ABSTRACT
Using a mass culture assay for the contact-dependent transfer of potassium among cells with intrinsic differences in ability to concentrate it, we have investigated the ability of several drugs to influence this form of cell communication. We concentrated on 12-O-tetradecanoylphorbol-13-acetate (TPA), which is known to interfere with gap junction-mediated communication and ion transport in several other systems, and compared its effects with those of its inactive derivative, 4-O-methyl-TPA. We found that the communication between mouse BALB/c 3T3 cells and human diploid fibroblasts was reduced in the presence of TPA but not O-methyl-TPA and that this inhibition was not obscured by small but measurable influences of TPA on steady-state content and transport of 86Rb+. We confirmed these findings using an autoradiographic assay for transfer of uridine derivatives among cells in contact. We also showed that retinoic acid had no effect on communication in the ion transfer assay but that furosemide, an inhibitor of Na(+)-K(+)-2Cl- cotransport, stimulated ion transfer dramatically both in the presence and absence of TPA. These results indicate both the promise and the limitations of the potassium transfer assay for identifying potential modulators of gap junction-mediated cell communication.
Subject(s)
Cell Communication/drug effects , Furosemide/pharmacology , Potassium/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tretinoin/pharmacology , 3T3 Cells , Animals , Biological Transport , Cells, Cultured , Humans , Infant, Newborn , Mice , Ouabain/pharmacology , Rubidium/metabolism , Tetradecanoylphorbol Acetate/analogs & derivativesABSTRACT
Carcinoembryonic antigen (CEA) is a glycoprotein metabolized primarily by the liver. Subcellular fractions of rat liver were examined for CEA binding activity. Hepatocyte plasma membrane and microsome fractions bound CEA, and this binding shared the calcium requirement, neuraminidase sensitivity, and carbohydrate specificity of the hepatocyte asialoglycoprotein receptor. CEA had previously been shown to react with this galactose-specific receptor, in vivo, only following neuraminidase treatment. Galactose receptor binding of CEA was measured in three different purified CEA preparations. The fraction of CEA capable of binding to excess levels of galactose receptor on membranes varied (46.5%, 40.2%, and 4.7% for CEA-1, -2, and -3, respectively). These CEAs were shown to be 2.3%, 7.9%, and 0.7% as effective, respectively, as asialo-alpha 1-acid glycoprotein in inhibiting the binding of radiolabeled asialo-alpha 1-acid glycoprotein to liver cell membranes. Each of the three CEA preparations showed different clearance kinetics from the circulation of mice. Coinjection of asialo-alpha 1-acid glycoprotein with the CEAs revealed differing inhibition of the clearances. These results show that differences in the carbohydrate components of purified CEA preparations affect their rate of removal from circulation and thus possibly the relationship between CEA production and observed plasma levels in patients. The possible origin of these CEA differences is discussed with their clinical implications.
Subject(s)
Asialoglycoproteins , Carcinoembryonic Antigen/metabolism , Liver/metabolism , Animals , Cell Membrane/metabolism , In Vitro Techniques , Kinetics , Male , Metabolic Clearance Rate , Mice , Orosomucoid/analogs & derivatives , Orosomucoid/metabolism , Rats , Rats, Inbred StrainsABSTRACT
Asialoglycoproteins are removed from the circulation by the carbohydrate-specific hepatic binding protein in the rat. Asialoglycoprotein in human serum can be detected by an inhibition assay in which the binding of 125I-labeled asialo-alpha 1-acid glycoprotein (ASAG) to purified hepatocyte membranes is inhibited by known amounts of ASAG or patient's serum. We were able to reproducibly measure inhibition equivalent to that of 1 to 20 ng of ASAG. The mean concentration of inhibitor in 11 healthy control patients was equivalent to 0.153 (SD = 0.051) mg of ASAG per liter, measured in 20 microL of serum. Significant increases of inhibitor were observed in patients with malignant extrahepatic biliary obstruction, alcoholic liver disease, viral hepatitis, or liver metastases, but not in those with benign biliary obstruction. For a set of six control sera the intra- and interassay CVs were 9.1% and 25.2%, respectively (n = 3 each).