h-89 and Inflammation

Macrophages are central to inflammation resolution, an active process aimed at restoring tissue homeostasis following an inflammatory response. Here, the effects of db-cAMP on macrophage phenotype and function were investigated. Injection of db-cAMP into the pleural cavity of mice induced monocytes recruitment in a manner dependent on PKA and CCR2/CCL2 pathways. Furthermore, db-cAMP promoted reprogramming of bone-marrow-derived macrophages to a M2 phenotype as seen by increased Arg-1/CD206/Ym-1 expression and IL-10 levels (M2 markers). Db-cAMP also showed a synergistic effect with IL-4 in inducing STAT-3 phosphorylation and Arg-1 expression. Importantly, db-cAMP prevented IFN-γ/LPS-induced macrophage polarization to M1-like as shown by increased Arg-1 associated to lower levels of M1 cytokines (TNF-α/IL-6) and p-STAT1. In vivo, db-cAMP reduced the number of M1 macrophages induced by LPS injection without changes in M2 and Mres numbers. Moreover, db-cAMP enhanced efferocytosis of apoptotic neutrophils in a PKA-dependent manner and increased the expression of Annexin A1 and CD36, two molecules associated with efferocytosis. Finally, inhibition of endogenous PKA during LPS-induced pleurisy impaired the physiological resolution of inflammation. Taken together, the results suggest that cAMP is involved in the major functions of macrophages, such as nonphlogistic recruitment, reprogramming and efferocytosis, all key processes for inflammation resolution.

Topics: Animals; Annexin A1; Apoptosis; Arginase; Bucladesine; CD36 Antigens; Cell Polarity; Cellular Reprogramming; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Inflammation; Interleukin-4; Isoquinolines; Lipopolysaccharides; Macrophages; Male; Mice, Inbred BALB C; Models, Biological; Monocytes; Neutrophils; Phagocytosis; Phenotype; Phosphorylation; Pleural Cavity; Receptors, CCR2; STAT3 Transcription Factor; Sulfonamides; Time Factors

Calcitonin gene-related peptides (CGRP), an endogenous neuropeptide, play an important role in the development of neuroinflammation by acting upon its receptor. The CGRP receptor immunoreactivity was identified on Schwann cells. However the effects of CGRP on Schwann cells are unknown and the exact signaling mechanisms associated with CGRP receptor activation related to Schwann cells inflammatory responses are not well understood. We investigated the effect of CGRP on CGRP receptor activation mediates a proinflammatory signaling response in Schwann cells.. CGRP-induced ERK-MAPK phosphorylation and proinflammatory cytokines, interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) expressions were measured by immune blotting. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including CGRP (8-37), SQ 22536 and H-89.. Treatment with CGRP demonstrated a significant generation of IL-1β and IL-6 but not in the level of TNF-α. In addition, there was a temporal increase in the activated form of ERK caused by CGRP that was prevented after pretreatment with CGRP (8-37), SQ 22536 and H-89. Furthermore, use of the CGRP (8-37), ERK inhibitor PD 98059, SQ 22536 or H-89 abolished the CGRP mediated increase in IL-1β.. This investigation provides evidence for a novel CGRP activation on Schwann cells that mediates inflammatory response by increasing of IL-1β and IL-6 expression. CGRP activates the cAMP-PKA-ERK signaling cascade leading to IL-1β production. These results support the notion that CGRP may play a direct role to initiate inflammatory processes in the peripheral nervous system.

Topics: Adenine; Calcitonin Gene-Related Peptide; Cell Line; Cyclic AMP; Cytokines; Dose-Response Relationship, Drug; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Isoquinolines; MAP Kinase Signaling System; Peptides; Phosphorylation; Receptors, Calcitonin Gene-Related Peptide; Schwann Cells; Sulfonamides; Tumor Necrosis Factor-alpha

Protein phosphatase 2Cβ (PP2Cβ) was found to act as a negative regulator of NF-κB-mediated inflammatory signaling; however, its regulatory mechanism has not been examined. Here, we show that protein kinase A (PKA) phosphorylates the PP2Cβ, which was inhibited by PKA-specific inhibitor, H89. Mutation analysis of serine residues in PP2Cβ revealed that Ser-195 in PP2Cβ is phosphorylated by PKA. Importantly, PKA inhibition by H89 abrogated the Forskolin-induced destabilization of PP2Cβ against ubiquitin-dependent proteosomal degradation pathway. Furthermore, H89 treatment efficiently reversed the negative effect of Forskolin on the anti-inflammatory function of PP2Cβ. Collectively, these data suggest that PKA destabilizes PP2Cβ upon inflammatory stimuli via phosphorylation of Ser-195 in PP2Cβ.

