Phorbol Ester Binding Sites Research Articles

Protein kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [ 3H]phorbol 12,13-dibutyrate

The influence of transient forebrain ischemia on the remporal alteration of protein kinase C (PKC) activity in the rat hippocampus was analyzed by quantitative autoradiography using [ 3H]phorbol 12,13-dibutyrate ([ 3H]PDBu). As reported previously, the grain density was highest in the strata oriens and radiatum in the CA1 subfield. After transient forebrain ischemia (20 min), the [ 3H]PDBu binding in the CA1 subfield gradually increased during early recirculation, and became maximum 6–12 h after ischemia, when no microscopic damage of the CA1 pyramidal cells was obvious. Thereafter, grain density decreased and binding activity in the CA1 was lost by approximately 40% 7 days after ischemia, when CA1 pyramidal cells had become necrotic. This indicated a close association of phorbol ester binding sites with CA1 pyramidal cells. By contrast, [ 3H]PDBu binding sites were unchanged in the stratum radiatum in the CA3 throughout the recirculation, although the number of binding sites in the stratum oriens of the CA3 was decreased during early recirculation period. Seven days after recirculation, in the molecular layer of the dentate gyrus, where granule cells remained intact, [ 3H]PDBu binding activity increased by 33%, with a higher grain density in the inner region (supragranular layer). These results suggest that enhancement of PKC activity and/or translocation of the enzyme play an important role in the postischemic modulation of synaptic efficacy in the hippocampal formation and neuronal death of CA1 pyramidal cells.

Inositol 1,4,5-trisphosphate receptor binding: autoradiographic localization in rat brain

Inositol 1,4,5-trisphosphate is a second messenger generated by stimulation of the phosphoinositide cycle, thought to release calcium from intracellular stores. We have mapped the distribution of 3H-inositol 1,4,5-trisphosphate receptor binding sites in rat brain by autoradiographic techniques. The cerebellum contains the highest level of inositol 1,4,5-trisphosphate binding sites in brain, which appear to be selectively localized to Purkinje cells. Moderate levels of binding sites are present in the hippocampus, cerebral cortex, caudate, and substantia nigra. Lesion studies indicate that binding in the hippocampus is restricted to intrinsic neuronal elements and in the nigra is found on terminals of the striatonigral projection. Overall, the autoradiographic distribution of inositol 1,4,5-trisphosphate receptors resembles the distribution of phorbol ester binding sites associated with protein kinase C. However, the inositol 1,4,5-trisphosphate receptor has a more restricted distribution since it is not detectable in the spinal cord or olfactory bulb, regions with substantial levels of protein kinase C.

Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C

We have identified a murine T lymphocyte clone that apparently lacks diacylglycerol- and phospholipid-activated protein kinase C (PKC): cell extracts do not display phosphatidylserine, Ca 2+, or phorbol ester-dependent phosphotransferase activity; the enzyme was not detected in immunoblots with PKC-specific antibodies; phorbol ester binding sites are not detectable in intact cells; and activators of PKC do not stimulate proliferation or Na + H + exchange in intact cells. Only PKC beta mRNA was detected in normal murine T lymphocytes. The mutant T lymphocytes contained amounts of 4.4 kb PKC beta message similar to those in normal murine lymphocytes, but the 2.9 kb and 1.2 kb messages found in normal lymphocytes were barely detectable. No abnormalities were detected on Southern analysis, suggesting that the abnormality may be at the level of message splicing or stability. Since PKC-deficient cells proliferate in response to the T lymphocyte growth factor, interleukin-2, we conclude that activation of PKC is not essential for the growth-promoting action of interleukin-2.

Development of phorbol ester (protein kinase C) binding sites in cat visual cortex

