Methionine-enkephalin in a porcine endometrial cell line and its responsiveness to potassium depolarization.
Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize ir-MENK present in PE-1 cells, and to investigate the effect of KCl depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)-HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCl stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCl allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete ir-MENK upon KCl stimulation.
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Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens 30602.
D000818:Animals; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004279:DNA, Viral / Q000235:genetics; D004717:Endometrium / Q000187:drug effects* / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects; D005260:Female; D006358:Hot Temperature; D007700:Kinetics; D011189:Potassium Chloride / Q000494:pharmacology*; D011863:Radioimmunoassay; D013539:Simian virus 40 / Q000235:genetics; D013552:Swine; D014162:Transfection
D004279:DNA, Viral; D004744:Enkephalin, Methionine; D011189:Potassium Chloride