Rapid,transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter

Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V_max of choline uptake, but had no effect on K_m for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.     

Trilinolein potentiates the pro-aggregating effect of phorbol-12-myristate 13-acetate in human polymorphonuclear leukocytes

Trilinolein, a triacylglycerol with linoleic acid as the only type of fatty acid residue in all three of the glycerol esterified positions, was recently reported to have an antiplatelet effect, mediated through stimulating nitric oxide and cyclic guanosine monophosphate (GMP) formation. In our study, trilinolein induced aggregation of human polymorphonuclear neutrophils (PMNs) and, pretreatment with 0.1 nM trilinolein enhanced phorbol-12-myristate 13-acetate (PMA) induced aggregation. Further investigation showed that trilinolein at concentrations ranging from 0.1 nM to 10 microM increased cyclic GMP formation after 10 min of incubation with PMNs. Pretreatment of trilinolein with 10 microM d-sphingosine, before being incubated with PMNs, attenuated the stimulatory effect of trilinolein on cyclic GMP formation, and pretreatment of 10 microM d-sphingosine also attenuated the aggregation induced by PMA and trilinolein. We conclude that trilinolein can induce the aggregation of human PMNs, and enhance the aggregation induced by PMA.     

蛋白激酶C在中性粒细胞凋亡信号转导中的作用

蛋白激酶C(PKC)是一类ca2+、磷脂依赖性的丝氨酸/苏氨酸蛋白激酶,由12种具有不同生物学特性的同工酶组成.PKC通过催化多种蛋白质上Ser/Thr的磷酸化,在细胞下游信号转导和调节细胞功能中发挥重要的作用.中性粒细胞(PMN)是参与机体固有免疫的主要细胞,一旦分化成熟便启动它的自发性凋亡程序,从而维持机体的内环境稳定和促成炎症反应的无损伤性收敛.PMN凋亡的调控是一个受多基因、多因子、多条信号转导通路独立或交联作用的复杂网络.作为细胞内信号转导重要递质的PKC,在调控PMN的自发性凋亡进程中起着重要作用.     

12-O-tetradecanoyl phorbol 13-acetate, protein kinase C (PKC) activator, protects human leukemia HL-60 cells from taxol-induced apoptosis: possible role for extracellular signal-regulated kinase

The purpose of this study was to evaluate whether the mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK) signaling pathway contributes to 12-O-tertadecanoyl phorbol 13-acetate (TPA)mediated protection from taxol-induced apoptosis of human leukemia HL-60 cells. Treatment of cells with taxol for 12 h resulted in apoptosis of HL-60 cells. TPA was protective against taxol-induced apoptosis and this anti-apoptotic effect was reversible when TPA was used in conjunction with staurosporine and H-7, PKC inhibitors, suggesting that TPA may protect HL-60 cells against taxol-induced apoptosis via the PKC-dependent pathway. Since TPA stimulates MEK signal transduction pathway in HL-60 cells, we postulated that MEK pathway may be playing a role in the ability of TPA to inhibit taxol-induced apoptosis. PD098059, a specific MEK kinase inhibitor, abolished the ability of TPA to inhibit taxol-induced apoptosis. These results suggest that activation of PKC in HL-60 cells confers protection against taxol-induced apoptosis and that MEK mediates anti-apoptotic signaling of PKC.     

On the role of hydroxyl radical and the effect of tetrandrine on nuclear factor--kappaB activation by phorbol 12-myristate 13-acetate

Nuclear factor kappaB (NF-kappaB) is considered to be an important target for therapeutic intervention because of its role in the regulation of proinflammatory and profibrotic mediators. The present study examined the role of hydroxyl (*OH) radical and the effect of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Stephania tetrandra, on NF-kappaB activation by a tumor promoter, phorbol 12-myristate 13-acetate (PMA) in human lymphoid T cells (ie, Jurkat cells). Exogenous superoxide dismutase (SOD) enhanced the NF-kappaB activation by PMA, while catalase blocked it. Formate, a scavenger of *OH radical, also was inhibitory, as was deferoxamine, a metal chelator. These data suggest an important role of *OH radical in PMA-induced NF-kappaB activation. Incubation of the cells with tetrandrine prior to the stimulation of the cells was found to inhibit PMA-induced NF-kappaB activation. Tetrandrine activity was so potent that 50 microM of tetrandrine was sufficient to inhibit activation of NF-kappaB completely. Electron spin resonance (ESR) spin trapping was used to investigate the antioxidant action of tetrandrine using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Tetrandrine is an antioxidant for both *OH and superoxide (O2-)radicals. The reaction rate constant of tetrandrine with *OH is 1.4 x 10(10) M(-1)sec(-1), which is comparable with several well established antioxidants, such as ascorbate, glutathione, and cysteine. The Fenton reaction (Fe(II) + H2O2-->Fe(III) + *OH + OH-) and xanthine/xanthine oxidase were used as sources of *OH and O2- radicals. The free radical scavenging activity of tetrandrine is responsible for its inhibition of PMA-induced NF-kappaB activation.     

Pharmacological studies on the role of protein kinase C in signal transduction of human basophils.

Recent investigations have demonstrated that activation of basophils involves the activation of protein kinase C (PKC). In the present study the effects of different nonselective and selective PKC inhibitors on IgE-mediated histamine release from human basophils were investigated. While potent but nonselective inhibitors such as staurosporine exerted a dose-dependent inhibition of Fc epsilon-receptor-mediated histamine release, staurosporine derivatives with high selectivity for PKC potentiated the IgE-mediated response. The results provide evidence that the histamine release-inhibiting activity of protein kinase inhibitors is inversely correlated with their specificity for PKC. This may confirm the hypothesis that PKC exerts a negative modulatory role during the process of stimulus secretion-coupling following receptor aggregation in basophils. Moreover, investigations with phorbol esters and diacylglycerol derivatives as potent PKC activators show that direct cellular PKC activation and antigen-stimulated mediator release are not closely correlated.     

Chemotactic peptide enhancement of phorbol ester-induced protein kinase C activity in human neutrophils

Chemotactic factors and phorbol esters may act synergistically to evoke neutrophil responses, but the mechanism of such interaction is not entirely clear. We investigated the combined effects of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on protein kinase C (PKC) activity in human neutrophils. FMLP had little effect on the sharp decrease in cytosolic PKC activity induced by PMA. However, cells exposed to FMLP and PMA exhibited a synergistic increase in particulate PKC activity (1 +/- 1 pmol 32P/10(7) PMNs/min in unstimulated cells, 53 +/- 12 pmol 32P with 20 ng/ml PMA, 6 +/- 3 pmol 32P with 10(-7) M FMLP, and 191 +/- 17 pmol 32P with FMLP and PMA). FMLP also markedly increased calcium/phospholipid-independent protein kinase activity in particulate fractions of control and PMA-treated cells. Enhancement of PKC activity required the presence of cytochalasin B during cell stimulation. Cellular calcium was crucial to the FMLP effect since enhancement was decreased in cells incubated with EGTA or Quin2. These results suggest that chemotactic factors and phorbol esters may mediate synergistic effects on neutrophil responses through enhancement of particulate PKC activity. The enhancing effect is probably mediated through chemoattractant-mediated increases in intracellular calcium.     

Comparison of interleukin 2 and 12-O-tetradecanoyl-phorbol 13-acetate as signals for protein kinase C activation in purified human T lymphocytes

