epidermal-growth-factor and 1-oleoyl-2-acetylglycerol

Regulated translocation of canonical transient receptor potential (TRPC) proteins to the plasma membrane has been proposed as a mechanism of their activation. By using total internal reflection fluorescence microscopy (TIRFM), we monitored green fluorescent protein-labeled TRPC3 (TRPC3-GFP) movement to the plasma membrane in HEK293 cells stably expressing this fusion protein. We observed no increase in TRPC3-GFP TIRFM in response to the muscarinic receptor agonist methacholine or the synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol, despite activation of TRPC3 by these agents. We did, however, observe a TIRFM response to epidermal growth factor (EGF). This TIRFM response to EGF was accompanied by increased Ba2+ entry and TRPC3 currents. However, 1-oleoyl-2-acetyl-sn-glycerol-induced TRPC3 activity was not increased. TIRFM also increased in response to Gd3+, a competitive inhibitor of TRPC3 channels. This may be indicative of constitutive trafficking of TRPC3, with Gd3+ acting to "trap" cycling TRPC3 molecules in the plasma membrane. Consistent with this interpretation, TRPC3-expressing cells exhibited large variance in membrane capacitance, and this variance was decreased by both Gd3+ and EGF. These results indicate the following: (i) trafficking of TRPC3 may play a role in regulating the concentration of channels in the plasma membrane but is not involved in activation through the phospholipase C pathway; (ii) TRPC3 undergoes constitutive cyclical trafficking in the plasma membrane, and the mechanism by which growth factors increase the number of plasma membrane channels may involve stabilizing them in the plasma membrane.

Topics: Barium; Cell Membrane; Cells, Cultured; Diglycerides; Epidermal Growth Factor; Green Fluorescent Proteins; Humans; Kidney; Methacholine Chloride; Muscarinic Agonists; Protein Transport; Recombinant Fusion Proteins; TRPC Cation Channels; Type C Phospholipases

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca(2+) release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.

Topics: Calcium; Calcium Channels; Cells, Cultured; Diglycerides; Epidermal Growth Factor; GTP-Binding Protein alpha Subunits, Gq-G11; Heterotrimeric GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Methacholine Chloride; Protein Kinase C; Receptors, Cytoplasmic and Nuclear; TRPC Cation Channels; Type C Phospholipases

Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1(-/-) cells could be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells was phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells showed Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells was activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-gamma1, PLD activity was restored. The targeted disruption of Plcg1 did not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase was similar in Plcg1(+/+) and Plcg1(-/-) cells. Because increased plasma membrane ruffles occurred in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs.

Topics: Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cell Membrane; Diglycerides; Embryo, Mammalian; Enzyme Activation; Epidermal Growth Factor; Fibroblasts; Gene Targeting; Inositol Phosphates; Ionomycin; Isoenzymes; Mice; Mice, Knockout; Microscopy, Fluorescence; Phospholipase C gamma; Phospholipase D; Phosphorylation; Platelet-Derived Growth Factor; Signal Transduction; Type C Phospholipases

It has been reported that keratinocytes possess phospholipase C (PLC)-mediated signal transduction system(s), that can be triggered by histamine, bradykinin, thrombin, platelet-activating factor (PAF), and epidermal growth factor (EGF)/transforming growth factor-alpha (TGF-alpha). Since the activation of PLC results in release of 1,2-diacylglycerol (DAG), the physiologic activator of protein kinase C (PKC) that modulates the epidermal adenylate cyclase, we investigated the effects of these PLC activating chemicals on the adenylate cyclase responses of dispase-separated normal pig epidermis. Among these chemicals and factors only histamine decreased the successive histamine-induced cyclic AMP accumulation and increased forskolin-, and cholera toxin-induced AMP accumulations. These effects were similar to those of PKC activators. However, in contrast to the PKC-activator-induced partial and receptor-non-specific desensitization, the histamine-induced desensitization was completely-inducible and specific to the histamine receptor system, and was not affected by the PKC inhibitor, H-7. Similar modulation of the epidermal adenylate cyclase was induced by other adenylate cyclase stimulators (epinephrine, adenosine and prostaglandin E2), but not by bradykinin, thrombin, PAF, or EGF. The combined addition of bradykinin, thrombin, PAF and EGF to the culture medium had no effect on the adenylate cyclase responses, either. Thus no evidence for receptor-agonist dependent PLC-induced modulation of the adenylate cyclase was obtained in the normal pig epidermis. Although keratinocytes might contain PLC-mediated signal transduction systems, that are triggered by histamine, bradykinin, thrombin, PAF, and EGF/TGF-alpha, none of the activators singly or in combination appear to activate PKC sufficiently for the modulation of adenylate cyclase responses of the normal pig epidermis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine; Adenylyl Cyclases; Animals; Bradykinin; Cell Division; Cholera Toxin; Colforsin; Cyclic AMP; Diglycerides; Dinoprostone; Enzyme Activation; Enzyme Inhibitors; Epidermal Cells; Epidermal Growth Factor; Epidermis; Epinephrine; Histamine; Isoquinolines; Keratinocytes; Piperazines; Protein Kinase C; Swine; Tetradecanoylphorbol Acetate; Thrombin; Type C Phospholipases

