Carsten Schade Larsen

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.

A study on the role of protein kinase C and intracellular calcium in the activation of superoxide generation

Accumulating evidence indicates that protein kinase C plays an essential role in the activation of NADPH oxidase. In the present study, the correlation between superoxide generation, intracellular calcium, activation of purified protein kinase C and stabilized membrane-bound protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) and 1-deacyl-2-acetyl-rac-glycerol (OAG) were found to induce equal activation of purified protein kinase C and translocation of protein kinase C to the membrane fraction, but differed significantly in their ability to induce superoxide generation. Intracellular calcium was varied using calcium ionophores and increasing the intracellular calcium concentration to more than 1 microM was found to induce increased superoxide generation in maximally OAG-stimulated cells; this contrasted to maximally PMA-stimulated leukocytes. Ionomycin and A23187 were both found to induce a translocation of protein kinase C to the membrane fraction. This translocation was highly dependent upon extracellular calcium. In contrast, PMA- and OAG-induced translocation of protein kinase C was not dependent upon extracellular calcium. In conclusion, our results indicate that although PMA, OAG and calcium ionophores seem to activate protein kinase C in human polymorphonuclear leukocytes these activators differ in their ability to induce superoxide generation.

Activation of human T lymphocytes by phorbol-12,13-dibutyrate and ionomycin

The calcium ionophore ionomycin and the phorbol ester phorbol‐12,13‐dibutyrate (PDBu) are shown to have a synergistic effect upon interleukin 2 (IL‐2) production, interleukin 2 receptor expression, and T‐lymphocyte proliferation.

Membrane-associated protein kinases in phorbol ester-activated human polymorphonuclear leukocytes

Membrane-associated protein kinases in human polymorphonuclear leukocytes were studied. In unstimulated polymorphonuclear leukocytes the protein kinase C was predominantly present in the cytosol but in phorbol 12-myristate 13-acetate- (PMA-) activated cells a time and dose-dependent translocation of the kinase to the particulate fraction occurred. Two new protein kinase activities also appeared in the particulate fraction upon PMA activation. The one had a Mr of 40,000 and its activity was independent of phospholipids. The other (Mr 90,000) as partially activated by phospholipids, but separated from protein kinase C on DEAE-cellulose chromatography.

Membrane-associated protein kinase C activity in superoxide-producing human polymorphonuclear leukocytes.

Protein kinase C activity was studied in superoxide‐producing human polymorphonuclear leukocytes. Using equivalent cell concentrations, superoxide production and particulate fraction‐associated protein kinase C activity increased in parallel in phorbol 12‐myristate 13‐acetate (PMA), oleoyl‐acetyl‐glycerol (OAG), opsonized zymosan, and A23187‐activated leukocytes. Also, an increase in particulate fraction‐associated phospholipid‐independent kinase activity was observed upon stimulation with these activators. In contrast, in formyl‐methionyl‐leucine‐phenylalanine (FMLP)‐activated cells the increase in superoxide production was only accompanied by an increase in particulate fraction‐associated protein kinase C activity if the cells were pretreated with cytochalasin B. Purified protein kinase C activity was stimulated by OAG and PMA, whereas no stimulation was observed using A23187 or opsonized zymosan. It is suggested that the activation induced in human neutrophils by PMA, OAG, opsonized zymosan, and A23187 involves a thight membrane association of phospholipid‐dependent and ‐independent protein kinase activity. This contrasts to FMLP‐activated neutrophils, in which a membrane‐bound form is only observed after pretreatment with cytochalasin B.

Protein kinase C activity in activated human T-lymphocytes stimulated by interleukin-2

Interleukin-2 and phorbol 12-myristate 13-acetate (PMA) are shown to induce DNA-synthesis in human T-lymphocytes activated with phytohaemagglutinin. However, whereas PMA induced a rapid and persistent translocation of protein kinase C from cytosol to particulate fraction, no translocation was observed upon stimulation with interleukin-2. Treatment with PMA for 72 h caused a slow down-regulation of protein kinase C activity to less than 10% of unstimulated T-lymphocytes and was mainly located in the particulate fraction. In contrast, stimulation with phytohaemagglutinin increased the total cellular protein kinase C activity by approx. 100% but with an unaltered subcellular distribution. However, interleukin-2-induced DNA synthesis in PMA- and phytohaemagglutinin-stimulated T-lymphocytes was comparable. Further, maximal DNA synthesis was shown to be dependent on the continuous presence of interleukin-2. These results indicate that interleukin-2-induced proliferation of activated human T-lymphocytes can occur without a translocation of protein kinase C from the cytosol to the particulate fraction and that interleukin-2 most likely functions as a progression factor.

