Biomedical subjects

E Viitanen

Publications and source records attributed to E Viitanen.

7 recordsLinked to original sources

PubMed1998

The beta-gal interferon assay: a new, precise and sensitive method.

Cells of a human glioblastoma line were stably transfected with a glial fibrillary acidic protein (GFAP) promoter sequence/lacZ reporter gene. Following this modification, they produced Escherichia coli beta-galactosidase constitutively in amounts that could be measured through their conversion of an added fluorophore into a product readily estimated by fluorimetry. Human interferons (IFN) selectively and in a dose-dependent manner reduce the formation of beta-galactosidase in this system. We have used it as the basis for a novel assay that is sensitive (4-40 pg/ml), precise, completed in 30 h, and applicable to both type I and type II human IFNs. Statistical analysis showed interassay relative standard deviations ranging from 5% to 11%, and most individual assays revealed potencies with limits of error within 85%-115%. Neither partially trypsin-digested IFN nor the other cytokines and mitogens we tested reacted in this system, except for tumor necrosis factor-alpha (TNF-alpha). The high selectivity was further shown by the loss of response to IFN in the presence of the appropriate specific anti-IFN or anti-IFN-gamma receptor antibodies.

Biological Assay↗

PubMed1997

Cholecystokinin-JMV-180 and pilocarpine are potent inhibitors of cholecystokinin and carbachol actions on guinea pig pancreatic acinar cells.

The release of amylase and the elevation of cytoplasmic Ca2+ concentration ([Ca2+]i) in response to cholecystokinin-octapeptide (CCK-8), the cholecystokinin analogue JMV-180, the stable choline ester carbamylcholine (carbachol) and the muscarinic agonist pilocarpine were studied in guinea-pig pancreatic acinar cells. The maximal amylase and [Ca2+]i responses to JMV-180 and pilocarpine were 12-15% of the corresponding responses to CCK-8 and carbachol. The amylase and [Ca2+]i responses to maximal concentrations of CCK-8 and carbachol were inhibited in concentration-dependent manners by JMV-180 and pilocarpine, respectively. In individual acinar cells, JMV-180 and pilocarpine like low concentrations of CCK-8 and carbachol caused oscillations of [Ca2+]i. The sustained [Ca2+]i responses to maximal concentrations of CCK-8 and carbachol were transformed into oscillatory responses during simultaneous exposure to JMV-180 and pilocarpine, respectively. Maximal concentrations of JMV-180 and pilocarpine did not cause homologous or heterologous desensitization of the [Ca2+]i responses but inhibited desensitization evoked by maximal concentrations of CCK-8 or carbachol. JMV-180 and pilocarpine acted as weak, partial agonists exhibiting effective inhibition of the acinar cell responses to full agonists. The effects appeared to be best explained by interactions with two forms of the respective receptor with JMV-180 and pilocarpine acting as partial agonists for one state of the receptor and as antagonist for the second state.

Amylases↗

PubMed1994

Rapid down-regulation of substance P binding to guinea-pig pancreatic acinar cells during homologous desensitization.

Binding of 125I-labelled peptides, cytoplasmic Ca2+ concentration ([Ca2+]i) and amylase release were studied in guinea-pig pancreatic acinar cells during exposure to substance P (SP), and cholecystokinin octapeptide (CCK-8). Pre-incubation of cells at 22 degrees C with 0.03 nM to 1 microM SP for 10 min or at 37 degrees C for 5 min followed by acid or neutral washes reduced subsequent binding of 125I-Bolton-Hunter reagent-labelled SP (125I-BH-SP) in a biphasic manner by up to 95%. Incubation at 4 degrees C eliminated high-affinity binding of 125I-BH-SP and concentrations of SP above 1 nM were required for inhibition of subsequent tracer binding. Pre-incubation of cells at 37 degrees C with 1 nM to 1 microM CCK-8 for 10 min followed by neutral washes reduced subsequent binding of 125I-BH-CCK-8 by up to 65%. In cell suspensions, the [Ca2+]i response to SP was gradually reduced by pre-exposure to increasing agonist concentrations from 0.2 to 20 nM. Pre-incubation with high SP concentrations for 10 min caused profound reduction of subsequent amylase responses to SP, whereas secretion was little affected in corresponding experiments with CCK-8. Down-regulation of receptor binding is not important during short exposure to CCK-8, but it is a pronounced and rapid phenomenon during SP exposure, which explains tachyphylaxis of [Ca2+]i and amylase responses.

