Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines.

The expression and function of gonadotropin receptors, and the secretion of steroids, transferrin, and cytokines were investigated in three immortalized (single transfection with v-myc) mouse granulosa cell lines (GRM01, GRM01L, and GRM02). A dose-dependent increase in progesterone production was obtained in GRM01 and GRM02 cells after addition of LH, FSH, modulators of the adenylate cyclase enzyme system, and cAMP analogues. The LH-induced release of progesterone was already detectable in GRM02 cells after 8 h and was related to incubation time and cell number. Both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) induced the secretion of progesterone in GRM02 cells, while no effect was obtained with TGF beta. LH receptor concentration was highest in the GRM02 cell line. FSH receptor mRNA was visualized in GRM01 and GRM02 cells. Aromatase activity in GRM02 cells was induced by androgens and inhibited by aromatase inhibitors. Whereas all cell lines were able to secrete transferrin, only in GRM01 cells was transferrin secretion increased significantly by LH. FSH did not affect transferrin secretion in the three cell lines, in contrast to forskolin or 8-bromo-cAMP. The immortalized mouse granulosa cell lines were able to express and release several growth factors. The expression and secretion of activin, inhibin, TGF beta, EGF, TGF alpha, insulin-like growth factor II, fibroblast growth factor (acidic and basic), platelet-derived growth factor, and interleukin-6 suggest an autocrine or paracrine role for these factors in follicular differentiation and function. In conclusion, these cells, derived from mural granulosa cells and immortalized in a preovulatory state, can be used to study granulosa cell physiology or to study the role of granulosa cells and their derivatives in the process of follicular maturation, fertilization, and early embryonic development.

Alpha 1-acid glycoprotein concentration and molecular heterogeneity: relationship to oxprenolol binding in serum from healthy volunteers and patients with lung carcinoma or cirrhosis.

1. alpha 1-acid glycoprotein (AAG) concentration and molecular heterogeneity, and oxprenolol protein binding were studied in serum of 15 healthy volunteers, 14 patients with lung carcinoma and 17 patients with liver cirrhosis. 2. The AAG serum concentration was increased to 180.7% in patients with lung cancer and decreased to 73.4% in cirrhotic patients as compared with controls (P less than 0.05). 3. The concanavalin A (conA) dependent heterogeneity of serum AAG was very similar in controls and patients with lung cancer: a ratio of 9/9/2 was obtained for the conA nonreactive, the conA weakly reactive and the conA strongly reactive subfraction respectively; in cirrhotic patients, the ratio shifted to 11/7/1. 4. The heterogeneity in electric charge, demonstrated by isoelectric focusing, was similar in the three groups of subjects: 70-80% of the focussed bands were found in the main three bands. 5. The binding of oxprenolol to serum proteins was increased in lung tumour patients and decreased in liver cirrhotic patients as compared with controls (P less than 0.05). There was no change in binding affinity and oxprenolol binding was significantly correlated to total AAG serum concentration and to the concentration of each of the conA dependent subtypes, in controls as well as in both patients groups.

Influence of inflammation on serum concentration, molecular heterogeneity and drug binding properties of canine alpha-1-acid glycoprotein.

The concentration and the heterogeneity of alpha-1-acid glycoprotein (alpha-1-AGP) and oxprenolol binding were determined in serum of healthy dogs and dogs with inflammatory disease. In inflammation, an increase in the mean alpha-1-AGP concentration from 0.47 to 2.85 g/l was accompanied by a reduction in the mean free oxprenolol fraction from 25% to 6%. alpha-1-AGP concentration and oxprenolol binding were inversely correlated. The heterogeneity of canine alpha-1-AGP remained essentially unchanged in dogs with inflammation and, in both these dogs and the controls, between five and seven forms with different isoelectric points and one single concanavalin A-reactive form were detected. It is concluded that in dogs, as in humans, oxprenolol binds to serum alpha-1-AGP. Changes in serum binding of oxprenolol during inflammation result from a change in the serum concentration of alpha-1-AGP rather than a change of molecular heterogeneity.

Dog alpha-1-acid glycoprotein: purification and biochemical characterization.

Dog alpha-1-acid glycoprotein was purified to homogeneity from dog serum in a three-step procedure involving precipitation with sulphosalicylic acid, isoelectric focusing and size exclusion chromatography. The molecular heterogeneity in the peptide part and in the carbohydrate part of the molecule was investigated with analytical isoelectric focusing in a narrow pH range and crossed immunoaffinity electrophoresis with concanavalin A (con A) in the first-dimension gel. Up to seven molecular forms with different isoelectric points were found, whereas only a single con A-dependent molecular form was detected.

Influence of enhanced alpha-1-acid glycoprotein concentration on protein binding, pharmacokinetics and antiarrhythmic effect of lidocaine in the dog.

The pharmacokinetics and the antiarrhythmic effect of lidocaine were studied in healthy dogs on three occasions: before administration of rifampicin, on the 14th day of treatment with rifampicin and 4 weeks after stopping rifampicin treatment. On each occasion a loading and a maintenance infusion of lidocaine were given to obtain steady-state concentrations. Blood and plasma concentrations of lidocaine, alpha-1-acid glycoprotein plasma concentrations and percentage of free lidocaine in plasma were determined at the end of the maintenance infusion. The antiarrhythmic effect of lidocaine was evaluated by measuring the arrhythmogenic dose of epinephrine. Blood concentrations and, consequently, the total blood clearance of lidocaine were comparable on the three occasions. Total plasma concentrations were significantly higher after rifampicin administration as compared to the two control periods. Percentage of free lidocaine decreased from about 50 to about 30%, accompanied by a nearly 3-fold increase of alpha-1-acid glycoprotein concentrations. Free plasma concentrations were slightly lower after rifampicin treatment. The epinephrine dose ratio (before/after) paralleled the changes in free lidocaine concentrations. The correlation between free plasma concentrations and epinephrine dose ratio was much stronger than between total plasma concentrations and epinephrine dose ratio. The plasma elimination half-life of lidocaine was markedly shortened after rifampicin treatment, due to a diminished volume of distribution. It is concluded that, under steady-state conditions, marked increases in the protein binding of lidocaine are accompanied by only slight decreases of free plasma concentrations and of antiarrhythmic effect.

Alpha 1-acid glycoprotein and serum binding of drugs in healthy and diseased dogs.

Inter-individual variation in drug serum protein binding was studied in healthy dogs and in dogs with inflammatory diseases for lidocaine, oxprenolol and propranolol, which bind mainly to alpha 1-acid glycoprotein (alpha 1-AGP), and for diazepam, digitoxin and phenytoin, which bind mainly to albumin. For the drugs mostly bound to alpha 1-AGP, in both groups of dogs binding varied considerably, and it was markedly higher in dogs with inflammatory disease. For the other drugs, the variation in binding was smaller and did not differ between the two groups of dogs. In both groups of dogs, the alpha 1-AGP concentration varied widely; it was higher in the serum of the dogs with inflammation, while the concentration of albumin was lower in these animals. There was a significant negative correlation between percentage free lidocaine, oxprenolol or propranolol and alpha 1-AGP concentration, suggesting that the inter-individual variation in binding of these drugs is due to the variation in alpha 1-AGP concentration. There was a marked intra-individual variation in lidocaine binding and in serum alpha 1-AGP concentration, studied over a period of 3 weeks in healthy dogs; a significant negative correlation between percentage free lidocaine and alpha 1-AGP concentration was obtained.