Concentrations Of Antagonists Research Articles

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding.

Potential Use of Dimethyl Sulfoxide in Treatment of Infections Caused by Pseudomonas aeruginosa.

Dimethyl sulfoxide (DMSO) is commonly used as a solvent to dissolve water-insoluble drugs or other test samples in both in vivo and in vitro experiments. It was observed during our experiment that DMSO at noninhibitory concentrations could significantly inhibit pyocyanin production in the human pathogen Pseudomonas aeruginosa Pyocyanin is an important pathogenic factor whose production is controlled by a cell density-dependent quorum-sensing (QS) system. Investigation of the effect of DMSO on QS showed that DMSO has significant QS antagonistic activities and concentrations of DMSO in the micromolar range attenuated a battery of QS-controlled virulence factors, including rhamnolipid, elastase, and LasA protease production and biofilm formation. Further study indicated that DMSO inhibition of biofilm formation and pyocyanin production was attained by reducing the level of production of an autoinducer molecule of the rhl QS system, N-butanoyl-l-homoserine lactone (C4-HSL). In a mouse model of a burn wound infection with P. aeruginosa, treatment with DMSO significantly decreased mouse mortality compared with that for mice in the control group. The capacity of DMSO to attenuate the pathogenicity of P. aeruginosa points to the potential use of DMSO as an antipathogenic agent for the treatment of P. aeruginosa infection. As a commonly used solvent, however, DMSO's impact on bacterial virulence calls for cautionary attention in its usage in biological, medicinal, and clinical studies.

Neutropenic sepsis is associated with distinct clinical and biological characteristics: a cohort study of severe sepsis.

BackgroundImmunocompromised patients who develop sepsis while neutropenic are at high risk for morbidity and mortality; however, it is unknown if neutropenic sepsis is associated with distinct clinical and biological characteristics.MethodsWe conducted a prospective cohort study of patients admitted to the medical intensive care unit of an academic medical center with severe sepsis. Patients were followed for the development of acute respiratory distress syndrome (ARDS), acute kidney injury (AKI), and mortality. Plasma proteins, representing the host inflammatory response, anti-inflammatory response, and endothelial leak were measured in 30 % of subjects. Clinical characteristics and plasma protein concentrations of patients with neutropenia at enrollment were compared to patients without neutropenia.ResultsOf 797 subjects enrolled, 103 (13 %) were neutropenic at ICU admission. The neutropenic subjects were more often in shock, admitted from the hospital ward, had higher APACHE III scores, and more likely bacteremic. Neutropenia was an independent risk factor for AKI (RR 1.28; 95 % CI 1.04, 1.57; p = 0.03), but not ARDS (RR 0.90; 95 % CI 0.70, 1.17; p = 0.42) or 30-day mortality (RR 1.05; 95 % CI 0.85, 1.31; p = 0.65). Neutropenic subjects had higher plasma interleukin (IL)-6 (457 vs. 249 pg/ml; p = 0.03), IL-8 (581 vs. 94 pg/ml; p <0.001), and granulocyte colony-stimulating factor (G-CSF) (3624 vs. 99 pg/ml; p <0.001). Angiopoietin-2 and IL-1 receptor antagonist concentrations did not differ between groups.ConclusionsNeutropenic sepsis is associated with a higher AKI risk and concentrations of inflammatory mediators IL-6, IL-8, and G-CSF relative to non-neutropenic patients. These differences may have implications for future therapies targeting neutropenic sepsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-016-1398-y) contains supplementary material, which is available to authorized users.

Assessment of Free Drug Concentration in Cyclodextrin Formulations Is Essential to Determine Drug Potency in Functional In Vitro Assays

Cyclodextrins (CD) have the ability to form inclusion complexes with drugs and can be used as excipients to enhance solubility of poorly soluble drugs. To make accurate estimations of the potency of the drug, knowledge of the free drug concentration is important. The aim of this study was to evaluate the applicability of calculated free drug concentrations toward response measurements in a transient receptor potential vanilloid receptor-1 cell-based in vitro assay. This included accounting for potential competitive CD binding of 2 transient receptor potential vanilloid receptor-1 active entities: 1 antagonist, and 1 agonist (capsaicin). Solubility of the CD-drug complexes was measured, and the ligand to substrate affinity in CD formulations was determined according to the phase-solubility technique. The total concentration of antagonist, agonist, CD, and the binding constants between ligands and CD were used to calculate the free concentration of CD ligands. For capsaicin and 2 of the 3 investigated model drugs, the calculated free drug concentration was consistent with the experimental in vitro data while it was overestimated for one of the compounds. In conclusion, the suggested approach can be used to calculate free drug concentration and competitive binding in CD formulations for the application of cell-based drug functionality assays.

