benzofurans and 4-bromophenacyl-bromide

The cross-talk of arachidonic acid (AA) and its lipoxygenase products with protein kinase C beta (PKC beta) mRNA levels during the action of gonadotropin-releasing hormone (GnRH) was investigated in the pituitary alpha T3-1 cell line. The addition of AA or its 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid (5-HETE) or leukotriene C4 (LTC4) for 30 or 60 min stimulated PCK beta, but not PKC alpha mRNA levels (3-5-fold); PCK gamma is not expressed by the cells. Other HETEs or leukotrienes tested showed no significant effect. The range of effective concentration for LTC4 and 5-HETE (around 10(-10) M) is the range found in GnRH-stimulated pituitary cells. Although PKC beta mRNA levels were preferentially elevated by LTC4 and 5-HETE at early time points, PKC alpha mRNA levels were elevated at 6-12 h of incubation when PKC beta mRNA levels returned to basal levels. The addition of the phospholipase A2 inhibitor 4-bromophenacyl bromide or the selective 5-lipoxygenase inhibitor L-656,224 abolished [D-Trp6]GnRH (GnRH-A) elevation of PKC beta mRNA levels, whereas PKC alpha mRNA levels were not increased by this neurohormone. The cyclo-oxygenase inhibitor indomethacin elevated basal PKC beta mRNA levels and potentiated the GnRH-A response. Cross-talk exists between AA and some of its lipoxygenase products and PKC beta gene expression during cell signalling. AA, 5-HETE and LTC4 participate in the rapid stimulation of PKC beta mRNA levels by GnRH.

Topics: Acetophenones; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Benzofurans; Cell Line; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Gonadotropin-Releasing Hormone; Hydroxyeicosatetraenoic Acids; Indomethacin; Kinetics; Leukotriene C4; Lipoxygenase; Lipoxygenase Inhibitors; Pituitary Gland; Protein Kinase C; Protein Kinase C beta; RNA, Messenger

Recent studies in our laboratory demonstrate that 1,25-dihydroxyvitamin D [1,25-(OH)2D] increases hepatocyte cytosolic calcium in the absence of extracellular calcium and activates phospholipase-A-induced deacylation of phosphatidylinositol within 5 min. To determine whether inhibition of phospholipase-A affects D-induced increments in cytosolic calcium and which metabolite of phosphatidylinositol mediates these increments, cytosolic calcium is measured in cultured hepatocytes, loaded with Fura 2AM. Cellular fluorescence is determined at excitation wavelengths of 340 and 380 nm, and cytosolic calcium is calculated. 1,25-(OH)2D treatment for 5 min increases cytosolic calcium levels in the cultured hepatocyte. Inhibition of phospholipase-A with bromophenacylbromide blocks the vitamin D effect on cytosolic calcium, indicating that phospholipase-A activation precedes increments in cytosolic calcium. Neither arachidonic acid nor sodium arachidonate affects cytosolic calcium. In contrast, LPI increases hepatocyte cytosolic calcium in the presence and absence of extracellular calcium. The effect is not blocked by bromophenacylbromide. Neither cell viability nor supernatant fluorescence is altered by LPI, indicating that LPI is increasing fluorescence within the cell. 1,25-(OH)2D treatment for 5 min increases cytosolic pH in cultured hepatocytes. Inhibition of cell alkalinization with amiloride blocks the vitamin D effect on both cytosolic pH and cytosolic calcium, but not the effect of LPI on calcium. NH4Cl increases hepatocyte pH but not cytosolic calcium. The results indicate that phospholipase-A activation is necessary for 1,25-(OH)2D-induced increments in hepatocyte cytosolic calcium and that LPI, a deacylation product of phosphatidylinositol, mediates these increments. Furthermore, inhibition of Na+/H+ exchange, while not blocking the ability of the cell to increase cytosolic calcium in response to LPI, prevents the 1,25-(OH)2D-induced increments in both pH and calcium. The data suggest that the rapid effects of 1,25-(OH)2D on hepatocyte calcium are preceded by cell alkalinization and phospholipase-A activation, and these effects appear to be linked to the Na+/H+ antiport system and not nonspecific cell alkalinization.

Topics: Acetophenones; Amiloride; Animals; Benzofurans; Calcitriol; Calcium; Cells, Cultured; Cytosol; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Liver; Lysophospholipids; Phospholipases A; Rats; Spectrometry, Fluorescence