Interleukin 2 (IL2) and 12-O-tetradecanoylphorbol 13-acetate (TPA) have been compared for their ability to induce translocation of protein kinase C (PKC) in T lymphocytes prestimulated with anti-CD3 monoclonal antibody (mAb), either in the presence or absence of monocytes. TPA alone did not promote purified T cell growth, but it was able to induce a transient, within 30 min, translocation of PKC activity. The profiles of PKC association with the membrane of the T cells under TPA stimulation were quite similar when either the anti-CD3 mAb or the fixed monocytes, or both, were added to the T cells. The decrease of cytosolic PKC under TPA stimulation was less pronounced for the purified T cells stimulated with anti-CD3 mAb, fixed monocytes alone or both than for unstimulated purified T cells. Even in the absence of monocytes, the addition of exogenous IL2 to the anti-CD3 mAb-treated T cells resulted in PKC translocation, with a transient increase in the PKC activity found in both the particulate and cytosolic fractions. When exogenous IL2 was added to the proliferating T cells, the association of PKC with the membrane was prolonged and the activity did not reach a plateau during the first 2 h after the IL2 stimulation. In parallel, the level of PKC associated with the membrane was higher in proliferating cells than in resting cells even 4 days after stimulation. These results suggest that activation of PKC by IL2 might be different from the direct activation of PKC by TPA and that a specific activation pathway, at least kinetically distinct from the classical phosphatidyl inositol diphosphate degradation by phospholipase C, might be involved during IL2 stimulation of T lymphocytes through high-affinity IL2 receptors.     

Dermal carcinogenicity in transgenic mice: effect of vehicle on responsiveness of hemizygous Tg.AC mice to phorbol 12-myristate 13-acetate (TPA).

The Tg.AC mouse is being evaluated for use in short-term carcinogenicity bioassays. Because the dermal test protocol necessitates dissolving test agents we determined the effects of several solvents on responsiveness of hemizygous mice to dermal applications of the classical skin tumor promoter. phorbol 12-myristate 13-acetate (TPA). Mice of both sexes received dermal applications of either acetone (negative control) or TPA in various vehicles [acetone, 100% methanol, 70% and 100% ethanol, DMSO and mixtures of acetone and ethanol (1:1), acetone and DMSO (4:1 and 1: 1). and acetone and olive oil (4:1)]. Negative control animals did not exhibit papillomas. When administered in acetone. ethanolic or methanolic vehicles TPA caused prompt and robust papillomatous responses. TPA was also tumorigenic in all nonalcoholic vehicles, except the acetone-olive oil mixture. Papilloma responses were generally delayed when TPA was applied in the nonalcoholic solvents but the distinction between TPA-dosed and negative control groups was unequivocal. These results show that choice of vehicle may affect the quantitative and qualitative nature of the response of Tg.AC mice to TPA, but 8 of 9 vehicles proved satisfactory for delivery of TPA.     

Effects of thymidine and phorbol 12-myristate 13-acetate on erythroid differentiation of K562 cells and their sensitivity to nonspecific lysis by rat splenocytes

Induction of hemoglobin synthesis in K562 cells by thymidine (2 mM)in vitro did not significantly enhance major histocompatibility complex antigen-unrestricted lysis of splenocyte-exposed K562 cells. Inhibition of thymidine-induced hemoglobin synthesis by simultaneous incubation of cells with thymidine and phorbol 12-myristate 13-acetate (100 nM) decreased cytolytic activity of splenocytes against K562 cells. Preincubation of tumor cells with phorbol ester alone did not affect major histocompatibility complex antigen-unrestricted lysis induced by rat splenocytes but decreased the basal level of hemoglobin synthesis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 11, pp. 521–524, November, 1999     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB₄ formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipiddependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B₄ (LTB₄) in FMLP-stimulated neutrophils, the ID₅₀ was approximately 4.5 g/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB₄ nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB₄ production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

Phospholipids and protein kinase C in acetylcholine-dependent signal transduction in Ascaris suum.

Quantitatively, the major phospholipid in the muscle of the nematode Ascaris suum was found to be phosphatidylcholine (lecithin). Stimulation of Ascaris muscle with acetylcholine or the agonists carbachol and levamisole increased the level of phosphorylcholine, 1,2-diacylglycerides and phosphatidic acid. Increased levels of these compounds, together with the demonstration of phospholipase C activity, suggest that phospholipid hydrolysis may be associated with the ACh response of the muscle via second messenger pathways. In other tissues, diacylglycerides and phosphatidic acid have been reported to regulate protein kinase C activity. Protein kinase C activity also was demonstrated in the muscle of Ascaris. For optimal activity the kinase was dependent upon Ca2+, unsaturated 1,2-diacylglyceride and phospholipid. All of the data are in accord with the possible involvement of a second messenger system being operative in the ACh-stimulated contraction of Ascaris muscle.     

