Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes.
While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.
D000818:Animals; D000831:Animals, Newborn; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D006023:Glycoproteins / Q000096:biosynthesis; D006531:HEPES / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects; D006493:Heparin / Q000494:pharmacology; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000096:biosynthesis / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D010879:Piperazines / Q000494:pharmacology*; D051381:Rats
D006023:Glycoproteins; D010879:Piperazines; D003994:Bucladesine; D006493:Heparin; D008071:Lipoprotein Lipase; D006531:HEPES