Novel relaxant effects of RPL554 on guinea pig tracheal smooth muscle contractility.

We investigated the effectiveness of RPL554, a dual PDE3 and 4 enzyme inhibitor, on airway smooth muscle relaxation and compared it with that induced by salbutamol, ipratropium bromide, glycopyrrolate or their combination on bronchomotor tone induced by different spasmogenic agents. Guinea pig tracheal preparations were suspended under 1g tension in Krebs-Henseleit solution maintained at 37°C and aerated with 95% O2 /5% CO2 and incubated in the presence of indomethacin (5μM). Relaxation induced by cumulative concentrations of muscarinic receptor antagonists (ipratropium bromide or glycopyrrolate), β2 -adrenoceptor agonists (salbutamol or formoterol), PDE3 inhibitors (cilostamide, cilostazol or siguazodan) or a PDE4 inhibitor (roflumilast) was evaluated in comparison with RPL554. Maximal relaxation was calculated (% Emax papaverine) and expressed as mean±SEM. Bronchomotor tone induced by the various spasmogens was reduced by the different bronchodilators to varying degrees. RPL554 (10-300μM) caused near maximum relaxation irrespective of the spasmogen examined, whereas the efficacy of the other relaxant agents varied according to the contractile stimulus used. During the evaluation of potential synergistic interactions between bronchodilators, RPL554 proved superior to salbutamol when either was combined with muscarinic receptor antagonists. RPL554 produced near maximal relaxation of highly contracted respiratory smooth muscle and provided additional relaxation compared with that produced by other clinically used bronchodilator drugs. This suggests that RPL554 has the potential to produce additional beneficial bronchodilation over and above that of maximal clinical doses of standard bronchodilators in highly constricted airways of patients.

A comparison of two semi-mechanistic models for prolactin release and prediction of receptor occupancy following administration of dopamine D2 receptor antagonists in rats

We compared the model performance of two semi-mechanistic pharmacokinetic-pharmacodynamic models, the precursor pool model and the agonist-antagonist interaction model, to describe prolactin response following the administration of the dopamine D2 receptor antagonists risperidone, paliperidone or remoxipride in rats. The time course of pituitary dopamine D2 receptor occupancy was also predicted.Male Wistar rats received a single dose (risperidone, paliperidone, remoxipride) or two consecutive doses (remoxipride). Population modeling was applied to fit the pool and interaction models to the prolactin data. The pool model was modified to predict the time course of pituitary D2 receptor occupancy. Unbound plasma concentrations of the D2 receptor antagonists were considered the drivers of the prolactin response. Both models were used to predict prolactin release following multiple doses of paliperidone.Both models described the data well and model performance was comparable. Estimated unbound EC50 for risperidone and paliperidone was 35.1nM (relative standard error 51%) and for remoxipride it was 94.8nM (31%). KI values for these compounds were 11.1nM (21%) and 113nM (27%), respectively. Estimated pituitary D2 receptor occupancies for risperidone and remoxipride were comparable to literature findings. The interaction model better predicted prolactin profiles following multiple paliperidone doses, while the pool model predicted tolerance better.The performance of both models in describing the prolactin profiles was comparable. The pool model could additionally describe the time course of pituitary D2 receptor occupancy. Prolactin response following multiple paliperidone doses was better predicted by the interaction model.

Gonadotropin-releasing hormone analogues lead to pro-inflammatory changes in T lymphocytes.

We aimed to investigate the effect of gonadotropin-releasing hormone (GnRH) analogues on T-cell immunity. TNF-α(+) -, INF-ɣ(+) -, IL-10(+) -, and IL-17(+) -expressing T cells in peripheral blood mononuclear cells (PBMCs) were treated with various concentrations (0.1, 1, 5, and 10μm) of GnRH agonist (buserelin acetate) and antagonist (cetrorelix acetate) for 4hours in vitro and they were analyzed with flow cytometry. TNF-α(+) /IL-10(+) T helper (TH) cell ratios were increased in PBMCs treated with 1, 5, and 10μm GnRH agonist when compared to controls (P=.006, P=.014 and P=.030, respectively). IFN-ɣ(+) /IL-10(+) TH cell ratios were significantly increased with 0.1, 1, 5, and 10μm GnRH agonist as compared with controls (P=.046, P=.004, P=.013, and P=.011, respectively). TNF-α(+) TH cell levels, and IFN-γ(+) /IL-10(+) TH cell ratios were significantly different (P<.001 and P<.004, respectively) between GnRH agonist- and antagonist-treated cells. GnRH analogues induce pro-inflammatory TH1 shift in T-cell immunity, in vitro. GnRH treatment during assisted reproductive technology cycle might explain a possible cause of inflammatory flare in women with inflammatory conditions.

TRPA1 is functionally co-expressed with TRPV1 in cardiac muscle: Co-localization at z-discs, costameres and intercalated discs

ABSTRACTTransient receptor potential channels of the ankyrin subtype-1 (TRPA1) and vanilloid subtype-1 (TRPV1) are structurally related, non-selective cation channels that show a high permeability to calcium. Previous studies indicate that TRP channels play a prominent role in the regulation of cardiovascular dynamics and homeostasis, but also contribute to the pathophysiology of many diseases and disorders within the cardiovascular system. However, no studies to date have identified the functional expression and/or intracellular localization of TRPA1 in primary adult mouse ventricular cardiomyocytes (CMs). Although TRPV1 has been implicated in the regulation of cardiac function, there is a paucity of information regarding functional expression and localization of TRPV1 in adult CMs. Our current studies demonstrate that TRPA1 and TRPV1 ion channels are co-expressed at the protein level in CMs and both channels are expressed throughout the endocardium, myocardium and epicardium. Moreover, immunocytochemical localization demonstrates that both channels predominantly colocalize at the Z-discs, costameres and intercalated discs. Furthermore, specific TRPA1 and TRPV1 agonists elicit dose-dependent, transient rises in intracellular free calcium concentration ([Ca2+]i) that are abolished in CMs obtained from TRPA1−/− and TRPV1−/− mice. Similarly, we observed a dose-dependent attenuation of the TRPA1 and TRPV1 agonist-induced increase in [Ca2+]i when WT CMs were pretreated with increasing concentrations of selective TRPA1 or TRPV1 channel antagonists. In summary, these findings demonstrate functional expression and the precise ultrastructural localization of TRPA1 and TRPV1 ion channels in freshly isolated mouse CMs. Crosstalk between TRPA1 and TRPV1 may be important in mediating cellular signaling events in cardiac muscle.

Interleukin-1 Receptor Antagonist and Interleukin-1 Beta Levels in Equine Synovial Fluid of Normal and Osteoarthritic Joints: Influence of Anatomic Joint Location and Repeated Arthrocentesis

This study was aimed to evaluate the concentrations of interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 beta (IL-1β) in normal and osteoarthritic (OA) joints as well as the influence of joint location and arthrocentesis on these concentrations. Interleukin-1 receptor antagonist and IL-1β levels were determined in the synovial fluid (SF) of 18 normal and 18 OA joints. In all normal joints, arthrocentesis was repeated after 1 hour. No significant difference of SF IL-1ra and IL-1β levels between metacarpophalangeal/metatarsophalangeal, radiocarpal, and talocrural joints was observed. There was no significant change in SF IL-1ra and IL-1β levels between first and second arthrocentesis detectable. Synovial fluid IL-1ra and IL-1β levels were significantly increased in OA joints compared to normal joints. Synovial fluid WBC count and protein concentration were not significant different between normal and OA joints. Synovial fluid WBC count and protein concentration as well as IL-1ra and IL-1β concentration were positive correlated. The anatomic location of high motion joints seems to have no influence on SF IL-1ra and IL-1β levels. Arthrocentesis did not increase SF IL-1ra and IL-1β levels within 1 hour after joint puncture. Increased SF IL-1ra, IL-1β, and protein concentrations as well as WBC counts seem to be indicators of joint inflammation, but on their own are not allowing an exact differentiation between healthy and mild OA joints due to great value ranges and value overlap. Yet it has to be further investigated if in combination with other biomarkers, a clearer differentiation of pathologic processes in the joint can be made.

