Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.

Stimulation of phosphatidylinositol hydrolysis, protein kinase C translocation, and mitogen-activated protein kinase activity by bradykinin in rat ventricular myocytes: dissociation from the hypertrophic response.

In ventricular myocytes cultured from neonatal rat hearts, bradykinin (BK), kallidin or BK(1-8) [(Des-Arg9)BK] stimulated PtdinsP2 hydrolysis by 3-4-fold. EC50 values were 6 nM (BK), 2 nM (kallidin), and 14 microM [BK(1-8)]. BK or kallidin stimulated the rapid (less than 30 s) translocation of more than 80% of the novel protein kinase C (PKC) isoforms nPKC-delta and nPKC-epsilon from the soluble to the particulate fraction. EC50 values for nPKC-delta translocation by BK or kallidin were 10 and 2 nM respectively. EC50 values for nPKC-epsilon translocation by BK or kallidin were 2 and 0.6 nM respectively. EC50 values for the translocation of nPKC-delta and nPKC-epsilon by BK(1-8) were more than 5 microM. The classical PKC, cPKC-alpha, and the atypical PKC, nPKC-zeta, did not translocate. BK caused activation and phosphorylation of p42-mitogen-activated protein kinase (MAPK) (maximal at 3-5 min, 30-35% of p42-MAPK phosphorylated). p44-MAPK was similarly activated. EC50 values for p42/p44-MAPK activation by BK were less than 1 nM whereas values for BK(1-8) were more than 10 microM. The order of potency [BK approximately equal to kallidin > BK (1-8)] for the stimulation of PtdInsP2 hydrolysis, nPKC-delta and nPKC-epsilon translocation, and p42/p44-MAPK activities suggests involvement of the B2 BK receptor subtype. In addition, stimulation of all three processes by BK was inhibited by the B2BK receptor-selective antagonist HOE140 but not by the B1-selective antagonist Leu8BK(1-8). Exposure of cells to phorbol 12-myristate 13-acetate for 24 h inhibited subsequent activation of p42/p44-MAPK by BK suggesting participation of nPKC (and possibly cPKC) isoforms in the activation process. Thus, like hypertrophic agents such as endothelin-1 (ET-1) and phenylephrine (PE), BK activates PtdInsP2 hydrolysis, translocates nPKC-delta, and nPKC-epsilon, and activates p42/p44-MAPK. However, in comparison with ET-1 and PE, BK was only weakly hypertrophic as assessed by cell morphology and patterns of gene expression. This difference could not be attributed to dissimilarities between the duration of activation of p42/p44-MAPK by BK or ET-1. Thus activation of these signalling pathways alone may be insufficient to induce a powerful hypertrophic response.

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 mu L) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced similar to 20% and 10 min after an acute it? vivo stimulus with insulin, the plasma membrane GLUT4 content was similar to 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (similar to 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright (C) 2010 John Wiley & Sons, Ltd.

Major determinants of photoinduced cell death: Subcellular localization versus photosensitization efficiency

We present a study on whether and to what extent subcellular localization may compete favorably with photosensitization efficiency with respect to the overall efficiency of photoinduced cell death. We have compared the efficiency with which two cationic photosensitizers, namely methylene blue (MB) and crystal violet (CV), induce the photoinduced death of human cervical adenocarcinoma (HeLa) cells. Whereas MB is well known to generate singlet oxygen and related triplet excited species with high quantum yields in a variety of biological and chemical environments (i.e., acting as a typical type II photosensitizer), the highly mitochondria-specific CV produces triplet species and singlet oxygen with low yields, acting mostly via the classical type I mechanism (e.g., via free radicals). The findings described here indicate that the presumably more phototoxic type II photosensitizer (MB) does not lead to higher degrees of cell death compared to the type I (CV) photosensitizer. In fact, CV kills cells with the same efficiency as MB, generating at least 10 times fewer photoinduced reactive species. Therefore, subcellular localization is indeed more important than photochemical reactivity in terms of overall cell killing, with mitochondrial localization representing a highly desirable property for the development of more specific/efficient photosensitizers for photodynamic therapy applications. (C) 2011 Elsevier Inc. All rights reserved.

Prevention of bacterial translocation using β-(1-3)-D-glucan in small bowel ischemia and reperfusion in rats

To investigate the role of β-(1-3)-D-glucan on 99mTc labelled Escherichia coli translocation and cytokines secretion in rats submitted to small bowel ischemia/reperfusion injury. Methods: Five groups (n=10 each) of Wistar rats were subjected to control(C), sham(S), group IR subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R), and group I/R+glucan subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R) and injected with 2mg/Kg intramuscular. Translocation of labelled bacteria to mesenteric lymph nodes, liver, spleen, lung and serum was determined using radioactivity/count and colony forming units/g(CFU/g). Serum TNFα, IL-1β, IL-6, IL-10 were measured by ELISA. Results: CFU/g and radioactivity/count were higher in I/R than in I/R+glucan rats. In C, S and S+glucan groups, bacteria and radioactivity/count were rarely detected. The I/R+glucan rats had enhancement of IL-10 and suppressed production of serum TNFα, IL-1β and, IL-6, compared to I/R untreated animals. Conclusion: The β-(1-3)-D-glucan modulated the production of pro-inflammatory and anti-inflammatory cytokines during bowel ischemia/reperfusion, and attenuated translocation of labelled bacteria