Topics: Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Stability; Gene Expression Regulation, Enzymologic; HEK293 Cells; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Isoquinolines; NF-kappa B; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2C; Proteolysis; Serine; Signal Transduction; Sulfonamides; Tumor Necrosis Factor-alpha; Ubiquitin

During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNFα on sugar transport and to identify the intracellular mechanisms involved.. Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNFα concentrations before measuring the apical uptake of galactose, α-methyl-glucoside (MG) or fructose for 15 min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot.. TNFα inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNFα only after long pre-incubation times (24 and 48 h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush border membrane of the cells, after 24h pre-incubation with the cytokine, revealed decrease on the amount of SGLT1 and increase on the amount of GLUT5 proteins. Short-term inhibition of MG transport by TNFα was not modified by H-89 but was blocked by chelerythrine.. SGLT1 and GLUT5 expression in the plasma membrane is regulated by TNFα in the human epithelial cell line Caco-2 cells, leading to alteration on sugars transport, suggesting that TNFα could be considered as a physiological local regulator of nutrients absorption in response to an intestinal inflammatory status.

Topics: Benzophenanthridines; Biological Transport; Caco-2 Cells; Cell Line; Fructose; Galactose; Glucose Transporter Type 5; Humans; Inflammation; Intestinal Mucosa; Isoquinolines; Methylglucosides; Monosaccharide Transport Proteins; Protein Kinase Inhibitors; Sodium-Glucose Transporter 1; Sulfonamides; Tumor Necrosis Factor-alpha

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor ? (TNF/TNF?)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 ? 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A 1 (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Autophagy; Carbazoles; Cyclic AMP; Endothelium, Vascular; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Isoquinolines; Resveratrol; Signal Transduction; Sirtuin 1; Stilbenes; Sulfonamides; Tumor Necrosis Factor-alpha; Vasculitis

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

Topics: Animals; Base Sequence; Chronic Pain; CpG Islands; Cyclic AMP-Dependent Protein Kinases; Cystathionine beta-Synthase; Cysteine; Disease Models, Animal; DNA Methylation; Epigenomics; Ganglia, Spinal; Hyperalgesia; Inflammation; Isoquinolines; Molecular Sequence Data; Patch-Clamp Techniques; Promoter Regions, Genetic; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Sulfides; Sulfonamides; Tetrodotoxin

H89 is a potent inhibitor of Protein Kinase A (PKA) and Mitogen- and Stress-Activated protein Kinase 1 (MSK1) with some inhibitory activity on other members of the AGC kinase family. H89 has been extensively used in vitro but its anti-inflammatory potential in vivo has not been reported to date. To assess the anti-inflammatory properties of H89 in mouse models of asthma.. Mice were sensitized intraperitoneally (i.p.) to ovalbumin (OVA) with or without alum, and challenged intranasally with OVA. H89 (10 mg/kg) or vehicle was given i.p. two hours before each OVA challenge. Airway hyperresponsiveness (AHR) was assessed by whole-body barometric plethysmography. Inflammation was assessed by the total and differential cell counts and IL-4 and IL-5 levels in bronchoalveolar lavage (BAL) fluid. Lung inflammation, mucus production and mast cell numbers were analyzed after histochemistry. We show that treatment with H89 reduces AHR, lung inflammation, mast cell numbers and mucus production. H89 also inhibits IL-4 and IL-5 production and infiltration of eosinophils, neutrophils and lymphocytes in BAL fluid.. Taken together, our findings implicate that blockade of AGC kinases may have therapeutic potential for the treatment of allergic airway inflammation.

