A selective effect of protein kinase C activators on noradrenaline release compared with subsequent contraction in canine isolated saphenous veins.

1. Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and subsequent contraction evoked by transmural nerve stimulation (TNS) were investigated in canine saphenous veins prelabelled with [3H]-noradrenaline. 2. Activation of PKC by stepwise increasing concentrations (0.01 nM-1 microM) of 12-O-tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) or mezerein caused a significant and concentration-dependent enhancement of the tritium overflow evoked by TNS, while the activators failed to affect the corresponding contraction except with the highest concentration of PDBu when the contraction was significantly reduced. Phorbol, which is inactive on PKC, had no effects on the tritium overflow and contraction induced by TNS. 3. PKC inhibitors, polymyxin B (1 and 10 microM) and the isoquinolinesulphonamide, H-7 (1 microM), inhibited significantly the phorbol ester-potentiated tritium overflow evoked by TNS with no effects on the contraction. H-7 and the related inhibitor H-8 at 10 microM reduced significantly both responses to TNS in the presence of TPA, while they suppressed only the TNS-induced contraction in the absence of TPA. 4. None of the PKC activators or inhibitors affected the spontaneous tritium overflow. 5. PDBu (0.01 and 0.1 microM) elevated resting tension of the veins more effectively than TPA and mezerein. 6. These results suggest that PKC may modulate electrically stimulated noradrenaline release from adrenergic nerve endings of the canine saphenous veins and the PKC activators may act more selectively on presynaptic than postsynaptic sites, but have no apparent effect on postjunctional noradrenergic mechanisms.     

Activation of protein kinase C potentiates postsynaptic acetylcholine response at developing neuromuscular synapses.

1. Phorbol 12-myristate 13-acetate (TPA, 1 microM) and phorbol 12,13-dibutyrate (PDBu, 2 microM), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve-muscle coculture, whereas, 4 alpha-phorbol (2 microM) which is an inactive phorbol analogue had no effect. 2. Staurosporine (0.5 microM) and H-7 (10 microM), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents. 3. Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1-day-old Xenopus cultures. TPA increased both the amplitude and decay time of ACh-induced whole-cell currents in isolated myocytes. 4. TPA concentration-dependently increased the mean open time of low-conductance ACh channels but did not affect those of high-conductance ACh channels. PDBu but not 4 alpha-phorbol exhibited similar effects to TPA. Staurosporine and H-7 inhibited the increasing effects of TPA. 5. These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity.     

The antiviral effect of human interferon alpha is dependent on phosphoinositide-derived messengers

Neomycin the putative blocker of membrane polyphosphoinositide hydrolysis, inhibited the antiviral activity of human interferon alpha, when tested on human quiescent fibroblasts challenged with vesicular stomatitis virus. The anti-interferon effect of neomycin could be correlated in terms of dose dependence for both neomycin (0.05-1 mM) and interferon (100-5,000 IU/ml). The results suggest that the antiviral activity of interferon alpha depends on diacylglycerol formation. Indeed, the synthetic diacylglycerol (50 microM) was as effective as 100 IU/ml interferon in inducing the antiviral state.     

Protein kinase C-dependent and -independent actions of a potent protein kinase C inhibitor, staurosporine.

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), induced ornithine decarboxylase (ODC) in primary cultured mouse epidermal cells. Staurosporine, a potent protein kinase C inhibitor, also induced ODC activity. Both TPA- and staurosporine-caused ODC inductions were markedly suppressed in the PKC-down-regulated cells. Another PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), inhibited both TPA- and staurosporine-caused ODC inductions. H-7 by itself never induced ODC activity. Under our experimental conditions, staurosporine induced no detectable phosphorylation of endogenous proteins. TPA induced a translocation of PKC from cytosol to membrane whereas an optimal concentration of staurosporine to induce ODC did not induce an obvious translocation of PKC. Indomethacin, a cyclooxygenase inhibitor, inhibited staurosporine-caused ODC induction, but not TPA-caused ODC induction. Staurosporine induced specific morphological changes of epidermal cells both in normal and in PKC-down-regulated cells. These results indicate that staurosporine induces ODC activity in a PKC-dependent manner and morphological changes possibly through a PKC-independent mechanism. The mechanism of ODC induction caused by staurosporine may be in some way different from that caused by TPA.     

The protein kinase C inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) disinhibits CA1 pyramidal cells in rat hippocampal slices.

1. The effects of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) on evoked synaptic potentials were investigated in the CA1 region of rat hippocampal slices by use of extracellular and intracellular recording techniques. 2. Extracellular recordings showed that superfusion with H-7 (10-100 microM) increased the amplitude of the population spike and the initial slope of the dendritic field e.p.s.p. H-7 also produced the appearance of multiple population spikes in the somatic region and in the dendritic field e.p.s.p. 3. H-7 (30 microM) induced the disappearance of intracellularly recorded inhibitory potentials elicited by orthodromic stimulation of CA1 pyramidal cells. At this concentration H-7 had no effect on resting membrane potential, input membrane resistance, and spike threshold. In voltage-clamped neurones H-7 blocked the antidromically evoked inhibitory currents and the spontaneous miniature inhibitory currents. 4. The hyperpolarizing effect of bath applied gamma-aminobutyric acid (GABA, 500 microM) or isoguvacine (30 microM) was not affected by 30 microM H-7. 5. Neither the PKC activity regulator sphingosine (10-40 microM) nor the H-7 analogue N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004, 20-50 microM) which is devoid of activity on PKC at these concentrations, affected the extracellularly recorded dendritic field e.p.s.p. or population spike. 6. It is concluded that the disinhibitory effect produced by H-7 is due to the block of a H-7-sensitive PKC which is involved in the spontaneous and evoked release of GABA.     

Enhancing effect of staurosporine on NO production in rat peritoneal macrophages via a protein kinase C-independent mechanism.

Staurosporine (3-100 nM), frequently used as a protein kinase C (PKC) inhibitor, increased accumulation of nitrite in the culture medium of rat peritoneal macrophages up to 6 times above the control level. Moreover, when used in combination with the stable analogue of cyclic AMP, dibutyrylcyclic AMP (db cyclic AMP; 0.1 mM), and/or a cytokine, tumour necrosis factor-alpha (TNF alpha; 100 u ml-1), staurosporine synergistically potentiated, up to 30 times, nitrite accumulation. On the other hand, the other PKC inhibitors, calphostin C and H-7 (10 nM-10 microM) were not effective under the same conditions. The staurosporine-induced nitrite accumulation, in both the presence and the absence of TNF alpha and/or db cyclic AMP was effectively inhibited by the protein synthesis inhibitor, cycloheximide, or by the nitric oxide (NO) synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA). Thus our data suggest that staurosporine may enhance NO production in macrophages via intracellular mechanisms unrelated to the PKC inhibition.     

H-7, a protein kinase C inhibitor, inhibits phorbol ester-caused ornithine decarboxylase induction but fails to inhibit phorbol ester-caused suppression of epidermal growth factor binding in primary cultured mouse epidermal cells

12-O-tetradecanoylphorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC) and suppressed 125I-epidermal growth factor (EGF) binding in primary cultured mouse epidermal cells. TPA (30 nM)-caused ODC induction was almost completely blocked by 30 microM H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], a well known protein kinase C inhibitor, but the same concentration of H-7 failed to restore the 125I-EGF binding suppressed by TPA (10 nM). On the other hand, sphingosine, another protein kinase C inhibitor, blocked not only TPA-caused ODC induction but also TPA-caused suppression of 125I-EGF binding. Concentration-response curves of sphingosine for these two TPA-caused cellular responses were almost identical. 1,2-Diacylglycerols such as 1,2-dioctanoylglycerol (30-300 microM) and 1-oleoyl-2-acetylglycerol (OAG) (30-300 microM) mimicked TPA actions. Similar to the case of TPA, suppression of 125I-EGF binding by OAG was barely inhibited by H-7, whereas sphingosine was more effective in inhibiting the OAG-caused suppression of 125I-EGF binding than was H-7. In TPA (50 nM)-pretreated epidermal cells, TPA (10 nM) failed to suppress 125I-EGF binding. H-7 (30 microM) did not affect TPA (30 nM)-caused translocation of protein kinase C. These results clearly demonstrate the differential inhibition by H-7 of the TPA-caused cellular responses and indicate that TPA-caused suppression of 125I-EGF binding to epidermal cells is mediated through protein kinase C function, which is barely inhibited by H-7.     

Endothelin-1 induced contraction of rat aorta in Ca2(+)-free medium independent of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown

1. The mechanism of endothelin-1 (ET)-induced contraction of rat aorta in Ca2(+)-free medium was investigated and compared with that of phenylephrine-induced contraction, measuring tension development and inositol 1,4,5-trisphosphate (IP3) formation. 2. After Ca2(+)-deprivation for 10 min, ET (10 nM) induced only a slow sustained contraction, whereas phenylephrine (10 microM) evoked a rapid phasic contraction followed by a small sustained one. Prolonged incubation of the strips in Ca2(+)-free medium (for 100 min) abolished the phasic contraction evoked by phenylephrine, but had no effect on the sustained contraction by either stimulant. 3. ET (100 nM) and phenylephrine (10 microM) stimulated inositol trisphosphate formation and these effects were inhibited by TPA (5 microM). 4. TPA (5 microM) had no effect on ET (10 nM)-induced contraction in Ca2(+)-free medium, but inhibited the contraction by phenylephrine (10 microM). 5. The ET- and phenylephrine-induced contractions in Ca2(+)-free PSS were inhibited by H-7, a protein kinase C inhibitor. 6. The difference and similarity of signal transduction pathways between alpha 1-adrenoceptor and ET receptor systems were discussed.     

Tumor cell adherence to cultured capillary endothelial cells is promoted by activators of protein kinase C

Stimulation of cells with protein kinase C (PKC)-specific activators such as phorbol esters increased in a reversible manner the rate of adherence of [3H]leucine-labelled L1210 cells to cultured bovine cerebral cortex capillary endothelial cells (CEC). This effect was not specific for L1210 cells since 12-O-tetradecanoyl phorbol 13-acetate (TPA) strongly increased the binding of various other tumor cell lines. Phorbol esters increased the rate of L1210 cell adhesion to CEC by enhancing their binding capacity without affecting the apparent affinity of L1210 cells for CEC. This stimulation was specific to the phorbol analogs which activate PKC since it was not effected by 4 alpha-phorbol didecanoate, known to be inactive for PKC. Down-regulation experiments showed that adhesion enhancement was entirely attributable to an effect on tumor cells without contribution of CEC intracellular PKC. PKC inhibitors like staurosporine, sphingosine and H-7 showed strong antagonistic activity towards TPA-induced L1210 cell adherence to CEC (IC50 = 0.5 nM, 160 nM and 10 microM, respectively). Adhesive proteins such as vitronectin, fibrinogen, fibronectin and the tetrapeptide RGDS, an active sequence from their cell-binding domains, exhibited potent, dose-dependent inhibition of PKC-induced tumor cell adhesion.     

Role of Ca2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells

The role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca2+. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca2(+)-evoked secretion. Polymyxin B at a low concentration (1 microM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca2+. However, polymyxin B at higher concentrations (10-100 microM) greatly reduced Ca2+-evoked catecholamine secretion. Sphingosine 10 microM-1 mM), Staurosporine (100 nM-1 microM, and H-7 (100-500 microM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca2(+)-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [3H]phorbol-12,13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca2(+)-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca2(+)-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.     

Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts.

1. Rheumatoid arthritis is associated with the accumulation and activation of selected populations of inflammatory cells within the arthritic joint. One putative signal for this process is the production, by resident cells, of a group of inflammatory mediators known as the chemokines. 2. The chemokines interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated on activation normal T-cell expressed and presumably secreted) are target-cell specific chemoattractants produced by synovial fibroblasts in response to stimulation with interleukin-1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha). The signalling pathways involved in their production are not well defined. We therefore used four different protein kinase C inhibitors to investigate the role of this kinase in the regulation of chemokine mRNA and protein expression in human cultured synovial fibroblasts. 3. The non-selective PKC inhibitor, staurosporine (1-300 nM) significantly increased the production of IL-1 alpha-induced IL-8 mRNA and protein. A specific PKC inhibitor, chelerythrine chloride (0.1-3 microM), also caused a small concentration-dependent increase in IL-8 mRNA and protein production. In contrast, 3-[1-[3-(amidinothio)propyl]-3-indoly]-4-(1-methyl-3-indolyl )- 1H-pyrrole-2,5-dione methanesulphonate (Ro 31-8220) and 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3- yl)-maleimide (GF 109203X), two selective PKC inhibitors of the substituted bisindolylmaleimide family had a concentration-dependent biphasic effect on IL-1 alpha or TNF alpha-induced chemokine expression. At low concentrations they caused a stimulation in chemokine production, which was especially evident at the mRNA level. At higher concentrations both inhibited IL-1 alpha or TNF alpha-induced chemokine mRNA and protein production. Ro 31-8220 was 10 fold more potent than GF 109203X, with an IC50 of 1.6 +/- 0.08 microM (mean +/- s.e.mean, n = 4) for IL-1 alpha induced IL-8 production. Ro 31-8220 also inhibited the expression of IL-1 alpha or TNF alpha-induced MCP-1 and RANTES mRNA with a similar potency. 4. The stimulatory effect of staurosporine is discussed in relation to the known poor selectivity of this inhibitor for PKC. It is proposed that activation of an isoform of PKC, possibly PKC epsilon or zeta, which is inhibited by higher concentrations of the bisinodolylmaleimides, plays a role in the regulation of chemokine expression induced by IL-1 alpha or TNF alpha in synovial cells. 5. The inhibition of chemokine production by bisindolylmaleimide compounds heralds a novel approach for future anti-inflammatory therapies.     

Effect of sphingosine and its N-methyl derivatives on oxidative burst, phagokinetic activity, and trans-endothelial migration of human neutrophils.

Neutrophils display three major functions: (i) oxidative burst, (ii) phagokinetic activity, and (iii) trans-endothelial migration. Sphingosine (SPN) is known to inhibit oxidative burst in human neutrophils via inhibition of protein kinase C (PKC). SPN is metabolically converted into N,N-dimethylsphingosine (DMS) in some tissues and cell lines. In previous studies, we have demonstrated that the PKC-inhibitory effect of DMS is stronger than that of SPN, and that of the synthetic analogue N,N,N-trimethylsphingosine (TMS) is even stronger. Therefore, in the present study, we compared the effects of SPN, DMS, and TMS on the neutrophil functions mentioned above. These three compounds, at 10-20 microM, showed equal inhibition of phorbol 12-myristate 13-acetate (PMA)-dependent superoxide (O2-) production and O2 consumption. They and other known PKC inhibitors (H-7, staurosporine, calphostin C), at 1-5 microM, showed equal inhibition of the phagokinetic activity of neutrophils. On the other hand, trans-endothelial migration of neutrophils was suppressed by SPN, DMS, and TMS at 5-10 microM, but was relatively unaffected by the other PKC inhibitors. All of these compounds inhibited PMA-induced phosphorylation of major neutrophil proteins with a M(r) of 60 and 47 kDa; this effect is ascribable to inhibition of PKC. Despite the similar effects of SPN, DMS, and TMS on neutrophil function, TMS was considerably less cytotoxic to neutrophils under the same experimental conditions. Furthermore, SPN and DMS at 10-20 microM caused obvious morphological changes of the endothelial cells, but TMS did not. SPN undergoes rapid metabolic conversion to various sphingolipid compounds, but TMS is stable. In view of all these findings, TMS appears to be a superior pharmacological agent, compared to SPN derivatives or other PKC inhibitors, for suppression of neutrophil overfunction associated with inflammatory processes and tissue injury.     

