Journal of pharmacological methods最新文献

{"title":"Rabbit aortic smooth muscle cell culture","authors":"C. Alipui ,&nbsp;T.E. Tenner Jr. ,&nbsp;K. Ramos","doi":"10.1016/0160-5402(91)90045-7","DOIUrl":"10.1016/0160-5402(91)90045-7","url":null,"abstract":"<div><p>Atherosclerotic vascular disease is the most common complication of diabetes mellitus. Enhanced vascular smooth muscle cell proliferation plays a central role in atherosclerotic lesion formation. Studies using explant cultures have demonstrated that aortic smooth muscle cells from rats with experimental or genetic diabetes have enhanced rates of proliferation when compared to controls. However, this method of culture may select for cells with enhanced migratory potential. In the present studies, aortic smooth muscle cells were successfully cultured from control and diabetic rabbits after enzymatic and mechanical dispersion from thoracic aortic segments. The proliferative patterns of control cells were characterized and growth rates of diabetic cells were compared to controls. Primary cultures from control rabbits grew after an initial 5-day lag period to achieve threefold increases in cell number by 9 days. Subcultures of aortic smooth muscle cells entered the logarithmic phase of growth after 2 days, reaching the plateau phase of growth in 5–7 days and achieving three to fourfold increases in cell number. The final density to which cultures grew was not affected by the number of cells attached on day 1 for the range studied. Cells from diabetic rabbits displayed shorter doubling times and reached greater densities at confluence than did cells from controls. These data support the hypothesis that diabetes induces an atherogenic response. The dissociated rabbit aortic smooth muscle cell culture provides a model in which to study diabetes-induced modulation of cell proliferation that is amenable to pharmacological manipulation to investigate agonist and growth factor-induced responses.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 3","pages":"Pages 211-222"},"PeriodicalIF":0.0,"publicationDate":"1991-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90045-7","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12917367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification and analysis of erythrocyte membrane Ca2+-ATPase from small samples of patient blood: Application to cystic fibrosis","authors":"Michael A. Bridges ,&nbsp;Sidney katz","doi":"10.1016/0160-5402(91)90042-4","DOIUrl":"10.1016/0160-5402(91)90042-4","url":null,"abstract":"<div><p>A method is presented for the micro-scale isolation and characterization of erythrocyte membrane Ca²⁺-ATPase from small samples (7 mL) of whole human blood. Ca²⁺-ATPase isolated by this technique was more than 92% pure and showed calcium-activation characteristics similar to enzyme purified by standard macroscale procedures—viz maximal velocity of activation (V_ca2+) = 15.5 ± 1.2 μmol ATP hydrolysed/mg/min, and reciprocal of apparent affinity (K_ca2⁺) = 0.73 ± 0.15 μM free calcium (mean ± SEM; <em>n</em> = 9). Using the isolation procedure described, purified Ca²⁺-ATPase could be prepared and assayed in a single working day. When the calcium-activation kinetics of cystic fibrosis erythrocyte membrane Ca²⁺-ATPase were reassessed using enzyme purified by this technique, V_ca2+ and K_ca2+ were not significantly different from normal values.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 3","pages":"Pages 173-185"},"PeriodicalIF":0.0,"publicationDate":"1991-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90042-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"12998274","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The recording of action potential currents as an assessment for drug actions on excitable cells","authors":"James G. McLarnon","doi":"10.1016/0160-5402(91)90059-E","DOIUrl":"10.1016/0160-5402(91)90059-E","url":null,"abstract":"<div><p>The use of the cell-attached patch clamp configuration to record action potential currents is shown to have utility in the testing for drug actions on ion channels in excitable cell membrane. A patch pipette was used to isolate a small patch of cell membrane on cultured hippocampal or hypothalamic neurons and spontaneous R-C coupled action potential currents, with well-defined Na⁺ and K⁺ components, were recorded. The addition of several potassium channel-blocking drugs to the bath solution completely abolished the after-hyperpolarization phase of the action potential currents while preserving the sodium spike. These drugs have previously been shown to block a calcium-dependent potassium channel in cultured hippocampal neurons, a channel that is responsible for the late slow after-hyperpolarization macroscopic current recorded in these cells. The addition of tetrodotoxin to the bath solution eliminated the R-C coupled currents. The novel approach of using the recording of action potential currents to assess drug actions on ion channels would be expected to be applicable to a variety of excitable cells.