Abstract
Cells in culture often undergo a "burst" of free sphingosine, sphingosine 1-phosphate, ceramide, and other bioactive lipids upon removal of "conditioned" medium, and at least one lipid signaling pathway (protein kinase C) has been shown to be affected by these changes (Smith, E. R. & Merrill A. H., Jr. (1995) J. Biol. Chem. 270, 18749-18758; Smith, E. R., Jones, P. L., Boss, J. M. & Merrill, A. H., Jr. (1997) J. Biol. Chem. 272, 5640-5646). Whereas increases in sphinganine and dihydroceramide are responses to provision of precursors for sphingolipid biosynthesis de novo in the new medium, the sphingosine burst is due to sphingolipid turnover upon removal of suppressive factor(s) in conditioned medium. This study describes the purification and characterization of these suppressive factors. Conditioned medium from J774 cells was fractionated into two components that suppress the burst as follows: ammonium ion, which reaches 2-3 mM within 48 h of cell culture; and a low molecular weight, cationic compound that has been assigned the structure 2, 6-bis(omega-aminobutyl)-3,5-diimino-piperazine (for which we suggest the name "batrachamine" based on its appearance) by (1)H and (13)C NMR, Fourier transform infrared spectroscopy, and mass spectrometric analyses. The physiological significance of these compounds as suppressors of sphingolipid metabolism is unclear; however, ammonium ion is a by-product of amino acid catabolism and reaches high concentrations in some tissues. Batrachamine is even more intriguing because this is, as far as we are aware, the first report of a naturally occurring compound of this structural type. Considering the many cell functions that are affected by sphingoid bases and their derivatives, the effects of NH(4) and batrachamine on sphingolipid metabolism may have important implications for cell regulation.
Highlights
Free sphingoid bases, sphingosine 1-phosphate, and ceramides affect numerous cell regulatory pathways when added exogenously or are formed endo
Sphingosine bursts have been seen with many types of cells in culture as follows: J774 cells [6, 9, 10], Swiss 3T3 cells [7], NIH-3T3 cells [8], A431 cells [8], NG108 –15 cells [8], and primary cultures of rat hepatocytes and mouse peritoneal
Studies with J774 cells have established that the sphingosine arises from sphingomyelin hydrolysis in what appears to be acidic intracellular compartment(s) because NH4ϩ and chloroquine are inhibitory [9]
Summary
Materials—J774A.1 cells (number TIB 67), a murine macrophagelike transformed cell line, were obtained from the American Type Culture Collection (Manassas, VA). Cells were used between passages 3 and 24. Analysis of the Sphingosine Burst—Unless indicated differently, suspended cells were removed from the spinner flask, collected by gentle centrifugation (in a table top centrifuge), resuspended in new medium, and added to 60-mm tissue culture dishes (Corning) at 5 to 7.5 ϫ 105 cells per 2 ml of medium. Under these conditions, the cells adhere to the dish rather than grow in suspension. The cells were incubated at 37 °C,
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