Calculated EC50 Values Research Articles

Effect of Amlodipine in Comparison to Nifedipine on Vascular Perfusion Pressure of Isolated Rat Kidney

Objective(s) This study aimed to investigate and to compare the effects of nifedipine and amlodipine, dihydropyridine (DHP) calcium channel blockers (CCBs) on perfusion pressure of isolated perfused rat kidney. Materials and Methods Following the establishment of renal perfusion with a constant baseline pressure of 85-95 mmHg, the renal vasculature was constricted by phenylephrine (PE) injection. Changes in the baseline perfusion pressure were recorded. Then nifedipine and amlodipine prepared in perfusion medium was fed to the kidney for 30 min. Finally alterations in the baseline pressure arising from PE administrations in the presence of CCBs were recorded and data analyses were done. Results PE-induced increases in perfusion pressure attenuated significantly in the presence of 5 and 10 µM of nifedipine and 1, 5, and 10 µM of amlodipine. Increases in perfusion pressure arising from PE (100 and 200 µM) in the presence of amlodipine (1, 5, and 10 µM) was significantly less than that in the presence of nifedipine (1, 5, and 10 µM). Calculated EC50 value of amlodipine for inhibition was significantly lower than that of nifedipine. Based on the EC50 values, the potency of amlodipine in inhibiting PE-induced responses is significantly higher compared to nifedipine. Conclusion

Effect of insecticide fenamiphos on soil microbial activities in Australian and Ecuadorean soils

The effect of fenamiphos, a widely used organophosphorus pesticide, on important soil microbial activities such as dehydrogenase, urease and potential nitrification in four soils from Australia and Ecuador were studied. The results showed fenamiphos in general was not toxic to dehydrogenase and urease up to 100 mg/Kg soil. However potential nitrification was found to be highly sensitive to fenamiphos with a significant inhibition recorded even at 10 mg/Kg soil. In general, the nitrification activity in soils was decreased with an increase in fenamiphos concentration. The calculated EC50 values for nitrification in all the tested soils ranged between 19 and 56 mg fenamiphos/kg dry soil. This study suggests that fenamiphos is likely to be detrimental to nitrification at field application rates.

First Report of Fludioxonil-Resistant Isolates of Fusarium spp. Causing Potato Seed-Piece Decay.

Potato (Solanum tuberosum L.) diseases incited by Fusarium spp. include postharvest dry rot and seed-piece decay. Fusarium seed-piece decay is commonly controlled by preplant applications of chemical seed treatments. However, isolates of Fusarium spp. resistant to benzimidazole fungicides have been reported (2,4). In the spring of 2007, samples of cut seed tubers (cvs. Shepody and Russet Burbank) showing extensive symptoms of decay were received from three seedlots in Prince Edward Island (PE) and one seedlot in Saskatchewan (SK), Canada. All seed tubers had been treated with fludioxonil (Maxim Potato Seed Protectant [PSP], 0.5% fludioxonil) following cutting and then stored for 10 to 14 days prior to planting. Using standard isolation protocols (4), the 19 potato tuber pieces examined from PE and 2 from SK yielded 21 Fusarium isolates for further study. Five isolates (including both isolates from SK) were identified as Fusarium sambucinum Fuckel and the remaining 16 isolates were identified as F. coeruleum (Libert) Sacc. (3). To confirm identifications, isolates were compared with two known standards of each of F. sambucinum and F. coeruleum identified by K. Seifert (Agriculture and Agri-Food Canada, Ottawa, ON) by DNA sequencing of the partial β-tubulin gene or the translation elongation factor 1-α ( http://fusarium.cbio.psu.edu ; [1]). These standard isolates were also used as fludioxonil-sensitive controls in amended agar assays for chemical sensitivity. Agar plugs (5 mm in diameter) taken from the margins of 7-day-old cultures of the Fusarium isolates were transferred to petri dishes containing ½-strength potato dextrose agar amended with 0, 0.1, 1.0, 10.0, or 100.0 mg/liter of fludioxonil. Fludioxonil (Maxim PSP, 0.5% a.i.) was prepared as a stock solution in sterile distilled water and added to the molten agar after autoclaving. Culture incubation and mycelial growth measurements were performed as described previously (4). Measurements from four replicate petri dishes per concentration of fludioxonil were taken. Calculated EC50 values (fludioxonil concentration inhibiting pathogen growth by 50%) were obtained. The trial was repeated three times. The two standard isolates of F. sambucinum were sensitive to fludioxonil, with mean EC50 values of 0.002 (±0.002 standard error [SE]) and 0.005 (±0.002 SE) mg/liter. The two standard isolates of F. coeruleum were also sensitive to fludioxonil, with mean EC50 values of 0.17 (±0.005 SE) and 0.19 (± 0.005 SE) mg/liter. All other tested isolates of F. sambucinum and F. coeruleum were resistant to fludioxonil and showed no growth inhibition even at 100 mg of fludioxonil per liter. To our knowledge, this is the first report of resistance to fludioxonil in isolates of Fusarium spp. causing potato seed-piece decay. Since the isolates of F. sambucinum were also resistant to thiophanate-methyl and thiabendazole (data not shown), multiclass (benzimidazole and pyrrole) resistance was also documented.