Induction of high-affinity interleukin 2 receptors on human T lymphocytes. The role of calcium and protein kinase C.

The relationship between free cytoplasmic calcium, activation of protein kinase C (PKC) and expression of high‐affinity interleukin 2 receptors (HA‐IL‐2R) on human T lymphocytes was studied. Induction of HA‐IL‐2R by phytohaemagglutinin (PHA) was associated with an increase in free cytoplasmic calcium and a transient increase in membrane‐associated PKC. However, whereas addition of EGTA inhibited induction of receptors by PHA, addition of the PKC‐inhibitor H7 did not. 12‐o‐tetradecanoyl‐phorbol‐13‐acetate (PMA) and 1‐oleoyl‐2‐acetyl‐racglycerol (OAG) were both found to activate and translocate PKC. However, only PMA induced expression of HA‐IL‐2R. Not surprisingly, the effect of PMA was independent of extracellular calcium, but was inhibited by H7. Furthermore, a correlation between the number of HA‐IL‐2R and free cytoplasmic calcium upon stimulation with ionomycin was observed. Associated with the rise in intracellular calcium, the ionophore caused a slight increase in membrane‐associat4ed PKC. Also, addition of 117 inhibited expression of HA‐IL‐2R. Finally, OAG and ionomycin acted synergistically on expression of HA‐IL‐2R. In conclusion, induction of HA‐IL‐2R requires at least two different signals and neither activation of PKC nor an increase in free cytoplasmic calcium is sufficient. However, these two signals may act synergistically. There is evidence for both a PKC‐ and calcium‐independent pathway.

Evidence that bacteria induce translocation of protein kinase C activity in human polymorphonuclear leukocytes

Particulate fraction associated protein kinase activity was studied in human polymorphonuclear leukocytes stimulated by bacteria. Staphylococcus aureus was found to increase particulate fraction associated protein kinase C activity in a time and concentration dependent manner. The increase comprised both the phospholipid dependent and independent kinase activity and was augmented by addition of serum. Similar observations were done using Staphylococcus epidermidis and Klebsiellae pneumoniae. However, Escherichia coli only increased the phospholipid independent kinase activity in the particulate fraction, which suggests the presence of protease activity.

Determination of interleukin-2 (IL-2) and soluble IL-2 receptors (S-IL-2R) in serum and cerebrospinal fluid does not discriminate purulent and aseptic meningitis.

Elevated levels of soluble interleukin-2 receptors (S-IL-2R) but not interleukin-2 (IL-2) activity were found in sera from patients with aseptic meningitis, purulent meningitis, and meningism. Elevated levels of S-IL-2R in serum was also observed in 4/4 patients with bacterial pneumonia and 2/2 patients with infectious mononucleosis. The inflammation of the meninges was only reflected by an increase in S-IL-2R in cerebrospinal fluid (CSF) in 1/14 patients with aseptic meningitis and 3/10 patients with purulent meningitis. Further, IL-2 activity was only demonstrated in CSF from 2 patients with aseptic meningitis and 3 patients with purulent meningitis. In conclusion, neither S-IL-2R nor IL-2 in serum or CSF seem to have any value in the diagnosis of or discrimination between purulent meningitis and aseptic meningitis. Further, the elevation of S-IL-2R in serum is not specific for infections primarily fought by cytotoxic T-lymphocytes such as viral infections, but seems merely to reflect an unspecific activation of the immune system.

Modulation of high-affinity interleukin 2 receptors on activated human T lymphocytes by activators of protein kinase C.

Phorbol myristate acetate (PMA) and 1‐oleoyl‐2‐acetyl‐rac‐glycerol (OAG) are shown to induce a rapid (within 30 min) down‐regulation of the capacity of activated human T lymphocytes to bind interleukin 2. This was associated with a manifold increase in membrane‐associated protein kinase C, whereas no change in free cytoplasmic calcium was observed. In contrast, a 10‐fold increase in free cytoplasmic calcium by ionomycin had no effect on interleukin 2 binding or sub‐cellular distribution of protein kinase C. The reduction of interleukin 2 binding was caused by a decreased number of high‐affinity interleukin 2 receptors, whereas the affinity of the remaining receptors was unchanged. However, PMA and OAG had no effect on the rate of initialization of the interleukin receptor. These data suggest that activation of protein kinase C. but not an increase in free cytoplasmic calcium, leads to a rapid decrease in the number of high‐affinity interleukin 2 receptors on activated human T lymphocytes. However, the mechanism and biological importance of this phenomenon have to be further elucidated.