Tritiated phorbol-12,13-dibutyrate [ 3H]PDBu), a phorbol ester, was utilized to autoradiographically localize protein kinase C (PKC) in the cat visual cortex. Thin, slide-mounted sections of adult cat brain were used to characterize binding of [ 3H]PDBu. This was found to be saturable, reversible, and more readily displaced by phorbol ester than by synthetic diacylglycerols. Binding sites displayed a tissue concentration of 20 pmol/mg protein, and a dissociation constant of 8.0 nM. [ 3H]PDBu was slow to associate with its receptor, requiring 9.5 h to reach equilibrium. Autoradiography revealed that PKC is heterogeneously distributed in the cat brain, and displays a laminar-specific pattern in the visual cortex. This laminar distribution undergoes marked changes during the first two months of postnatal life. In the visual cortex of neonatal kittens, [ 3H]PDBu binding is confined to layers I and V. Layer III acquires high levels of binding by postnatal day 15, layer II by 28 days, and layer VI becomes labelled by 40 days of age. Adult animals exhibit high levels of binding in all laminae except layer IV. Age-dependent changes in PKC's laminar distribution do not seem to be correlated with specific anatomical, neurochemical, or behavioural events during development. PKC appears to be associated with cell bodies or processes intrinsic to the visual cortex, and is probably not located on the terminals of cortical afferents.

Modulation of cellular phorbol ester binding and/or protein kinase C activity by human placental fractions

We describe two factors in human placenta that modulate the interaction of phorbol ester tumor promoters with cell membranes or with protein kinase C. One, phorbol ester binding inhibitory factor, can inhibit binding of [ 3H]phorbol-12,13-dibytyrate to cultured cells or to a membrane fraction but does not inhibit its binding to a homogeneous C kinase preparation (phorbol ester binding sites). The other, C kinase activating factor, stimulates C kinase activity in a calcium-dependent manner. We separated these two biochemical activities from a crude human placental fraction by gel filration.

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4β-phorbol myristoyl acetate. Specific [ 3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 μM 4β-phorbol myristoyl acetate and 1 μM 4β-phorbol didecanoate (but not 1 μM 4α-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4β-phorbol myristoyl acetate is ineffective at 20 nM. 1 μM 4β-phorbol myristoyl acetate slightly increases the cytosolic free Ca 2+ concentration of polarized synaptosomes, but not that following partial depolarization, 4β-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca 2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca 2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.

Suppression of nerve growth factor-directed neurite outgrowth in PC12 cells by sphingosine, an inhibitor of protein kinase C.

The formation of vertebrate neural circuitry is regulated in part by neurotrophic agents, such as nerve growth factor (NGF); however, the biochemical mechanisms involved in neurite outgrowth have yet to be completely resolved. Phorbol ester tumor promoters are known to influence the extension of neurites in a variety of neurodevelopmental systems, and protein kinase C, the major phorbol ester receptor, has been implicated in this process. In the present study, sphingosine, a specific pharmacological inhibitor of protein kinase C, was employed to investigate the role of this enzyme in the elaboration of neurites in PC12 pheochromocytoma cells. Normally, PC12 cells respond to NGF by morphologically differentiating into sympathetic neuron-like cells, exhibiting a marked hypertrophy, and extending slender neurites piloted by well defined growth cones. The elaboration of NGF-induced neurites was found to be reversibly inhibited by sphingosine in a dose-dependent manner (IC50 = 2.5-5 microM), while similar concentrations of several structural analogs were inactive. The suppression of neurite outgrowth by sphingosine was antagonized by the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA), which binds to and directly activates protein kinase C. In the presence of NGF, TPA treatment increased the incidence of neurite outgrowth, and this increase, in turn, was antagonized by sphingosine. The binding of [3H]phorbol 12,13-dibutyrate to specific phorbol ester binding sites in PC12 cells was inhibited by sphingosine at concentrations similar to those which inhibited neurite outgrowth. The effects of sphingosine on TPA-directed protein phosphorylation were examined in situ, revealing inhibition of [32P]phosphate incorporation into cellular proteins. The specific TPA-directed phosphorylation of tyrosine hydroxylase was inhibited by sphingosine, as was the resulting increase in enzyme activity. The effects of sphingosine on the levels of alpha- and beta-tubulin mRNAs were also examined in an effort to delimit the locus of protein kinase C action. Concentrations of sphingosine which suppressed neurite outgrowth did not inhibit the NGF-directed elevation of tubulin transcript levels. Taken together, these results reveal the presence of a sphingosine-sensitive pathway in neurite outgrowth and indicate that protein kinase C plays a role in mediating the neuritogenic effects of NGF. Furthermore, the results suggest that protein kinase C acts at a distal segment of the neurite growth pathway.

Mouse tumor-associated macrophages do not generate procoagulant activity in response to different stimuli.