Topics: Animals; Asthma; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Immunoglobulin E; Immunoglobulin G; Inflammation; Isoquinolines; Lung; Male; Mast Cells; Mice; Mucus; Ovalbumin; Protein Kinase Inhibitors; Sulfonamides; Th2 Cells

In the combat against inflammation, glucocorticoids (GCs) are a widespread therapeutic. These ligands of the glucocorticoid receptor (GR) inhibit the transactivation of various transcription factors, including nuclear factor-kappaB (NF-kappaB), and alter the composition of the pro-inflammatory enhanceosome, culminating in the repression of pro-inflammatory gene expression. However, pharmacological usage of GCs in long-term treatment is burdened with a detrimental side-effect profile. Recently, we discovered that GCs can lower NF-kappaB transactivation and pro-inflammatory gene expression by abolishing the recruitment of mitogen- and stress-activated protein kinase 1 (MSK1) (EC 2.7.11.1) to pro-inflammatory gene promoters and displacing a significant fraction of MSK1 to the cytoplasm. In our current investigation in L929sA fibroblasts, upon combining GCs and MSK1 inhibitors, we discovered a dose-dependent additive repression of pro-inflammatory gene expression, most likely due to diverse and multilayered repression mechanisms employed by GCs and MSK1 inhibitors. Therefore, the combined application of GCs and MSK1 inhibitors enabled a similar level of repression of pro-inflammatory gene expression, using actually a lower concentration of GCs and MSK1 inhibitors combined than would be necessary when using these inhibitors separately. Although H89 can inhibit both MSK1 and PKA, TNF does not activate PKA (EC 2.7.11.11) and as such PKA inhibition does not mediate H89-instigated repression of TNF-stimulated gene expression. Furthermore, the additional repressive effects of liganded GR and inhibition of MSK1, are not mediated via GR transactivation mechanisms. In conclusion, these results could entail a new therapeutic strategy using lower drug concentrations, potentially leading to a more beneficial side-effect profile.

Topics: Animals; Cells, Cultured; Drug Therapy, Combination; Glucocorticoids; Inflammation; Isoquinolines; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 8; Protein Kinase Inhibitors; Sulfonamides

Considerable evidence has indicated that the aberrant, sustained enhancement of spinal NMDA receptors (NMDARs) function is closely associated with behavioral sensitization during inflammatory pain. However, the molecular mechanisms underlying inflammation-induced NMDARs hyperfunction remain poorly understood. The present study performed immunoblotting analysis to evaluate the possible changes in the protein expression of spinal NMDARs after injection of complete Freund's adjuvant (CFA) in mice. We found that CFA did not affect the total protein level of NMDARs subunit NR1 in spinal dorsal horn. However, NR1 immunoreactivity at synapses significantly increased after CFA injection, which was correlated in the time course with the development of mechanical allodynia. Inhibition of spinal NMDARs with D-APV completely eliminated the CFA-induced increase in NR1 immunoreactive density at synapses, and direct application of NMDA onto the spinal cord of naïve mice mimicked the effects of CFA, suggesting the importance of NMDARs activity in regulating the synaptic content of NR1 during inflammatory pain. Moreover, cAMP-dependent protein kinase (PKA) downstream to NMDARs was also required for NR1 synaptic expression because inhibition of PKA activity abolished the enhancement of synaptic NR1 immunoreactivity evoked by either CFA or NMDA. Thus, our data suggested that NMDARs- and PKA-dependent increase in NR1 synaptic expression represented an important mechanism for the hyperfunction of spinal NMDARs following peripheral inflammation.

Topics: 2-Amino-5-phosphonovalerate; Animals; Behavior, Animal; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Freund's Adjuvant; Functional Laterality; Gene Expression Regulation; Inflammation; Isoquinolines; Male; Mice; Mice, Inbred C57BL; N-Methylaspartate; Pain Measurement; Posterior Horn Cells; Protein Kinase Inhibitors; Receptors, N-Methyl-D-Aspartate; Serine; Spinal Cord; Subcellular Fractions; Sulfonamides; Synapses

Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone with cellular protective actions. We hypothesized that GLP-1 would protect the endothelium from injury during inflammation. Our aims were to determine the: (1) effect of GLP-1 on basal microvascular permeability, (2) effect of GLP-1 on increased microvascular permeability induced by lipopolysaccaride (LPS), (3) involvement of the GLP-1 receptor in GLP-1 activity, and (4) involvement of the cAMP/PKA pathway in GLP-1 activity. Microvascular permeability (L(p)) of rat mesenteric post-capillary venules was measured in vivo. First, the effect of GLP-1 on basal L(p) was measured. Second, after systemic LPS injection, L(p) was measured after subsequent perfusion with GLP-1. Thirdly, L(p) was measured after LPS injection and perfusion with GLP-1+GLP-1 receptor antagonist. Lastly, L(p) was measured after LPS injection and perfusion with GLP-1+inhibitors of the cAMP/PKA pathway. Results are presented as mean area under the curve (AUC)+/-SEM. GLP-1 had no effect on L(p) (AUC: baseline=27+/-1.4, GLP-1=1+/-0.4, p=0.08). LPS increased L(p) two-fold (AUC: LPS=54+/-1.7, p<0.0001). GLP-1 reduced the LPS increase in L(p) by 75% (AUC: LPS+GLP-1=34+/-1.5, p<0.0001). GLP-1 antagonism reduced the effects of GLP-1 by 60% (AUC: LPS+GLP-1+antagonist=46+/-2.0, p<0.001). The cAMP synthesis inhibitor reduced the effects of GLP-1 by 60% (AUC: LPS+GLP-1+cAMP inhibitor=46+/-1.5, p<0.0001). The PKA inhibitor reduced the effects of GLP-1 by 100% (AUC: LPS+GLP-1+PKA inhibitor=56+/-1.5, p<0.0001). GLP-1 attenuates the increase in microvascular permeability induced by LPS. GLP-1 may protect the endothelium during inflammation, thus decreasing third-space fluid loss.

Topics: Animals; Capillary Permeability; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dideoxyadenosine; Endothelium, Vascular; Enzyme Inhibitors; Female; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Inflammation; Isoquinolines; Lipopolysaccharides; Mesentery; Peptide Fragments; Perfusion; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Glucagon; Rolipram; Sulfonamides; Venules

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of Ex-4 is due to increased adiponectin mRNA level through the GLP-1R. Both forskolin and 3-isobutyl-1-methylxanthine (IBMX), which may finally elevate cyclic adenosine monophosphate (cAMP) concentration, prevented the induction of adiponectin expression by Ex-4. Moreover, H89, a protein kinase A inhibitor, blocked the effect of Ex-4 on adiponectin. On the other hand, Ex-4 decreased the mRNA levels of inflammatory adipokines. The results indicate that Ex-4 directly promotes adiponectin secretion via the protein kinase A pathway in 3T3-L1 adipocytes and may ameliorate insulin resistance.

Topics: 1-Methyl-3-isobutylxanthine; 3T3-L1 Cells; Adipocytes; Adipokines; Adiponectin; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Inflammation; Insulin Resistance; Isoquinolines; Mice; Peptides; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Receptors, Glucagon; RNA, Messenger; Sulfonamides; Venoms

Glucocorticoids are widely used anti-inflammatory and immunomodulatory agents, of which the action mechanism is mainly based on interference of hormone-activated glucocorticoid receptor (GR) with the activity of transcription factors, such as nuclear factor-kappaB (NF-kappaB). In addition to the well described interaction-based mutual repression mechanism between the GR and NF-kappaB, additional mechanisms are at play, which help to explain the efficacy of glucocorticoid-mediated gene repression. In this respect, we found that glucocorticoids counteract the recruitment of activated Mitogen- and Stress-activated protein Kinase-1 (MSK1) at inflammatory gene promoters resulting in the inhibition of NF-kappaB p65 transactivation and of concurrent histone H3 phosphorylation. Additionally, we observed that activated GR can trigger redistribution of nuclear MSK1 to the cytoplasm through a CRM1-dependent export mechanism, as a result of an interaction between liganded GR and activated MSK1. These findings unveil a novel aspect within the GR-mediated NF-kappaB-targeting anti-inflammatory mechanism.

Topics: Cell Line; Cytokines; Cytoplasm; Enzyme Induction; Exportin 1 Protein; Gene Expression Regulation; Glucocorticoids; Histones; Humans; Inflammation; Isoquinolines; Karyopherins; Ligands; Phosphorylation; Promoter Regions, Genetic; Protein Transport; Receptors, Cytoplasmic and Nuclear; Receptors, Glucocorticoid; Ribosomal Protein S6 Kinases, 90-kDa; Serine; Subcellular Fractions; Sulfonamides; Transcription Factor RelA; Tumor Necrosis Factor-alpha