Validation of a prediction model for estimating serum concentrations of chemicals which are equivalent to toxic concentrations in vitro

The objective of the present study was to evaluate the validity of a recently developed extrapolation model for the prediction of concentrations of chemicals in serum which are equivalent to in vitro effective nominal concentrations. Necessary input data are in vitro toxic concentrations and distribution relevant system and substance specific parameters, e.g. lipid volume fractions and albumin concentrations, octanol/water partition coefficients and specific binding to albumin. It was investigated whether the influence of human and bovine serum, respectively, on nominal cytotoxic potencies (EC 50-values) of selected chemicals in vitro can be properly predicted using this algorithm. Cytotoxicity was determined as growth inhibition of proliferating Balb/c 3T3 cells after exposure for 72 h. Concentration-effect relationships were measured in the presence of 2% foetal bovine serum (FBS) and, additionally, 18% FBS or human serum (HS), or 1% (w/v) bovine (BSA) or human (HSA) albumin, respectively. Addition of HSA and BSA increased the EC 50-values of the different chemicals by factors of 2.1–22 and 1.7–29, respectively. From these measurements values for the specific binding of the test compounds to BSA and HSA were derived. Addition of 18% HS increased the EC 50-values by factors between 4.2 and 52, while addition of 18% FBS resulted only in 1.5–10.4-fold increases. A comparison of experimentally determined and calculated EC 50-values revealed that the differing influence of human and bovine serum was quite well predicted by the extrapolation model. Deviations did not exceed the factor 3 and were in most cases lower than 2. It is concluded that the extrapolation model is quite well suited to predict equivalent concentrations in serum from in vitro effective concentrations.

A dynamic artificial clam (Corbicula fluminea) allows parsimony on-line measurement of waterborne metals

We introduce a novel on-line biomonitoring system based on a valvometric conversion technique for clam Corbicula fluminea, allowing for rapid, continuous, and ecological relevant water quality control. Our model builds upon the basic principles of biological early warning system model in two ways. We first adopted a risk-based methodology to build a dynamic artificial clam for simulating how the bivalve closure rhythm in response to waterborne copper (Cu) and cadmium (Cd). Secondly, we integrated a probabilistic model associated with the time-varying dose–response relationships of valve closing behavior into the mechanisms of a dynamic artificial clam, allowing estimation of the time-varying waterborne Cu/Cd concentrations for on-line providing the outcomes of the toxicity detection technique. Measurements with Cu/Cd were performed and the calculated EC50 values were compared with published data for the valve movement test with C. fluminea. This proposed dynamic artificial clam provides a better quantitative understanding of on-line biomonitoring measurements of waterborne metals and may foster applications in clam farm management strategy and ecotoxicological risk assessment.