Comment on “A second trigeminal CGRP receptor: function and expression of the AMY1 receptor”

Dear Editor, We read with the great interest the recent publication on a second trigeminal CGRP receptor by Walker et al.1 The authors convincingly present the presence of a functional noncanonical CGRP receptor (AMY1) at neuronal sites in the trigeminal system. The presence of a second CGRP receptor has been debated and although it is not yet recognized by IUPHAR, several reports have previously suggested the presence of more than one CGRP receptor. We have previously published reports on the effect of olcegepant2 and telcagepant3 in the human coronary artery. In these studies, we evaluated several concentrations of both antagonists and observed clear differences in their respective Schild plots (Fig. ​(Fig.11). Figure 1 Left panels: Relaxant effect of h‐αCGRP on human distal coronary arteries in the absence (open circle) or presence (closed square, 1 nmol/L; open triangle, 3 nmol/L; closed triangle, 10 nmol/L; open square, 30 nmol/L; closed nabla, 100 ... According to the study by Walker et al., olcegepant should discriminate better between hCLR/RAMP1 (“CGRP receptor”) and hCTR/RAMP1 (“AMY1 receptor”) than telcagepant. This is remarkably similar to what we have observed on the CGRP responses in human isolated distal coronary artery, where the Schild plot slope of olcegepant was significantly different from unity, with a clearly biphasic antagonistic profile,2 while the Schild plot slope of telcagepant in this same preparation was not significantly different from unity and the antagonistic profile was not that apparent biphasic.3 This difference can be explained by the presence of two different CGRP receptors for which olcegepant and telcagepant have different affinities. In light of these new experiments on the cloned human receptors, it is even more convincing to us that the human coronary artery also expresses two functional CGRP receptors, most probably matching the presence of hCLR/RAMP1 and hCTR/RAMP1. It is in this setting relevant to mention that amylin indeed causes relaxation in human coronary arteries.4 The study by Walker et al. sheds further light on an additional CGRP receptor, which might have implications in migraine in view of therapeutic efficacy and combined with our studies, in view of cardiovascular side effects of CGRP‐blocking drugs. As pointed by Walker et al., the clinically studied doses of neither olcegepant nor telcagepant are selective for one these receptors. The CGRP receptor antibody (AMG334), however, selectively binds to hCLR/RAMP1 and not to hCTR/RAMP1.5 We are awaiting the results of the clinical trials with this antibody with great interest to learn whether this selectivity has any clinical implications.

IκBα mediates prostate cancer cell death induced by combinatorial targeting of the androgen receptor.

BackgroundCombining different clinical agents to target multiple pathways in prostate cancer cells, including androgen receptor (AR) signaling, is potentially an effective strategy to improve outcomes for men with metastatic disease. We have previously demonstrated that sub-effective concentrations of an AR antagonist, bicalutamide, and the histone deacetylase inhibitor, vorinostat, act synergistically when combined to cause death of AR-dependent prostate cancer cells.MethodsIn this study, expression profiling of human prostate cancer cells treated with bicalutamide or vorinostat, alone or in combination, was employed to determine the molecular mechanisms underlying this synergistic action. Cell viability assays and quantitative real time PCR were used to validate identified candidate genes.ResultsA substantial proportion of the genes modulated by the combination of bicalutamide and vorinostat were androgen regulated. Independent pathway analysis identified further pathways and genes, most notably NFKBIA (encoding IκBα, an inhibitor of NF-κB and p53 signaling), as targets of this combinatorial treatment. Depletion of IκBα by siRNA knockdown enhanced apoptosis of prostate cancer cells, while ectopic overexpression of IκBα markedly suppressed cell death induced by the combination of bicalutamide and vorinostat.ConclusionThese findings implicate IκBα as a key mediator of the apoptotic action of this combinatorial AR targeting strategy and a promising new therapeutic target for prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2188-2) contains supplementary material, which is available to authorized users.

Development of theta rhythm in hippocampal formation slices perfused with 5-HT1A antagonist, (S)WAY 100135

Numerous studies have revealed that median raphe nuclei stimulation induces desynchronization of hippocampal field activity in vivo. Some findings provide evidence for tonic regulation of the theta oscillation of the septo-hippocampal system via the serotonergic system. To date the involvement of serotonergic transmission in theta rhythm generation in hippocampal slices has never been investigated. Thus the aim of the present study was to test whether HPC in vitro preparation is capable of producing theta in the presence of compounds modulating the activity of 5-HT1A receptors. To achieve this a series of experiments designed to determine the effect of different concentrations of the 5-HT1A receptor antagonist (S)WAY 100135 on HPC field activity was carried out. The dominant field potential pattern recorded within HPC slices was epileptiform activity, with a maximum frequency ranging from 0.19±0.06Hz to 0.69±0.10Hz. In addition, after the bath application of (S)WAY 100135 in concentrations 3 and 10µM rhythmic epochs in the theta frequency range were also noted. The highest probability of theta rhythm production was observed after the bath perfusion with a solution of (S) WAY 100135 at a concentration of 10μM. These theta rhythm epochs were characterized by a higher-than-average amplitude compared to carbachol-induced theta epochs and shorter time duration, with no apparent differences in the average frequency and duration of intervals between theta epochs. These results obtained herein of in vitro studies provide direct evidence for the involvement of serotonergic receptors in the depression of oscillatory activity in the HPC theta band.

Synovial Fluid and Serum Concentrations of Interleukin-1 Receptor Antagonist and Interleukin-1ß in Naturally Occurring Equine Osteoarthritis and Septic Arthritis

The objective of this study was to investigate the concentrations of interleukin-1 receptor antagonist (IL-1Ra) and interleukin-1ß (IL-1ß) in synovial fluid (SF) and serum (SE) of horses with different grades of osteoarthritis (OA) and septic arthritis. Based on SF analysis, radiographic, and arthroscopic scores, 40 horses were classified into three groups as follows: mild OA, advanced OA, and septic arthritis. Horses without orthopedic problems served as a control group. Equine-specific antibody enzyme-linked immunosorbent assays were used to determine the concentration of IL-1Ra and the catabolic cytokine IL-1ß in SF and SE. Results were further compared with levels of the previously described biomarkers C-telopeptide fragments of type II collagen (CTX-II) and myeloperoxidase. In the present study, the SF of healthy joints, those with nonseptic OA, and those with septic arthritis contained significantly different levels of IL-1Ra. Serum concentrations of IL-1Ra were only significantly elevated in horses with septic arthritis when compared with the control group. Different levels of IL-1ß were detected in SE of control horses compared with those with various degrees of joint disease. Synovial fluid concentrations of IL-1ß were only moderately elevated in the groups of horses with joint disease. In addition to lameness examination and standard diagnostic procedures, the determination of IL-1Ra concentration in SF, in combination with further biomarkers, might be useful to assess the extent of intrasynovial inflammation.

Growth hormone and cancer: GH production and action in glioma?

The hypersecretion of pituitary growth hormone (GH) is associated with an increased risk of cancer, while reducing pituitary GH signaling reduces this risk. Roles for pituitary GH in cancer are therefore well established. The expression of the GH gene is, however, not confined to the pituitary gland and it is now known to occur in many extrapituitary tissues, in which it has local autocrine or paracrine actions, rather than endocrine function. It is, for instance, expressed in cancers of the prostate, lung, skin, endometrium and colon. The oncogenicity of autocrine GH may also be greater than that induced by endocrine or exogenous GH, as higher concentrations of GHR antagonists are required to inhibit its actions. This may reflect the fact that autocrine GH is thought to act at intracellular receptors directly after synthesis, in compartments not readily accessible to endocrine (or exogenous) GH. The roles and actions of extrapituitary GH in cancer may therefore differ from those of pituitary GH. The possibility that GH may be expressed and act in glioma tumors was therefore examined by immunohistochemistry. These results demonstrate, for the first time, the presence of abundant GH− and GH receptor (GHR−) immunoreactivity in glioma, in which they were co-localized in cytoplasmic but not nuclear compartments. These results demonstrate that glioma differs from most cancers in lacking nuclear GHRs, but GH is nevertheless likely to have autocrine or paracrine actions in the induction and progression of glioma.

Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Oxaliplatin evokes P2X7-dependent glutamate release in the cerebral cortex: A pain mechanism mediated by Pannexin 1