Previous studies from our laboratory have demonstrated that epidermal growth factor (EGF), induces intracellular alkalinization in chicken granulosa cells by activating a sodium-dependent and amiloride-sensitive Na+/H+ antiporter. In the present investigation we have examined the possible involvement of protein kinase C (PKC) in the regulation of intracellular pH (pHi) by EGF in chicken granulosa cells. Intracellular pH in granulosa cells obtained from the two largest preovulatory follicles was determined spectrofluorometrically using the dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein. The resting pHi was 6.81 +/- 0.01 (n = 30) when the extracellular pH and sodium concentration were 7.3 and 144 mM, respectively. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA; 50-400 ng/ml) and 1-oleoyl-2-acetylglycerol (OAG; 1-75 micrograms/ml) mimicked the actions of EGF by inducing a concentration-dependent increase in pHi which reached a maximum of 0.25-0.30 pH units. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester with no tumor promoting activity had no effect on pHi. Cytosolic alkalinization was observed within 10 min of the addition of each agent and increased over the 60-min observation period. Like EGF-induced cytosolic alkalinization, the increases in pHi in response to TPA or OAG were dependent on the presence of sodium concentration and were inhibited by amiloride, an inhibitor of the Na+/H+ antiporter. The effects of EGF, TPA, and OAG were attenuated by the PKC inhibitors 5-isoquinolinylsulfonyl-2-methyl piperazine and trifluoperazine. Down-regulation of granulosa cell PKC by pretreatment with TPA (200 ng/ml) for 2.5 h inhibited EGF-, TPA-, and OAG-induced cytosolic alkalinization. The effects of maximally stimulatory concentrations of EGF and TPA on cytosolic alkalinization were not additive. The increases in pHi induced by TPA and OAG, but not by EGF, were dependent on the presence of extracellular Ca++. These studies suggest that the EGF-induced intracellular alkalinization in chicken granulosa cells involves a PKC-mediated activation of the Na+/H+ antiporter.

Topics: Animals; Calcium; Carrier Proteins; Chickens; Diglycerides; Drug Interactions; Enzyme Activation; Epidermal Growth Factor; Female; Granulosa Cells; Hydrogen-Ion Concentration; Protein Kinase C; Sodium; Sodium-Hydrogen Exchangers; Spectrometry, Fluorescence; 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

We have previously characterized a hormonally regulated soluble form of phospholipase A2 (PLA2) in the cultured renal mesangial cell which is similar and possibly identical to the major form in rat kidney. In an attempt to further characterize the mechanisms of regulation of this enzyme we have used epidermal growth factor (EGF), which does not activate polyphosphoinositide-specific phospholipase C in these cells. EGF-enhanced PLA2 activity as assayed by the ability of the soluble extracts of cells to cleave arachidonic acid from the sn-2 position of phosphatidylcholine and phosphatidylethanolamine. This represents a direct demonstration of EGF-induced PLA2 activation which is preserved in a cell-free extract. Phorbol myristate acetate (PMA), as well as 1-oleoyl-2-acetylglycerol, also enhanced PLA2 activity. By contrast, the calcium ionophore A23187 had no effect on extract PLA2 activity. The EGF- and PMA-induced enhanced activity was recovered following fractionation by Mono-Q anion exchange chromatography. The peak of activity comigrated for both agonists, suggesting that both EGF and PMA stimulated the same form of the enzyme. Down-regulation of protein kinase C by pretreatment with PMA resulted in loss of the PMA-induced, but not the EGF-induced, enhancement in PLA2 activity. 8-Bromo-cAMP had no effect upon the PLA2 activity, and did not modulate the EGF effect. Pertussis toxin induced G protein ADP-ribosylation but had no effect upon PLA2 activity, and did not alter the EGF effect. In summary, EGF results in a stable modification of PLA2 activity in glomerular mesangial cells. This enhanced activity is independent of polyphosphoinositide hydrolysis, insensitive to protein kinase C down-regulation, and is not affected by cAMP or pertussis toxin pretreatment of the cells.

Topics: Animals; Arginine Vasopressin; Calcimycin; Cells, Cultured; Chromatography, Ion Exchange; Cytosol; Diglycerides; Enzyme Activation; Epidermal Growth Factor; Glomerular Mesangium; Inositol; Inositol Phosphates; Kinetics; NAD; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

