The use of drugs to dissect the pathway for secretion of the glycoprotein hormone chorionic gonadotropin by cultured human trophoblastic cells.
Agents that affect intracellular cation and pH gradients and inhibit energy production have been tested for their ability to modulate the processing and secretion of the free alpha subunit and the alpha beta dimer of human chorionic gonadotropin (hCG) by cultured human trophoblastic cells (JAR). Incubation of JAR cells with monensin or nigericin, monovalent cation ionophores that produce equilibration of Na+ and K+ across cellular membranes, dicyclohexylcarbodiimide, an agent that inhibits intracellular membrane ATPases, and methylamine, which neutralizes intracellular pH gradients, produced similar effects on hCG processing and secretion. All these agents inhibited the processing of the asparagine-linked oligosaccharide chains of free alpha subunit and the alpha and beta subunits contained in the hCG dimer. Moreover, after treatment of JAR cells with these agents, there was an intracellular accumulation of precursor forms and an inhibition of secretion of "mature" forms of hCG. Monensin affected the processing and secretion of hCG subunits differently at different concentrations. At 5 X 10(-7) M, monensin inhibited the processing of the asparagine-linked oligosaccharides of hCG without altering the rate-limiting step in the secretory pathway or blocking hCG secretion. The intracellular hCG subunit precursors in both control and monensin-treated cells contained a similar array of high mannose oligosaccharides, predominantly of the Man8GlcNAc2 and Man9GlcNAc2 types. However, monensin-treated cells secreted hCG subunits that contained endo H-sensitive oligosaccharides of the high mannose (mostly Man5GlcNAc2) and hybrid types rather than the endo H-resistant complex chains synthesized by control cells. Nevertheless, a full complement of serine-linked oligosaccharides was added to the hCG-beta subunit in monensin-treated cells. These results indicate that the intracellular movement of hCG from the rough endoplasmic reticulum to the cell surface was not inhibited by monensin at a concentration that impaired Golgi-localized steps in the processing of asparagine-linked oligosaccharides. At 5 X 10(-6) M, monensin significantly inhibited secretion of hCG and created a new rate-limiting step in the processing pathway. hCG subunits bearing Man5GlcNAc2 units accumulated intracellularly, suggesting that the equilibration of intracellular Na+/K+ pools blocked oligosaccharide processing at an intra-Golgi point, perhaps by inhibiting movement of the glycoprotein hormone from the "cis" to the "trans" Golgi compartment. Since the other drugs mentioned above produced similar effects on hCG processing and secretion, it appears that maintenance of intracellular cation and pH gradients is necessary for the intra-Golgi transport of glycoprotein hormones.(ABSTRACT TRUNCATED AT 400 WORDS)
D000001:Calcimycin / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008744:Methylamines / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D014327:Trophoblasts / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D008744:Methylamines; D009840:Oligomycins; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000001:Calcimycin; D004024:Dicyclohexylcarbodiimide; D008985:Monensin; C027451:methylamine; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase