Effects of suramin, an anti-human immunodeficiency virus reverse transcriptase agent, on protein kinase C. Differential activation and inhibition of protein kinase C isozymes.

Suramin inhibited protein kinase C (PKC) type I-III activity in a concentration-dependent manner. Similar inhibitory effects were observed with M-kinase, the constitutively active catalytic fragment of PKC, and autophosphorylation of PKC types I-III. Kinetic experiments indicated that suramin competitively inhibits activity with respect to ATP (Ki = 17, 27, and 31 microM, respectively) and that it can also inhibit by interaction with the substrate histone III-S. With protamine as the Pi acceptor, suramin inhibition was dependent on lipid, being approximately 4-fold less sensitive to inhibition in the absence of phosphatidylserine and diacylglycerol than in their presence. Suramin at low concentrations (10-40 microM), in the presence of Ca2+ and absence of lipid, was able to stimulate kinase activity (approximately 200-400%) in a type-dependent manner and at higher concentrations inhibited activity with histone III-S as substrate. These results indicate that suramin, a hexa-anionic hydrophobic compound, can act as a negatively charged phospholipid analog in activating PKC in the presence of Ca2+ and absence of lipid and can inhibit Ca2+/phosphatidylserine/diacylglycerol-stimulated kinase activity at higher concentrations by competing with ATP or by interaction with the exogenous substrate. Suramin inhibited cAMP-dependent protein kinase much less potently (IC50 = 656 microM) than PKC. The ability of suramin to inhibit PKC-mediated processes in intact cells was tested using the phorbol ester-stimulated respiratory burst of neutrophils as a model system. The respiratory burst of human neutrophils, when preincubated with suramin and then stimulated with phorbol ester, was inhibited in a concentration-dependent manner, suggesting that suramin may also be able to inhibit PKC-mediated processes in intact cells.     

Halothane, an inhalation anesthetic, activates protein kinase C and superoxide generation by neutrophils

The rate of superoxide generation of guinea pig intraperitoneal neutrophils by a chemotactic peptide or 12-O-tetradecanoylphorbol-13-acetate (TPA) was increased by 2-bromo-2-chloro-1,1,1,-trifluoroethane (halothane), an inhalation anesthetic. This increase was inhibited by 1-(5-isoquinolinesulfonyl)methylpiperazine dihydrochloride (H-7), a specific inhibitor of Ca2+- and phospholipid-dependent protein kinase C (PKC). Halothane was found to significantly activate partially purified PKC. The activation required phosphatidylserine (PS) and Ca2+. Dioleoylglycerol- or TPA-activated PKC activity was further increased by halothane. The cytoplasmic proteins of guinea pig neutrophils phosphorylated by halothane-activated PKC were similar to those phosphorylated by PMA-activated PKC. The phosphorylation of a 48 kDa protein, a phosphorylated protein required for NADPH oxidase activation, was also increased by halothane. These data suggest that the increase of superoxide production by halothane is correlated with its activation of PKC.     

Effect of tumor necrosis factor-alpha on the stimulus-coupled responses of neutrophils and their modulation by various inhibitors.

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.     

Identification of phosphatidylserine-binding proteins in human white blood cells.

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.     

Dual role of platelet protein kinase C in thrombus formation: stimulation of pro-aggregatory and suppression of procoagulant activity in platelets

Protein kinase C (PKC) isoforms regulate many platelet responses in a still incompletely understood manner. Here we investigated the roles of PKC in the platelet reactions implicated in thrombus formation as follows: secretion aggregate formation and coagulation-stimulating activity, using inhibitors with proven activity in plasma. In human and mouse platelets, PKC regulated aggregation by mediating secretion and contributing to alphaIIbbeta3 activation. Strikingly, PKC suppressed Ca(2+) signal generation and Ca(2+)-dependent exposure of procoagulant phosphatidylserine. Furthermore, under coagulant conditions, PKC suppressed the thrombin-generating capacity of platelets. In flowing human and mouse blood, PKC contributed to platelet adhesion and controlled secretion-dependent thrombus formation, whereas it down-regulated Ca(2+) signaling and procoagulant activity. In murine platelets lacking G(q)alpha, where secretion reactions were reduced in comparison with wild type mice, PKC still positively regulated platelet aggregation and down-regulated procoagulant activity. We conclude that platelet PKC isoforms have a dual controlling role in thrombus formation as follows: (i) by mediating secretion and integrin activation required for platelet aggregation under flow, and (ii) by suppressing Ca(2+)-dependent phosphatidylserine exposure, and consequently thrombin generation and coagulation. This platelet signaling protein is the first one identified to balance the pro-aggregatory and procoagulant functions of thrombi.     

