epidermal-growth-factor and 1-2-dioctanoylglycerol

The effects of epidermal growth factor (EGF) on intracellular calcium ([Ca(2+)](i)) responses to the muscarinic agonist carbachol were studied in a human salivary cell line (HSY). Carbachol (10(-4) M)-stimulated [Ca(2+)](i) mobilization was inhibited by 40% after 48-h treatment with 5 x 10(-10) M EGF. EGF also reduced carbachol-induced [Ca(2+)](i) in Ca(2+)-free medium and Ca(2+) influx following repletion of extracellular Ca(2+). Under Ca(2+)-free conditions, thapsigargin, an inhibitor of Ca(2+) uptake to internal stores, induced similar [Ca(2+)](i) signals in control and EGF-treated cells, indicating that internal Ca(2+) stores were unaffected by EGF; however, in cells exposed to thapsigargin, Ca(2+) influx following Ca(2+) repletion was reduced by EGF. Muscarinic receptor density, assessed by binding of the muscarinic receptor antagonist L-[benzilic-4,4'-(3)HCN]quinuclidinyl benzilate ([(3)H]QNB), was decreased by 20% after EGF treatment. Inhibition of the carbachol response by EGF was not altered by phorbol ester-induced downregulation of protein kinase C (PKC) but was enhanced upon PKC activation by a diacylglycerol analog. Phosphorylation of mitogen-activated protein kinase (MAP kinase) and inhibition of the carbachol response by EGF were both blocked by the MAP kinase pathway inhibitor PD-98059. The results suggest that EGF decreases carbachol-induced Ca(2+) release from internal stores and also exerts a direct inhibitory action on Ca(2+) influx. A decline in muscarinic receptor density may contribute to EGF inhibition of carbachol responsiveness. The inhibitory effect of EGF is mediated by the MAP kinase pathway and is potentiated by a distinct modulatory cascade involving activation of PKC. EGF may play a physiological role in regulating muscarinic receptor-stimulated salivary secretion.

Topics: Binding, Competitive; Calcium; Calcium Signaling; Carbachol; Cell Line; Diglycerides; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Extracellular Space; Humans; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscarinic Agonists; Muscarinic Antagonists; Phosphorylation; Protein Kinase C; Quinuclidinyl Benzilate; Receptors, Muscarinic; Salivary Glands; Signal Transduction; Tetradecanoylphorbol Acetate; Thapsigargin

Epidermal growth factor (EGF) increases 12-lipoxygenase mRNA by about 2-fold with a lag period of 4 to 8 hr, which precedes the increase in 12-lipoxygenase activity by 2 to 4 hr in human epidermoid carcinoma A431 cells. Induction of 12-lipoxygenase expression in human erythroleukemia cells by phorbol 12-myristate 13-acetate (PMA) has been reported previously. The present report describes a study of the involvement of protein kinase C (PKC) in EGF-induced 12-lipoxygenase expression in A431 cells. EGF-induced 12-lipoxygenase expression was inhibited by methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor. Staurosporine and calphostin C, which are two PKC inhibitors, inhibited EGF-induced enzyme activity and mRNA expression of 12-lipoxygenase. 1,2-Dioctanoyl-sn-glycerol (a membrane-permeant diacylglycerol) and PMA significantly induced enzyme activity and mRNA expression. Simultaneous treatment of cells with EGF and PMA did not exhibit an additive effect, suggesting that EGF and PMA share a common biochemical pathway in 12-lipoxygenase induction. Expression of mRNA for PKC alpha, delta and zeta was detected in A431 cells, whereas no mRNA expression for PKC beta 1, gamma and epsilon was observed. Taken together, these results suggest that EGF-induced 12-lipoxygenase expression is at least in part mediated by the PKC signal transduction pathway.

