Dose-response Curves For Inhibition Research Articles

A novel biomimetic nanoplasmonic sensor for rapid and accurate evaluation of checkpoint inhibitor immunotherapy

Immune checkpoint inhibitors (ICIs) emerged as promising immunotherapies for cancer treatment, harnessing the patient’s immune system to fight and eliminate tumor cells. However, despite their potential and proven efficacies, checkpoint inhibitors still face important challenges such as the tumor heterogeneity and resistance mechanisms, and the complex in vitro testing, which limits their widespread applicability and implementation to treat cancer. To address these challenges, we propose a novel analytical technique utilizing biomimetic label-free nanoplasmonic biosensors for rapid and reliable screening and evaluation of checkpoint inhibitors. We have designed and fabricated a low-density nanostructured plasmonic sensor based on gold nanodisks that enables the direct formation of a functional supported lipid bilayer, which acts as an artificial cell membrane for tumor ligand immobilization. With this biomimetic scaffold, our biosensing approach provides real-time, highly sensitive analysis of immune checkpoint pathways and direct assessment of the blocking effects of monoclonal antibodies in less than 20 min/test. We demonstrate the accuracy of our biomimetic sensor for the study of the programmed cell death protein 1 (PD1) checkpoint pathway, achieving a limit of detection of 6.7 ng/mL for direct PD1/PD-L1 interaction monitoring. Besides, we have performed dose–response inhibition curves for an anti-PD1 monoclonal antibody, obtaining a half maximal inhibitory concentration (IC50) of 0.43 nM, within the same range than those obtained with conventional techniques. Our biomimetic sensor platform combines the potential of plasmonic technologies for rapid label-free analysis with the reliability of cell-based assay in terms of ligand mobility. The biosensor is integrated in a compact user-friendly device for the straightforward implementation in biomedical and pharmaceutical laboratories.Graphical

Renal Function and Transporters in the Na/K-ATPase α1S/S Mouse Model of High Affnity for Cardiotonic Steroids

Genetic ablation of ATP1A1 in the mouse renal proximal tubule (RPT) revealed that Na/K-ATPase (NKA) α1 tonically inhibits sodium and water reabsorption. Mechanistically, findings in KO mouse RPT and renal epithelial cells were consistent with a prevalent role of a NKA/Src-dependent downregulation of Na+/H+ exchanger 3 (NHE3). Pharmacologically, functional activation of this pathway has been obtained in vitro and ex vivo using low concentrations of NKA archetypal ligands cardiotonic steroids (CTS). However, the physiological implication of an activation of the endogenous CTS (eCTS)/NKA/Src on renal transporters including NHE3 has not been explored. We assessed the renal impact of CTS/NKA α1 receptor activation by comparing mice expressing the naturally resistant NKA α1 and age-matched mice genetically engineered to express a mutated NKA α1 with high affnity for CTS (R111Q/D122N double substitution). Mice obtained from established colonies at the University of Cincinnati were a gift from Dr. Jerry Lingrel. CTS-sensitive (α1S/S) male mice aged 3-6 months on a C57Bl6 background and age-matched controls (α1R/R) were used. Mice were born with a normal Mendelian ratio, with no observable difference between α1S/S and α1R/R. Renal Na/K-ATPase inhibitory dose-response curve to the CTS ouabain was shifted to the left, as expected, and occurred without detectable change of total renal NKA activity, NKA α1 or β1 protein contents. Metabolic cage studies revealed that increased α1 sensitivity to CTS resulted in a significant increase in urine output in α1S/S mice compared to age-matched α1R/R controls (3.9±0.3 vs. 2.4±0.2 ml/24h; n=6, p<0.01). Consistent with an activation of the NKA/Src receptor by eCTS, western blot analyses of renal cortical preparation revealed an increased phosphorylation of NKA α1 at Y260 (Src target residue) and decreased NHE3 content (43.6 %; n=5, p<0.01). RNA-seq analysis of whole kidneys from α1S/S and α1R/R littermate mice using the Illumina NovaSeq 6000 system (Novogene) and Volcano Plot (DEG data using Python) further showed differential expression of 296 genes (147 upregulated and 149 downregulated). Gene Set Enrichment Analysis (GSEA) using the WebGestalt toolkit showed that the altered pathways were mostly involved in solute transport. Taken together, these results provide genetic evidence of the physiological role of eCTS/NKAα1 on renal function in the intact mouse. Increased affnity to eCTS is suffcient to lead to an activation of NKAα1/Src, a downregulation of NHE3, and transcriptional regulation of renal transporters associated with increased urine output. This work was supported by AHA, Predoctoral fellowship #19PRE34450095, NIH DK129937, and NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Broad-Spectrum Antimicrobial Activity of Synthetic Peptides GV185 and GV187.

