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Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca 2+ transients in human RPE cells by activating the kinin B 2 receptor. Pretreatment of the cells with TNF-α and/or IL-1β enhanced Ca 2+ response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B 2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B 1 , acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B 2 receptor and COX-2 expression and with the secretion of prostaglandin E 1 and E 2 into the extracellular medium. BK, through activation of the kinin B 2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca 2+ response. In conclusion, overall data revealed a cross-talk between the kinin B 2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema. [ABSTRACT FROM AUTHOR]

Abstract: Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca2+]i mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B1 receptor agonist, Lys-[des-Arg9]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B2 receptor antagonist, fasitibant chloride, but not by the B1 selective antagonist, Lys-[Leu8][des-Arg9]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B2 receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B2 receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride. [Copyright &y& Elsevier]

Abstract: The present study investigated the effect of HDAC inhibitors on the differentiation of human subcutaneous white adipocytes. Results showed that trichostatin A, suberoylanilide hydroxamic acid, valproic acid and MS-275 inhibited triglyceride accumulation, GPDH activity and FABP4 protein expression in adipocytes, as well as leptin and VEGF release, while cells retained a fibroblast-like morphology. HDAC inhibitors exerted their antiadipogenic effect without inducing apoptosis or affecting cell viability and number, while 1–2 log unit higher concentrations were mostly required to exert an antiproliferative effect or to reduce LDH activity. A brief exposure to HDAC inhibitors at the beginning of the differentiation program was sufficient to observe the antiadipogenic effect while differentiation restarted after compound withdrawal and further exposure to inducers of differentiation demonstrating reversibility of the events. HDAC inhibitors hyperacetylated histone H4, but only hydroxamate-based compounds produced a massive acetylation of α-tubulin, indicating that this latter event is not required to prevent adipose conversion. HDAC inhibitors induced a significant reduction of the expression of the transcription factor C/EBPα, an early marker of differentiation, and a diminution of fibronectin immunoreactivity was also observed. In conclusion, HDAC inhibitors from different chemical classes potently inhibited human adipose conversion at an early stage of the differentiation program. [Copyright &y& Elsevier]

The present study investigates the effects of ethanol and hydrogen peroxide (H2O2) on the barrier function and prostaglandin E2 (PGE2) release in differentiated Caco-2 cells. Epithelial barrier integrity was estimated by measuring transepithelial electrical resistance (TEER), the transport of reference compounds and lactate dehydrogenase leakage, the PGE2 release by enzyme immunoassay. Ethanol and H2O2 decreased TEER and increased the transport of lucifer yellow without affecting that of propranolol and phenylalanine. Only the effects of ethanol were accompanied by PGE2 production and were reversible without causing long-term cytotoxicity. The cyclooxygenase-2 inhibitor, NS-398, prevented the effect of ethanol on both PGE2 release and TEER, while inhibition of both cyclooxygenase-2 and tyrosine kinase drastically compromised cell viability and TEER recovery. Hepatocyte growth factor, keratinocyte growth factor or insulin prevented the effect of ethanol on cell permeability, but not on PGE2 release. Their combination prevented the effect of H2O2. In conclusion, ethanol and H2O2 increased paracellular permeability in differentiated Caco-2 cells without affecting transcellular and active transport. Cyclooxygenase-2 stimulated PGE2 release mediated the reversible effect of ethanol on tight junctions and, meanwhile, contributed to cell survival. Growth factors, normally present in the intestine, exerted a selective protective effect toward paracellular permeability increase induced by irritants. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:713–727, 2009 [ABSTRACT FROM AUTHOR]

The present study investigates the intestinal permeability of otilonium bromide, a spasmolytic drug used to treat irritable bowel syndrome, across Caco-2 cell monolayers. The amount of otilonium bromide transported was determined by high-performance liquid chromatography-mass spectrometry. Epithelial barrier integrity was estimated by measuring transepithelial electrical resistance and the transport of reference compounds, P-glycoprotein activity by measuring rhodamine 123 efflux. Results showed that the apparent permeability of otilonium bromide was comparable to that of our zero permeability marker, inulin, in the apical-to-basal direction and similar to that of rhodamine 123 in the basal-to-apical direction. The P-glycoprotein substrate, verapamil, prevented otilonium bromide efflux and, conversely, otilonium bromide inhibited P-glycoprotein activity. Bile salts induced a transient opening of tight junctions, as measured by selective increase of paracellular transport, and significantly enhanced the absorption of otilonium bromide. In turn otilonium bromide potentiates the effect of bile salts on tight junctions without modifying their critical micellar concentration or altering cell viability. In conclusion, otilonium bromide is a paracellularly transported drug whose absorption, in amounts sufficient to exert a spasmolytic effect, is favoured by bile salts. P-glycoprotein, by stimulating efflux, contributes to remove excess compound, restraining its distribution and site of action to the intestinal wall. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:4087–4100, 2008 [ABSTRACT FROM AUTHOR]

