Recent studies have demonstrated cytochrome P450 CYP1-mediated metabolism and CYP1-enzyme induction by naturally occurring flavonoids in cancer cell line models. The arising metabolites often exhibit higher activity than the parent compound. In the present study we investigated the CYP1-mediated metabolism of the citrus polymethoxyflavone nobiletin by recombinant CYP1 enzymes and MCF7 breast adenocarcinoma cells. Incubation of nobiletin in MCF7 cells produced one main metabolite (NM1) resulting from O-demethylation in either A or B rings of the flavone moiety. Among the three CYP1 isoforms, CYP1A1 exhibited the highest rate of metabolism of nobiletin in recombinant CYP microsomal enzymes. The intracellular CYP1-mediated bioconversion of the flavone was reduced in the presence of the CYP1A1 and CYP1B1-selective inhibitors α-napthoflavone and acacetin. In addition nobiletin induced CYP1 enzyme activity, CYP1A1 protein and CYP1B1 mRNA levels in MCF7 cells at a concentration dependent manner. MTT assays in MCF7 cells further revealed that nobiletin exhibited significantly lower IC50 (44 μM) compared to cells treated with nobiletin and CYP1A1 inhibitor (69 μM). FACS analysis demonstrated cell a cycle block at G1 phase that was attenuated in the presence of CYP1A1 inhibitor. Taken together the data suggests that the dietary flavonoid nobiletin induces its own metabolism and in turn enhances its cytostatic effect in MCF7 breast adenocarcinoma cells, via CYP1A1 and CYP1B1 upregulation.

Agonist-induced internalisation of receptors may lead to the formation of signalling endosomes. There is little evidence relating to whether this occurs to native receptors in non-transformed cells, and no previous studies asking whether this endosomal signalling can promote cell cycle progression in non-transformed cells. We investigated the hypothesis that in primary hepatocytes clathrin-dependent epidermal growth factor (EGF)-induced internalisation of the EGF receptor leads to signalling from endosomal EGF-EGF receptor complexes which may support EGF-stimulated cell cycle progression. We used EGF-stimulation of rat hepatocytes followed by confocal microscopy, and Western blots for phosphoproteins. [(3)H]thymidine incorporation into DNA was used as a indicator of progression to S-phase. Confocal microscopy demonstrated co-internalisation of EGF, EGF receptors and transferrin into endosomes. Internalisation of EGF/EGF receptor/transferrin was blocked by expression of dominant-negative dynamin, but not by the tyrosine kinase inhibitor AG 1478. Dominant-negative dynamin expression reduced EGF-stimulated extracellular signal-related kinase and Akt signalling, but increased tyrosine phosphorylated EGF receptor. EGF-stimulated cell cycle progression requires stimulation of EGF receptors during an initial period (e.g. 1h) and also later during a 24h incubation. EGF receptor internalisation in the presence of AG 1478 followed by removal of the inhibitor resulted in signalling from internalised EGF receptors that is sufficient for the initial stimulation to provide progression to S-phase of the cell cycle. These observations on hepatocytes characterise, for the first time in non-transformed cells, endosomal signalling from internalised EGF receptors, and provide evidence that this endosomal signalling may support the early phase of EGF-stimulated cell cycle progression.

Epidermal growth factor (EGF) stimulation of cell cycle progression in cultured primary hepatocytes has previously been reported to be dependent on the mammalian target of rapamycin (mTOR) elements of the phosphoinositide 3-kinase (PI3K) signaling cascade and not the Akt pathway. Here we have established conditions of combined treatment of rat hepatocytes with insulin and EGF that favor cell cycle progression. The resulting cell population expresses albumin and retains receptor regulation of the signaling pathways leading to glycogen phosphorylase activation. We then investigated the hypothesis that the Akt limb of the PI3K pathway plays a central role in this insulin/EGF enhancement of cell cycle progression. The phosphorylation of Akt, central to the PI3K pathway, was increased by both insulin (sustained) and EGF (transient). The stimulation of Akt phosphorylation was inhibited in a concentration-dependent manner by the PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Cell cycle progression in these cultures was reduced, but not abolished, by this inhibitor. The mTOR inhibitor, rapamycin, also inhibited entry into S phase. The novel Akt inhibitor A-443654 [(S)-1-(1H-indol-3-ylmethyl)-2-[5-(3-methyl-1H-indazol-5-yl)-pyridin-3-yloxy]-ethylamine] blocked both EGF-stimulated cell cycle progression and phosphorylation of the Akt substrate glycogen synthase kinase-3. Infection of cells with an adenoviral vector expressing a constitutively active form of Akt but not a kinase-dead form increased hepatocyte proliferation probably through enhanced cell cycle progression and reduced apoptosis. These results show that the Akt element of the PI3K cascade is necessary for EGF-stimulated cell cycle progression and provide evidence that the sustained elevation of Akt alone generates a hyperproliferative window in hepatocyte cultures.