Intravenous Anesthetics Are More Effective than Volatile Anesthetics on Inhibitory Pathways in Rat Hippocampal CA1

In this study, we have examined the effects of both volatile and IV general anesthetics on excitatory synaptic transmission, with and without recurrent inhibition, to clarify whether excitatory or inhibitory synapses are the major targets of action. Field population spike amplitudes (fPSs) of CA1 pyramidal neurons were recorded in rat hippocampal slices. Schaffer-collateral-commissural fibers (Sch) were stimulated orthodromically, and the evoked fPSs (PS[Sch]) in CA1 area were measured. In addition, the fPSs (PS[Alv+Sch]) elicited by stimulation of the Sch after antidromic stimulation of the alveus hippocampi (Alv) to produce recurrent inhibition were determined. It was observed that sevoflurane (0.5%-5%) and isoflurane (0.5%-5%) primarily inhibited PS[Sch] and also produced additive inhibition on the PS[Alv+Sch] in a concentration-dependent manner. The calculated 50% effective concentration (EC50) values for PS[Sch] and PS[Alv+Sch] were 5.3 vol% and 3.9 vol% (sevoflurane) and 1.7 vol% and 1.1 vol% (isoflurane), respectively. In comparison, thiopental (2.0 x 10(-5)-5.0 x 10(-4) mol/L) reduced both the PS[Sch] and PS[Alv+Sch] in a concentration-dependent manner. The calculated EC50 values for thiopental on PS[Sch] and PS[Alv+Sch] were 3.4 x 10(-4) and 5.7 x 10(-5) mol/L, respectively. Propofol (2.0 x 10(-5)-3.5 x 10(-4) mol/L) had little effect on the PS[Sch] but reduced PS[Alv+Sch] with a calculated EC(50) value of 5.1 x 10(-4) mol/L. The effects of the IV anesthetics with recurrent inhibition were antagonized in the presence of the gamma-aminobutyric acid-A-receptor antagonist bicuculline methiodide. In addition, all anesthetics prolonged recurrent inhibition from 100 ms (sevoflurane and isoflurane) to 400 ms (propofol). The results suggest that sevoflurane and isoflurane inhibit mainly on glutamate-mediated orthodromic pathways, whereas thiopental and propofol enhance gamma-aminobutyric acid-A-mediated recurrent inhibitory pathways in CA1 neurons, thus providing further evidence that the mechanisms of general anesthetics are drug- and pathway-specific.

Characterization of the Gαs Regulator Cysteine String Protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Inheritance of mefenoxam resistance in selfed populations of the homothallic oomycetePhytophthora erythroseptica (Pethybr.), cause of pink rot of potato

The inheritance of mefenoxam resistance in the oomycetePhytophthora erythroseptica, causal organism of pink rot of potato, was investigated in two successive selfed generations of single oospore isolates. Fourteen parental isolates from three mefenoxam-sensitivity phenotypes (sensitive, intermediately resistant, and resistant) were used to produce S1 and S2 progeny isolates by selfing each isolate and germinating single oospore cultures from each isolate. The two generations of isolates were tested for mefenoxam sensitivity using the radial growth inhibition assay and calculating EC50 values. Twenty-four-hundred isolates from S1 and S2 generations were tested for mefenoxam sensitivity. Results demonstrated the lack of segregation for mefenoxam sensitivity among S1 and S2 progeny isolates from the resistant and sensitive parents. The majority of the S1 progeny isolates from the parents with intermediate resistance also possessed intermediate resistance, but demonstrated substantial quantitative shifts in response to mefenoxam more often toward increased insensitivity. A few isolates from the S1 progeny were sensitive to mefenoxam, and one isolate was mefenoxam-resistant. Similarly, the majority of the S2 progeny isolates from intermediately resistant parents also possessed intermediate resistance with some quantitative shifts in sensitivity to mefenoxam similar to those observed in S1 progeny, as well as a few isolates that were sensitive to mefenoxam. These results do not support the hypothesis that resistance to mefenoxam inP. erythroseptica is controlled by a single gene exhibiting incomplete dominance. Alternative hypotheses are proposed such as the genetics of mefenoxam resistance inP. erythroseptica is probably under the control of more than one major gene and perhaps some minor genes of additive effect.

