Epidermal growth factor stimulates the phosphorylation of pyruvate kinase in freshly isolated rat hepatocytes.

Epidermal growth factor (EGF) has previously been shown to stimulate gluconeogenesis in rat liver by decreasing the activity of pyruvate kinase [(1988) Biochem. J. 255, 361-364]. Here we investigate the mechanism underlying the inactivation of the enzyme. EGF was found to increase the incorporation of phosphate into pyruvate kinase, with maximal phosphorylation achieved only after 10 min in the presence of the growth factor. The increase in phosphorylation was not additive with that caused by cyclic AMP. Phosphoamino acid analysis of pyruvate kinase isolated from cells treated with EGF indicated that EGF increases phosphorylation solely on serine residues. The exact site of EGF-mediated phosphorylation has yet to be identified.     

Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.     

Tumour promotor 12-O-tetradecanoylphorbol 13-acetate inhibits angiotensin II-induced inositol phosphate production and cytosolic Ca2+ rise in rat renal mesangial cells

Preincubation of rat renal mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) strongly inhibited the increases of inositol phosphates and of free cytosolic Ca2+ induced by angiotensin II (10(-7) M). TPA had no significant effect on the basal values of inositol phosphates and of free cytosolic Ca2+. Inhibition appeared already after 1 min and was maximal after 5 min. These effects occur without significant changes on angiotensin II binding in intact cells. The concentration of TPA needed (10(-9)-10(-7) M) was in the range believed to cause specifically an activation of protein kinase C. Furthermore the biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. From the entirety of these results it is likely that protein kinase C inhibits angiotensin II activation of phospholipase C at a stage distal to receptor occupancy.     

Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate

Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 microM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethanolamine. Exposure of cells to phorbol 12-myristate 13-acetate induces an increase of the activity of the enzymes CTP: ethanolaminephosphate cytidylyltransferase and ethanolaminephosphotransferase. These effects are accompanied by a decrease of the pool size of ethanolaminephosphate and CDPethanolamine and an increase of the level of diacylglycerols after 30 min of incubation in the presence of phorbol 12-myristate 13-acetate. Upon prolonged incubation, the CDPethanolamine and diacylglycerol pools are restored to the level found in untreated cells. These results indicate that stimulation of phosphatidylethanolamine synthesis by phorbol 12-myristate 13-acetate is probably exerted at the level of CTP : ethanolaminephosphate cytidylytransferase, although there may be an additional effect on the subsequent step of phosphatidylethanolamine synthesis, the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerols.     

Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells

The effect of mitogens on phospholipase D activity was investigated in NIH-3T3 fibroblasts by measuring the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity when 1-propanol acts as the phosphatidyl group acceptor. Platelet-derived growth factor (PDGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated phosphatidylpropanol production by the cells. The dose-response relationships for activation of phospholipase D and stimulation of thymidine incorporation by PDGF and TPA were comparable. The possibility that activation of phospholipase D is utilized by mitogens as a trans-membrane pathway for signalling cell growth is discussed.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.     

Metabolism via the pentose phosphate pathway in rat pheochromocytoma PC12 cells: Effects of nerve growth factor and 6-aminonicotinamide

Exposure of rat pheochromocytoma PC12 cells to 0.1 mM 6-aminonicotinamide (6AN) for 24 hours resulted in a 500-fold increase in 6-phosphogluconate indicating active metabolism of glucose via the oxidative enzymes of the pentose phosphate pathway. Amounts of 6-phosphogluconate that accumulated in 6AN-treated cells at 24 hours were significantly increased by treatment of the cells with nerve growth factor (NGF) (100 ng 7S/ml) suggesting that metabolism of glucose via the pentose pathway at this time was enhanced by NGF. This stimulation of metabolism via the pentose pathway is probably a late response to NGF because initial rates of 6-phosphogluconate accumulation in 6AN-treated cells were the same in the presence and absence of NGF. Moreover, amounts of¹⁴CO₂ generated from 1-[¹⁴CO₂]glucose during the initial six hour incubation period were the same in control and NGF-treated cells. Specific activities of hexose phosphates labeled from 1-[¹⁴CO₂]glucose were also the same in control and NGF-treated cells. The observation that 6AN inhibited metabolism via the pentose phosphate pathway but failed to inhibit NGF-stimulated neurite outgrowth suggests that NADPH required for lipid biosynthesis accompanying NGF-stimulated neurite outgrowth from PC12 cells can be derived from sources other than, or in addition to, the oxidative enzymes of the pentose phosphate pathway.Special Issue dedicated to Dr. O. H. Lowry.     

Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei

Fluorescence photobleaching was used to measure the effect of epidermal growth factor (EGF), insulin, and glucagon on the nuclear transport of fluorescent-labeled dextrans across the nuclear pore complex. EGF and insulin were found to stimulate transport approximately 200%, while boiling these polypeptide growth factors greatly diminished this enhancement activity. Glucagon demonstrated no enhancement effect. The nuclear transport enhancement effects were observed at EGF and insulin concentrations that elicit the various physiological responses, e.g., nanomolar range.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Synergism between subthreshold concentrations of thrombin and 12-O-tetradecanoylphorbol 13-acetate in platelet activation

A comparison was made between the time courses and interdependence of platelet aggregation, serotonin release, and cytosolic free Ca2+ concentration in the same sample of platelets loaded with [14C]-serotonin and Ca2+-sensitive photoprotein aequorin. In 100 micrograms/ml aspirin-treated platelets, neither 0.01 U/ml thrombin nor 50nM TPA, an active phorbol ester, induced significant aggregation, serotonin release, or a rise in the intracellular calcium concentration. However, when these two agents were added together, marked aggregation and release were observed without a change in the cytosolic free Ca2+ concentration. No correlation was observed between the extent of the synergistic effects and time of preincubation with TPA. Potentiatory effects of protein kinase C on receptor-mediated agonists need to be considered in platelet activation.     

Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes.

The effects of hepatocyte growth factor (HGF) on intracellular Ca2+ mobilization were studied using fura-2-loaded single rat hepatocytes. Hepatocytes microperfused with different amounts of HGF responded with a rapid concentration-dependent rise in the cytosolic free Ca2+ concentration with a maximum increase of 142% at 80 ng/ml of HGF. The lag period of the Ca2+ response was decreased with increasing HGF concentrations, being 64 +/- 12 s, 42 +/- 6 s, and 14 +/- 2 s, respectively, with 8, 20, and 80 ng/ml of HGF. The detailed pattern of Ca2+ transients, however, was variable. Out of 16 cells tested using 20 ng/ml of HGF, 68% showed sustained oscillatory responses, whereas other cells showed a sustained increase in the cytosolic-free Ca2+ upon exposure to HGF, which was dependent on the presence of extracellular Ca2+. HGF also induced Ca2+ entry across the plasma membrane. Mobilization of Ca2+ by HGF was accompanied by a rapid accumulation of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3). The effects of HGF and epidermal growth factor (EGF) were comparable and partly additive for Ins 1,4,5-P3 production and for the sustained phase of Ca2+ mobilization. Preincubation of cells with 10 microM of genistein to inhibit protein tyrosine kinases abolished the HGF-induced Ca2+ response and also inhibited HGF-induced Ins 1,4,5-P3 production in rat liver cells. These data indicate that early events in the signal transduction pathways mediated by HGF and EGF have in common the requirements for tyrosine kinase activity, Ins 1,4,5-P3 production, and Ca2+ mobilization.     

12-O-tetradecanoylphorbol 13-acetate potentiates the action of cAMP in inducing DNA cleavage in thymocytes

12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the action of cAMP in DNA cleavage in thymocytes induced by a low concentration of adenosine receptor-site agonists such as adenosine, 2-chloroadenosine and forskolin. The enhancement of DNA cleavage by TPA was also observed in dibutyryl cAMP-treated thymocytes. On the other hand, TPA suppressed accumulation of cAMP by the adenosine receptor-site agonists. These results suggest that activation of protein kinase C inhibits cAMP production, but stimulates cAMP-triggered process to induce DNA cleavage and death of thymocytes.     

Epidermal growth factor mimics insulin effects in rat hepatocytes.

Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.     