TCDD (dioxin) induces a rapid inflammatory response from 3T3-L1 adipocytes as judged by prominent induction of the mRNA expression of prostaglandin-endperoxide synthase 2 (Cox-2) along with other inflammation markers within 1 h. This action of TCDD is clearly antagonized by cell pretreatment with AACOCF3 (an inhibitor of cPLA2), nifedipine (a Ca(2+) channel blocker), or 3'-methyl-4'-nitroflavone (MNF), an antagonist of the Ah receptor (AhR), suggesting the possible involvement of the nongenomic pathway of action of TCDD as shown previously in MCF10A cells [Dong, B., and Matsumura, F. (2008) Mol. Pharmacol. 74 (1), 255-263]. This early inflammatory action of TCDD is clearly different from that mediated by its classical action pathway in that the former is mediated by protein kinases such as PKC, PKA, and tyrosine kinases, but not by ARNT. Furthermore, the former is not blocked by two "DRE-decoy" treatments. Such an inflammatory effect of TCDD on 3T3-L1 adipocyes persists at least for 5 days, when the affected adipocytes exhibit significant reduction in their adipocyte characteristics. To assess the cause for the long-lasting influence of this nongenomic action of TCDD, we tested the effects of AACOCF3, exogenous arachidonic acid (AA), and H89 (an inhibitor of PKA) on the 5 day action of TCDD. These agents clearly antagonized all the long-term actions of TCDD except that on CYP1A1 induction, indicating that the influence of the nongenomic action of TCDD lasts a long time in this cell material. One of the major factors mediating its long-lasting effects has been identified to be PKA.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Arachidonic Acid; Arachidonic Acids; Cyclic AMP-Dependent Protein Kinases; Inflammation; Isoquinolines; Mice; Polychlorinated Dibenzodioxins; Sulfonamides; Time Factors

The roles of central protein kinases A and C (PKA and PKC) in various pain states have intensively been investigated during the past decade. The aim of the present study was to investigate the peripheral involvement of PKA and PKC in persistent nociceptive response, evoked pain behaviors, and inflammation induced by subcutaneous (s.c.) injection of bee venom (BV, 0.2mg/50 microl) in rats. The effects of intraplantar injection of H-89 (a PKA inhibitor, 5-100 microg/50 microl) and chelerythrine chloride (a PKC inhibitor, 5-100 microg/50 microl) on BV-elicited persistent nociception (nociceptive flinching reflex), mechanical hyperalgesia, and inflammation were systematically investigated. Pre-treatment with H-89 dose-dependently inhibited only BV-induced mechanical hyperalgesia, but not the persistent nociception and inflammation. In contrast, pre-treatment with chelerythrine chloride dose-dependently inhibited BV-induced sustained nociception and inflammation, but not the mechanical hyperalgesia. Topical pre-treatment of the sciatic nerve with 1% capsaicin significantly blocked the inhibitory effects of the PKC inhibitor on BV-induced inflammation, but not the persistent flinching response. These results indicate that peripheral PKA and PKC involvements in BV-induced pain behaviors differ, and capsaicin-sensitive afferents appear to participate in the pro-inflammatory role of PKC in the BV pain model. Findings from the present study also suggest that targeting specific peripheral protein kinases might prove effective in the treatment of persistent pain and inflammation.

Topics: Analysis of Variance; Animals; Bee Venoms; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Functional Laterality; Hyperalgesia; Inflammation; Injections, Subcutaneous; Isoquinolines; Male; Pain; Pain Measurement; Plethysmography; Protein Kinase C; Random Allocation; Rats; Rats, Sprague-Dawley; Reaction Time; Sulfonamides