A practical note on the use of cytotoxicity assays

In this study, four cytotoxicity detection assays and four cytotoxic mechanisms were compared in one cellular system. Cellular responses and their effects were characterized. The assays used are based on different modes of detection like LDH release, MTT metabolism, neutral red uptake and the ATP content of treated cells. As cytotoxic mechanisms were used the model agents triton X-100, chloroquine and sodium azide (which are common in cell culture) as well as an ion channel (NMDA) mediated excitotoxicity cell death (which is specific for the cell line used). We found major differences in the calculated EC 50-values for the cytotoxic effect of choroquine (0.1 up to 200 mM) and for sodium azide (4 up to 1300 mM) depending on the assay used. Therefore, it is important to choose a suitable cytotoxicity assay depending on the supposed cell death mechanism. As this study compares the strengths and weaknesses of the most common assays, it can help to find the appropriate one.

Ring-testing and Field-validation of a Terrestrial Model Ecosystem(TME) – An Instrument for Testing Potentially Harmful Substances: Effects of Carbendazim on Organic Matter Breakdown and Soil Fauna Feeding Activity

Organic matter (OM) decomposition and soil fauna feeding activity were integrated as functional endpoints into ecotoxicological tests with intact-soil-core Terrestrial Model Ecosystems (TMEs). Cellulose filter paper served as standardized OM and was either inserted into the top soil or placed on the soil surface for a period of up to 16 weeks. Faunal feeding activity was assessed by the bait-lamina method. The fungicide carbendazim, applied at six dosages ranging from 0.36 kg/ha to 87.5 kg a.i./ha, served as a model chemical. To validate the results from the TME test, a field study was run in parallel. In TMEs the cellulose paper inserted into the soil was decomposed faster than under field conditions. The carbendazim-induced effects on OM decomposition in TMEs and in the field were comparable and followed a clear dose-response relationship. The calculated EC50 values after 8 weeks of incubation were 9.5, 7.1 and 2.1 kg carbendazim/ha for grassland TMEs, grassland field and arable TMEs, respectively. The feeding activity of the soil fauna showed a large variability. The EC50 values for the effect of carbendazim on bait-lamina consumption ranged between 2.0 and 56 kg a.i./ha. Effects on decomposition were correlated with effects on enchytraeids and earthworms but not with effects on bait-lamina consumption.

Spiral Gradient Dilution, a Rapid Method for Determining Growth Responses and 50% Effective Concentration Values in Fungus-Fungicide Interactions

ABSTRACT A new technique, the spiral gradient dilution method, was evaluated for determining 50% effective concentrations (EC(50) values) of fungicides for the inhibition of mycelial growth and conidial germination of various fungi. In this method, an agar medium is plated with a fungicide solution by means of a spiral plater, which applies the fungicide in a 2.5-log dilution in a continuous radial concentration gradient. Fungal inoculum is then placed along radial lines across the gradient. After incubation of the plates, distinct growth shapes were observed in different fungus-fungicide interactions. Mycelial growth responses to increasing fungicide concentrations ranged from abrupt to gradual transitions. Conidial germination responses were similar; in addition, distinct zones of confluent growth, nonconfluent growth, and outlier colonies were also identified, depending on the fungus-fungicide interaction. The fungicide concentration at the radial distance at which 50% reduction of growth or spore germination occurred, compared with growth or germination on unamended media, was calculated by a computer program. EC(50) values were obtained for mycelial growth in 22 fungus-fungicide interactions and for conidial germination in five interactions. The fungi evaluated were members of the Zygomycota, Ascomycota, Basidiomycota, and Deuteromycota. Nine fungicides, belonging to six different chemical classes, were tested. EC(50) values were compared with those obtained by the traditional agar dilution method. In linear regression analyses of the two methods, the models were highly significant (P < 0.01), and coefficients of determination (r(2)) were 0.92 for the mycelial growth assays and 0.94 for the conidial germination assays. Regression slopes were not significantly different from 1 (P > 0.05) with optimal program settings in the software. Estimated bias, coefficients of variation, and actual confidence intervals for the regression slope were 13.5%, 6.5%, and 1.14 +/- 0.14 for the mycelial growth assays and 7.5%, 14.5%, and 1.08 +/- 0.37 for the conidial germination assays. These analyses indicate that the spiral gradient dilution method is accurate and precise compared with the agar dilution method for calculating EC(50) values of fungicides in continuous growth responses to fungicide concentration gradients.

