muramidase and 1-oleoyl-2-acetylglycerol

We have previously shown that HL-60 cells treated with 1 alpha, 25-(OH)2D3 in magnesium-deficient medium are committed to differentiate but do not express differentiation-related phenotypes. In the present study, we demonstrated that Mg2+ deprivation blocked the process of differentiation before the induction of lysozyme mRNA and that the process of HL-60 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step. We studied the effects of protein kinase A (PKA) and calcium/phospholipid-dependent protein kinase (PKC) modulators at each step. The results indicated that agonists of PKA enhanced both steps but that N-(2-[methylamino]ethyl-5-isoquinolinesulfonamide inhibited them. On the other hand, 1-oleyl-2-acetylglycerol and 12-O-tetradecanoylphorbol-13-acetate enhanced the commitment step but inhibited that of phenotypic expression. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine inhibited the commitment step and enhanced that of phenotypic expression. These results indicate that PKA acts as a positive regulatory signal and that PKC has a dual role in the process of HL-60 cell differentiation, i.e., as a positive regulatory signal in the commitment step and as a negative one in the phenotypic expression step. Recently, we have also shown that in K-562 cell differentiation into erythroid lineage, PKA may serve as a negative regulatory signal in both steps; however, PKC may act dually, namely as a negative regulatory signal in the commitment step and as a positive one in the phenotypic expression step.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaloids; Calcium; Cell Differentiation; Cell Line; Cholecalciferol; Diglycerides; Enzyme Activation; Gene Expression Regulation; Isoquinolines; Leukemia, Promyelocytic, Acute; Magnesium; Muramidase; Phenotype; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate

The generally accepted sequence of intracellular signal transduction involves: (1) cell surface receptor-ligand interactions; (2) activation of G-proteins; (3) activation of phospholipase C, leading to inositol phosphate (IP3), and diacylglycerol production; (4) parallel mobilization of intracellular Ca2+ by IP3, and; (5) activation of protein kinase C (PKC) by diacylglycerol and Ca2+, leading to; (6) cellular responses. Human neutrophils appear to utilize this cascade, at least in general, and some, but not all, elements of the intracellular signal cascade known to be operating in intact cells also function in permeabilized cell systems. We have previously shown that permeabilized neutrophils can be induced to secrete lysosomal enzymes in response to elevated levels of Ca2+ alone and this secretion can be synergistically enhanced by the presence of guanine nucleotides. We now show that Ca2+, in the presence and absence of guanine nucleotides, can stimulate the production of soluble inositol phosphates. Furthermore, neomycin, a putative inhibitor of phospholipase C, can block Ca2(+)-induced secretion. These data thus suggest a role for phospholipase C activity or its products in the transduction process. The next enzymatic activity 'downstream' is PKC. Consequently, we looked at the role Mg-ATP, one of the substrates of PKC, plays in degranulation by permeabilized neutrophils, We found no obligatory role for this nucleotide in the secretory process. We then looked at the activity of oleoyl-acetyl-glycerol (OAG), a synthetic diacylglycerol and PKC agonist, on degranulation. We found that OAG was largely additive with Ca2+. Another PKC agonist, phorbol myristate acetate (PMA), also did not display notable synergy. Finally, inhibitors of PKC activity were not capable of blocking secretion, either in the presence or absence of guanine nucleotides. Thus, while circumstantial evidence seems to point towards a requirement for phospholipase C activation and diacylglycerol production in secretion, we were unable to demonstrate the next putative step in signal transduction, namely activation of PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Cell Membrane Permeability; Diglycerides; Glucuronidase; Humans; Isoquinolines; Lysosomes; Magnesium; Muramidase; Neutrophils; Piperazines; Protein Kinase C; Tetradecanoylphorbol Acetate; Transcobalamins; Type C Phospholipases

The effects of two co-carcinogenic phorbol esters (phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu] and a synthetic diacylglycerol (OAG, 1-oleoyl-2-acetyl-glycerol), which all stimulate protein kinase C, were compared with two inactive phorbol compounds (4 alpha-phorbol and 4 alpha-phorbol didecanoate (4 alpha-PDD)) on three functional properties of stimulated human polymorphonuclear leukocytes (PMNs): release of granular enzymes lysozyme and beta-glucuronidase, chemokinesis, and changes in cytoplasmic free calcium [Ca2+]i. PMA, PDBu and the diacylglycerol, OAG, all caused a dose-dependent and slow (max by 15 min) release of small amounts of lysozyme with much less beta-glucuronidase and no release of cytoplasmic lactate dehydrogenase. Release was unaffected by removal of extracellular Ca2+. PMA, PDBu and OAG inhibited random movement of the cells, did not cause chemokinesis and induced a slow reduction in the basal [Ca2+]i, as measured by the quin-2 method. PMA, PDBu and OAG increased the capacity of five independently-acting stimulants (N-formyl-Met-Leu-Phe, leukotriene B4, C5a des-Arg, platelet activating factor and A23187) to cause release of lysozyme and beta-glucuronidase but strongly inhibited PMN chemokinesis induced by the same five agents and reduced the stimulant-induced increases in [Ca2+]i. PMA was always more potent than PDBu and much more potent than OAG in eliciting these stimulatory or inhibitory effects on human PMNs. In all tests, 4 alpha-phorbol and 4 alpha-PDD were inactive. The results confirm that stimulation of the diacylglycerol/protein kinase C system in human PMN, either by active phorbol esters or the synthetic diacylglycerol, causes bidirectional effects on human PMN function. In particular, activation of the C-kinase causes inhibition of stimulated neutrophil motility, whereas the secretory functions of the cells are enhanced.

Topics: Calcium; Chemotaxis, Leukocyte; Cocarcinogenesis; Cytoplasmic Granules; Diglycerides; Glycerides; Humans; Muramidase; Neutrophils; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate

Diglyceride activators of protein kinase C (i.e., 1-0-myristoyl-, 1-0-palmitoyl-, and 1-0-oleoyl-2-acetylglycerol) interacted synergistically with an arachidonate metabolite, 5-hydroxyicosatetraenoate, to stimulate neutrophil degranulation and superoxide anion generation. Contrastingly, combinations of 15-hydroxyicosatetraenoate with the glycerides or 5-hydroxyicosatetraenoate with a dialkylglyceride (1-0-hexadecyl-2-ethylglycerol) produced no such synergy. The data support a view of stimulus-response coupling wherein protein kinase C is activated in parallel with the mobilization of arachidonate. Respective products of these events, e.g., phosphorylated proteins and hydroxyicosatetraenates, then interact to mediate function.

Topics: Arachidonic Acid; Arachidonic Acids; Cytoplasmic Granules; Diglycerides; Drug Synergism; Enzyme Activation; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Muramidase; Neutrophils; Protein Kinase C; Protein Kinases; Superoxides

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.

Topics: Bucladesine; Calcimycin; Calcium; Calmodulin; Cytoplasmic Granules; Dibutyryl Cyclic GMP; Diglycerides; Drug Interactions; Egtazic Acid; Enzyme Activation; Glucuronidase; Lactoferrin; Muramidase; Neutrophils; Protein Kinase C; Sulfonamides; Tetradecanoylphorbol Acetate; Vacuoles