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 2","pages":"Pages 105-111"},"PeriodicalIF":0.0,"publicationDate":"1991-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90059-E","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13100866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of a microassay for human kininogens as cysteine protease inhibitors","authors":"Tove S. Karlsrud ,&nbsp;Ansgar O. Aasen ,&nbsp;Harald T. Johansen","doi":"10.1016/0160-5402(91)90060-I","DOIUrl":"10.1016/0160-5402(91)90060-I","url":null,"abstract":"<div><p>Several methods have been described for the identification and quantification of kininogens based on both immunochemical and functional characteristics. This article presents a rapid, cheap and simple microplate assay of kininogens based on their ability to inhibit cysteine proteases. The target enzyme papain is activated by cysteine HCl and the activated enzyme will be inhibited by added kininogens. The residual enzyme activity that is not inhibited in this reaction subsequently hydrolyzes the added substrate, S-2302, generating a yellow color that is read in a microplate reader at 405 nm. This method is very sensitive, the smallest amount of kininogen that causes significant inhibition of papain is established to be 0.01 μg. As a quantitative method, the assay performs accurately when approximately 0.1 μg of low molecular weight kininogen or high molecular weight kininogen is added to the test system. The within-run coefficient of variation (%) of the method was 1.7% when the inhibition of papain was in the range 45–70% and the day to day variation as low as 2.3% when performed with a papain inhibition of 80%. Applications of the method are presented, studying chromatographic separated kininogens in plasma, ascites, and urine.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 2","pages":"Pages 113-124"},"PeriodicalIF":0.0,"publicationDate":"1991-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90060-I","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13100867","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Method for studying the in vivo accumulation of inorganic mercury in segments of the nephron in the kidneys of rats treated with mercuric chloride","authors":"Rudolfs K. Zalups","doi":"10.1016/0160-5402(91)90058-D","DOIUrl":"10.1016/0160-5402(91)90058-D","url":null,"abstract":"<div><p>A method for studying the <em>in vivo</em> accumulation of inorganic mercury along the nephron of Sprague-Dawley rats pretreated with a radiolabelled 0.66 μmol/kg dose of mercuric chloride is described in this article. Forty-eight hr after rats received the radiolabelled dose of mercuric chloride intravenously the kidneys of the animals were perfused <em>in situ</em> with a collagenase solution in order to dissect and isolate various readily assessable segments of the nephron and collecting duct. Three different categories of tubular segments were isolated; proximal convoluted tubules, proximal straight tubules and combined segments of the distal nephron and collecting duct. A group of isolated tubular segments were measured in length, drawn up and placed in counting tubes, and placed in a gamma counter for the determination of the content of inorganic mercury that accumulated in them during the 48 hr subsequent to the administration of the dose of mercuric chloride. In a separate set of animals, the intrarenal distribution of inorganic mercury was determined 48 hr after the intravenous dose of mercuric chloride was asministered. Inorganic mercury accumulated mainly in the renal cortex and outer stripe of the outer medulla. In addition, the concentration of inorganic mercury in the outer stripe of the outer medulla was twice that in the cortex. The findings obtained with the isolated tubular segments revealed that most of the accumulated inorganic mercury in the kidneys of the rats was in the proximal tubule. The content of inorganic mercury in the proximal straight tubules was significantly greater than that in the proximal convoluted tubules. Negligible amounts of inorganic mercury were detected in the distal segments of the nephron and collecting duct. The data obtained from the isolated tubular segments indicate that the increased accumulation of inorganic mercury that occurs in the outer stripe of the outer medulla is due specifically to increased accumulation of inorganic mercury in the proximal straight tubules. The method used to study the accumulation of inorganic mercury in segments of the nephrons proves to be sensitive enough to explain the mechanism for various patterns of accumulation of inorganic mercury in the kidneys of rats.