First Report of Thiabendazole-Resistant Isolates of Fusarium sambucinum Infecting Stored Potatoes in Nova Scotia, Canada.

Fusarium dry rot is a significant postharvest disease of potato (Solanum tuberosum L.) and is often controlled by applying thiabendazole to tubers prior to storage. However, thiabendazole-resistant isolates of Fusarium spp. have been reported from Europe (2), the United States (1), and Canada (1,4). To address concerns, samples of potato tubers showing symptoms of dry rot caused by Fusariumspp. were collected from three storage bays in a commercial storage facility in Nova Scotia, Canada, in February 2001. All tubers had been treated with thiabendazole after harvest and prior to storage. Tubers were cut longitudinally, and small tissue samples (10 × 5 × 3 mm) were taken from the margins of internal necrotic regions with a sterile scalpel, surface-sterilized in 0.6% sodium hypochlorite for 15 s, rinsed twice in sterile distilled water (SDW), and blotted dry on sterile filter paper. Tissue pieces were plated on 0.5-strength potato dextrose agar (PDA) amended with tetracycline (0.05 g/liter) and streptomycin sulfate (0.1 g/liter). Petri dishes were incubated in the dark at 22°C for 4 to 7 days. After incubation, hyphal tips from the margins of actively growing isolates were removed with a sterile probe and plated on 0.5-strength PDA to generate pure cultures. Of 35 potato tubers examined, 10 (29%) yielded Fusarium isolates for further study. All 10 isolates were identified as F. sambucinum Fuckel according to Nelson et al. (3). Agar plugs (5 mm diameter) taken from the margins of 7- to 10-day-old cultures of F. sambucinum isolates were transferred to petri dishes containing 0.5-strength PDA amended with thiabendazole at 0, 1, 5, 10, 20, 50, or 100 mg/liter. Thiabendazole was prepared as a stock solution in SDW and added to molten agar after autoclaving. Cultures were grown in the dark for 7 days at 22°C, after which mycelial growth diameter was measured using digital calipers. Two measurements, along orthogonal diameters, were taken from each of three replicate plates for a total of six measurements per thiabendazole concentration. Means were calculated, and the diameter of the inoculation plug was subtracted from each mean. Calculated EC50 values (thiabendazole concentration inhibiting pathogen growth by 50%) were obtained by regression of the log of the chemical concentration against the corresponding probit of percent fungal inhibition. All isolates of F. sambucinum were resistant to thiabendazole, with EC50 values ranging from 7 to 82 mg/liter. Six isolates had EC50 values between 40 and 82 mg/liter. Control isolates of F. sambucinum, F. avenaceum, F. solani, and F. oxysporum were sensitive to thiabendazole, with EC50 values of <1 mg/liter. Although isolates of F. sambucinum resistant to thiabendazole have been recovered from eastern Canada (1,4), this is the first report of thiabendazole resistance in F. sambucinum isolates from tubers in commercial storage in the Annapolis Valley of Nova Scotia, Canada, a production region that concentrates on growing processing potatoes for the potato chip industry and is several hundred kilometers from other potato-growing regions of Prince Edward Island and New Brunswick.