Properties of a Protein Kinase C Activity in Synaptic Plasma Membrane and Postsynaptic Density Fractions Isolated from Canine Cerebral Cortex

Protein kinase C (PKC) activity (phosphorylation increased by addition of Ca2+/phosphatidylserine or Ca2+/phosphatidylserine/phorbol ester) was found in both a synaptic plasma membrane (SPM) and a postsynaptic density (PSD) fraction. The SPM fraction had as endogenous substrates 87K-, 60K-, 50K-, and 20K-Mr proteins, whereas the PSD fraction had only the 20K-Mr protein. The PKC activity was also detected using histone III-S as a substrate, in SPM but much less in PSD. Phosphorylations of histone and the endogenous substrates of PKC, assayed in the absence of Ca2+, were enhanced in the SPM prepared after treatment of brain homogenate with phorbol 12-myristate 13-acetate (TPA), but very little enhancement was found in PSD after such treatment. The SPM PKC activity (both for endogenous substrate proteins and for histone), which was enhanced by TPA treatment of brain homogenate, was inhibited by calcium (IC50, 3 x 10(-7) M). The phosphorylations of the 20K-Mr protein in PSD, and in SPM prepared with and without TPA treatment, were all inhibited by H-7. The 20K-Mr protein in the PSD fraction is also phosphorylated by a PSD Ca2+/calmodulin-dependent protein kinase II. The evidence indicates that both SPM and PSD fractions contain a PKC activity. Detergent treatment of SPM, to produce a purified PSD fraction, results in a PSD fraction that has lost most of the endogenous substrates, lost the TPA-induced enhanced activity assayed in the absence of Ca2+, and lost the inhibitory effect of low Ca2+ concentration.     

Nonsteroidal antiestrogen suppresses protein kinase C--its inhibitory effect on interaction of substrate protein with membrane.

The mechanism by which nonsteroidal antiestrogen inhibits Ca(2+)- and phospholipid-dependent protein kinase (PKC) activity was investigated. Antiestrogenic agents, clomiphene and tamoxifen, inhibited the PKC-dependent phosphorylation of histone and r-annexin I in a dose-dependent manner. Ki values for the agents were different for two substrate proteins. The inhibitory action of the agents depended on the membrane-substrate protein interaction. Phosphorylation of cytoplasmic proteins obtained from rat uterus and mammary gland, including annexin I, by endogenous PKC was also inhibited by low concentrations of these agents. These results suggest that the inhibitory action of nonsteroidal antiestrogens occurs through their inhibitory effect on the membrane-substrate protein interaction.     

Activation of protein kinase C by myristate and its requirements of Ca2+ and phospholipid

Myristate (C14:0) was found to significantly activate partially purified rat brain Ca(2+)- and phospholipid-dependent protein kinase (PKC). The Ka value, the concentration needed for half maximum activation, for C14:0 in the presence of 1 microM Ca2+ and 20 microM phosphatidylserine (PS) was 20 microM. This activation required Ca2+ and acidic phospholipid and was associated with a decreased Ka for Ca2+ of the enzyme to 10 microM in an analogous fashion as dioleoylglycerol (DO) or phorbol myristate acetate (PMA). The phospholipid requirement for the activation was concentration dependent and was inhibited by 1-(5-isoquinolinesulfonyl)-methylpiperazine dihydrochloride (H-7), a inhibitor of this enzyme. The concentration of H-7 required for half inhibition of the enzyme was about 15 microM and maximum inhibition was about 75%. The concentration profile of cytoplasmic proteins phosphorylated by C14:0-activated PKC was similar to that by PMA-activated PKC. The 47 kDa protein of guinea pig neutrophil was also phosphorylated by the C14:0-activated PKC. It is further discussed whether PKC can function as signal transduction for stimulus-mediated generation of superoxide in neutrophils.     

Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.     

Regulation of T-cell-derived protein kinase C activity by vitamin A derivatives

Protein kinase C (PKC) is a ubiquitous enzyme linked to transmembrane signal transduction. It regulates agonist-mediated activation of intracellular events that result in growth and differentiation in a variety of cells and tissues. PKC is the cellular receptor for phorbol ester tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), that bind to, and directly activate, this enzyme. Vitamin A analogs (retinoids) have been known to antagonize biologic effects of phorbol esters, e.g., promotion of skin tumor formation; however, the extract mechanism(s) of this action is not clear. To analyze the effects of retinoids on T-cell-derived PKC, we partially purified the enzyme from human leukemic T cells (Jurkat) and examined the effects of different vitamin A analogs on its activity. Furthermore, the regulatory effects of retinoids on PKC activity were compared with those of common membrane phospholipids. Retinal inhibited PKC activation induced by TPA, as well as by diacylglycerol, the physiologic activator of PKC. The observed inhibition resulted from competition with phospholipid (phosphatidylserine) and was selective for the phospholipid-dependent C kinase; cAMP-dependent protein kinase, which is phospholipid-independent, was not affected by retinal. The inhibitory effect of retinal on PKC activity was similar to that of phosphatidylcholine. Retinoic acid, in contrast to retinal, induced a Ca2+-dependent activation of PKC, thus substituting for phosphatidylserine. Furthermore, PKC activation by retinoic acid was similar to that by phosphatidylserine, the natural phospholipid cofactor, in that both could be inhibited by phosphatidylcholine and augmented by phosphatidylinositol. The inhibition or activation of PKC by retinal or retinoic acid, respectively, was independent of whether the terminal aldehyde (retinal) or carboxyl (retinoic acid) groups were in the trans or cis configuration. Other vitamin A analogs tested did not affect PKC activity. The results demonstrate that different retinoids and phospholipids may have positive or negative cooperativity in PKC activation, thereby regulating its enzymatic activity and affecting the resulting intracellular activation events. These findings suggest that at least part of the biologic effects of retinoids in general, and their modulation of T-cell function in particular, may be mediated via the influence of their intracellular metabolites on PKC, and that this mechanism may account for some of the antagonistic effects of retinoids on TPA-mediated responses in cells.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

A synthetic peptide homologous to retroviral transmembrane envelope proteins depresses protein kinase C mediated lymphocyte proliferation and directly inactivated protein kinase C: a potential mechanism for immunosuppression

CKS-17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS-17-BSA). To define its site of action, we determined if CKS-17 conjugated to human serum albumin (CKS-17-HSA) could block the direct activation of lymphocytes by phorbol-12-myristate-13-acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiC8). CKS-17-HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose-dependent manner with up to 88% inhibition occurring with 15 microM CKS-17-HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 microM CKS-17-HSA. Based on these findings we investigated the effect of CKS-17-HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS-17-HSA caused a dose-dependent enzyme inhibition with a concentration giving half-maximal inhibition (IC50) of ca.3 microM and greater than 95% inhibition at 15 microM CKS-17-HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS-17-HSA did not inhibit the activity of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PK-A) nor the calcium and phospholipid-independent form of PKC (PK-M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA-stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS-17-HSA may be due to the direct inactivation of PKC.     

2-Hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.     

Expression and properties of two distinct classes of the phorbol ester receptor family, four conventional protein kinase C types, and a novel protein kinase C