In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK2 receptor splice variant named β isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the α isoform. Moreover, neurokinin A was unable to inhibit the [3H]SR48968 binding to the β isoform up to μM concentrations. In cells expressing the human tachykinin NK2 receptor β isoform, contrary to those expressing the α isoform, natural or selective tachykinin receptor agonists (1 μM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca2+]i. The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca2+]i increase in cells expressing the α or β isoform of the human tachykinin NK2 receptor. The present data indicate that the human tachykinin NK2 receptor β isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK2 receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands. [Copyright &y& Elsevier]

Non peptide products have been found to show nanomolar binding and functional affinities at the human tachykinin NK-2 receptor. The new antagonists do not possess stereogenic centers and their thermal behaviour in solution is featured by a peculiar set of conformational stereoisomers. A macroscopic viewpoint is preferentially adopted to rationalize the obtained results. [Copyright &y& Elsevier]

Characterizes the Caco-2 cell culture absorption properties. Use of fluorescent or radioactive chemicals to test the parameters; Description of liquid chromatography-mass spectrometry method; Application of method in laboratory to check on reproducibility and consistency of the results.

Abstract: The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pK i 10.1) and potently antagonized (pK B 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pK i 6.1) and NK3 (pK i 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pK B 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pK B 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2 ≤6). In anaesthetized guinea-pigs, MEN15596 inhibited in a dose-related and persistent manner colon contractions induced by the selective tachykinin NK2 receptor agonist, [βAla8]neurokinin A(4–10) (3 nmol/kg i.v.), either after intravenous (ED50 0.18 μmol/kg), intraduodenal (ED50 3.16 μmol/kg) or oral administration (10–30 μmol/kg) without affecting, at 3 μmol/kg, i.v., the colonic contractions produced by the NK1 receptor selective agonist [Sar9]substance P sulfone (3 nmol/kg i.v.). In addition MEN15596 was effective in inhibiting bronchoconstriction produced by i.v. administration of [βAla8]neurokinin A(4–10). Overall the results indicate that MEN15596 is a potent and selective tachykinin NK2 receptor antagonist possessing high affinity and potency for guinea-pig, pig and human receptor, long duration of action in in vivo experiments and good oral bioavailability. [Copyright &y& Elsevier]

We have studied the effect of selective tachykinin NK1 and NK2 receptor antagonists on airway hyperreactivity to acetylcholine and increase of inflammatory cells on bronchoalveolar lavage fluid induced by sephadex beads (20 mg/kg, i.v.) in guinea pigs. Airway hyperreactivity was assessed by measuring the increase of bronchial insufflation pressure to acetylcholine (0.01–30 μmol/kg, i.v.) at 3 h (early phase) and 24 h (late phase) after sephadex administration. An increase in inflammatory cells in bronchoalveolar lavage fluid (eosinophils and macrophages) was detected at 24 h (from 11.6·106 to 49.3·106 cells) but not at 3 h from sephadex administration. Neurokinin A and substance P levels in bronchoalveolar lavage fluid showed a significant increase at 24 h (from 31.7±11.6 to 561±231 pg/ml and from 5.9±2.6 to 29.3±4.1 pg/ml for neurokinin A and substance P, respectively). At this time point, the tachykinin in bronchoalveolar lavage cellular content was depleted from 232±43 to 21±20 pg/sample and from 56.6±6.7 to 2±2 pg/sample for neurokinin A and substance P, respectively. Capsaicin pretreatment abolished the early but not the late phase of airway hyperreactivity induced by sephadex without modifying bronchoalveolar lavage total cells number and bronchoalveolar lavage levels of neurokinin A and substance P. Administration of the tachykinin NK2 (nepadutant) and/or the NK1 receptor antagonist (MEN 11467 or (1R,2S)-2-N[1(H)indol-3-yl-carbonyl]-1-N[N-(p-tolylacetyl)-N-(methyl)-d-3(2-naphthyl)alanyl}diaminocyclohexane)), 5 min before sephadex, prevented the early phase of airway hyperreactivity to acetylcholine but only nepadutant prevented the late phase. Nepadutant was able to abolish the early phase of airway hyperreactivity if given after sephadex administration and reduced by about 50% the increase of cell number in bronchoalveolar lavage fluid during the late phase, without affecting the levels of neurokinin A and substance P. These findings indicate an involvement of endogenous tachykinins in the genesis of airway hyperreactivity in a guinea-pig model of non-allergic asthma. Early airway hyperreactivity apparently involves release of tachykinins from capsaicin-sensitive afferent nerves acting via tachykinin NK1/NK2 receptors. Late airway hyperreactivity involves tachykinins acting via tachykinin NK2 receptors: inflammatory cells activated/recruited in response to sephadex challenge appear a likely source of tachykinins involved in the late phase of the response. [Copyright &y& Elsevier]

Abstract: NK2 antagonists have been reported to be potentially useful for the treatment of a number of chronic diseases, such as asthma, irritable bowel syndrome, cystitis, and depression. Starting from an in-house prepared library of capped dipeptides, we have identified a series of molecules with subnanomolar binding affinity for the hNK2 receptor. These molecules are composed by three well-defined regions: a planar aromatic acyl system as N-terminal capping group, a rigid and quite lipophilic core, and a flexible and relatively hydrophilic C-terminal capping group. Here we report how we were able to manipulate the N-terminal capping group to obtain significant in vivo activity after iv and id administration. [Copyright &y& Elsevier]