A Novel Acinetobacter Sp. For 'Treating Highly Acidic Rubber Latex Cen'trifugation Effluent

A novel Acinetobacter sp. BTJR-IO isolated from highly acidic (pH 2.5-4.5) rubber latex centrifugation effluent with high COD (22000 rng/L) and BOD (5000 rng/L). This strain could effect 39.5% COD reduction on free cell inoculation of effluent without incorporation of additional nutrients after 8 days. CalciLnn alginate irrmobilized cells showed 16.4% and 25% COD reduction after 6 hra, without aeration and after 1 hr. with mild aeration under batch process respectively. Whereas 44.0% COD reduction could be achieved after 6 hrs. on continuous treatment in a packed bed reactor with mild aeration. Further, even after 3 cycles 37% COD reduction was recorded with continuous treatment

Apatite-Polymer Composites for the Controlled Dual Delivery of BMP-2 and BMP-6 for Bone Tissue Engineering

The release of growth factors from tissue engineering scaffolds provides signals that influence the migration, differentiation, and proliferation of cells. The incorporation of a drug delivery platform that is capable of tunable release will give tissue engineers greater versatility in the direction of tissue regeneration. We have prepared a novel composite of two biomaterials with proven track records - apatite and poly(lactic-co-glycolic acid) (PLGA) – as a drug delivery platform with promising controlled release properties. These composites have been tested in the delivery of a model protein, bovine serum albumin (BSA), as well as therapeutic proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and rhBMP-6. The controlled release strategy is based on the use of a polymer with acidic degradation products to control the dissolution of the basic apatitic component, resulting in protein release. Therefore, any parameter that affects either polymer degradation or apatite dissolution can be used to control protein release. We have modified the protein release profile systematically by varying the polymer molecular weight, polymer hydrophobicity, apatite loading, apatite particle size, and other material and processing parameters. Biologically active rhBMP-2 was released from these composite microparticles over 100 days, in contrast to conventional collagen sponge carriers, which were depleted in approximately 2 weeks. The released rhBMP-2 was able to induce elevated alkaline phosphatase and osteocalcin expression in pluripotent murine embryonic fibroblasts. To augment tissue engineering scaffolds with tunable and sustained protein release capabilities, these composite microparticles can be dispersed in the scaffolds in different combinations to obtain a superposition of the release profiles. We have loaded rhBMP-2 into composite microparticles with a fast release profile, and rhBMP-6 into slow-releasing composite microparticles. An equi-mixture of these two sets of composite particles was then injected into a collagen sponge, allowing for dual release of the proteins from the collagenous scaffold. The ability of these BMP-loaded scaffolds to induce osteoblastic differentiation in vitro and ectopic bone formation in a rat model is being investigated. We anticipate that these apatite-polymer composite microparticles can be extended to the delivery of other signalling molecules, and can be incorporated into other types of tissue engineering scaffolds.

New mono- and dinuclear ruthenium complexes containing the 3,5-bis(2-pyridyl)pyrazole ligand. Synthesis, characterization and applications