Optimizing synthetic antimicrobial peptides for safe and enhanced activity against fungal and bacterial pathogens is useful for genetic engineering of plants for resistance to plant pathogens and their associated mycotoxins. Nine synthetic peptides modeled after lytic peptides tachyplesin 1, D4E1 from cecropin A, and protegrin 1 were added to germinated spores of fungal species Aspergillus flavus, Rhizopus stolonifer, Fusarium oxysporum f. sp. vasinfectum, F. verticillioides, F. graminearum, Claviceps purpurea, Verticillium dahliae, and Thielaviopsis basicola and bacterial cultures of Pseudomonas syringae pv. tabaci and Xanthomonas campestris pv. campestris at different doses and inhibitory dose response curves, and were modeled to assess antimicrobial activity. Peptides GV185 and GV187, modified from tachyplesin 1, had superior abilities to inhibit fungal and bacterial growth (50% inhibitory concentrations [IC50] ranging from 0.1 to 8.7 µM). R. stolonifer (IC50 = 8.1 µM), A. flavus (IC50 = 3.1 µM), and F. graminearum (IC50 = 2.2 µM) were less inhibited by GV185 and GV187 than all the remaining fungi (IC50 = 1.4 µM) and bacteria (IC50 = 0.1 µM). Of the remaining peptides, GV193, GV195, and GV196 (IC50 range of 0.9 to 6.6 µM) inhibited fungal growth of A. flavus, F. verticillioides, and F. graminearum less than GV185 and GV187 (IC50 range of 0.8 to 3.9 µM), followed by GV197 (IC50 range of 0.8 to 9.1 µM), whereas GV190 and GV192 inhibited poorly (IC50 range of 28.2 to 36.6 µM and 15.5 to 19.4 µM, respectively) and GV198 stimulated growth. GV185 and GV187 had slightly weaker hydrophobic and cationic residues than other tachyplesin 1 modified peptides but still had unexpectedly high lytic activity. Germinated fungal spores of R. stolonifer and F. graminearum exposed to these two peptides and D4E1 and AGM182 appeared wrinkled, with perforations near potential cytoplasmic leakage, which provided evidence of plasma membrane and cell wall lysis. We conclude that peptides GV185 and GV187 are promising candidates for genetic engineering of crops for resistance to plant-pathogenic bacteria and fungi, including A. flavus and aflatoxin contamination.

Monosomy 7 and Del(7q) Cause Selective Sensitivity to Inhibitors of Nicotinamide Phosphoribosyltransferase in Acute Myeloid Leukemia

Monosomy 7 and deletion of chromosome arm 7q (-7/-7q) occur in up to 10% of patients with acute myeloid leukemia (AML). In AML, -7/-7q are associated with resistance to chemotherapy and adverse prognosis. As such, treatment outcomes for this subgroup of patients remain particularly dismal, and targeted therapies are urgently needed. To identify targeted therapies for AML with -7/-7q we analyzed ex vivo drug sensitivities in 158 AML samples. The presence of -7/-7q (n=18) was determined using karyotyping and exome-sequencing of bone marrow (BM) mononuclear cells and a matched skin biopsy control. Mononuclear cells from fresh bone marrow aspirates of AML patients were incubated with a library of up to 525 oncology drugs for 72h, with each drug pre-plated in 5 concentrations. Cell viability was measured using the CellTiter Glo (CTG) assay, and the response of each sample was summarized as a drug sensitivity score (DSS), which is a modified integration of the dose-response inhibition curve, directly proportional to the sensitivity of a sample. We found that AML samples with -7/-7q were highly sensitive to the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor daporinad (p-value ~ 4.2e-06) (Figure A). NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway. The NAMPT gene is located at 7q22.3. Monosomy 7 and del(7q) lead to the deletion of one copy (i.e., haploinsufficiency) of NAMPT. Notably, a rare AML with del(7q) in which the deletion did not include the NAMPT gene (and which was thus diploid for NAMPT) was not sensitized to NAMPT inhibition. Taken together, this data demonstrates a significant association between the NAMPT haploinsufficiency in AML with -7/-7q and increased sensitivity to NAMPT inhibition. To further support our findings, we analyzed the sensitivity of AML cells without or with -7/-7q to four NAMPT inhibitors (daporinad, GMX1778, KPT-9274, and LSN3154567) using multiparametric flow cytometry. Viably frozen BM mononuclear cells from AML patients with (n=11) and without (n=51) -7/-7q were thawed and incubated with drugs in 8 concentrations for 72 hours. After incubation, cell viability was analyzed using the iQue Screener Plus flow cytometer with a panel consisting of CD45, CD3, CD34, CD117, CD14, and CD38 antibodies, to identify myeloid and lymphoid cell populations, as well as Annexin V and 7-AAD to discriminate live from dead cells. We found that CD34+ myeloblasts from AML with -7/-7q were selectively killed by all four NAMPT inhibitors, validating our initial results (p-value ~ 1.4e-02). An in-depth analysis of cellular subpopulations revealed that while CD34+CD38+ cells (representing more differentiated myeloblasts) were significantly more sensitive to NAMPT inhibition in -7/-7q samples compared to wild-type samples (p-value ~ 4.5e-04), the CD34+CD38- compartment (anticipated to contain the immature leukemic stem and progenitor cells) was not sensitized in this group (Figure B). Interestingly, the analysis of ex vivo drug sensitivities also revealed that while the majority of AML with -7/-7q are resistant to the FDA-approved BCL2 inhibitor venetoclax, they are highly sensitive to daporinad (Figure A). This finding proposes NAMPT inhibitors as a suitable alternative to venetoclax for -7/-7q patients. Additionally, since venetoclax has previously shown significant efficacy against the highly BCL2-dependent undifferentiated (CD34+CD38-) myeloblasts, its combination with NAMPT inhibitors represents a promising strategy for achieving total leukemic cell death in these patients. In conclusion, we have found that AML with NAMPT haploinsufficiency caused by -7/-7q shows selective sensitivity to NAMPT inhibition. This suggests that NAMPT inhibitors have the potential to be a potent targeted therapy for AML with -7/-7q, a disease with a particularly adverse prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Oxytocin Receptor Activation: An Alternative to Naloxone for Rescuing Opioid‐Induced Respiratory Depression by Systemic Fentanyl