Epidermal growth factor (urogastrone)-stimulated gluconeogenesis in isolated mouse hepatocytes

In freshly isolated mouse hepatocytes obtained from fasted animals, we have studied the receptors for epidermal growth factor urogastrone (EGF-URO) in terms of the electrophoretic profile, ligand affinity, and numbers of EGF-URO receptors present on the cells, and also in terms of the ability of EGF-URO to stimulate gluconeogenesis, as reflected by the increased incorporation of [3-14C]pyruvate into glucose. The effects of EGF-URO were compared with those of glucagon. Ligand-binding studies revealed that the mouse hepatocytes possess an unusually high number of EGF-URO receptors (about 3 X 10(6) binding sites/cell), with a ligand dissociation constant of 4.4 nM. The binding of EGF-URO by mouse hepatocytes was more than 10-fold higher than the previously measured binding of EGF-URO by rat hepatocytes. Crosslink-labeling studies, coupled with gel electrophoretic analysis, demonstrated the presence of intact EGF-URO receptors, although some receptor processing had occurred during the isolation procedure. EGF-URO was able to stimulate the incorporation of 3-14C-labeled pyruvate into glucose; glucagon was unable to do so. In contrast, in rat hepatocytes isolated and assayed under identical conditions, glucagon (10 nM) caused a marked (250%) stimulation of the incorporation of pyruvate into glucose. Maximally, EGF-URO caused a 34% increase in the incorporation of [3-14C]pyruvate into glucose; a half-maximal effect was observed at a concentration of 2.5 nM EGF-URO. The stimulatory effect of EGF-URO was not dependent on the concentration of pyruvate, lactate, glucose, or calcium in the incubation medium. Although raising the concentration of pyruvate in the incubation medium increased the incorporation of [3-14C]pyruvate into glycogen, EGF-URO did not cause any change in the incorporation of radioactivity into glycogen. Overall, our data point to marked differences between rat and mouse liver preparations, in terms of the hormonal regulation of glucose metabolism, and our work documents a potential role for the remarkably high number of mouse hepatocyte EGF-URO receptors in terms of the modulation of gluconeogenesis in the mouse.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

The activity of the pentose phosphate pathway in isolated liver cells

Isolated liver cells have been used to assess the relative contribution of the pentose phosphate pathway to glucose metabolism. The incorporation of carbon from specifically labelled glucose into ¹⁴CO₂ by isolated cells gave values (μg.atoms/g.cells/hr) of: C-1, 7.9; C-6, 1.3; C-U, 3.4. The corresponding figures for liver slices were: C-1, 2.3; C-6, 1.6; C-U, 3.0. The most striking difference was the 3.5-fold increase in the oxidation of C-1 of glucose. Isolated cells retain more than 50% of ATP and have a content of intermediates of the glycolytic pathway closely similar to freeze-clamped liver. The relative importance of the pentose phosphate pathway in isolated liver cells, approximately 16% of glucose catabolised, is consistent with the enzyme profile of liver and the reductive synthetic reactions of the tissue.     

Epidermal growth factor and interleukin-1beta synergistically stimulate the production of nitric oxide in rat intestinal epithelial cells

Epidermal growth factor (EGF) is one of the trophic factors for intestinal adaptation after small bowel transplantation (SBT). A recent report indicates that nitric oxide (NO) has cytoprotective effects on bacterial translocation (BT) after SBT. We hypothesized that EGF stimulates the expression of the inducible NO synthase (iNOS) gene in the graft after SBT, followed by increased production of NO, resulting in the decrease of BT. Intestinal epithelial cells (IEC)-6 were treated with EGF and/or IL-1beta in the presence and absence of phosphatidylinositol 3-kinase (PI3-kinase) and EGF receptor kinase inhibitors (LY-294002 and tyrphostin A25). The induction of NO production and iNOS and its signal molecules, including the inhibitory protein of NF-kappaB (IkappaB), NF-kappaB, and Akt, were analyzed. IL-1beta stimulated the degradation of IkappaB and the activation of NF-kappaB but had no effect on iNOS induction. EGF, which had no effect on the NF-kappaB activation and iNOS induction, stimulated the upregulation of type 1 IL-1 receptor (IL-1R1) through PI3-kinase/Akt. Simultaneous addition of EGF and IL-1beta stimulated synergistically the induction of iNOS, leading to the increased production of NO. Our results indicate that EGF and IL-1beta stimulate two essential signals for iNOS induction in IEC-6 cells: the upregulation of IL-1R1 through PI3-kinase/Akt and the activation of NF-kappaB through IkappaB kinase, respectively. Simultaneous addition of EGF and IL-1beta can enhance the production of NO, which may contribute to the cytoprotective effect of EGF against intestinal injury.