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 2","pages":"Pages 89-104"},"PeriodicalIF":0.0,"publicationDate":"1991-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90058-D","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13102244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Time course of spontaneous ventricular arrhythmias following acute coronary occlusion in the dog","authors":"Yvette Trolese-Mongheal,&nbsp;Jacques Barthelemy,&nbsp;Jean-Francois Trolese,&nbsp;Pierre Duchene-Marullaz","doi":"10.1016/0160-5402(91)90061-9","DOIUrl":"10.1016/0160-5402(91)90061-9","url":null,"abstract":"<div><p>In this study, the arrhythmias occurring in dogs between 4 and 15 hr after occlusion of the left anterior descending coronary artery were continuously monitored by recording the electrocardiogram from bipolar leads. At 4.5 hr the number of dogs with less than 50% of sinus beats had increased and at 5 hr 15 min sinus beats represented on average 80% of total heart beats. In the period up to 6 hr isolated ventricular beats and ventricular salvos were seen in 95% and 63% of the dogs respectively and at 7 hr there were, on average, 50% of sinus beats and monomorphic ventricular rhythm was observed in 58% of the dogs. From 7 hr half the dogs had over 50% of ventricular ectopic beats and by 9 hr ventricular rhythm disturbances were permanently present in all the dogs. The ventricular arrhythmias reached a peak at about 11–12 hr (mean % sinus beats &lt; 10) when all dogs had a predominantly monomorphic (42%) and/or polymorphic (63%) ventricular heart rhythm.</p><p>The characteristic time course of these cardiac disturbances suggest that it may form the basis for an experimental model that may be useful in analyzing the effects of potential antiarrhythmic drugs.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 2","pages":"Pages 125-137"},"PeriodicalIF":0.0,"publicationDate":"1991-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90061-9","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13102241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neuroblastoma cells for testing neuroprotective drug effects","authors":"Barbara Peruche,&nbsp;Josef Krieglstein","doi":"10.1016/0160-5402(91)90062-A","DOIUrl":"10.1016/0160-5402(91)90062-A","url":null,"abstract":"<div><p>An attempt was made to use neuroblastoma cells for testing neuroprotective drug effects. To achieve cellular damage, cytotoxic hypoxia was induced in neuroblastoma cells after 10 days in culture by addition of sodium cyanide (1 nmol/L) to the culture medium and was terminated after 6 hr by replacing the cyanide-containing fresh nutrient medium. During this hypoxic period cells were additionally deprived of glucose. They were allowed to recover for another 7 days. Drugs were available to the cells from 30 min prior to hypoxia until 24 hr after hypoxia. Cell concentration of high-energy phosphates and culture protein content were determined as representatives for the posthypoxic development of cell damage, cell activity and viability.</p><p>While barbiturates and phenytoin revealed neurotoxic effects when applied in doses higher than 300 μmol/L, chlorpromazine, dizocilipine, ketamine, ketzocine, naftidrofuryl, and flunarizine protected neuroblastoma cells against hypoxic damage. These results were comparable to those obtained from primary cultures of neurons under similar experimental conditions. In addition, they were in keeping with neuroprotective drug effects obtained from in vivo experiments. It is suggested that neuroblastoma cells are suitable for testing neuroprotective drug effects.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 2","pages":"Pages 139-148"},"PeriodicalIF":0.0,"publicationDate":"1991-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90062-A","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13102242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A revised rotarod procedure for measuring the effect of antinociceptive drugs on motor function in the rat","authors":"Steven M. Cartmell,&nbsp;Linda Gelgor,&nbsp;Duncan Mitchell","doi":"10.1016/0160-5402(91)90063-B","DOIUrl":"10.1016/0160-5402(91)90063-B","url":null,"abstract":"<div><p>Tests of putative antinociceptive agents that rely on a motor response of an experimental animal to a noxious stimulus will give false positive results, and may be unethical, if the agent compromises motor function. We report a procedure for measuring potential effects of antinociceptive agents on motor function in the trained rat, using an 80 mm diameter rotarod.</p><p>Rats were selected for ability to exercise on the rotarod and trained to increasing speeds. In test trials, we measured the time that trained rats could stay on the rod, rotating at 25 rpm, with a cut off at 60 min. Morphine administration decreased rotarod performance significantly at doses of 5.0 mg/kg (<em>P</em> &lt; 0.05, <em>n</em> = 10) and 7.5 mg/kg (<em>P</em> &lt; 0.005, <em>n</em> = 10). We also assessed the response to a noxious thermal stimulus by measuring tail flick latency following tail immersion in water at 49°C. A significant dose-dependent increase in tail flick latency was found for dosages of morphine between 2.5 mg/kg and 7.5 mg/kg (<em>P</em> &lt; 0.005, <em>n</em> = 10).