Topics: Alkaloids; Arachidonate 12-Lipoxygenase; Carcinoma, Squamous Cell; Diglycerides; Enzyme Induction; Epidermal Growth Factor; Humans; Naphthalenes; Phosphatidylinositol Phosphates; Polycyclic Compounds; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Studies were performed to identify the site at which activation of protein kinase C (PKC) inhibits arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cultured rat inner medullary collecting tubule (RIMCT) cells. Neither endogenous stimulation of PKC by epidermal growth factor (EGF) nor the addition of exogenous 1,2-dioctanoyl-sn-glycerol (DOG) impaired forskolin-stimulated cAMP accumulation. Similarly, neither EGF nor DOG altered cAMP generation in response to cholera toxin. However, pretreatment of RIMCT cells with pertussis toxin resulted in loss of inhibition of AVP-stimulated cAMP accumulation by DOG. Likewise, the ability of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), to inhibit AVP-stimulated cAMP accumulation was eliminated by pretreatment with pertussis toxin. PMA also inhibited AVP-stimulated adenylyl cyclase activity in plasma membranes prepared from rat inner medullas. In contrast to its effects on AVP, activation of PKC did not impair cAMP accumulation in response to isoproterenol or prostaglandin E2. These studies demonstrate that PKC-mediated inhibition of AVP-stimulated cAMP accumulation in cultured RIMCT cells requires the intact inhibitory guanine nucleotide binding protein Gi.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Arginine Vasopressin; Colforsin; Cyclic AMP; Diglycerides; Enzyme Activation; Epidermal Growth Factor; GTP-Binding Proteins; Kidney Tubules, Collecting; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Animals; Arachidonic Acids; Calcimycin; Calcium; Carcinogens; Chickens; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Male; Pituitary Gland; Protein Kinase C; Second Messenger Systems; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Transforming Growth Factor alpha

A single VL30 element present in the RVL-3 cell line was transcriptionally induced by both epidermal growth factor (EGF) and the protein kinase C (pkC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and sn-1,2-dioctanoylglycerol within 5 min of stimulation. Following TPA-induced depletion of protein kinase C activity, EGF stimulation of VL30 transcription and accumulation was unaffected while TPA effects were inhibited, implying that EGF and TPA act by separable pathways.

Topics: Animals; Cell Line; Diglycerides; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation; Glycerides; Mice; Multigene Family; Protein Kinase C; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Transcription, Genetic

We have found many compounds that amplify the action of glucocorticoid without themselves having any glucocorticoid-like action and have proposed the concept of 'Glucocorticoid Action Biomodulators'. These biomodulators consist of 'Glucorticoid Sensitivity Amplifiers', which greatly amplify the action of glucocorticoid at doses of glucocorticoid that alone have minimal effects, and 'Glucocorticoid Potency Amplifiers', which markedly enhance the effect of glucocorticoid at doses that have maximal effects. Potent activators of protein kinase C, such as 1,2-racemic dioctanoylglycerol, 12-o-tetradecanoyl-phorbol-13-acetate, and epidermal growth factor (EGF), markedly enhanced the induction of tyrosine aminotransferase and ornithine decarboxylase by dexamethasone in adrenalectomized rats in vivo and in primary cultures of adult rat hepatocytes in vitro. They amplified enzyme induction by even a large amount of dexamethasone that had a maximal effect, but had no effect in the absence of glucocorticoid. These modes of amplification show that these compounds are 'Glucocorticoid Potency Amplifiers'. They amplified not only enzyme induction in liver but also growth inhibition by glucocorticoid of solid tumor L5178Y lymphoblasts. They specifically amplified the actions of glucocorticoids and did not amplify the actions of other steroids, such as 17-beta estradiol, glucagon and insulin. The induction of tyrosine aminotransferase by glucocorticoid and its amplification by EGF were both inhibited by 1-(5-iso-quinoline-sulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, and not by N-[2-(methylamino)-ethyl]-5-isoquinoline-sulfonamide, an inhibitor of cyclic nucleotide dependent protein kinases, suggesting that the induction and the amplification are mediated by protein kinase C.