Anti-proliferative and antiviral effects of human alpha-interferon on tumor cells

Antiproliferative and antiviral activities of a type alpha human leukocytic interferon on several heteroploidic cell lines: HeLa, HEp-2, and T-10, a cell line of malignant origin (glioblastoma) were investigated, as compared to subcultures of human embryo fibroblasts. The tumor cell multiplication rate decreased proportionally to the amount of interferon in the culture medium. The highest interferon concentration used in our experiments (1,000 mu/ml) induced a decrease of the normal cell multiplication rate (human embryo fibroblasts). The same amount of interferon had a cytotoxic effect against the T-10 cells, but this phenomenon is reversible if the interferon is excluded after 24 h from the culture medium. There was no quantitative relation between the magnitude of the antiviral and of the cytotoxic effects of the type alpha human interferon on the tested cellular substrates.     

Protein kinase C does not mediate the inhibitory action of lead on vitamin D3-dependent production of osteocalcin in osteoblastic bone cells

The level of osteocalcin in serum is lower in lead-intoxicated children than in their normal counterparts. To explain this clinical observation, we investigated the mechanism of action of lead on vitamin D3-dependent osteocalcin production. Lead (5-20 microM) blocked the stimulating effects of vitamin D3 on osteocalcin production in cultured rat osteosarcoma cells (ROS 17/2.8). It is often suggested that activation of protein kinase C (PKC) is a critical mediator of the toxic actions of lead. Treatment of ROS cells with G?6976, an inhibitor of PKC alpha and beta isozymes, produced similar effects as lead on vitamin D3-dependent osteocalcin production, while activation of PKC by phorbol-12-myristate-13-acetate (TPA) did not reverse or mimic this effect of lead. Thus activation of PKC is not consistent with the actions of lead on vitamin D3-dependent osteocalcin production. Measurement of PKC enzyme activity showed that 10 microM lead treatment does not activate or inhibit the activity of PKC in ROS cells. Western blot analysis indicated that lead treatment does not translocate PKC alpha, beta, or zeta from cytosol to membrane. Therefore, we concluded that PKC does not mediate the cellular toxicity of lead on vitamin D3-dependent osteocalcin production.     

Activation of human leukemia protein kinase C by tumor promoters and its inhibition by N-trifluoroacetyladriamycin-14-valerate (AD 32).

N-Trifluoroacetyladriamycin-14-valerate (AD 32), a lipophilic, DNA non-binding analog of Adriamycin (ADR), was found to be a potent inhibitor of the membrane-bound enzyme, protein kinase C (PKC). PKC was isolated and purified from human leukemia ML-1 cells, and the enzyme activity was shown to be activated by the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu). AD 32, nevertheless, inhibited the activation of PKC by TPA or PDBu. The IC50 values for AD 32 inhibition of PKC activation were 0.85 microM for TPA and 1.25 microM for PDBu. Under the same assay conditions, ADR demonstrated much higher IC50 values: 550 microM for TPA and greater than 350 microM for PDBu. The inhibition of PKC by AD 32 was further shown to be competitive in nature; AD 32 inhibited the binding of [3H]PDBu to PKC. Therefore, AD 32 competes with the tumor promoter for the PKC binding site and prevents the latter from both interacting with the phospholipid and binding to PKC. These effects of AD 32 were reproduced in situ; incubation of human leukemia ML-1 cells with TPA showed an increased phosphorylation of cellular proteins, and the TPA-induced protein phosphorylation was inhibited by the addition of AD 32 to the cultured cells.     

