phosphorus-radioisotopes and chelerythrine

The activated leukocyte cell adhesion molecule (ALCAM) is dynamically regulated by the actin cytoskeleton. In this study we explored the molecular mechanisms and signaling pathways underlying the cytoskeletal restraints of this homotypic adhesion molecule. We observed that ALCAM-mediated adhesion induced by cytoskeleton-disrupting agents is accompanied by activation of the small GTPases RhoA, Rac1 and Cdc42. Interestingly, unlike adhesion mediated by integrins or cadherins, ALCAM-mediated adhesion appears to be independent of Rho-like GTPase activity. By contrast, we demonstrated that protein kinase C (PKC) plays a major role in ALCAM-mediated adhesion. PKC inhibition by chelerythrine chloride and myristoylated PKC pseudosubstrate, as well as PKC downregulation by PMA strongly reduce cytoskeleton-dependent ALCAM-mediated adhesion. Since serine and threonine residues are dispensable for ALCAM-mediated adhesion and ALCAM is not phosphorylated, we can rule out that ALCAM itself is a direct PKC substrate. We conclude that PKCalpha plays a dominant role in cytoskeleton-dependent avidity modulation of ALCAM.

Topics: Activated-Leukocyte Cell Adhesion Molecule; Alkaloids; Antibodies, Monoclonal; Benzophenanthridines; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cytochalasin D; Cytoskeleton; Dose-Response Relationship, Drug; Down-Regulation; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; K562 Cells; Microscopy, Fluorescence; Phenanthridines; Phosphorus Radioisotopes; Protein Kinase C; Protein Kinase C-alpha; Retroviridae; rho GTP-Binding Proteins; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidines

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.

Topics: Alkaloids; Animals; Antineoplastic Agents, Phytogenic; Benzophenanthridines; Cytochrome P-450 Enzyme System; DNA Adducts; Intercalating Agents; Isoquinolines; Male; Microsomes, Liver; Phenanthridines; Phosphorus Radioisotopes; Quantum Theory; Rats; Rats, Sprague-Dawley

The aim of this study was to investigate whether mitochondria from human placenta contain phosphorylated proteins and kinases. Interestingly, the placenta contains two types of mitochondria with different sizes. These are 'heavy' mitochondria which sediment at a much lower g force than 'light' mitochondria. Mitochondria were incubated with [gamma32]P-ATP and labelled proteins analysed by electrophoresis and autoradiography. A major protein band of 20 kDa was detected with minor bands at 22, 38 and 85 kDa. The 20 kDa band was attenuated by 83 per cent by the co-incubation of mitochondria with Herbimycin, a tyrosine kinase inhibitor. A 20 kDa protein was also identified using an anti-tyrosine phosphate antibody and detection of this protein was significantly higher in heavy mitochondria as opposed to light mitochondria. Protein kinase A enzyme activity was also detected in mitochondria at a level not significantly different than that found in whole non-fractionated cells. These data indicate that mitochondria from human placenta contains kinase activity and phosphoproteins. These molecules may have functions in signalling systems in this organelle. Phosphoprotein signalling systems may be differentially modulated in heavy mitochondria as compared with light mitochondria.

Topics: Adenosine Triphosphate; Alkaloids; Autoradiography; Benzophenanthridines; Cell Fractionation; Electrophoresis; Enzyme Inhibitors; Female; Humans; Immunosorbent Techniques; Mitochondria; Phenanthridines; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Placenta; Pregnancy; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases

The partially purified myosin-bound phosphatase had an associated protein kinase that phosphorylated the holoenzyme, primarily on the large (130-kDa) subunit. Phosphorylation of the 130-kDa subunit resulted in inhibition of phosphatase activity. The major site of phosphorylation was threonine 654 of the 130-kDa subunit or threonine 695 of the 133-kDa isoform. Phosphorylation of the large subunit did not dissociate the holoenzyme. Dephosphorylation of the large subunit was achieved by the holoenzyme, and addition of the catalytic subunit of the type 2A enzyme did not increase the rate of dephosphorylation. The associated kinase was inhibited by chelerythrine, with half-maximal inhibition at approximately 5 microM (in 150 microM ATP). The associated kinase phosphorylated two synthetic peptides, one corresponding to the sequence flanking the phosphorylated threonine, i.e. 648-661 of the 130-kDa subunit, and the other to a known protein kinase C substrate, i.e. a modified sequence from the autoinhibitory region of epsilon protein kinase C. The associated kinase was activated by arachidonic and oleic acid and to a lesser extent by myristic acid. The protein kinase that phosphorylated the 130-kDa subunit and resulted in inhibition of myosin phosphatase activity was not identified.

Topics: Adenosine Triphosphate; Alkaloids; Amino Acid Sequence; Animals; Autoradiography; Benzophenanthridines; Chickens; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fatty Acids, Nonesterified; Gizzard, Avian; Kinetics; Macromolecular Substances; Molecular Sequence Data; Muscle, Smooth; Myosin-Light-Chain Phosphatase; Phenanthridines; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Turkeys

The efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alkaloids; Benzophenanthridines; Blood Proteins; Humans; Male; Molecular Weight; Naphthalenes; Phenanthridines; Phosphorus Radioisotopes; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Polycyclic Compounds; Prostaglandin Endoperoxides, Synthetic; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboxane A2