Amylases↗

PubMed1992

Binding of epidermal growth factor to receptors in preparations of enriched porcine parietal cells and inhibition of aminopyrine uptake.

Preparations of isolated porcine gastric cells, enriched in parietal cells, were used to study binding of epidermal growth factor (EGF) to receptors and subsequent inhibition of [14C]aminopyrine uptake. EGF in concentrations from 10(-10) to 10(-7) M inhibited aminopyrine uptake stimulated by 10(-5) M histamine with an IC50 of 3 x 10(-10) M. [125]EGF bound in a saturable and specific manner to sites on cells in preparations containing 40-90% parietal cells. Mean apparent dissociation constant for the sites was 1.6 x 10(-9) M, with an average number of approximately 20,000 sites per cell. Endocytosis of ligand by parietal cells was limited, amounting to 10-20% of bound EGF after 1 h of incubation at 37 degrees C. Occupation of a fraction of the receptors caused a maximal reduction by 40% of aminopyrine uptake in histamine-stimulated cells, suggesting the occurrence of spare receptors. The results indicate the existence of specific receptors for EGF on porcine parietal cells exerting a regulatory influence on acid secretion.

Aminopyrine↗

PubMed1990

Quantitation of human chorionic gonadotrophin (hCG) by radioreceptor assay.

A sensitive radioreceptor assay was developed for pharmaceutical preparations of human chorionic gonadotrophin with the use of rat testicular membranes as receptor preparation and human 125I-chorionic gonadotrophin as tracer. The addition of unlabelled human chorionic gonadotrophin or luteinizing hormone inhibited the binding of 125I-chorionic gonadotrophin to the receptors in a concentration dependent way. Concentrations of human chorionic gonadotrophin between 30-300 mIU ml(-1) were normally used for a three-dose assay fulfilling pharmacopoeial statistical requirements for assay validity. The relative standard deviation for five assays was 7%. Estimates of potency of commercial preparations of human chorionic gonadotrophin obtained with the radioreceptor assay correlated well with corresponding estimates from in vivo assays. The proposed radioreceptor assay, however, provides a considerable saving in the number of animals required, requires less technical support, and is more precise than the in vivo method.

Animals↗

PubMed1989

In-vitro bioassays for cholecystokinin.

Two in-vitro methods for the bioassay of cholecystokinin (CCK) are described. They should be useful for determinations of potencies of pharmaceutical formulations of CCK. Both are based on the hormone's ability to stimulate exocrine pancreas. The stimulatory effect on 45Ca efflux from preloaded suspended acinar cells or on release of amylase from pancreatic acini from the guinea-pig has been recorded. The assay based on stimulation of 45Ca outflux is more specific and precise than the method using measurements of amylase secretion. Therefore, the calcium assay was further validated against the conventional guinea-pig gall-bladder contraction assay. Both methods gave similar potency readings but the results from the in-vitro assay were more precise.

Amylases↗

PubMed1987

Radioreceptor assay for insulin formulations.

A sensitive radioreceptor assay was developed for pharmaceutical insulin formulations with the use of rat liver membranes and 125I-insulin. The addition of unlabeled insulin or insulin analogues inhibited the binding of 125I-insulin to the receptors in a concentration-dependent way. Insulin concentrations between 3 x 10(-9) and 3 x 10(-8) M were used for a three-point assay which fulfilled pharmacopoeial statistical requirements for validity of the assay. The coefficient of variation for five assays was 4%. Potency estimates of insulin preparations obtained with the radioreceptor assay correlated well with corresponding estimates from in vivo assays. Both assays also gave similar potency readings for insulin preparations that were partly degraded during storage at elevated temperatures, but the radioreceptor method was more precise.

Animals↗