Se han sintetizado dos nuevos complejos mononucleares de Ru, con formula [RuCl2(Hbpp)(dmso)2], a partir de la reacción entre [RuCl2(dmso)4] y Hbpp (3,5-bis(2-piridil)pirazola). El hecho que sólo tres de los seis posibles estereoisómeros se obtengan a partir de esta reacción, se ha racionalizado en base a factores estructurales y electrónicos. Estos complejos se han caracterizado de forma estructural, espectroscópica y electroquímica. En acetonitrilo en medio básico, el isómero trans,cis-[RuCl2(Hbpp)(dmso)2] da lugar a procesos de isomerización de enlace de un ligando dmso cuando el Ru(II) se oxida a Ru(III). Las constantes termodinámicas y cinéticas para el proceso se han determinado por voltametria cíclica. La irradiación de trans,cis-[RuCl2(Hbpp)(dmso)2] y cis(out),cis-[RuCl2(Hbpp)(dmso)2] con luz UV o solar da lugar a reacciones de fotosustitución de un ligando dmso por una molécula de acetonitrilo para dar un nuevo compuesto el cual ha sido caracterizado en solución por técnicas espectroscópicas y electroquímicas. Ambos complejos resultan catalizadores útiles en la transferencia de hidrógeno de isopropanol a acetofenona, obteniéndose 1-feniletanol como único producto y un 42.1% de conversión (36.1 ciclos metálicos) a 80ºC con el isómero trans,cis-[RuCl2(Hbpp)(dmso)2], que resulta significativamente más eficaz que el complejo cis(out),cis-[RuCl2(Hbpp)(dmso)2]. La reacción de cis(out),cis-[RuCl2(Hbpp)(dmso)2] con trpy (2,2':6',2"-terpiridina) da lugar a los dos isómeros geométricos del complejo [Ru(Hbpp)(trpy)(Cl)]+, el in y el out. Estos complejos se han aislado y caracterizado por técnicas estructurales, espectroscópicas y electroquímicas. Estos cloro complejos han sido utilizados como precursores para la síntesis de los complejos análogos con ligandos aqua (in,out-[Ru(Hbpp)(trpy)(H2O)]2+) y piridina (in,out-[Ru(Hbpp)(trpy)(py)]2+), los cuales también han sido aislados y caracterizados. Las propiedades ácido-base de los aqua complejos, y del complejo out-py se han estudiado detalladamente por voltametria cíclica y mediante valoraciones espectrofotométricas ácido-base. El tratamiento matemático de los datos así obtenidos nos ha permitido determinar los valores de pKa para los distintos equilibrios de protonación de los complejos en los estados de oxidación II y III. El complejo out-aqua ha demostrado ser un buen catalizador para la oxidación electroquímica del alcohol benzílico, presumiblemente a benzaldehido. La constante de velocidad de segundo orden para el proceso ha sido determinada como 17.1 M-1 s-1, por simulación matemática. El dímero con un puente cloro, [Ru2Cl(bpp)(trpy)2]2+ ha sido preparado por dos rutas sintéticas diferentes. El dímero análogo con un puente acetato se ha obtenido por reacción del cloro dímero con un exceso de acetato sódico. El dímero con dos ligandos aqua [Ru2(bpp)(trpy)2(OH2)2]3+ puede obtenerse por hidrólisis ácida del complejo con un acetato puente o por hidrólisis básica del complejo con un puente cloro. Estos complejos han sido caracterizados por técnicas estructurales, espectroscópicas y electroquímicas. Las soluciones del dímero con dos ligandos aqua en medio ácido resultan inestables a la coordinación de aniones de la solución con el tiempo. Las propiedades ácido-base del dímero con dos aguas coordinadas han sido estudiadas por voltametria cíclica y mediante experimentos de electrólisis a potencial controlado. El pKa para la desprotonación de uno de los ligandos aqua ha sido determinado mediante una valoración espectrofotométrica ácido-base como 6.7. Este valor tan bajo de pKa se atribuye a la formación de la entidad {Ru2O2H3}, favorable termodinámicamente. Los espectros UV-vis para los distintos estados de oxidación del aqua dímero, de RuIIRuII a RuIIIRuIV, han sido obtenidos por oxidación química y electroquímica del complejo. Se han llevado a cabo estudios cinéticos de la oxidación, paso a paso, de RuII,II a RuIV,IV , y se han determinado las constantes de oxidación de segundo orden para los distintos procesos de oxidación. La capacidad del aqua dímero en la oxidación del agua a oxígeno molecular ha sido investigada en solución homogénea utilizando CeIV como oxidante. La evolución de oxígeno se ha demostrado por cromatografia de gases. Se ha obtenido una eficiencia del 73% y 18.6 ciclos catalíticos, cuando 1.83 x 10-6 moles de dímero se han mezclado con un exceso de 100 equivalentes de cerio. El dímero con dos aguas cataliza también la oxidación del agua de forma heterogénea, con el complejo adsorbido sobre una membrana de nafion, aunque la eficiencia es menor. Se ha propuesto un mecanismo intramolecular para la reacción de oxidación del agua. Consiste en la oxidación a 4 electrones del dímero, de RuII,II a RuIV,IV, el cual reacciona con el agua para formar oxígeno y revierte nuevamente al estado de oxidación II,II. Este modelo es consistente con estudios catalíticos de la evolución de oxígeno en función de las concentraciones de cerio y catalizador, llevados a cabo en solución ácida homogénea, que demuestran que la oxidación a 4 electrones del agua se encuentra catalizada por una sola molécula de complejo bajo concentraciones elevadas de cerio. La constante de pseudo-primer-orden para la evolución de oxígeno tiene un valor de 1.4 x 10-2 s-1, que es uno de los valores de constante más elevados obtenidos hasta la fecha. Desafortunadamente, el aqua dímero se desactiva durante el proceso de catálisis dando lugar a una especie naranja, la cual estamos actualmente tratando de caracterizar.