Mononuclear phagocytes, an integral part of the lymphoreticular infiltrate of human and experimental tumors, might contribute to fibrin deposition within malignant tissues through the production of procoagulant activity (PCA). We have studied the PCA of tumor-associated macrophages (TAM) in 2 poorly immunogenic, metastatic murine sarcomas (mFS6 and MN/MCA1); peritoneal macrophages (PM) from tumor-bearing and control animals were also studied, as reference cell populations. PCA was evaluated by a one-stage clotting assay immediately after isolation (basal PCA) and following in vitro stimulation. Basal PCA was very low (less than 1 U/10(4) macrophages) in all cell preparations. Exposure of PM from both normal and tumor-bearing animals to bacterial endotoxin (lipopolysaccharide, LPS), phorbol myristate acetate (PMA) or the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in 10-, 7- and 3-fold increases in PCA, respectively. In contrast, TAM from mFS6 and MN/MCA1 consistently failed to generate PCA in response to different concentrations of the same stimuli. Treatment of TAM with aspirin did not affect cell unresponsiveness. Fluorescence microscopy showed that almost all PM were stained with fluorescein isothiocyanate (FITC)-LPS, while less than 10% of the TAM were stained. These data, coupled with previous evidence that TAM have a lower number of specific binding sites for phorbol esters than PM, suggest that the defective responsiveness of TAM to endotoxin, PMA and, possibly, FMLP, is due to the lack, or very low expression, of binding sites for these agents on the cell surface. The tumor environment may orient the functional status of in situ macrophages in a direction less favorable to the host.

O-238 - Alterations of phorbol ester binding sites in neurodegenerative disease

O-238 - Alterations of phorbol ester binding sites in neurodegenerative disease

Defective production of reactive oxygen intermediates by tumor-associated macrophages exposed to phorbol ester.

Macrophages were isolated from poorly immunogenic metastatic sarcomas (mFS6 and MN/MCA1) of C57BL/6 origin. Tumor-associated macrophages (TAM) showed little release of superoxide when exposed to phorbol myristate acetate. When exposed to a phagocytic stimulus (zymosan), TAM released appreciable amounts of superoxide. TAM had a lower number of specific binding sites for phorbol esters than resident or caseinate-elicited peritoneal macrophages, but had normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated products of neoplastic cells, may influence the functional status of in situ macrophages and, thus, impair host anti-tumor and anti-microbial defense mechanisms.

TPA induces subcellular translocation and subsequent down-regulation of both phorbol ester binding and protein kinase C activities in MCF-7 cells.

Phorbol ester TPA has been previously shown to induce a rapid translocation, followed by a progressive decline of protein kinase C activity in MCF-7 cells (J.M. Darbon et al, 1986, Biochem. Biophys. Res. Comm. 137: 1159-1166). We show now a parallel TPA-induced movement of phorbol ester binding sites from the cytosolic to the particulate fraction with no change in the binding affinities for the (3H) PDBu probe (KD congruent to 2 nM). The subcellular redistribution process is followed by a rapid decrease of the phorbol ester binding capacity at the membrane level. The concomitant decline in both phorbol ester binding and protein kinase C activities that we observed during the course of TPA treatment strongly argues for a real down-regulation of the enzyme in phorbol ester-treated MCF-7 cells. The molecular mechanisms of these events and their relations to the inhibition of cell growth remain to be clarified.

Tumor promoter chloroform is a potent protein kinase C activator

The major interaction site for tumor-promoting phorbol esters is the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), a key-element in signal transduction. Binding of phorbol esters results in enzyme activation which mediates, at least in part, the action of these agents. We have investigated the effects of tumor promoter chloroform on protein kinase C activity. Like thrombin and 12-0-tetradecanoylphorbol-13-acetate (TPA), chloroform was able to activate protein kinase C in intact rabbit platelets. In addition, chloroform stimulated enzyme activity as well as TPA binding capacity in cell-free system. Scatchard analysis of the data has shown that chloroform increased the number of phorbol ester binding sites. Structurally related compounds, carbon tetrachloride and methylene chloride, activated the enzyme similarly.

Protein kinase C in the nervous system of insects: effects of phorbol esters on cholinergic synapses

Specific high affinity binding sites for phorbol esters have been demonstrated in the nervous tissue of locust. The binding was reduced by the kinase C-inhibitor polymyxin B. Phorbol esters in micromolar concentrations activated protein kinase C in subcellular preparations from locust ganglia. The same concentrations of phorbol esters significantly enhanced the high affinity transport of choline and the release of acetylcholine by locust synaptosomes.