</p><p>Our rotarod procedure, which incorporates selection, training, and a 60 min trial, provides a sensitive and consistent means of assessing motor function. Our results, implying that morphine indeed compromises motor function in rats at doses at which it is antinociceptive, confirm the necessity for investigating the motor effects of any putative antinociceptive agent.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 2","pages":"Pages 149-159"},"PeriodicalIF":0.0,"publicationDate":"1991-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90063-B","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13102243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Methods and limitations of an experimental model of long QT syndrome","authors":"J. Weissenburger ,&nbsp;F. Chezalviel ,&nbsp;J.M. Davy ,&nbsp;P. Lainée ,&nbsp;C. Guhennec ,&nbsp;E. Penin ,&nbsp;F. Engel ,&nbsp;L. Cynober ,&nbsp;G. Motté ,&nbsp;G. Cheymol","doi":"10.1016/0160-5402(91)90051-6","DOIUrl":"10.1016/0160-5402(91)90051-6","url":null,"abstract":"<div><p>An experimental model of the long QT syndrome has been developed in conscious dogs. This report discusses the methods used in its preparation and the strengths and weaknesses of the model. This new model is suitable for screening the bradycardia-dependent proarrhythmic effects of drugs and for studying the electrophysiology of “torsades de pointes.”</p><p>Permanent bradycardia (RR: 1558 ± 83 ms) was obtained in 37 dogs by chemically-induced complete atrioventricular block. A 10% further increase of ventricular repolarization (QT: 306 ± 7.0 ms to 331 ± 5.5 ms) was obtained in 28 of these dogs by diuretic-induced hypokalemia. Diuretics, despite saline replacement, induced some degree of functional renal failure and extracellular volume losses. The QT interval increased although ventricular cycle length decreased slightly. These biological and electrophysiological parameters were reproducible except for a slow increase in plasma creatinine.</p><p>Cardiac failure and sudden death rarely occurred. The most severe, but reversible, renal failure occurred in some dogs given the highest diuretic doses. Hypokalemia resulted in ventricular arrhythmias in only 6 dogs, 2 of them exhibiting runs of ventricular tachycardia and even “torsade de pointes” as their potassium levels fell below 2 mmol/L.</p><p>The results of studies with several drugs using the model, with or without hypokalemia, or with bradycardia worsened by propranolol are analysed.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 1","pages":"Pages 23-42"},"PeriodicalIF":0.0,"publicationDate":"1991-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90051-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13080005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid and selective measurement of platelet-activating factor using a quantitative bioassay of platelet aggregation","authors":"Alaina Jean Ammit,&nbsp;Chris O'Neill","doi":"10.1016/0160-5402(91)90050-F","DOIUrl":"10.1016/0160-5402(91)90050-F","url":null,"abstract":"<div><p>A bioassay for the measurement of platelet-activating factor (PAF) based on the quantification of platelet aggregation was developed. The method used a platelet analyzer in conjunction with a multiwelled micromixing device and whole blood collected from male New Zealand White rabbits. This bioassay can be nonselective and used to quantitate platelet aggregation induced by any activator. The EC₅₀ for platelet-activating factor, arachidonic acid, and adenosine diphosphate were 0.0232, 55, and 10 μM, and at these concentrations the half maximal aggregation response occurred at 7.5, 10.0, and 12.5 min, respectively. The bioassay was capable of the sensitive quantification of platelet aggregation in small volumes (50 μL) of citrated rabbit whole blood, using a short (i.e., 15-min) incubation period. This enabled multiple bioassays to be performed without the need for a large volume of whole blood to be collected from the rabbit. Selective measurement of platelet-activating factor was achieved by adding inhibitors of arachidonic acid-and adenosine diphosphate-dependent pathways of platelet activation, that is, acetylsalicylic acid and phosphoenolpyruvate/pyruvate kinase, respectively, to the citrated rabbit whole blood immediately before bioassay. These inhibited arachidonic acid- and adenosine diphosphate-induced platelet aggregation, but had no affect on platelet aggregation induced by platelet-activating factor. Platelet-activating factor was selectively inhibited by its receptor antagonist BN 52021. This method for measuring platelet-activating factor was reproducible; at the EC₅₀, the inter- and intrabioassay coefficients of variation were within acceptable limits at 13.17% and 9.75%, respectively.</p></div>","PeriodicalId":16819,"journal":{"name":"Journal of pharmacological methods","volume":"26 1","pages":"Pages 7-21"},"PeriodicalIF":0.0,"publicationDate":"1991-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/0160-5402(91)90050-F","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13080009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}