Experimental and mathematical/computational assessment of the acute toxicity of chemical mixtures from the Microtox® assay

A mathematical algorithm has been developed to compute the combined toxicity of mixtures of metal contaminants using toxicity data for the individual components of the mixture. The Microtox® bioassay was used to experimentally obtain the toxicity data for a suite of metals (Pb, As, Cd, Cu, Hg, Zn, Cr) that could be potentially present in the environment. Statistically significant deviation of the experimental toxicity of mixtures from the calculated EC50 value using the algorithm represents a departure from an additive response, and is classified as synergism or antagonism, as appropriate. Results from binary and ternary metal combinations indicate that all three mixture situations — additive, synergistic, and antagonistic — are possible. The approach described here may also be applicable to any other assay capable of generating the required algorithm parameters.

Automated Biomonitoring Using Real Time Movement Analysis of Euglena gracilis

An automated biomonitoring system for early warning of pollutants in aquatic environments is described and characterized. The system uses sublethal changes in the movement behavior of the flgellate Euglena gracilis as biological endpoints. The movement is determined by real time image analysis. All parameters describing motility, velocity, orientation, and form of the cells are calculated during measurement, and changes of these parameters are interpreted as effect. By automatic dilution of the water sample, dose-effect relationships can be recorded automatically. A total measurement procedure, including control and sample measurement and filling and rinsing of the system, typically requires 8 min. Measurements with different organic and inorganic toxic compounds were performed and the calculated EC50 values compared with literature data for the bioluminescence test with Vibrio fischeri. Also, measurements with waste water samples from different industrial plants were performed. The fast response time, the small size, the reliable image analysis system, the calculation of several endpoints, and the automatic measuring procedure are major advantages compared to other biological test systems.

Randomized trial of low molecular weight heparin (enoxaparin) vs. unfractionated heparin for unstable coronary artery disease: 1-year results of the ESSENCE study

A mathematical algorithm has been developed to compute the combined toxicity of mixtures of metal contaminants using toxicity data for the individual components of the mixture. The Microtox® bioassay was used to experimentally obtain the toxicity data for a suite of metals (Pb, As, Cd, Cu, Hg, Zn, Cr) that could be potentially present in the environment. Statistically significant deviation of the experimental toxicity of mixtures from the calculated EC50 value using the algorithm represents a departure from an additive response, and is classified as synergism or antagonism, as appropriate. Results from binary and ternary metal combinations indicate that all three mixture situations — additive, synergistic, and antagonistic — are possible. The approach described here may also be applicable to any other assay capable of generating the required algorithm parameters.

Modulation of Gluconeogenesis by Epinephrine in Hepatocytes Isolated from Genetically Obese (fa/fa) Zucker Rats

The obese (fa/fa) Zucker rat shows an impaired sympathetic tone which is accompanied by an altered thermogenesis and changes in both lipid and carbohydrate metabolism. In this work, we have investigated the regulatory effects of epinephrine on the rate of gluconeogenesis from a mixture of [14C]lactate/pyruvate, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/−) littermates. Epinephrine caused a dose-dependent stimulation of the rate of [14C]glucose formation in both obese and lean rat hepatocytes, the maximal rates being five- and twofold higher than the corresponding basal values (0.50 ± 0.06 and 1.96 ± 0.15 μmol of lactate converted to glucose/g of cell × 20 min, respectively). No significant differences were found between the calculated half-maximal effective concentrations (EC50) for epinephrine in obese and lean rat liver cells. The stimulation of gluconeogenesis by epinephrine was accompanied by a decrease in the cellular concentration of fructose 2,6-bisphosphate, and an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, to similar extents in both types of hepatocytes. Epinephrine also significantly raised the hepatocyte content of cyclic AMP, with about a twofold increase at a saturating concentration of the catecholamine (1 μM), in both lean and obese rat liver cells. However, at suboptimal concentrations of epinephrine, the rise in cyclic AMP levels was significantly less marked in obese than in lean rat hepatocytes. Nevertheless, no significant differences were found in either the affinity or the number of β-adrenergic receptors, in radioligand binding studies carried out in liver plasma membranes obtained from obese and lean Zucker rats. In conclusion, compared to the corresponding basal values, the response of gluconeogenesis from lactate to the stimulatory effect of epinephrine is higher in obese (fa/fa) than in lean (Fa/−) Zucker rat hepatocytes, with no significant differences in the calculated EC50 values for this hormone. This occurs in spite of an apparent decreased sensitivity of the adenylate cyclase system to the stimulatory effect of epinephrine in obese rat liver cells.