Opioid overdose intervention by naloxone, a high affinity receptor antagonist, reverses opioid-induced respiratory depression (OIRD) and analgesia by displacing opioids. Systemic naloxone stimulates release of the hypothalamic neuropeptide oxytocin (OXTN), which has analgesic properties and participates in cardiorespiratory homeostasis. To test the hypothesis that OXTN can reverse OIRD, we assessed the rescue potential of graded doses (0, 0.1, 2, 5, 10, 50 nmol/kg, i.v., n=5-6/dose) of OXTN to counteract fentanyl (60 nmol/kg, i.v.)-induced depression of neural inspiration indexed by recording phrenic nerve activity (PNA) in anesthetized (urethane/α-chloralose), vagotomized, and artificially ventilated rats. OXTN rescued fentanyl OIRD by reducing fentanyl-induced burst arrest (P=0.0057) and recovery time of burst frequency (P=0.0016) and amplitude (P=0.0025), resulting in parabolic inhibitory dose-response curves.OXTN receptor (OXTNR) antagonism (40 nmol/kg, i.v., n=5) impaired OXTN restoration of PNA post-fentanyl (P=0.0207) and prolonged recovery time of PNA frequency (P=0.0066) and amplitude (P=0.0022). Vasopressin (AVP) receptor 1A (V1aR) antagonism restored the efficacy of high dose OXTN to rescue fentanyl OIRD (P<0.0001, n=4-6/dose), resulting in classic sigmoidal inhibitory dose-response curves, and prevented (P>0.9999) transient hypertension from V1aR cross-activation (P=0.0173). Consistent with this finding, post-fentanyl AVP (5 nmol/kg, i.v., n=5) produced acute hypertension (P=0.0004) and failed to reverse OIRD (PNA arrest P=0.6184, PNA freq. P=0.2369, PNA ampl. P=0.9571). The non-peptide OXTNR agonist WAY-267464 (75 nmol/kg, i.v., n=5), which also has V1aR antagonist properties, quickly reversed fentanyl OIRD (P<0.0001), with rapid recovery to baseline PNA frequency (P=0.0011) and amplitude (P=0.0044) without adverse hemodynamic consequences (P=0.4022). Findings indicate that peptide and non-peptide agonist activation of OXTNR without cross-activation of V1aR rescues fentanyl OIRD.OXTNR agonists could be lifesaving resuscitation agents that enhance rather than interrupt opioid analgesia.

Partial Characterization of Protease Inhibitors of Ulva ohnoi and Their Effect on Digestive Proteases of Marine Fish

This piece of research evaluates the presence of protease inhibitors in the macroalga Ulva ohnoi and provides an initial overview of their mode of action. The ability of Ulva protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when Ulva concentration increased in the assay. The thermal treatment of Ulva reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver–Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on Ulva concentration. Overall, the results confirmed the presence of protease inhibitors in Ulva, though heat treatment was enough for inactivating these compounds.

