epidermal-growth-factor and 2-5-di-tert-butylhydroquinone

Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I(sc)) by 15-25%, whereas the addition of ATP to the apical bathing solution decreased I(sc) by 40-60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I(sc) in mCT12 monolayers by 46 +/- 4% (n = 8) and 22 +/- 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 microM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I(sc). In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 microM) almost completely blocked the PMA-induced decrease in I(sc), but did not alter the EGF- or ATP-induced inhibition of I(sc). The DBHQ-mediated decrease in I(sc) was due to inhibition of basolateral Na(+)-K(+)-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na(+) channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia.

Topics: Adenosine Triphosphate; Amiloride; Animals; Biological Transport; Cells, Cultured; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Hydroquinones; Indoles; Kidney Tubules, Collecting; Maleimides; Mice; Phorbol Esters; Phosphorylation; Protein Kinase C; Sodium

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Female; Hydroquinones; Imidazoles; Immunohistochemistry; Mammary Glands, Animal; Mice; Mice, Inbred ICR; Pregnancy; Proliferating Cell Nuclear Antigen; Uridine Triphosphate

The role of heterotrimeric GTP-binding proteins in the process of store-operated Ca2+ inflow in hepatocytes was investigated by testing the ability of pertussis toxin to inhibit thapsigargin- and 2,5-di-tert-butylhydroquinone (DBHQ)-induced bivalent cation inflow. Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited markedly inhibited rates of both Ca2+ and Mn2+ inflow when these were stimulated by vasopressin, angiotension II, epidermal growth factor, thapsigargin and DBHQ. Pertussis toxin had little effect on the basal intracellular free Ca2+ concentration ([Ca2+]i), basal rates of Ca2+ and Mn2+ inflow, the abilities of vasopressin, angiotensin II, thapsigargin and DBHQ to induce the release of Ca2+ from intracellular stores, and the maximum value of [Ca2+]i reached following agonist-induced release of Ca2+ from intracellular stores. It is concluded that store-operated Ca2+ inflow in hepatocytes employs a slowly ADP-ribosylated trimeric GTP-binding protein and is the physiological mechanism, or one of the physiological mechanisms, by which vasopressin and angiotensin stimulate plasma membrane Ca2+ inflow in this cell type.

Topics: Angiotensin II; Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Calibration; Epidermal Growth Factor; GTP-Binding Proteins; Hydroquinones; Injections, Intraperitoneal; Liver; Manganese; Pertussis Toxin; Rats; Spectrometry, Fluorescence; Terpenes; Thapsigargin; Vasopressins; Virulence Factors, Bordetella