Bradykinin B2-receptor-mediated modulation of membrane currents in guinea-pig cardiomyocytes.

1. In order to define the electrophysiological mechanism(s) responsible for bradykinin (BK)-induced positive inotropic and chronotropic responses in isolated guinea-pig atria, effects of BK on the membrane currents were examined in isolated atrial cells using patch clamp techniques. 2. BK (0.1-1000 nM) increased the L-type Ca2+ current (I(Ca)), which was recorded from enzymatically-dissociated atrial myocytes by the nystatin-perforated patch method, in a concentration-dependent fashion, and the calculated EC50 value for increasing I(Ca) was 5.2 nM. In conventional ruptured patch experiments, BK inhibited the muscarinic acetylcholine receptor-operated K+ current (I(K.ACh)) that was activated by the muscarinic agonist carbachol (1 microM) with an EC50 value of 0.57 nM. Both the increase in I(Ca) and the decrease in I(K.ACh) were blocked by HOE140, a selective bradykinin B2 receptor antagonist. 3. The BK-induced inhibition of I(K.ACh) was significantly attenuated by staurosporine and calphostin C, protein kinase C inhibitors. In addition, the I(K.ACh) inhibition by BK was also attenuated by the tyrosine kinase inhibitor genistein or tyrphostin but not by daidzein, an inactive analogue of genistein. However, neither protein kinase C inhibitor nor tyrosine kinase inhibitor affected the BK-induced increase in I(Ca). 4. In the presence and absence of muscarinic stimulation, BK prolonged the action potential recorded from the atrial cells in the current clamp mode. 5. We conclude that BK increases I(Ca) and decreases I(K.ACh) in atrial cells, resulting in positive inotropic and chronotropic responses in atrial preparations. Protein kinase C activation, and possibly tyrosine kinase activation, may be involved in the B2-receptor-mediated I(K.ACh) inhibition.

Effects of Some Persistent Halogenated Environmental Contaminants on Aromatase (CYP19) Activity in the Human Choriocarcinoma Cell Line JEG-3

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126), a technical PCB mixture (Aroclor 1016), and a technical toxaphene mixture (Camphechlor) on aromatase (CYP19) activity were investigated in human choriocarcinoma JEG-3 cells. After 18 h incubation with TCDD, PCB126, Aroclor 1016 or toxaphene, ethoxyresorufin-O-deethylase (EROD), and aromatase activity were determined. To exclude serum effects, incubations were carried out with or without fetal calf serum in the medium. EROD activity was induced by both TCDD and PCB126 in the presence or absence of serum, which indicates that JEG-3 cells are responsive toward dioxin-like chemicals. Neither Aroclor 1016 nor toxaphene affected EROD activity in these cells. Calculated EC50 values for induction of EROD activity were 0.71 and 0.40 nmfor TCDD, and 48 and 20 nmfor PCB126 in presence or absence of serum, respectively. Incubation with TCDD or PCB126 with or without serum caused a concentration-dependent decrease in the aromatase activity of up to 4.9-fold. Calculated EC50 values for this effect were 52 pmand 13 nmfor TCDD, and 75 and 48 nmfor PCB126 in the presence and absence of serum, respectively. Aroclor 1016 and toxaphene had no effect on aromatase activity at concentrations up to 1.0 μmfor Aroclor 1016 or 3.0 μmfor toxaphene. These results show that aromatase activity can be decreased in a concentration dependent way within the same range where EROD activity is increased. In view of these results, possible effects of dioxin-like compounds on estrogen producing and androgen target cells should be studied in more detail.