A lowered threshold to the cough response frequently accompanies chronic airway inflammatory conditions. However, the mechanism(s) that from chronic inflammation results in a lowered cough threshold is poorly understood. Irritant agents, including capsaicin, resiniferatoxin, and citric acid, elicit cough in humans and in experimental animals through the activation of the transient receptor potential vanilloid 1 (TRPV1). Protease-activated receptor-2 (PAR2) activation plays a role in inflammation and sensitizes TRPV1 in cultured sensory neurons by a PKC-dependent pathway. Here, we have investigated whether PAR2 activation exaggerates TRPV1-dependent cough in guinea pigs and whether protein kinases are involved in the PAR2-induced cough modulation. Aerosolized PAR2 agonists (PAR2-activating peptide and trypsin) did not produce any cough per se. However, they potentiated citric acid- and resiniferatoxin-induced cough, an effect that was completely prevented by the TRPV1 receptor antagonist capsazepine. In contrast, cough induced by hypertonic saline, a stimulus that provokes cough in a TRPV1-independent manner, was not modified by aerosolized PAR2 agonists. The PKC inhibitor GF-109203X, the PKA inhibitor H-89, and the cyclooxygenase inhibitor indomethacin did not affect cough induced by TRPV1 agonists, but abated the exaggeration of this response produced by PAR2 agonists. In conclusion, PAR2 stimulation exaggerates TRPV1-dependent cough by activation of diverse mechanism(s), including PKC, PKA, and prostanoid release. PAR2 activation, by sensitizing TRPV1 in primary sensory neurons, may play a role in the exaggerated cough observed in certain airways inflammatory diseases such as asthma and chronic obstructive pulmonary disease.

Topics: Animals; Capsaicin; Citric Acid; Cough; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase Inhibitors; Diterpenes; Guinea Pigs; Indomethacin; Inflammation; Isoquinolines; Male; Neurons, Afferent; Protein Kinase C; Protein Kinase Inhibitors; Receptor, PAR-2; Saline Solution, Hypertonic; Sulfonamides; TRPV Cation Channels; Trypsin

Cadmium is an environmentally widely dispersed and highly toxic heavy metal that has been classified as a human carcinogen. Using the suppression subtractive hybridization technique, we identified previously 29 cadmium-inducible genes, primarily involved in inflammation, cell survival and apoptosis. Among these genes, we are particularly interested in Nor-1, because this gene belongs to the Nur77 family, which plays a key role in the apoptotic processes of a variety of cells and tissues, including the lung. In the present study, we characterized the induction of the Nur77 family genes in the lungs after cadmium exposure. Nur77, Nor-1 and Nurr1 were all induced after cadmium treatment in a dose- and time-dependent manner in WI-38 and A549 lung cell lines. Treatment with inhibitors of signaling pathways, such as PD98059 and H89, almost completely blocked the expression of Nur77, indicating that the extracellular signal-regulated kinase and protein kinase A signaling pathways are important in cadmium-induced Nur77 expression. When a plasmid encoding dominant-negative Nur77 was transfected into A549 cells, cadmium-induced apoptotic changes, such as chromosomal condensation and Bax expression, were significantly reduced, suggesting that the expression of Nur77 plays an important role in cadmium-induced apoptosis. Furthermore, the number of apoptotic cells and the expression of Nur77 was increased in lung tissues collected from cadmium-treated (30 micromol/kg body wt) Wistar rats. Taken together, these results demonstrate that cadmium induces the expression of Nur77 family genes, leading to apoptosis in lung cells, which may cause pulmonary toxicity in response to cadmium exposure.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cadmium; Cell Line, Tumor; Cell Survival; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation; Genes, Reporter; Humans; Immunohistochemistry; Inflammation; Isoquinolines; Lung; Mitogen-Activated Protein Kinases; Nuclear Receptor Subfamily 4, Group A, Member 1; Nucleic Acid Hybridization; Plasmids; Precipitin Tests; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfonamides; Time Factors; Transcription Factors; Transfection

In this study, we investigated the role of PGE(2) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE(2) accelerated ProNectin F(TM) (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin F(TM). We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE(2) effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin F(TM) and the pharmacological inhibitor of PKA, H-89, blocked PGE(2)-mediated adhesion. Consistent with mRNA expression of the G(s)-coupled EP4- and G(i)-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1-329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1-329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated G(s)-activity, which was inhibited by a G(i) inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE(2) accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclases; Animals; Cell Adhesion; Cyclic AMP; Dinoprostone; Dose-Response Relationship, Drug; Fibronectins; Inflammation; Isoquinolines; Mastocytoma; Mice; Protein Binding; Protein Kinases; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides; Tumor Cells, Cultured