We studied the effects of epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate (TPA), and 1-oleoyl-2-acetyl-glycerol (OAG) on cytoplasmic pH (pHi) and cell growth in cultured porcine thyroid cells. pHi was measured using 2',7'-bis(2-carboxyethyl-5,6-carboxyfluorescein (BCECF), an internalized fluorescent pH indicator. EGF, TPA, and OAG alkalinized the thyroid cells and stimulated their growth. These EGF-, TPA-, and OAG-stimulated cell alkalinization and growth depended on extracellular Na concentrations and were inhibited by amiloride, an inhibitor of Na(+)-H+ exchanger, indicating that EGF-, TPA-, and OAG-stimulated cell alkalinization and growth may occur through activation of Na(+)-H+ exchange. Alkalinization seems to be involved in thyroid cell growth. TPA (a tumor-promoting phorbol ester) and OAG (synthetic diacylglycerol), both potent activators of protein kinase C, imitate the action of EGF in rapidly elevating pHi and stimulating cell growth in thyroid cells. Trifluoperazine, an inhibitor of protein kinase C, inhibited EGF-, TPA-, and OAG-stimulated cell alkalinization and growth. The data suggest that activation of protein kinase C may be involved in the mechanism of EGF-stimulated cell alkalinization and growth of the thyroid cells.

Topics: Amiloride; Animals; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Epidermal Growth Factor; Glycerides; Hydrogen-Ion Concentration; Kinetics; Sodium; Swine; Tetradecanoylphorbol Acetate; Thyroid Gland; Trifluoperazine

Newborn rat keratinocytes, the NBR cell line, synthesized the cyclooxygenase metabolic products, prostaglandins E2 and F2 alpha, and the lipoxygenase metabolic product, hydroxyeicosatetraenoic acid. This metabolism was stimulated by incubation of the cells with the Ca++ ionophore, A23187; melittin; bradykinin; recombinant human f-met epidermal growth factor; the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate; and the synthetic analog of diacylglycerol, 1-oleoyl-2-acetyl glycerol. Production of the cyclooxygenase products was inhibited by the synthetic glucocorticoid, dexamethasone. The stimulation appeared to be modulated by deesterification of arachidonic acid from the cellular lipids, presumably by phospholipase A2. Increased intracellular levels of Ca++ and phosphorylating activities that result from polyphosphoinositol turnover as well as phosphorylating activities independent of phosphatidylinositol turnover appear to be regulating phospholipase A2 hydrolysis of phospholipids.

Topics: Animals; Animals, Newborn; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Cell Line; Dexamethasone; Diglycerides; Epidermal Growth Factor; Melitten; Prostaglandin-Endoperoxide Synthases; Rats; Skin; Tetradecanoylphorbol Acetate

Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2-3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF-stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2-acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor.

Topics: Calcium; Calcium Radioisotopes; Carcinoma, Squamous Cell; Cell Line; Diglycerides; Epidermal Growth Factor; Humans; Kinetics; Phosphatidylinositols; Protons; Sodium; Sodium Radioisotopes; Tetradecanoylphorbol Acetate; Vanadates; Vanadium

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

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 implication of protein phosphorylation in the mitogenic action of high density lipoproteins (HDL) on bovine vascular endothelial cells was investigated by incubating endothelial cell cultures in the presence of 32P-labeled phosphoric acid. The incorporation of 32P into proteins was measured after fractionation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and autoradiography of the gel. In endothelial cells seeded at low density and made quiescent by serum starvation, HDL markedly and consistently enhanced the degree of phosphorylation of a Mr 27,000 protein in a time- and dose-dependent manner. Using 500 micrograms/ml HDL, 32P labeling of the 27-kDa protein was already measurable after 10 min of incubation and reached a maximum at 20-30 min. Minimal effective dose of HDL during a 30-min incubation period was in the range of 5-10 micrograms/ml. While the apolipoprotein moiety of HDL was able to mimic the effect of total HDL, the lipid part of HDL was not. Furthermore, fibroblast growth factor appeared to potentiate the effect of HDL on 27-kDa protein phosphorylation, in agreement with the synergism observed between fibroblast growth factor and HDL on endothelial cell proliferation. Two activators of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetylglycerol also induced the phosphorylation of the 27-kDa protein. These results suggest that the 27-kDa protein may be a physiological substrate for protein kinase C and that HDL could exert their mitogenic effect on endothelial cells through activation of protein kinase C and subsequent protein phosphorylation.

Topics: Amino Acids; Animals; Aorta, Thoracic; Cattle; Cells, Cultured; Diglycerides; Endothelium; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Kinetics; Lipoproteins, HDL; Phosphorylation; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.

Topics: Animals; Binding Sites; Diglycerides; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Glycerides; Kinetics; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Cell Surface; Temperature; Time Factors

The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c-myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c-myc expression is sufficient for the mitogenic action of PDGF.

Topics: Animals; Bradykinin; Cell Division; Cell Line; Diglycerides; DNA; Enzyme Activation; Epidermal Growth Factor; Humans; Mitogens; Phosphatidylinositols; Platelet-Derived Growth Factor; Protein Kinase C; Proto-Oncogenes; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymidine; Vasopressins

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.

Topics: Animals; Bombesin; Cells, Cultured; Diglycerides; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Models, Biological; Phosphatidylinositols; Platelet-Derived Growth Factor; Protein Kinase C; Protein Kinases; Receptors, Cell Surface; Vasopressins