Effect of Roflumilast in airways disorders via dual inhibition of phosphodiesterase and Ca2+-channel

The bronchodilator effects of Roflumilast “a selective phosphodiesterase type-4 (PDE4)” inhibitor studied in this experimental protocol. The spiral strips of isolated guinea-pig tracheal chains mounted in organ bath and maintained in Krebs solution ventilated with carbogen at 32 °C and in Ca++ restricted krebs solution. PDE inhibitory activity was evaluated by recording dose response curves using inhibitory effect of isoprenaline on CCh induced contractions. For confirmation of PDE inhibition the intracellular cAMP levels were also estimated. Roflumilast resulted a sharp inhibition in contractile responses of carbachol (CCh, 1 µM) and K+ (80 mM) and the results were almost similar to verapamil. In Ca++ restricted Krebs solution, a rightward shift in the Ca++ response curves observed in the tracheal chain strips which were pretreated with Roflumilast (0.001–0.003 mg/mL) and the maximum response was suppressed, similarly as with verapamil. PDE inhibitory effect of Roflumilast evaluated by recording dose-dependent (0.03–0.1 mg/mL) responses, the isoprenaline-induced inhibitory dose response curves shifted leftward similar to papaverine (PDE inhibitor). Pretreatment with Roflumilast exhibited elevated intracellular cAMP levels in tracheal strips. Findings of the experiment conclude bronchodilatory influence of Roflumilast via PDE and Ca++ channel inhibition. Results of current experiment offers comprehensive mechanistic background of Roflumilast in future as therapeutic bronchodilator for hyperactive bronchial airway diseases.

2348Novel direct thrombin inhibitor achieves superior antithrombotic effect with lower bleeding risk than heparin or bivalirudin

Abstract Background We have isolated, variegin, a unique direct thrombin inhibitor (DTI) from tropical bont tick Amblyomma variegatum. Variegin inhibits thrombin active site and exosite-1 with an inhibitory constant of 0.3 nM (9-fold better than bivalirudin). It is also &gt;5 orders of magnitude more selective for thrombin than other blood coagulation serine proteases. Variegin has a plasma half-life of 50 minutes (compared with bivalirudin 25 minutes and heparin ∼ hours). Purpose We aimed to develop variegin into a parenteral anticoagulant for percutaneous coronary intervention (PCI) and tested variegin in several pre-clinical models. Methods In rats, variegin was tested for efficacy (anticoagulation intensity) in a FeCl3-induced carotid artery thrombosis model while safety (bleeding risk) was tested in a tail incision model that recapitulated the time-frame of PCI (∼1 hour) in humans (time-response model). In pigs, an extracorporeal circuit with modified Badimon chambers containing coronary stents was used to assess efficacy, while bleeding risk was evaluated through needle-induced injury on a superficial ear vein, with or without concurrent administration of aspirin and ticagrelor (DAPT). Unfractionated heparin (UFH) and bivalirudin at dosages recommended for PCI were used as references. Ex vivo clotting analyses including thrombin generation test, rotational thromboelastometry, activated partial thromboplastin time and clot waveform analysis were performed in human blood spiked with DAPT and the three anticoagulants. Results In the rat time-response model, a single variegin bolus conferred better antithrombotic effect than a continuous infusion of bivalirudin and more rapid recovery of haemostasis than a single bolus of heparin. In the porcine ex vivo model, without DAPT, UFH, bivalirudin and 1 mg/kg variegin reduced stent thrombus by 35% (P&lt;0.001), 60% (P&lt;0.0001), and 80% (P&lt;0.0001), compared with saline, respectively. In the presence of DAPT, UFH, bivalirudin and only 0.1 mg/kg of variegin (10-fold lowered dose) reduced stent thrombus by 65% (P&lt;0.01), 75% (P&lt;0.001), and 87% (P&lt;0.0001), respectively (Fig. 1A). However, in the presence of DAPT, standard-dose UFH and bivalirudin prolonged bleeding times far longer than low-dose variegin (Fig. 1B). In human platelet rich plasma treated with DAPT, UFH showed a much more precipitous decline in thrombin generation potential than variegin (Fig. 1C). Dose response curves for inhibition of thrombin generation are also steeper in UFH and bivalirudin than in variegin, suggesting larger safety dose margin for variegin (Fig. 1D). These observations potentially account for the better preservation of haemostasis with low-dose variegin in combination with DAPT. Figure 1 Conclusion In the presence of aspirin and ticagrelor, a low dose of variegin, a novel direct thrombin inhibitor, achieved superior antithrombotic effect with significantly lower bleeding risk than heparin or bivalirudin in pre-clinical PCI models.

