ucn-1028-c and phorbol-12-13-didecanoate

During angiogenesis in the chick chorioallantoic membrane (CAM), microvascular proliferation continues through day 12 of the 18-day CAM lifespan. Up to day 4.5, the neovascularization is associated with endothelial hyperpermeability and differentiation of restrictive barrier function occurs abruptly at day 5.0. Although exogenous activation of cAMP/protein kinase A (PKA) signaling served to decrease macromolecular extravasation at day 4.5, endogenous signaling cascades responsible for the temporal hyperpermeability remain uncertain. Here, we evaluated protein kinase C (PKC) function in the CAM endothelium at day 4.5 and day 5.0. The specific, broad-based PKC inhibitor calphostin C reduced basal levels of FITC-dextran 40 extravasation at day 4.5. Bisindolymaleimide (BIM), which inhibits selective PKC isoforms, also reduced temporal FITC-dextran 40 efflux, but to a lesser extent than calphostin C. Activation of PKC activity by phorbol-12, 13-didecanoate (PDD) or phorbol-12, 13-dibutyrate (PDBu) at day 5.0 served to partially de-differentiate barrier properties of the angiogenic endothelium. The associated elevation of FITC-dextran 40 extravasation occurred without interendothelial gap formation along the junctional clefts. Together, these results are consistent with the interpretation that PKC activity contributes, in part, to CAM endothelial hyperpermeability at day 4.5. Furthermore, down-regulation of PKC signaling correlates temporally with the ontogeny of restrictive barrier function at day 5.0.

Topics: Allantois; Animals; Capillary Permeability; Carcinogens; Chick Embryo; Dextrans; Endothelium, Vascular; Enzyme Inhibitors; Fluorescein-5-isothiocyanate; Intercellular Junctions; MAP Kinase Signaling System; Microscopy, Electron; Naphthalenes; Neovascularization, Physiologic; Phorbol Esters; Protein Kinase C

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.

Topics: Animals; Endothelial Growth Factors; Endothelin-1; Enzyme Inhibitors; Estrenes; Flavonoids; Gene Expression Regulation; Imidazoles; Lymphokines; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Naphthalenes; Osteoblasts; p38 Mitogen-Activated Protein Kinases; Phorbol Esters; Protein Kinase C; Pyridines; Pyrrolidinones; Receptor, Endothelin A; Receptors, Endothelin; Tetradecanoylphorbol Acetate; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Nerve terminals ("synaptosomes") isolated from rat brain hippocampus were loaded with the fluorescent Ca2+ indicator fura-2 and were subjected to depolarization with an elevated K+ concentration in a stopped-flow spectrophotometer to measure the activity of voltage-gated Ca2+ channels in the presynaptic membrane. Three components of Ca2+ influx were seen, which were tentatively identified as two classes of voltage-dependent Ca2+ channels with different inactivation kinetics (tau of approximately 60 ms and 1 s, respectively) and Na+/Ca2+ exchange working in the "reverse" mode. The activity of both classes of voltage-dependent Ca2+ channels was slightly augmented by the phorbol ester phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), but the effect of PMA was markedly enhanced by the protein phosphatase inhibitor okadaic acid (OKA). The PKC inhibitors calphostin C and dihydrosphingosine (DHS) caused a prompt decrease in voltage-dependent Ca2+ channel activity, but the effect of DHS could be slowed by coaddition of OKA. These results suggest that the activity of presynaptic voltage-dependent Ca2+ channels in the hippocampus is under a dynamic balance between PKC phosphorylation (leading to activation) and protein phosphatase dephosphorylation (leading to inactivation) and that both of these metabolic pathways are tonically active in the nerve terminals.

Topics: Animals; Calcium; Calcium Channels; Fura-2; Hippocampus; Kinetics; Naphthalenes; Phorbol Esters; Phosphorylation; Polycyclic Compounds; Potassium; Presynaptic Terminals; Protein Kinase C; Rats; Sodium; Spectrometry, Fluorescence; Sphingosine; Tetradecanoylphorbol Acetate