Displacement of Zn through acidic light-textured soils

In order to gain understanding of the movement of pollutant metals in soil. the chemical mechanisms involved in the transport of zinc were studied. The displacement of zinc through mixtures of sand and cation exchange resin was measured to validate the methods used for soil. With cation exchange capacities of 2.5 and 5.0 cmol(c) kg(-1). 5.6 and 8.4 pore volumes of 10 mM CaCl2, respectively, were required to displace a pulse of ZnCl2. A simple Burns-type model (Wineglass) using an adsorption coefficient (K-d) determined by fitting a straight line relationship to an adsorption isotherm gave a good fit to the data (K-d=0.73 and 1.29 ml g(-1), respectively). Surface and subsurface samples of an acidic sandy loam (organic matter 4.7 and 1.0%. cation exchange capacity (CEC) 11.8 and 6.1 cmol(c) kg(-1) respectively) were leached with 10 mM calcium chloride. nitrate and perchlorate. With chloride. the zinc pulse was displaced after 25 and 5 pore volumes, respectively. The Kd values were 6.1 and 2.0 ml g(-1). but are based on linear relationships fitted to isotherms which are both curved and show hysteresis. Thus. a simple model has limited value although it does give a general indication of rate of displacement. Leaching with chloride and perchlorate gave similar displacement and Kd values, but slower movement occurred with nitrate in both soil samples (35 and 7 pore volumes, respectively) which reflected higher Kd values when the isotherms were measured using this anion (7.7 and 2.8 ml g(-1) respectively). Although pH values were a little hi-her with nitrate in the leachates, the differences were insufficient to suggest that this increased the CEC enough to cause the delay. No increases in pH occurred with nitrate in the isotherm experiments. Geochem was used to calculate the proportions of Zn complexed with the three anions and with fulvic acid determined from measurements of dissolved organic matter. In all cases, more than 91% of the Zn was present as Zn2+ and there were only minor differences between the anions. Thus, there is an unexplained factor associated with the greater adsorption of Zn in the presence of nitrate. Because as little as five pore volumes of solution displaced Zn through the subsurface soil, contamination of ground waters may be a hazard where Zn is entering a light-textured soil, particularly where soil salinity is increased. Reductions in organic matter content due to cultivation will increase the hazard. (C) 2004 Elsevier B.V. All rights reserved.

Effect of pH on the development of acidic sites in clayey and sandy loam Oxisol from the Cerrado Region, Brazil

Titration curves were determined for soil from horizon samples of a clayey and a sandy loam Oxisol by (a) adding NaOH to soil suspensions and (b) incubating moist soils with Ca(OH)(2). The organic fraction was primarily responsible for buffering in both soils. Humic acids were more important than fulvic acids in buffering against NaOH additions. With Ca(OH)(2), greater buffer capacities were found due to carboxyl sites, primarily on fulvic acids, becoming complexed with Ca2+ so that in the clay soil humic and fulvic acids were equally important as buffering components while fulvic acids were more important in the sandy loam soil. The buffer capacity of organic matter against Ca(OH)(2) additions was 1.1 mol(c) kg(-1) pH(-1). In the incubated soils, exchangeable cations were also determined and changes in the amounts of exchangeable and non-exchangeable Ca2+ acidity and effective cation exchange capacity were calculated. Up to half the added Ca2+ became complexed and was nonexchangeable. Aluminum complexed by organic matter appears to be an important buffering component, together with non exchangeable H+. With the increase of pH the dissociated sites from the carboxyl groups could complex Ca2+. (c) 2005 Elsevier B.V. All rights reserved.