In the rat both short-term liver function, such as glycogen metabolism, and long-term events such as proliferation after partial hepatectomy, are in part controlled by release of nucleotides such as ATP acting on hepatocyte P2Y1 and P2Y2 receptors (members of a family of P2Y receptors for extracellular nucleotides such as ATP and UTP). Here, we have studied P2Y receptor regulation of signaling pathways involved in glycogen phosphorylase activation and proliferation of primary human hepatocytes. Stimulation of cultured hepatocytes with either ATP and UTP, but not UDP or 2-methylthio ADP, led to concentration-dependent increases in cytosolic free Ca 2 concentration ([Ca 2 ]c ;E C50 for ATP 3.3 M, for UTP 2.3 M) and [ 3 H]inositol (poly)phosphates (EC50 for ATP 9.4 M, for UTP 15.4 M). ATP and UTP also stimulated glycogen phosphorylase in human hepatocytes, each with a threshold for activation of less than 1 M. Application of 2-methylthio ADP up to 100 M was ineffective. Phosphorylation of both extracellular signal-related kinase and c-Jun N-terminal kinase was stimulated by ATP and UTP, but not by 2-methylthio ADP or UDP, either alone or when costimulated with epidermal growth factor. In conclusion, in human hepatocytes P2Y receptors control both glycogen metabolism and proliferation-associated responses such as increased [Ca 2 ]c and mitogen-activated protein kinase cascades. Regulation seems to be primarily through P2Y2 receptors. In contrast with previous studies on rat hepatocytes, there is an absence of responses mediated by P2Y1 receptors.

1 We have investigated increases in cytosolic Ca(2+) in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron-free glial cultures. 2 In both cultures, the agonist-response profile was 2-methylthioADP(2MeSADP)>2-methylthioATP(2MeSATP)>ADP>ATP>adenosine 5'-O-(3-thiotriphosphate), consistent with a P2Y(1) receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. 3 Suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA(2) values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. 4 Single cell imaging divided the cells from the mixed neuronal-glial cultures into two populations: responsive (neurons) and unresponsive (glial cells) to high [K(+)]. The response of cells to nucleotides was almost exclusively limited to those not responsive to high K(+). 5 In the presence of extracellular Mn(2+), the response of the mixed cultures to 30 mM K(+) and 20 micro M Bay K 8644 was attenuated. However, when 2MeSADP was added there was no reduction in response in cultures previously loaded with Mn(2+). This further indicated that the 2MeSADP response was not in the neurons. 6 Reverse transcriptase-polymerase chain reaction studies detected transcripts for P2Y(1), P2Y(4) and P2Y(6) in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y(2) transcripts were not detected in the embryonic cortex. 7 Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca(2+) in glial cells and presynaptic sites on neurons.

Using primary cultures of bovine adrenal chromaffin cells labelled with 32 P i , we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [ 32 P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of nor‐ adrenaline release from Chromaffin cells. Using 45 Ca 2+ influx studies, we show that the nicotine‐evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [ 32 P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine‐ and potassium‐stimulated [ 32 P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.

Journal of NeurochemistryVolume 53, Issue 1 p. 1-11 Presynaptic Aspects of Cotransmission: Relationship Between Vesicles and Neurotransmitters Michael R. Boarder, Corresponding Author Michael R. Boarder Department of Pharmacology and Therapeutics, University of Leicester, Leicester, EnglandAddress correspondence and reprint requests to Dr. M. R. Boarder at Department of Pharmacology and Therapeutics, University of Leicester, P.O. Box 138, Medical Sciences Building, University Road, Leicester LE1 9HN, U.K.Search for more papers by this author Michael R. Boarder, Corresponding Author Michael R. Boarder Department of Pharmacology and Therapeutics, University of Leicester, Leicester, EnglandAddress correspondence and reprint requests to Dr. M. R. Boarder at Department of Pharmacology and Therapeutics, University of Leicester, P.O. Box 138, Medical Sciences Building, University Road, Leicester LE1 9HN, U.K.Search for more papers by this author First published: July 1989 https://doi.org/10.1111/j.1471-4159.1989.tb07288.xCitations: 23Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume53, Issue1July 1989Pages 1-11 RelatedInformation