The lipophilic muramyltripeptide MTP-PE, a biological response modifier, is an activator of protein kinase C

The lipophilic immunomodulator MTP-PE is able to activate purified protein kinase C (PKC) by substituting phosphatidylserine (PS) or the synthetic diacylglycerol, DiC 8, in the assay system. In addition, MTP-PE inhibited [ 3H]-phorbol-12, 13-dibutyrate ([ 3H]-PDBu) binding to PKC in a reconstituted receptor system as well as on intact cells (MCF-7). Furthermore, MTP-PE was also able to reduce the epidermal growth factor binding of MCF-7 cells to an extent similar to that found with DiC 8 or PDBu. These data indicate that MTP-PE is able to compete for the phorbol ester binding site on PKC both in vivo and in vitro. The components of the MTP-PE molecule, MTP (muramyltripeptide) and PE (phosphatidylethanolamine) exerted only marginal effects on PKC activity, did not affect the phorbol ester binding of PKC and the EGF binding of intact MCF-7 cells. Our results suggest that only the complete molecule of the immunomodulator MTP-PE is able to interact with PKC.

Transient modulation and internalization of T4 antigen induced by phorbol esters.

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.

Phorbol ester receptors and protein kinase C in primary neuronal cultures: development and stimulation of endogenous phosphorylation.

Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.

Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells.

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.

Identification of phorbol ester binding activity in the particulate and soluble fractions of human fetal tissues.

The presence of specific binding sites for phorbol esters was demonstrated in various human fetal tissues by binding assays of [3H]12-O-tetradecanoylphorbol-13-acetate (TPA) to particulate and cytosol fractions. Maximal binding activity of the soluble receptor is dependent on calcium and phosphatidylserine. Marked differences in receptor levels were found in various human fetal tissues. However, all the tissues tested contained binding sites for TPA. Particulate receptor concentration was highest in the brain, while the cytosolic receptor concentration was high in brain, stomach and liver. Human fetal tissues bound TPA with a Kd between 10 and 30 nM.

Specific binding and biological effects of tumor promoting phorbol esters on sponges

Sponges grown in the presence of 12-O-tetradecanoyl phorbol-13-acetate (TPA) show deep alterations of their structure and development. Their aquiferous system (flagellated cells and canals) is largely altered and the tissues show an unusually high cell density. This focalized effect of TPA on the aquiferous system seems specific and is reversible at low concentrations (100 ng/ml). A toxic, non-specific effect is also noted, particularly at high concentrations (5000 ng/ml). Using 3H-phorbol-12, 13-dibutyrate (3H-PDBu), we demonstrate a class of specific binding sites for phorbol esters in the homogenates of sponges. These binding sites have high affinity (Kd = 26.0 nM) for PDBu and at saturation about 20 pmoles of 3H-PDBu is bound per mg protein of sponge homogenates. The binding of 3H-PDBu was inhibited by other phorbol esters and their congeners, and there was a good correlation between their potency in binding inhibition and their tumor promoting activity. It is concluded that sponges have a class of specific saturable and high affinity receptors for phorbol esters and that there is a very high conservation of these receptors during evolution. Such specific binding may be responsible for subsequent biological effect of TPA on sponges.

Phorbol esters, phospholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells.

Addition of biologically active phorbol esters to intact quiescent 3T3 mouse cells stimulates an extremely rapid (detectable within seconds) phosphorylation of a Mr 80,000 cellular protein (termed "80k"). Phorbol 12,13-dibutyrate enhances 80k phosphorylation in a dose-dependent manner; half-maximal effect is obtained at 32 nM. The possibility that this phosphorylation is related to the activation of Ca2+-activated phospholipid-dependent protein kinase is suggested by the fact that phospholipid breakdown induced by exogenous treatment of the cells with phospholipase C from Clostridium perfringens or with platelet-derived growth factor, which is a potent activator of endogenous phospholipase C activity, also causes a rapid enhancement of 80k phosphorylation. Moreover, prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which leads to a marked decrease in the number of specific phorbol ester binding sites, prevents the phosphorylation of 80k stimulated by phorbol esters, phospholipase C, and platelet-derived growth factor. These findings provide evidence obtained with intact cells that implicate the stimulation of Ca2+-activated phospholipid-dependent protein kinase in the action of phorbol esters and other growth factors.