OR16-5 Feedback on LH in Testosterone-Clamped Men Depends on the Mode of Testosterone Administration and Body Composition

Context. Investigations of testosterone (T)-mediated feedback on LH secretion are few and contradictory. Discrepancies may reflect the choice of androgen, as well as dose, route and/or pattern (continuous vspulsatile) of administration. Moreover, for controlled T feedback studies, endogenous T secretion must be shut down and replaced by iv T infusion. Objective. The goal of this study was to quantify feedback in relation to the infused T pattern (pulsatile vs nonpulsatile) with the hypotheses that: 1) feedback by T pulses is less inhibitory, 2) T feedback operates partly at the pituitary level, 3) T feedback is controlled in part by body composition. Design. This was a placebo-controlled, blinded, and prospectively randomized crossover study comprising 16 men (age range 23-54 yr and BMI between 22.3-34.2 kg/m2). Subjects received ketoconazole (1600 mg over 22 h to block endogenous T secretion: serum T below 85 ng/dL) followed by pulsatile or continuous iv T addback (total T dose 5 mg/22 h). Cortisol inhibition was treated with low-dose dexamethasone. Setting. The study was performed in a Clinical Translational Research Unit. Interventions. Subjects underwent 14 h of blood sampling at 10-min intervals, starting at 0600 h. T was given continuously starting at 0900 h or as 45-min squarewave pulses every 90 min. To test pituitary feedback, a bolus iv injection of 33 ng/kg GnRH was given at 1630 h. Outcome measures. Log-transformed LH and T concentration ratios before and after GnRH administration. In addition, a four-parameter logistic regression between serum T concentration and LH secretion rate (calculated by deconvolution analysis) was used to independently estimate feedback of T on LH. Results. Despite higher T (total, bioavailable and free T) concentrations during pulsatile feedback, LH concentrations and secretion rates, whether driven by endogenous or exogenous GnRH, were similar to those during continuous infusion, indicating diminished pulsatile T feedback. This interpretation was corroborated by the right-shift (i.e. towards higher T concentrations) of the T-LH inhibitory dose-response curve. During continuous and pulsatile T infusion, LH concentrations and LH:T ratios depended positively on BMI to a similar extent before and after GnRH administration. Under both feedback regimens, basal but not pulsatile LH secretion correlated negatively with CT-estimated abdominal visceral fat mass. Age was not a significant covariate over the limited range explored. Conclusion. In summary, pulsatile T is less effective as a negative feedback signal on LH secretion than continuous T administration, as demonstrated by two independent methods. This difference in feedback strength was evident for exogenous GnRH-stimulated LH release as well, and thus occurs probably in part at the pituitary level, while not excluding additional hypothalamic involvement.

Lethal and sublethal responses in the fish, Odontesthes bonariensis, exposed to chlorpyrifos alone or under mixtures with endosulfán and lambda-cyhalothrin.

Need for ecotoxicological information on local species has been recently highlighted as a priority issue in Latin America. In addition, little information has been found on concentration distances between lethal and sublethal effects, and the effect of mixtures at these two levels of analysis. Chlorpyrifos (CPF) is an organophosphate insecticide broadly used in soybean crops which has dramatically expanded in Latin America and other regions of the world. The aim of the present study was to evaluate lethal and sublethal effects of CPF, singly or in mixtures, on the inland "Pejerrey" (Odontesthes bonariensis) under laboratory conditions. Bioassays were performed using 15-30 d post hatch Pejerrey larvae. Six toxicity tests were run for estimating the average inter-assay dose-response curve of CPF and other six for assessing the effects of mixtures of CPF with endosulfan (EN) or lambda-cyhalothrin (LC), at three toxic units (TU) proportions (25:75, 50:50, 75:25). In addition, four assays were performed to describe the average inter-assay dose-response inhibition curve of acetylcholinesterase (AchE) for CPF alone and two for assessing the mixtures. The estimated 96 h-LC50 for CPF was 2.26 ± 1.11 µg/L and the incipiency value was 0.048 ± 0.012 µg/L, placing this Neotropical species among the 13% of worldwide fish more sensitive to CPF. In addition, the 96 h-LC50 for EN and LC were 0.30 ± 0.012 µg/L and 0.043 ± 0.031 µg/L, respectively. Therefore, relative toxicity of the three soybean insecticides for O. bonariensis was LC > EN > CPF. Effects of mixtures with EN and LC were variable, but in general fitted to both, independent action (IA) and concentration addition (CA) models. Slight antagonism was found when CPF TU proportions were above 50%. Therefore, from the regulatory point of view, the use of both mixture models, CA or IA, would be precautionary. Differential sensitivity to CPF was found for AchE inhibition at the head (96 h-IC50 = 0.065 ± 0.058 µg/L) and the body (96 h-IC50 = 0.48 ± 0.17 µg/L). In addition, whereas no significant effects induced by mixtures was observed in body AchE activity, antagonism was induced in head AchE inhibition in presence of both, EN and LC in the mixture. The lethal to sublethal ratio was close to 25.2 and 3.4 when comparing the CPF-LC50 and IC50s for head and body AchE activity, respectively. However, considerable overlapping was observed between concentration-response curves, indicating that the use of AchE as biomarker for environmental monitoring would be limited.