1. Roles of histamine in the production of vascular endothelial growth factor (VEGF) in the carrageenin-induced granulation tissue in rats were analysed in vitro and in vivo. 2. Incubation of the minced granulation tissue in the presence of histamine (1 and 10 microM) increased the content of VEGF protein in the conditioned medium in a time- and concentration-dependent manner. The levels of VEGF mRNA in the minced granulation tissue were also increased by histamine in a concentration-dependent manner. 3. The increase in the content of VEGF protein in the conditioned medium by histamine (10 microM) was suppressed by the H(2) receptor antagonist cimetidine (IC(50) 0.37 microM), but not by the H(1) receptor antagonist pyrilamine maleate, the H(3) receptor antagonist thioperamide or the cyclo-oxygenase inhibitor indomethacin. 4. The histamine-induced increase in the content of VEGF protein in the conditioned medium was inhibited by the cyclic AMP antagonist Rp-cAMP (IC(50) 6.8 microM), and the protein kinase A inhibitor H-89 (IC(50) 12.5 microM), but not by the protein kinase C inhibitors Ro 31-8425 and calphostin C or the tyrosine kinase inhibitor genistein. 5. Simultaneous injection of cimetidine (400 microg) and indomethacin (100 microg) into the air pouch of rats additively reduced the carrageenin-induced increase in VEGF protein levels and angiogenesis in the granulation tissue as assessed by using carmine dye. 6. These findings indicate that histamine has an activity to induce VEGF production in the granulation tissue via the H(2) receptor-cyclic AMP-protein kinase A pathway and augments angiogenesis in the granulation tissue.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cells, Cultured; Cimetidine; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endothelial Growth Factors; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Granulation Tissue; Histamine; Histamine Antagonists; Immunohistochemistry; Indoles; Indomethacin; Inflammation; Isoquinolines; Lymphokines; Macrophages, Peritoneal; Male; Maleimides; Naphthalenes; Neovascularization, Pathologic; Piperidines; Protein Kinase C; Pyrilamine; Rats; Rats, Sprague-Dawley; Receptors, Histamine H2; RNA, Messenger; Specific Pathogen-Free Organisms; Sulfonamides; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

1. The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation. 2. Injection of PGE2 (10-100 ng paw-1) evoked a dose-dependent hyperalgesic effect which was mediated via a cyclic AMP-activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89. 3. The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4. 4. To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2. 5. The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down-regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine. 6. In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the intracellular levels of cyclic GMP results from activation of a soluble guanylate cyclase.

Topics: Animals; Bradykinin; Carrageenan; Cyclic AMP; Cyclic GMP; Dinoprostone; Dopamine; Hyperalgesia; Inflammation; Interleukin-1; Interleukin-6; Interleukin-8; Isoquinolines; Male; Oxadiazoles; Phosphodiesterase Inhibitors; Pyrrolidinones; Quinoxalines; Rats; Rats, Wistar; Rolipram; Second Messenger Systems; Sulfonamides; Tumor Necrosis Factor-alpha

1. In an air pouch-type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2. To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3. When the infiltrated leucocytes were incubated for 4 h in medium containing the non-selective protein kinase inhibitor K-252a (1-100 ng ml-1, 2.14-214 nM), the tyrosine kinase inhibitor genistein (1-50 micrograms ml-1, 3.7-185 microM), and the more selective protein kinase C inhibitor H-7 (5-100 micrograms ml-1, 13.7-274 microM); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration-dependent manner, but the adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor H-89 (1-1000 ng ml-1, 2.24-2240 nM) showed no effect. 4. Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte-derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF-2. Treatment of the leucocytes with K-252a, genistein, and H-7, but not H-89, inhibited production of both LDNCF-1 and LDNCF-2. 5. These results suggest that activation of tyrosine kinase and protein kinase C, but not cAMP-dependent protein kinase, is responsible for the production of LDNCF-1 and LDNCF-2. 6. The steroidal anti-inflammatory drug dexamethasone and the protein synthesis inhibitor cycloheximide inhibited neutrophil chemotactic factor production in a concentration-dependent manner. Time-course experiments showed that the inhibitory effect by dexamethasone was apparent even 30 min after the incubation.7. Mechanism for inhibiting the production of LDNCF-1 and LDNCF-2 by dexamethasone is also discussed.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Carbazoles; Cells, Cultured; Chemotactic Factors; Culture Media, Conditioned; Cycloheximide; Depression, Chemical; Dexamethasone; Genistein; Hypersensitivity; Indole Alkaloids; Inflammation; Isoelectric Focusing; Isoflavones; Isoquinolines; Leukocytes; Male; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Sulfonamides