A specific and sensitive radioimmunoassay procedure for Met-enkephalin[Arg6,Phe7] which allows its measurement in regions of the rat brain is described. The antiserum as raised against the methionine sulphoxide derivative of the peptide, and all samples and standards were oxidized with hydrogen peroxide prior to use in the assay with chloramine T-oxidized 125I-labelled Met(O)-enkephalin[Arg6,Phe7]. The only significant cross-reactivity was 30% with the reduced heptapeptide Met-enkephalin[Arg6,Phe7]. The assay showed less than 0.15% cross-reactivity with fragments of the heptapeptide and with leucine-enkephalin-containing peptides. Acid acetone extraction of rat striatum followed by Sephadex G-50 chromatography and reverse-phase high pressure liquid chromatography showed that essentially all immunoreactivity co-chromatographed with Met-enkephalin[Arg6,Phe7]. This confirmed the specificity of the assay and showed that the striatum does not contain a high concentration of larger molecular weight forms with the heptapeptide at the COOH terminus. Distribution of the heptapeptide followed that of methionine enkephalin, with highest concentrations in the globus pallidus, intermediate levels in caudate-putamen and hypothalamus, and low levels in cortex and cerebellum.

The opioid peptides have been related to behavior in both animal and human studies. Further investigation can be anticipated which could lead to the elucidation of genetic controls over enzymes which process these peptides and the receptors upon which the peptides act. The enzymes, both synthetic and degradative, can lead to the formation of different forms of the opiate peptides. Differential control of these enzymes or of the multiple forms of opiate receptors could lead to discrete changes in opiate status and subsequent behavioral changes. Conversely, genetically regulated behavioral modification could also lead secondarily to opiate changes.

Studies were designed to assess whether the enkephalin-containing peptides and proteins present in the chromaffin granules of the adrenal medulla and in other secretory tissues, such as the neurohypophysis, could be found circulating in human blood. We analyzed human plasma acid acetone extracts chromatographed on Sephadex G-75 in acetic acid and found evidence for the existence of opioid peptides of several different molecular weights and a large number of peptides and small proteins which generate opioid activity after tryptic digestion. These compounds are different from and present in much greater quantities than previously described opioid peptides in human plasma, and are separate from dynorphin-immunoreactive compounds, which we also report in the blood. Expressed in leucine-enkephalin equivalents on a radioreceptor assay, we found 63.2 +/- 6.5 (n = 4; mean +/- SEM) pmol/ml plasma. One active peak from the Sephadex G-75 chromatography of human plasma (apparent mol wt, 3000) was examined by reverse phase high pressure liquid chromatography, tryptic digestion, and Sephadex G-50 chromatography. The results were consistent with the notion that this opioid active peptide contains an enkephalin sequence at its N-terminal, followed by a basic residue. Mild stress (2 min of deep knee bends) produced a 2-fold elevation in overall circulating opioid activity. The possibility is considered that the large enkephalin-containing peptides may have an endocrine function, independent of a role as enkephalin precursors.

A procedure for the dimethylation of the amino groups of human β-endorphin by reductive methylation is described. A single product with the theoretical maximum degree of methylation was produced and the consequences of dimethylation on proteolytic attack are reported. The derivative was shown to be resistant to tryptic digestion and to attack by leucine aminopeptidase. The nonmethylated β-endorphin was rapidly degraded by incubation with pituitary homogenate; under these conditions methylated β-endorphin was degraded at a slower rate. In the presence of bacitracin, the methylated peptide was essentially resistant to degradation by the pituitary homogenate. The methylated peptide may be expected to have a longer in vivo half-life.

The biologically active peptides human β-endorphin and corticotropin as well as the β-endorphin precursor β-lipotropin could be phosphorylated by either the purified catalytic subunit of cyclic AMP-dependent protein kinase or a cyclic nucleotide-independent kinase partially purified from rat brain. Parathyroid hormone and secretin served also as substrates for either kinase suggesting that phosphorylation may be a commonly occurring covalent modification of peptides. The potential role of phosphorylation for peptide processing and function is discussed.