Five rabbit cDNAs, encoding four conventional protein kinase Cs (PKCs), alpha, beta I, beta II, and gamma, and a novel PKC-related protein (nPKC epsilon) were transfected into COS cells. Antisera raised against a bacterially synthesized fragment of PKC alpha or nPKC epsilon and against a chemically synthesized peptide of PKC beta I or beta II, specifically identified the corresponding species in the transfected cells. All four PKCs and nPKC epsilon expressed by transfection served as phorbol ester receptors. Phorbol 12,13-dibutyrate (PDBu)-binding activities of all PKCs and nPKC epsilon required phospholipid but not magnesium. The phosphatidylserine requirement for the activity of nPKC epsilon is independent of Ca2+ and similar to that for PKC alpha observed at 0.03 mM Ca2+. Calcium dependence of the binding activity was observed only for the four conventional PKCs. Scatchard plot analysis clearly showed that the dissociation constants of PDBu for all four PKCs were nearly the same (approximately 25 nM) in the presence of Ca2+, and that the value for nPKC epsilon was slightly higher (84 nM) and independent of Ca2+. The latter value is comparable to those observed in several cell types under conditions of Ca2+ chelation. Translocation of conventional PKC alpha to the membranes was induced with phorbol ester in a Ca2+-dependent manner, whereas the PDBu-stimulated translocation of nPKC epsilon did not require Ca2+. These results, together with previous studies on the enzymological characteristics of nPKC epsilon (Ohno, S., Akita, Y., Konno, Y., Imajoh, S., and Suzuki, K. (1988) Cell 53, 731-741), suggest that nPKC epsilon plays an important role in a transmembrane signaling pathway distinct from that involving conventional PKCs.     

Synthetic branched-chain analogues of distearoylphosphatidylcholine: structure-activity relationship in inhibiting and activating protein kinase C

A series of distearoylphosphatidylcholine (DSPC) analogues having various branched alkyl chains were synthesized and tested for their abilities to regulate protein kinase C (PKC). The greatest improvement (about 3-fold) in the PKC inhibitory activity over that seen for the parental lipid (i.e., DSPC) was accomplished by substitution of 8-methylstearate at sn-2 and 16-methylstearate at both sn-1 and sn-2 positions of glycerol; substitutions at both sn-1 and sn-2 with 8-methylstearate, on the other hand, caused a decrease (about 4-fold) in its inhibitory activity. Introduction of butyl, phenyl, or keto functions to various positions in the fatty alkyl chain substituted at both sn-1- and sn-2 positions imparted upon the DSPC analogues an ability to potently stimulate PKC to an extent comparable to those attainable by diacylglycerol or phorbol ester; the analogues having substitution only at the sn-2 position, in comparison, had no or reduced stimulatory activity. The butyl, phenyl, and keto analogues of DSPC, as with DSPC itself and its methyl analogues, inhibited PKC at high concentrations. Kinetic analysis indicated that the methyl DSPC analogues inhibited the enzyme competitively with respect to phosphatidylserine (PS; a phospholipid cofactor) and Ca2+. The butyl analogues activated the enzyme without affecting its affinity for PS or Ca2+, indicating a mechanism different from that seen for diacylglycerol or phorbol ester. The inhibitory activity of the methyl DSPC analogues and the stimulatory activity of the butyl DSPC analogues were reduced when PKC was activated by phorbol ester. Both classes of the analogues were unable to compete for the binding of [3H]phorbol dibutyrate to PKC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutritional lipid emulsions modulate cellular signaling and activation of human neutrophils

Although numerous studies suggest that nutritional lipids modulate human immune responses, the mechanism behind this observation remains unclear. On the basis of the hypothesis that lipids might affect cellular signaling we evaluated the effects of various lipid emulsions on two major pathways involved in neutrophil activation: second messenger (Ca(2)+) mobilization and protein kinase C (PKC) activation. Activation by opsonized yeast particles (serum-treated zymosan; STZ) increased cytosolic [Ca(2)+] ([Ca(2)+](i)) in neutrophils, with an initial slow rise that turned into a fast phase until a plateau was reached. The PKC activator 4-alpha-phorbol 12-myristate 13-acetate (PMA) markedly increased the initial STZ-induced [Ca(2)+](i) rise. This PMA effect was mimicked by emulsions containing medium-chain triglycerides (MT), but not by long-chain triglycerides (LT) or structured lipids (SL). However, like PMA, all emulsions decreased the STZ-induced [Ca(2)+](i) plateau and all activated purified PKC, suggesting that only MT emulsions activate PKC in the context of the intact cell. MT, like PMA, evoked a leftward shift of the dose-response curve for the STZ-induced [Ca(2)+](i) rise, indicating PKC-dependent sensitization of neutrophils for stimulation by STZ. This study is the first to show that nutritional lipids distinctively modulate cellular signaling and stimulation of neutrophils through effects on calcium mobilization and PKC activation: i) MT emulsions sensitize neutrophils for STZ in a PKC-dependent manner, and ii) MT, LT, and SL emulsions all reduce the stimulatory effect of STZ in a nonspecific manner. -- Wanten, G., S. van Emst-de Vries, T. Naber, and P. Willems. Nutritional lipid emulsions modulate cellular signaling and activation of human neutrophils. J. Lipid Res. 2001. 42: 428--436.     