Topics: Animals; Dexamethasone; Diglycerides; Drug Synergism; Enzyme Induction; Epidermal Growth Factor; Glucocorticoids; Liver; Male; Mice; Mice, Inbred DBA; Neoplasms, Experimental; Phorbols; Rats; Rats, Inbred Strains; Tyrosine Transaminase

The phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced several rapid changes in the HEL-37 mouse epidermal cell line. These included an alteration in cell morphology, inhibition of cell-cell communication, inhibition of epidermal growth factor (EGF) binding and a stimulation of phosphatidylcholine (PC) synthesis. The synthetic diacylglycerol sn 1-oleoyl-2-acetylglycerol (OAG) and sn 1,2-dioctanoylglycerol (diC8) caused similar changes, implying an involvement of the Ca2+- and phospholipid-dependent protein kinase (C-kinase). Treatment of the cells with the cAMP-generating agents db-cAMP and isoproterenol together with the phosphodiesterase inhibitors aminophylline and isobutyl-methylxanthine (IBMX) prior to and during TPA, OAG or diC8 treatment protected the cells against the inhibition of both junctional communication and EGF binding. TPA-induced morphological changes and enhanced PC synthesis, however, were unaffected by elevated levels of intracellular cAMP. These experiments provide evidence for the existence of a dual regulatory system controlling some (but not all) tumour promoter effects.

Topics: Aminophylline; Animals; Bucladesine; Carcinogens; Cell Communication; Cell Line; Cyclic AMP; Diglycerides; Epidermal Growth Factor; Glycerides; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Phosphatidylcholines; Protein Kinase C; Tetradecanoylphorbol Acetate

We have shown previously that in rat tracheal epithelial 2C5 cells the induction of ornithine decarboxylase (ODC) activity and the reduction in the binding of epidermal growth factor (EGF) by diacylglycerol is related to the activation of protein kinase C. In this paper we analyse the action of retinoic acid (RA) on these two parameters in order to determine whether RA acts on the level of protein kinase C. RA inhibits the induction of ODC activity by diacylglycerol (sn-1,2-dioctanoylglycerol) in a dose- and time-dependent manner. A biologically inactive analog of RA has no effect on this induction. RA does not affect the activation of protein kinase C by diacylglycerol in an in vitro assay. In contrast to the effect on ODC induction, RA does not counteract the reduction in EGF binding induced by diacylglycerol. These results are consistent with the concept that RA does not act at the level of protein kinase C and inhibits ODC induction during a stage following protein kinase C activation.

Topics: Animals; Cell Line; Diglycerides; Enzyme Activation; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Ornithine Decarboxylase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Rats; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Trachea; Tretinoin

The biosynthesis of phosphatidylcholine (PC) in HEL-37 cells was followed by measuring the incorporation of [32P]Pi into PC. Incorporation was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), but not by sn-1-oleoyl-2-acetylglycerol or sn-1,2-dihexanoylglycerol (diC6). DiC8 was rapidly metabolised by HEL-37 cells to the corresponding PC and phosphatidic acid derivatives. diC8, diC6 and oleoylacetylglycerol effectively displaced [3H]phorbol-12,13-dibutyrate bound to a soluble cell extract from HEL-37 cells, but only diC8 was able to displace the labelled phorbol ester from prelabelled cells. TPA, diC8, diC6 and oleoylacetylglycerol were all effective inhibitors of 125I-labelled epidermal growth factor binding to, and gap junctional communication between, HEL-37 cells. It is concluded that only cell-permeable diacylglycerols stimulate PC biosynthesis which may therefore require interaction with membranes other than the plasma membrane.

Topics: Animals; Cell Communication; Cell Line; Diglycerides; Epidermal Growth Factor; Epidermis; Glycerides; Intercellular Junctions; Mice; Phorbols; Phosphatidylcholines; Protein Kinase C; Tetradecanoylphorbol Acetate

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Diglycerides; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Glycerides; Humans; Mice; Mitosis; Phorbol Esters; Phorbols; Phospholipids; Phosphoproteins; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface; Tetradecanoylphorbol Acetate