR4 regulate malignant brain tumor growth and are potential chemotherapeutic targets. However, the molecular basis for CXCL12-induced tumor growth remains unclear, and the optimal approach to inhibiting CXCR4 function in cancer is unknown. To develop such a therapeutic approach, we investigated the signaling pathways critical for CXCL12 function in normal and malignant cells. We discovered that CXCL12-dependent tumor growth is dependent upon sustained inhibition of cyclic AMP (cAMP) production, and that the antitumor activity of the specific CXCR4 antagonist AMD 3465 is associated with blocking cAMP suppression. Consistent with these findings, we show that pharmacologic elevation of cAMP with the phosphodiesterase inhibitor Rolipram suppresses tumor cell growth in vitro and, upon oral administration, inhibits intracranial growth in xenograft models of malignant brain tumors with comparable efficacy to AMD 3465. These data indicate that the clinical evaluation of phosphodiesterase inhibitors in the treatment of patients with brain tumors is warranted.