Acid-base reactions between an acidic soil and plant residues

The elemental composition of residues of maize (Zea mays), sorghum (S. bicolor), groundnuts (Arachis hypogea), soya beans (Glycine max), leucaena (L. leucocephala), gliricidia (G. sepium), and sesbania (S. sesban) was determined as a basis for examining their alkalinity when incorporated into an acidic Zambian Ferralsol. Potential (ash) alkalinity, available alkalinity by titration to pH 4 and soluble alkalinity (16 It water extract titrated to pH 4) were measured. Potential alkalinity ranged from 3 73 (maize) to 1336 (groundnuts) mmol kg(-1) and was equivalent to the excess of their cation charge over inorganic anion charge. Available alkalinity was about half the potential alkalinity. Cations associated with organic anions are the source of alkalinity. About two thirds of the available alkalinity is soluble. Residue buffer curves were determined by titration with H2SO4 to pH 4. Soil buffer capacity measured by addition of NaOH was 12.9 mmol kg(-1) pH(-1). Soil and residue (10 g:0.25 g) were shaken in solution for 24 h and suspension pH values measured. Soil pH increased from 4.3 to between 4.6 (maize) and 5.2 (soyabean) and the amounts of acidity neutralized (calculated from the rise in pH and the soil buffer capacity) were between 3.9 and 11.5 mmol kg(-1), respectively. The apparent base contributions by the residues (calculated from the buffer curves and the fall in pH) ranged between 105 and 350 mmol kg(-1) of residue, equivalent to 2.6 and 8.8 mmol kg(-1) of soil, respectively. Therefore, in contact with soil acidity, more alkalinity becomes available than when in contact with H2SO4 solution. Available alkalinity (to pH 4) would be more than adequate to supply that which reacts with soil but soluble alkalinity would not. It was concluded that soil Al is able to displace cations associated with organic anions in the residues which are not displaced by H+, or that residue decomposition may have begun in the soil suspension releasing some of the non-available alkalinity. Soil and four of the residues were incubated for 100 days and changes in pH, NH4+ and NO3- concentrations measured. An acidity budget equated neutralized soil acidity with residue alkalinity and base or acid produced by N transformations. Most of the potential alkalinity of soyabean and leucaena had reacted after 14 days, but this only occurred after 100 days for gliricidia, and for maize only the available alkalinity reacted. For gliricidia and leucaena, residue alkalinity was primarily used to react with acidity produced by nitrification. Thus, the ability of residues to ameliorate acidity depends not only on their available and potential alkalinity but also on their potential to release mineral N. (C) 2004 Elsevier B.V. All rights reserved.

Platelet adhesion to collagen and collagen-related peptide under flow. Roles of the alpha(2)beta(1) integrin, GPVI, and Src tyrosine kinases

Objective - Platelet stimulation by collagen and collagen-related peptides (CRPs) is associated with activation of protein tyrosine kinases. In the present study, we investigated the role of Src family tyrosine kinases in the initial adhesion events of human platelets to collagen and cross-linked CRP. Methods and Results - Under arterial flow conditions, a glycoprotein VI - specific substrate, cross-linked CRP, caused rapid (<2 second) platelet retention and protein tyrosine phosphorylation that were markedly decreased by the Src family kinase inhibitor pyrozolopyrimidine (PP2) or by aggregation inhibitor GRGDSP. CRP-induced platelet retention was transient, and 90% of single platelets or aggregates detached within seconds. PP2, although having no effect on RGD peptide-binding to CRP, completely blocked aggregation and tyrosine phosphorylation of Syk and phospholipase Cγ2 (PLCγ2). In contrast, PP2 weakly (<30%) suppressed firm adhesion to collagen mediated primarily by the alpha(2)beta(1) integrin. Although PP2 prevented activation of Syk and PLCgamma2 in collagen-adherent platelets, tyrosine phosphorylation of several unidentified protein bands persisted, as did autophosphorylation of pp125(FAK). Conclusions - These findings indicate that activation of Src-tyrosine kinases Syk and PLCgamma2 is not required for the initial stable attachment of human platelets to collagen and for FAK autophosphorylation. However, Src-tyrosine kinases are critical for glycoprotein VI - mediated signaling leading to platelet aggregation.

Photocatalytic degradation of humic acid in saline waters part 2. Effects of various photocatalytic materials

The present study explores for the first time, the effectiveness of photocatalytic oxidation of. humic acid (HA) in the increasingly important highly saline water. TiO2 (Degussa P25), TiO2 (Anatase), TiO2 (Rutile), TiO2 (Mesoporous) and ZnO dispersions were used as catalysts employing a medium pressure mercury lamp. The effect of platinum loading on P25 and zinc oxide was also investigated. The zinc oxide with 0.3% platinum loading was the most efficient catalyst. The preferred medium for the degradation of HA using ZnO is alkaline, whereas for TiO2 it is acidic. In addition, a comparative study of HA decomposition in artificial seawater (ASW) and natural seawater (NSW) is reported, and the surface areas and band gaps of the catalysts employed were also determined. A spectrophotometric method was used to estimate the extent of degradation of HA. (C) 2003 Elsevier Science B.V. All rights reserved.