Equivalent intraperitoneal doses of ibuprofen supplemented in drinking water or in diet: a behavioral and biochemical assay using antinociceptive and thromboxane inhibitory dose-response curves in mice.

Background. Ibuprofen is used chronically in different animal models of inflammation by administration in drinking water or in diet due to its short half-life. Though this practice has been used for years, ibuprofen doses were never assayed against parenteral dose–response curves. This study aims at identifying the equivalent intraperitoneal (i.p.) doses of ibuprofen, when it is administered in drinking water or in diet.Methods. Bioassays were performed using formalin test and incisional pain model for antinociceptive efficacy and serum TXB2 for eicosanoid inhibitory activity. The dose–response curve of i.p. administered ibuprofen was constructed for each test using 50, 75, 100 and 200 mg/kg body weight (b.w.). The dose–response curves were constructed of phase 2a of the formalin test (the most sensitive phase to COX inhibitory agents), the area under the ‘change in mechanical threshold’-time curve in the incisional pain model and serum TXB2 levels. The assayed ibuprofen concentrations administered in drinking water were 0.2, 0.35, 0.6 mg/ml and those administered in diet were 82, 263, 375 mg/kg diet.Results. The 3 concentrations applied in drinking water lay between 73.6 and 85.5 mg/kg b.w., i.p., in case of the formalin test; between 58.9 and 77.8 mg/kg b.w., i.p., in case of the incisional pain model; and between 71.8 and 125.8 mg/kg b.w., i.p., in case of serum TXB2 levels. The 3 concentrations administered in diet lay between 67.6 and 83.8 mg/kg b.w., i.p., in case of the formalin test; between 52.7 and 68.6 mg/kg b.w., i.p., in case of the incisional pain model; and between 63.6 and 92.5 mg/kg b.w., i.p., in case of serum TXB2 levels.Discussion. The increment in pharmacological effects of different doses of continuously administered ibuprofen in drinking water or diet do not parallel those of i.p. administered ibuprofen. It is therefore difficult to assume the equivalent parenteral daily doses based on mathematical calculations.

Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors.

Background:The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described.Methods:QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 μM everolimus, respectively, 1000-fold and 250-fold initial IC50. Using total DNA content as a measure of cell number, growth inhibitory dose–response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT–qPCR).Results:Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10–12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed.Conclusions:Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1.

1011 EFFECT OF MELANOTAN II, A MELANOCORTIN AGONIST, ON SEXUAL BEHAVIOR SUPPRESSED BY PSYCHOLOGICAL STRESS IN MALE RATS

acetylcholine (Ach, 10 to 3 x 10M) were added with various concentrations into organ bath to induce isometric contraction of the isolated rat seminal vesicles. The dose response curve of the isolated rat seminal vesicles reacting to PE and Ach, the inhibitory dose response curve to the different adrenergic antagonists (prazosin for 1, WB-4101 for 1a, Chloroethylclonidine CEC for 1b), muscarinic antagonists (atropine for M, pirenzepine for M1, methoctramine for M2, and 4-DAMP for M3) and cross-over reaction were recorded. The twitch pattern of SVSM contraction was analyzed according to its phasic and tonic components. RESULTS: PE and Ach could both achieve good contraction of rat seminal vesicle. The IC50 of prazosin and WB-4101 were far greater than that of CEC. The IC50 of 4-DAMP was very close to atropine. PE and Ach could cause different patterns of SVSM twitch. PE induced SVSM contraction had more tonic components, but Ach induced SVSM contraction had more phasic components. CONCLUSIONS: The predominant subtypes of adrenergic and cholinergic receptors on rat seminal vesicle are £ 1a and M3 respectively. Different drugs also cause different pattern of SVSM twitch. In our speculation, sympathetic effect is more dominant during emission stage to squeeze seminal fluid out; however, parasympathetic effect is more dominant during ejection stage and provides an anti-reflux effect on ejaculatory duct. Further studies for realizing physiology of ejaculation are worthy.