Muscarine activates a nonselective cation current through a M3 muscarinic receptor subtype in rat dorsolateral septal nucleus neurons.

1. In the present study, we examined the cellular mechanism and receptor type responsible for a muscarine-induced inward current (Imi) in neurons of rat dorsolateral septal nucleus (DLSN) using single-microelectrode voltage-clamp and "slice" patch-clamp techniques. 2. Imi was associated with an increase of membrane conductance in 75% of DLSN neurons. There was no voltage-dependence of Imi between -60 and -140 mV; it exhibited a reversal potential of -17.0 +/- 5.3 mV (n = 14) determined by extrapolation of Imi and voltage relationship recorded using whole cell patch recording. Lowering extracellular sodium (26 mM) or potassium (1.4 mM) ions depressed Imi. 3. Imi was concentration dependent; 3 and 100 microM muscarine produced the minimum [22 +/- 4.6 pA, (mean +/- SE) n = 8] and maximum (167 +/- 28 pA, n = 7) responses, respectively. An EC50 was determined to be 15 microM (n = 8). Oxotremorine-methiodide (1-100 microM) also produced an inward current with similar potency compared with muscarine. On the other hand, McN-A-343 and pilocarpine (3-100 microM) did not produce any inward current in DLSN neurons. 4. Atropine (1 microM) completely reduced Im produced by 30 microM muscarine, whereas pirenzepine (PZP) shifted the concentration-response curve for muscarine in a parallel manner to the right. The EC50 for muscarine was shifted to 32, 52, and 204 microM by 0.2, 0.5, and 2 microM PZP, respectively. The apparent Kd value for PZP estimated by Schild plot analysis was 190 nM (n = 5). 5. Methoctramine (1 microM) also competitively depressed Imi; the calculated EC50 values were 26, 41, and 107 microM in concentrations of 0.2, 2, and 10 microM methoctramine, respectively. The apparent Kd for methoctramine was 420 nM. In contrast, AF-DX 116 (1 microM) did not significantly inhibit Imi. 6. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, suppressed irreversibly Imi. Pretreatment of DLSN neurons with pertussis toxin (PTX) did not prevent Imi (n = 8). 7. We suggest that muscarine causes this inward current by activating a M3 subtype of muscarinic receptor, which is coupled to a PTX-insensitive GTP-protein in rat DLSN neurons.

Antagonistic effect of Na+ and Mg2+ on P2z purinoceptor-associated pores in dibutyryl cyclic AMP-differentiated NG108-15 cells.

The effect of replacement of extracellular Na+ with N-methyl-D-glucamine (NMG) on P2 receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15, cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca2+ concentration ([Ca2+]i) with an EC50 value of 230 microM. Replacement of Na+ with NMG as well as removal of Mg2+ from the bathing buffer potentiated ethidium bromide uptake, [Ca2+]i increase, and 45Ca2+ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg2+ to limit the amount of ATP4-, replacement of Na+ with NMG had no effect on the ATP-induced [Ca2+]i increase but caused a markedly larger [Ca2+]i increase when the calculated concentration of ATP4- was > 10 microM. The calculated EC50 value for ATP4- stimulation of the [Ca2+]i increase was 23 microM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca2+ release was the major pathway for the ATP-induced [Ca2+]i increase; both removal of Mg2+ and replacement of Na+ with NMG did not affect the action of ATP. These data suggest that ATP(4-)-promoted pores are antagonized by Na+ and Mg2+ in dibutyryl cyclic AMP-differentiated NG108-15 cells.