Translocation of 99mTc labelled bacteria after intestinal ischemia and reperfusion

Ischemia and reperfusion of the small intestine disrupts gut barrier, causes bacterial translocation and activates inflammatory responses. An experimental study was planned to evaluate if 99mTc labelled Escherichia coli translocates to mesenteric lymph nodes, liver, spleen, lung and serum of rats submitted to mesenteric ischemia/reperfusion. Additionally, it was observed if the time of reperfusion influences the level of translocation. METHODS: Forty male Wistar rats underwent 45 minutes of gut ischemia by occlusion of the superior mesenteric artery. The translocation of labelled bacteria to different organs and portal serum was determined in rats reperfused for 30 minutes, 24 hours, sham(S) and controls(C), using radioactivity count and colony forming units/g (CFU). RESULTS: All the organs from rats observed for 24 hours after reperfusion had higher levels of radioactivity and positive cultures (CFU) than did the organs of rats reperfused for 30 minutes, C and S, except in the spleen (p<0,01). CONCLUSION: The results of this study indicated that intestinal ischemia/reperfusion led to bacterial translocation, mostly after 24 hours of reperfusion

Bacterial translocation in rats nonfunctioning diverted distal colon

To investigate whether the alterations of the diverted colon segment mucosa, evidenced in fecal colitis, would be able to alter Bacterial Translocation (BT). Methods: Sixty-two Wistar male rats ranging from 220 to 320 grams of weight, were divided in two groups: A (Colostomy) and B (Control), with 31 animals each one. In group A, all animals underwent end colostomy, one stoma, in ascending colon; and in the 70th POD was injected in five rats, by rectal route – diverted segment - 2ml of a 0.9% saline solution in animals (A1 subgroup); in eight it was inoculated, by rectal route, 2ml of a solution containing Escherichia coli ATCC 25922 (American Type Culture Collection), in a concentration of 108 Colony Forming Unit for milliliters (CFU/ml) - A2 Subgroup; in ten animals the same solution of E. coli was inoculated, in a concentration of 1011 CFU/ml (A3 Subgroup); and in eight it was collected part of the mucus found in the diverted distal colonic segment for neutral sugars and total proteins dosage (A4 subgroup). The animals from the group B underwent the same procedures of group A, but with differences in the colostomy confection. In rats from subgroups A1, A2, A3, B1, B2, and B3 2ml of blood were aspirated from the heart, and fragments from mesenteric lymphatic nodule, liver, spleen, lung and kidney taken for microbiological analysis, after their death. This analysis consisted of evidencing the presence of E. coli ATCC 25922 CFU. Mann-Whitney and ANOVA Tests were applied as analytic techniques for association of variables. Results: The occurrence of BT was evidenced only in those animals in which inoculated concentration of E. coli ATCC 25922, reached levels of 1011CFU/ml, i.e. in Subgroups A3 and B3, although, being significantly greater (80%) in those animals without colostomy (subgroup B3) when compared to the ones with colostomy (20%) from the subgroup A3 (P <0.05). Lung, liver and mesenteric lymphatic nodules were the tissues with larger percentile of bacterial recovery, so much in subgroup A3, as in B3. Blood culture was considered positive in 60% of the animals from subgroup B3 and in 10% of those from subgroup A3 (p <0.05). There was greater concentration of neutral sugars, in subgroup A4 - mean 27.3mg/ml -, than in subgroup B4 - mean 8.4mg/ml - (P <0.05). Conclusion: The modifications in the architecture of intestinal mucosa in colitis following fecal diversion can cause alterations in the intestinal barrier, but it does not necessarily lead to an increased frequency of BT

Prevention of bacterial translocation using β-(1-3)-D-glucan in small bowel ischemia and reperfusion in rats

To investigate the role of β-(1-3)-D-glucan on 99mTc labelled Escherichia coli translocation and cytokines secretion in rats submitted to small bowel ischemia/reperfusion injury. Methods: Five groups (n=10 each) of Wistar rats were subjected to control(C), sham(S), group IR subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R), and group I/R+glucan subjected to 45 min of bowel ischemia/60 min of reperfusion(I/R) and injected with 2mg/Kg intramuscular. Translocation of labelled bacteria to mesenteric lymph nodes, liver, spleen, lung and serum was determined using radioactivity/count and colony forming units/g(CFU/g). Serum TNFα, IL-1β, IL-6, IL-10 were measured by ELISA. Results: CFU/g and radioactivity/count were higher in I/R than in I/R+glucan rats. In C, S and S+glucan groups, bacteria and radioactivity/count were rarely detected. The I/R+glucan rats had enhancement of IL-10 and suppressed production of serum TNFα, IL-1β and, IL-6, compared to I/R untreated animals. Conclusion: The β-(1-3)-D-glucan modulated the production of pro-inflammatory and anti-inflammatory cytokines during bowel ischemia/reperfusion, and attenuated translocation of labelled bacteria