Tetrandrine and transmembrane signal transduction: effect on phosphoinositide metabolism, calcium flux and protein kinase C translocation in human lymphocytes

Time-course and dose-dependent studies showed consistent suppression of phosphoinositide turnover in Con A-stimulated human lymphocytes in the presence of the plant alkaloid, tetrandrine. Significant inhibition of Con A-stimulated calcium flux was also observed. Furthermore, protein kinase C activity was also significantly inhibited by tetrandrine irrespective of whether Con A or phorbol myristate acetate was the stimulant. These results suggest that the immunosuppressive properties of tetrandrine are in part mediated by the capacity of tetrandrine to interfere with transmembrane signalling.     

Stimulation of protein phosphorylation in mixed glial cell primary cultures and subcultures by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate

Glial cell primary cultures consisting of protoplasmic and fibrous astrocytes, oligodendrocytes and progenitor glial cells incubated in medium containing 0.5% foetal calf serum and treated with 25 nM 12-o-tetradecanoylphorbol-13-acetate (TPA) for periods between 15 and 60 min showed a stimulation of protein phosphorylation which was most prominent in a polypeptide with a molecular weight of about 80,000 Da. Glial subcultures consisting mainly of Type 2 astrocytes, oligodendrocytes and progenitor glia showed a similar TPA stimulation of 80,000 Da protein phosphorylation detectable within 1 min of phorbol ester addition. TPA treatment of primary glial cultures led to an enhancement of phospholipid turnover but exposure of primary glial cultures to concentrations of TPA up to 250 nM caused no morphological change in protoplasmic astrocytes. 4-Phorbol (4-PH) or dimethylsulfoxide (DMSO) was without effect on protein phosphorylation or lipid turnover in glial cultures.     

Effect of 12-O-tetradecanoyl phorbol 13-acetate on solute transport and production of cAMP in isolated frog skin

In the present study we have examined the action of the phorbol diester tetradecanoyl phorbol acetate, an activator of protein kinase C, on the transepithelial transport of sodium, chloride and water and the production of cAMP in the isolated frog skin epithelium (Rana esculenta). Addition of tetradecanoyl phorbol acetate to the mucosal solution resulted initially in an increase in the short-circuit current, which was followed by a progressive decrease. If the short-circuit current was first activated by addition of the antidiuretic hormone, arginine vasotocin, then the addition of tetradecanoyl phorbol acetate resulted only in a pronounced inhibition. The changes in the short-circuit current were the result of changes in the active influx of Na+. The effect of tetradecanoyl phorbol acetate on the intracellular potential measured under short-circuited conditions (Vscc) was time-dependent. Just after addition of tetradecanoyl phorbol acetate to the mucosal solution, Vscc depolarized; this was followed by a slight hyperpolarization, after which Vscc continued to decline. The inhibition of the Na+ transport by tetradecanoyl phorbol acetate was associated with a decline in the response to the antidiuretic hormone (arginine vasotocin), but the ability of arginine vasotocin to increase the cellular level of cAMP and to stimulate the osmotic water flow was not affected by the presence of tetradecanoyl phorbol acetate. In skin halves in which the short-circuit current was stimulated with arginine vasotocin, addition of tetradecanoyl phorbol acetate resulted in a dose-dependent inhibition of the short-circuit current, but only minor changes in Vscc were observed. The results presented suggest that the addition of tetradecanoyl phorbol acetate to the isolated frog skin first increases and then decreases the arginine vasotocin-sensitive sodium permeability of the apical membrane. This might be due to a stimulating effect of tetradecanoyl phorbol acetate on both the activation and deactivation (turnover) of the sodium channels.