Cellulose Biosynthesis Inhibitors: Comparative Effect on Bean Cell Cultures

The variety of bioassays developed to evaluate different inhibition responses for cellulose biosynthesis inhibitors makes it difficult to compare the results obtained. This work aims (i) to test a single inhibitory assay for comparing active concentrations of a set of putative cellulose biosynthesis inhibitors and (ii) to characterize their effect on cell wall polysaccharides biosynthesis following a short-term exposure. For the first aim, dose-response curves for inhibition of dry-weight increase following a 30 days exposure of bean callus-cultured cells to these inhibitors were obtained. The compound concentration capable of inhibiting dry weight increase by 50% compared to control (I50) ranged from subnanomolar (CGA 325′615) to nanomolar (AE F150944, flupoxam, triazofenamide and oxaziclomefone) and micromolar (dichlobenil, quinclorac and compound 1) concentrations. In order to gain a better understanding of the effect of the putative inhibitors on cell wall polysaccharides biosynthesis, the [14C]glucose incorporation into cell wall fractions was determined after a 20 h exposure of cell suspensions to each inhibitor at their I50 value. All the inhibitors tested decreased glucose incorporation into cellulose with the exception of quinclorac, which increased it. In some herbicide treatments, reduction in the incorporation into cellulose was accompanied by an increase in the incorporation into other fractions. In order to appreciate the effect of the inhibitors on cell wall partitioning, a cluster and Principal Component Analysis (PCA) based on the relative contribution of [14C]glucose incorporation into the different cell wall fractions were performed, and three groups of compounds were identified. The first group included quinclorac, which increased glucose incorporation into cellulose; the second group consisted of compound 1, CGA 325′615, oxaziclomefone and AE F150944, which decreased the relative glucose incorporation into cellulose but increased it into tightly-bound cellulose fractions; and the third group, comprising flupoxam, triazofenamide and dichlobenil, decreased the relative glucose incorporation into cellulose and increased it into a pectin rich fraction.

Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream (Sparus auratus L.)

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml(-1), which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 μmol l(-1)), PI3K (LY-294002, 75 μmol l(-1)) or MEK (U-012610, 10 μmol l(-1)) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.

Immunological and physiological validation of an enzyme-linked immunosorbent assay (ELISA) for the measurement of growth hormone in goat (Capra hircus) plasma

The current study describes the optimization and validation of an enzyme-linked immunosorbent assay (ELISA) for the quantification of growth hormone (GH) in blood plasma of goats, Capra hircus. A microtiter plate based competitive assay was developed using a capture ELISA for anti-ovine GH and biotinylated ovine GH. Ovine GH standards were prepared in charcoal–dextran stripped goat plasma. The assay was optimized in terms of sensitivity, specificity and precision. The sensitivity of the assay was 50pg GH/100μl, which corresponds to 0.5ng/ml plasma. A dose–response inhibition curve resulting from serially diluted goat plasma was parallel to the standard curve using ovine GH and thus it confirmed the similar specificity of ovine GH standard and endogenous GH in goat plasma for ovine GH antibody. The precision of the assay, i.e., the intra- and inter-assay coefficients of variations (for repeatability and reproducibility, respectively) for the pooled plasma samples containing two GH concentrations (32 and 2ng/ml) were 6.72%, 11.2%, 7.1% and 12.8%, respectively. To physiologically validate the assay, goats were treated with human growth hormone-releasing factor (hGRF) and there was an immediate rise in plasma GH levels in hGRF-treated goats. This ELISA is economic, safe, quick (requires only 48h), convenient and the first competitive enzyme immunoassay described for caprine GH.

Study of the reversal effect of NF449 on neuromuscular blockade induced by d-tubocurarine

AimsThe aim of this study was to investigate the mechanism for the reversal effect of NF449 (a suramin analogue) on the neuromuscular block induced by d-tubocurarine (d-TC). Main methodsNerve-stimulated muscle contractions and end-plate potentials were performed in mouse phrenic nerve-diaphragm preparations. Acetylcholine (ACh)-induced muscle contractions were performed in the chick biventer cervicis preparations. Presynaptic nerve terminal waveform recordings were performed in mouse triangularis sterni preparations. Key findingsAmongst the suramin analogues in this study, only the NF449 and suramin were able to reverse the blockade effect produced by d-TC on nerve-stimulated muscle contractions. Each of these suramin analogues (NF007, NF023, NF279 and NF449) alone has no significant effect on the amplitude of nerve-stimulated muscle contractions. NF449 and suramin also showed the antagonising effects on the inhibition of end-plate potentials induced by d-TC. Furthermore, pre-treatment with NF449 can antagonise the inhibition of d-TC in ACh-induced contractions of chick biventer cervicis muscle. NF449 produced a greater rightward shift of the dose–response inhibition curve for d-TC than did suramin. Because other purinergic 2X (P2X) receptor antagonists, NF023 and NF279, do not have the reverse effects on the neuromuscular blockade of d-TC, the effect of NF449 seems irrelevant to inhibition of P2X receptors. SignificanceThese data suggest that NF449 was able to compete with the binding of d-TC on the nicotinic ACh receptors, and the effect of NF449 was more potent than suramin in reducing the inhibition of d-TC. The structure of NF449 may provide useful information for designing potent antidotes against neuromuscular toxins.