Ligand effects in cis-Ru-II(N-N)(2)L-2 species where N-N is a substituted 2,2 '-bisoxazoline and L is monodentate

Starting from previously reported cis-Ru(MeL)(2)Cl-2, where MeL is 4,4,4',4'-tetramethyl-2,2'-bisoxazoline, cis-Ru(MeL)(2)Br-2 (1), cis-Ru( MeL)(2)I-2 (2), cis-Ru(MeL)(2)(NCS)(2) center dot H2O (3), cis-Ru(MeL)(2)(N-3)(2) (4) and cis-[Ru(MeL)(2)(MeCN)(2)](PF6)(2) center dot (CH3)(2)CO (5) are synthesised. The X-ray crystal structures of complexes 1, 2, 3 and 5 have been determined. All the five new complexes have been characterized by FTIR, ESIMS and H-1 NMR. In cyclic voltammetry in acetonitrile at a glassy carbon electrode, the complexes display a quasireversible Ru(II/III) couple in the range 0.32-1.71 V versus NHE. The Ru(II/III) potentials yield a satisfactorily linear correlation with Chatt's ligand constants P-L for the monodantate ligands. From the intercept and by comparing the known situation in Ru(2,2'-bipyridine)(2)L-2, it is concluded that MeL, a non-aromatic diimine, is significantly more pi-acidic than 2,2'-bipyridine. (c) 2008 Elsevier B.V. All rights reserved.

Palladium and platinum complexes of 2-(2 '-carboxyphenylazo)-4-methylphenol: synthesis, structure and spectral properties

Reaction of 2-(2'-carboxyphenylazo)-4-methylphenol (H2L) with [M(PPh3)(2)Cl-2] (M = Pd, Pt) affords mixed-ligand complexes of type [M(PPh3)(L)]. Structures of both the complexes have been determined by X-ray crystallography. Both the complexes are square planar, where the 2-(2'-carboxyphenylazo)-4-methylphenol is coordinated to the metal center, via dissociation of the two acidic protons, as a dianionic tridentate O,N,O-donor, and the fourth position is occupied by the triphenylphosphine. These complexes show intense MLCT transitions in the visible region.

Electronic properties, redox behavior, and interactions with H2O2 of pH-sensitive hydroxyphenyl-1,2,4-triazole-based oxovanadium(V)complexes

The syntheses and spectroscopic characterization of two 1,2,4-triazole-based oxovanadium(V) complexes are reported: 1(-)[VO(2)L1](-) and 2 [(VOL2)(2)(OMe)(2)] (where H(2)L1 = 3-(2'-hydroxyphenyl)-5-(pyridin-2"-yl)-H-1-1,2,4-triazole, H3L2 = bis-3,5-(2'-hydroxyphenyl)-1H-1,2,4-triazole). The ligand environment (N,N,O vs O,N,O) is found to have a profound influence on the properties and reactivity of the complexes formed. The presence of the triazolato ligand allows for pH tuning of the spectroscopic and electrochemical properties, as well as the interaction and stability of the complexes in the presence of hydrogen peroxide. The vanadium(IV) oxidation states were generated electrochemically and characterized by UV-vis and EPR spectroscopies, For 2, under acidic conditions, rapid exchange of the methoxide ligands with solvent [in particular, in the vanadium(IV) redox state] was observed.

Investigation of the factors influencing the survival of Bifidobacterium longum in model acidic solutions and fruit juices

The survival of Bifidobacterium longum NCIMB 8809 was studied during refrigerated storage for 6 weeks in model solutions, based on which a mathematical model was constructed describing cell survival as a function of pH, citric acid, protein and dietary fibre. A Central Composite Design (CCD) was developed studying the influence of four factors at three levels, i.e., pH (3.2–4), citric acid (2–15 g/l), protein (0–10 g/l), and dietary fibre (0–8 g/l). In total, 31 experimental runs were carried out. Analysis of variance (ANOVA) of the regression model demonstrated that the model fitted well the data. From the regression coefficients it was deduced that all four factors had a statistically significant (P < 0.05) negative effect on the log decrease [log10N0 week−log10N6 week], with the pH and citric acid being the most influential ones. Cell survival during storage was also investigated in various types of juices, including orange, grapefruit, blackcurrant, pineapple, pomegranate and strawberry. The highest cell survival (less than 0.4 log decrease) after 6 weeks of storage was observed in orange and pineapple, both of which had a pH of about 3.8. Although the pH of grapefruit and blackcurrant was similar (pH ∼3.2), the log decrease of the former was ∼0.5 log, whereas of the latter was ∼0.7 log. One reason for this could be the fact that grapefruit contained a high amount of citric acid (15.3 g/l). The log decrease in pomegranate and strawberry juices was extremely high (∼8 logs). The mathematical model was able to predict adequately the cell survival in orange, grapefruit, blackcurrant, and pineapple juices. However, the model failed to predict the cell survival in pomegranate and strawberry, most likely due to the very high levels of phenolic compounds in these two juices.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

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.