Anticancer therapy based on the repeated administration of oxaliplatin is limited by the development of a neuropathic syndrome difficult to treat. Oxaliplatin neurotoxicity is based on complex nervous mechanisms, the comprehension of the role of single neurotransmitters and the knowledge of the signal flow among cells is matter of importance to improve therapeutic chances.In a rat model of oxaliplatin-induced neuropathy, we report increased P2X7-evoked glutamate release from cerebrocortical synaptosomes. The release was abolished by the P2X7 receptor (P2X7R) antagonists Brilliant-Blue-G (BBG) and A-438079, and significantly reduced by Carbenoxolone and the Pannexin 1 (Panx1) selective inhibitors Erioglaucine and 10Panx suggesting the recruitment of Panx1. Aimed to evaluate the significance of P2X7R-Panx1 system activation in pain generated by oxaliplatin, pharmacological modulators were spinally infused by intrathecal catheter in oxaliplatin-treated animals. BBG, Erioglaucine and 10Panx reverted oxaliplatin-dependent pain. Finally, the influence of the P2X7R-Panx1 system blockade on oxaliplatin anticancer activity was evaluated on the human colon cancer cell line HT-29. Prevention of HT-29 apoptosis and mortality was dependent by kind and concentration of P2X7R antagonists. On the contrary, the inhibition of Panx1 did not alter oxaliplatin lethality in tumor cells.It is concluded that glutamate release dependent on P2X7R is increased in cerebrocortical nerve terminals from oxaliplatin-treated rats; the increase is mediated by functional recruitment of Panx1; P2X7R antagonists and Panx1 inhibitors revert oxaliplatin-induced neuropathic pain; Panx1 inhibitors do not alter the oxaliplatin-induced mortality of cancer cells HT-29. The inhibition of Panx1 channel is suggested as a new and safe pharmacological target.

Biosynthesis and actions of 5-oxoeicosatetraenoic acid (5-oxo-ETE) on feline granulocytes

The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially hampered progress in this area. As an alternative, we have explored the possibility that the cat could serve as an appropriate animal model to investigate the role of 5-oxo-ETE. We found that feline peripheral blood leukocytes synthesize 5-oxo-ETE and that physiologically relevant levels of 5-oxo-ETE are present in bronchoalveolar lavage fluid from cats with experimentally induced asthma. 5-Oxo-ETE (EC50, 0.7nM) is a much more potent activator of actin polymerization in feline eosinophils than various other eicosanoids, including leukotriene (LT) B4 and prostaglandin D2. 5-Oxo-ETE and LTB4 induce feline leukocyte migration to similar extents at low concentrations (1nM), but at higher concentrations the response to 5-oxo-ETE is much greater. Although high concentrations of selective human OXE receptor antagonists blocked 5-oxo-ETE-induced actin polymerization in feline granulocytes, their potencies were about 200 times lower than for human granulocytes. We conclude that feline leukocytes synthesize and respond to 5-oxo-ETE, which could potentially play an important role in feline asthma, a common condition in this species. The cat could serve as a useful animal model to investigate the pathophysiological role of 5-oxo-ETE.

Triphenyl phosphate-induced developmental toxicity in zebrafish: Potential role of the retinoic acid receptor

Using zebrafish as a model, we previously reported that developmental exposure to triphenyl phosphate (TPP) – a high-production volume organophosphate-based flame retardant – results in dioxin-like cardiac looping impairments that are independent of the aryl hydrocarbon receptor. Using a pharmacologic approach, the objective of this study was to investigate the potential role of retinoic acid receptor (RAR) – a nuclear receptor that regulates vertebrate heart morphogenesis – in mediating TPP-induced developmental toxicity in zebrafish. We first revealed that static exposure of zebrafish from 5–72h post-fertilization (hpf) to TPP in the presence of non-toxic concentrations of an RAR antagonist (BMS493) significantly enhanced TPP-induced toxicity (relative to TPP alone), even though identical non-toxic BMS493 concentrations mitigated retinoic acid (RA)-induced toxicity. BMS493-mediated enhancement of TPP toxicity was not a result of differential TPP uptake or metabolism, as internal embryonic doses of TPP and diphenyl phosphate (DPP) – a primary TPP metabolite – were not different in the presence or absence of BMS493. Using real-time PCR, we then quantified the relative change in expression of cytochrome P450 26a1 (cyp26a1) – a major target gene for RA-induced RAR activation in zebrafish – and found that RA and TPP exposure resulted in a ∼5-fold increase and decrease in cyp26a1 expression, respectively, relative to vehicle-exposed embryos. To address whether TPP may interact with human RARs, we then exposed Chinese hamster ovary cells stably transfected with chimeric human RARα-, RARβ-, or RARγ to TPP in the presence of RA, and found that TPP significantly inhibited RA-induced luciferase activity in a concentration-dependent manner. Overall, our findings suggest that zebrafish RARs may be involved in mediating TPP-induced developmental toxicity, a mechanism of action that may have relevance to humans.

Measurement of smooth muscle function in the isolated tissue bath-applications to pharmacology research.

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum – if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum – if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.