Pharmacological Characterization of GaTx2, a Peptide Inhibitor of ClC-2 Chloride Channels

ClC-2 chloride channels are voltage-gated ion channels that are expressed in neuronal and epithelial cells where they are critical mediators for the passive diffusion of Cl− across the plasma membrane. Although ClC-2 is nearly ubiquitously expressed in mammalian cells, many details regarding channel biophysics and the physiological role that ClC-2 channels play remain undefined. We recently isolated the first peptide toxin active against ClC channels: Georgia anion toxin 2 (GaTx2), a 3.2 kDa peptide composed of 29 residues with three disulfide bonds. Here, we describe the basic pharmacological features of the inhibitory activity of GaTx2 against ClC-2, including affinity, mechanism of inhibition, and specificity. Using two-electrode voltage-clamp, we created a dose-response curve for inhibition of ClC-2 by GaTx2 at VM = -100 mV, and calculated a KD of 22 ± 10 pM. This value was very similar to the value obtained from dose-response curves created from multi-channel patches, which gave a KD of 12 ± 5 pM. Additionally, from TEVC recordings we measured kon = 43 x 106 M−1s−1, and koff = 0.0034 s−1, which is consistent with rate constants for other peptide inhibitors. Single channel recording showed that the latency to first opening is increased nearly 8 fold in the presence of 20 pM GaTx2. Also, outside-out macropatches revealed that GaTx2 is unable to inhibit open ClC-2 channels; thus, this toxin may act as a gating modifier. Finally, we found that GaTx2 is specific for ClC-2, being unable to inhibit other ClC channels or transporters, other major classes of Cl− channels, or voltage-dependent K+ channels. This high affinity, highly specific inhibitor of ClC-2 will provide an excellent tool for studies designed to understand the function and regulation of this channel, and will help define its physiological role(s).

Trimetazidine modulates AMPA/kainate receptors in rat vestibular ganglion neurons

Trimetazidine (1[2,3,4-trimethoxy-benzyl] piperazine, 2 HCl) is an anti-ischemic agent frequently administered as a prophylactic treatment for episodes of angina pectoris and chorioretinal disturbances. It is also employed as a symptomatic treatment of vertigo but its mechanism of action is yet to be defined. Using Fura-2 fluorescence photometry and whole-cell patch-clamp recordings we investigated the effect of trimetazidine on the [Ca(2+)](i) and current responses induced by the application of non-N-methyl-D-aspartate (NMDA) receptor agonists on low density vestibular ganglion neuronal cultures explanted from 3 day s postnatal rats. Trimetazidine blocked the [Ca(2+)](i) and current responses induced by 100 microM applications of both kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). These responses were dependent on external Ca(2+) and were blocked by the voltage-dependent Ca(2+) channel blockers Ni(2+) and Cd(2+) . Trimetazidine only acts on the AMPA/kainate receptors and had no effect on K(+)-induced depolarizations. Dose-dependent curves were obtained for the inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and trimetazidine (IC(50) 7 microM and 0.7 microM) of kainate stimulations. After AMPA stimulation, dose-response inhibition curves showed an IC(50) of 3 microM for CNQX and 25 microM for trimetazidine. These results indicate that trimetazidine could be a potent antagonist of AMPA/kainate receptors in vestibular ganglion neurons. This may explain the protective role of trimetazidine in the inner ear suggesting an anti-excitotoxic activity.

Ouabain stimulates proliferation of epithelial cells from kidneys of patients with autosomal dominant polycystic kidney disease via the Na,K‐ATPase and MEK‐ERK pathway.

The hormone ouabain, through its receptor the Na,K-ATPase, stimulates proliferation of certain cell types. The objective of the present work was to determine the effect and intracellular signaling of ouabain in epithelial cells from kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Ouabain, in nanomolar concentrations, such as those circulating in blood, induced growth in ADPKD cells, increasing the rate of proliferation and mitosis in the cells. In contrast, ouabain did not significantly affect growth of normal human kidney cells (NHK cells). In ADPKD cells, ouabain also stimulated phosphorylation of the extracellular regulated kinase ERK. AG1478, an inhibitor of the epidermal growth factor receptor (EGFR) and PP2, an inhibitor of Src kinase blocked the effects of ouabain in the cystic cells. U0126, an inhibitor of the mitogen activated protein kinase MEK caused a similar effect. Ouabain stimulated phosphorylation of ERK occurred only when the hormone was applied to the basolateral surface of the cells, where the Na,K-ATPase is localized. Interestingly, dose response curves for inhibition of Na,K-ATPase activity by ouabain showed that approximately 20% of the Na,K-ATPase of ADPKD cells exhibits an abnormally increased affinity for ouabain. Altogether, these results suggest that ouabain is able to bind to the Na,K-ATPase of ADPKD cells with high affinity to activate the MEK-ERK pathway to stimulate proliferation of the cells. This presents ouabain as a factor with a positive effect in the pathophysiology of autosomal dominant polycystic kidney disease. [Supported by NIH grant HD043044-01 and a grant from the PKD Foundation].