Potentiation of miniature endplate potential frequency by ATP in Xenopus tadpoles.

1. Extracellular application of ATP (1 mM), a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiated the spontaneous secretion of acetylcholine (ACh) but had no effect on the amplitude and decay time constant of miniature endplate potentials (m.e.p.ps) at neuromuscular synapses in Xenopus tadpoles. 2. alpha,beta-Methylene ATP (0.3 mM) and GTP (1 mM) were also effective in increasing m.e.p.p. frequency. On the other hand, ADP, AMP and adenosine (all at 1 mM) decreased m.e.p.p. frequency. 3. Unlike the transient effect of ATP analogue and GTP on m.e.p.p. frequency, the phorbol ester TPA (2 microM) which is a protein kinase C activator, increased m.e.p.p. frequency consistently and the effects lasted as long as the presence of TPA. 4. Staurosporine (0.5 microM) and H-7 (10 microM), which are protein kinase C inhibitors, each decreased the basal level of m.e.p.p. frequency and markedly inhibited the effects of both ATP and TPA. 5. These results suggest that there is a basal activity of cytosolic protein kinases in the nerve terminals of Xenopus tadpoles and the effect of ATP is probably mediated by the binding of membrane surface purinoceptors which in turn activates cytosolic protein kinases and increases ACh release.     

Synergistic effect of palmitoylcarnitine and the ionophore A23187 on isolated rat mast cells

Dl-palmitoylcarnitine in combination with low concentrations (0.2 microM) of the ionophore A23187 induced a pronounced non-cytotoxic histamine release, with maximal response at 10 microM palmitoylcarnitine and a lower response at 20 microM. The concentration-response curve was shifted to the right when the mast cells were preincubated with palmitoylcarnitine before exposure to the ionophore. Palmitoylcarnitine alone was without effect at concentrations below 20 microM but cytotoxic at higher concentrations. The response to the combination of palmitoylcarnitine and the ionophore was highly sensitive to changes in the ionophore concentration. The results indicate that conditions allowing physicochemical interactions between the two drugs led to greatest potency and effectiveness of palmitoylcarnitine. Preincubation with the phorbol ester TPA potentiated the response. The release induced by the combination of palmitoylcarnitine and the ionophore was completely inhibited by low concentrations of the flavonoid phloretin (IC50 of 0.5 - 2 microM) whereas the protein kinase inhibitor H-7 enhanced the response. The synergistic response to palmitoylcarnitine and the ionophore and its affection by phloretin and H-7 resembles previous findings with TPA and the ionophore. Although not conclusive the results indicate that palmitoylcarnitine can stimulate mast cells by activation of protein kinase C.     

Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.     

Hydrogen peroxide-induced arachidonic acid release in L929 cells; roles of Src, protein kinase C and cytosolic phospholipase A2alpha

Hydrogen peroxide (H(2)O(2)) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of alpha-type cytosolic phospholipase A(2) (cPLA(2)alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA(2)alpha) and a stable clone of C12 cells expressing cPLA(2)alpha (C12-cPLA(2)alpha cells). In the presence of 10 muM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H(2)O(2) synergistically stimulated arachidonic acid release from L929 cells and C12-cPLA(2)alpha cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H(2)O(2) without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H(2)O(2)/A23187 in CaCl(2)-free buffer and the release was inhibited by a selective cPLA(2)alpha inhibitor (3 microM pyrrophenone). Addition of 10 microM H(2)O(2), which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H(2)O(2)/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 microM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H(2)O(2)/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H(2)O(2) with Ca(2+) stimulates arachidonic acid release via cPLA(2)alpha in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.