Lidocaine-induced apoptosis of gingival fibroblasts: participation of cAMP and PKC activity

Local anaesthetics are drugs that prevent or relieve pain by interrupting nervous conduction and are the most commonly used drugs in dentistry. Their main targets of action are voltage-dependent Na+ channels. The Na+ channel is modulated by phosphorylation of two enzymes: PKA (protein kinase A) and PKC (protein kinase C). We studied the ability of lidocaine to modulate programmed cell death of human gingival fibroblasts and the mechanisms involved in this process. Lidocaine (10-5 to 10-7 M) stimulated apoptosis in primary cultures and the caspase-3 activity in a concentration-dependent manner. The stimulatory effect of lidocaine on apoptosis was attenuated in the presence of HA 1004 (PKA inhibitor) and stimulated by staurosporine and Go 6976 (PKC inhibitors). Lidocaine-induced apoptotic nuclei correlated positively with cAMP accumulation and negatively with PKC activity. These results show that lidocaine promotes apoptosis in human gingival fibroblasts at concentrations used for local anaesthesia. The mechanism involves PKA stimulation and PKC inhibition, which in turn stimulates caspase-3 and leads to programmed cell death.     

Continuous fluorometric assay of Ca2+ transport by liposomes with Quin 2 entrapped: effect of phospholipase A2 and unsaturated long-chain fatty acids

The amount of free calcium in the cytoplasm is important in stimulation coupled with a number of cellular functions. The putative ionophoretic action of membrane lipid metabolites on Ca2+ offers convenient explanation of the stimulation-coupled mobilization of cytoplasmic Ca2+. To analyze the ionophoretic action of the lipid metabolites, we devised a sensitive method to study Ca2+ transport that uses liposome-entrapped Quin 2. A calcium ionophore, A23187, increased the fluorescence intensity of the Ca2+-Quin 2 complex as a function of Ca2+ transport into liposomes. A similar Ca2+ flux into the liposomes was induced by phospholipase A2 (PLA2) and by various long-chain fatty acids in liposomes that consist of phospholipids containing unsaturated fatty acids. The potencies of the fatty acids for Ca2+ transport is inversely correlated with their melting points. The oxidized products of the unsaturated fatty acids increased the Ca2+ and nonspecific permeability of the biological membranes. These results suggest that stimulation-coupled PLA2 activation might mediates the mobilization of cytoplasmic Ca2+.     

Enzymatic properties of a novel phorbol ester receptor/protein kinase, nPKC

A protein kinase C-related cDNA encodes a novel phorbol ester receptor/protein kinase, nPKC epsilon, clearly distinct from the four "conventional" PKCs [Ohno, S., Akita, Y., Konno, Y., Imajoh, S., & Suzuki, K. (1988) Cell 53, 731-741]. We purified nPKC epsilon from COS cells transfected with nPKC cDNA and compared its enzymatic properties with a conventional PKC, PKC alpha. nPKC epsilon was eluted from a hydroxyapatite column at a position coincident with type II PKC and thus was separated from type III PKC (PKC alpha), the only PKC expressed in COS cells. The protein kinase activity of nPKC epsilon is activated by phospholipids and diacylglycerols (or phorbol esters) in a manner similar to conventional PKCs. However, the cofactor dependencies and substrate specificities were clearly different from PKC alpha. A phospholipid, cardiolipin, enhances the kinase activity three- to fourfold compared with phosphatidylserine. The optimum Mg2+ concentration (3 mM) is clearly different from those of conventional PKCs (10-20 mM). The activation of nPKC epsilon by these cofactors is totally independent of Ca2+. Similar to conventional PKCs, nPKC epsilon autophosphorylates serine and threonine residues, indicating the specificity of the kinase to these amino acid residues. However, it shows a clearly different substrate specificity against exogenous substrates in that myelin basic proteins rather than histone are good substrates. These properties of nPKC epsilon permit clear discrimination of nPKC epsilon from conventional PKCs.     

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Ctheta

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.