EGF has been reported to promote oocyte maturation in several species, although the mechanism of action is not yet known. The present study is designed to determine the pathway used by EGF to enhance porcine oocyte maturation. Oocytes were aspirated from 2-5 mm follicles and cultured with various treatments in Medium-199 at 37 degrees C, 100% relative humidity, and 5% CO2 for 48 hr for the maturation study and 3 hr for intracellular cAMP measurement. Although treatment with 100 IU/ml hCG stimulated both intracellular cAMP formation and oocyte maturation, 10 ng/ml EGF stimulated oocyte maturation only. Dibutyryl cAMP (dbcAMP) inhibited oocyte maturation at 10(-5), 10(-4), and 10(-3) M concentration s in the control medium. However, in the presence of 10 ng/ml EGF, dbcAMP inhibited oocyte maturation only at a concentration of 10(-3) M. Increasing concentrations of EGF (i.e., 25 and 50 ng/ml) were ineffective in overcoming the inhibitory effect of dbcAMP at 10(-3) M. In contrast, EGF reversed the decreased maturation rate caused by transforming growth factor-beta. Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, enhanced the spontaneous maturation rate; 4 alpha-phorbol dideconoate, an inactive phorbol ester, did not show this effect. PMA- and EGF-stimulated porcine oocyte maturation is reversed by calphostin-C, a PKC inhibitor. In conclusion, EGF's promotional activity on porcine oocyte maturation is independent of the cAMP pathway and probably mediated by the PKC pathway.

Topics: Animals; Bucladesine; Cyclic AMP; Dose-Response Relationship, Drug; Epidermal Growth Factor; Mice; Naphthalenes; Oocytes; Oogenesis; Phorbol Esters; Swine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

In cultured glomerular mesangial cells, interleukin 1 beta (IL-1 beta) has been shown to induce a dose- and time-dependent accumulation of nitrite, a stable metabolite of nitric oxide (NO). In parallel, increased levels of mRNA of an inducible macrophage-type of nitric oxide synthase (iNOS) were observed after incubating mesangial cells with IL-1 beta. Here we report that addition of the biologically active phorbol esters, phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), dose-dependently inhibited the IL-1 beta-stimulated increase in iNOS mRNA levels and nitrite production. In contrast, the biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate, had no effect on cytokine induction of iNOS and nitrite formation. Incubation of mesangial cells with PMA or PDBu alone, in the absence of IL-1 beta, did not trigger any iNOS expression. Time-course studies indicated that phorbol ester needs to be added for only 1 h in order to maximally inhibit cytokine-induced nitrite production. Down-regulation of protein kinase C (PKC)-alpha and -delta isoenzymes by 8 h PMA or PDBu treatment before stimulation with IL-1 beta still resulted in full inhibition of iNOS induction. In contrast, a 24 h treatment of mesangial cells with PMA or PDBu, a regimen that also causes depletion of PKC-epsilon, abolished inhibition of IL-1 beta-induced iNOS expression and nitrite production. In addition, the selective PKC inhibitor calphostin C potentiated IL-1 beta induction of iNOS activity. In summary these data suggest that IL-1 beta induction of iNOS expression is tonically suppressed by PKC and the epsilon-isoenzyme is the most likely candidate mediating this effect.

Topics: Amino Acid Oxidoreductases; Animals; Blotting, Northern; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Induction; Glomerular Mesangium; Interleukin-1; Isoenzymes; NADH Dehydrogenase; Naphthalenes; Nitric Oxide Synthase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Polycyclic Compounds; Protein Kinase C; Rats; RNA, Messenger; Tetradecanoylphorbol Acetate

The formation of constitutive transport vesicles involves the association of non-clathrin coat proteins to transport organelles. Here we report that IgE receptors and protein kinase C (PKC) regulate the GTP-dependent binding of the two coat proteins ADP-ribosylation factor (ARF) and beta-COP to Golgi membranes in rat basophilic leukaemia cells. Activation of IgE receptors and PKC prevented the ARF and beta-COP dissociation from Golgi membranes that occurs in permeabilized cells in the absence of GTP and potentiated the association-promoting effects of GTP and the G protein activator fluoroaluminate. In contrast, PKC downregulation and PKC inhibition abolished the activity of GTP and fluoroaluminae in promoting ARF binding to the Golgi complex. Studies of ARF binding to isolated Golgi membranes gave similar results. These findings suggest that coat assembly on Golgi membranes, and thus possibly constitutive secretory traffic, is modulated by membrane receptors and second messengers.

Topics: ADP-Ribosylation Factors; Aluminum; Animals; Brefeldin A; Cell Membrane Permeability; Coatomer Protein; Cyclopentanes; Enzyme Activation; Fluorine; Glycosaminoglycans; Golgi Apparatus; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Microtubule-Associated Proteins; Naphthalenes; Phorbol Esters; Polycyclic Compounds; Protein Binding; Protein Kinase C; Rats; Receptors, IgE; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured