[On the chemistry of phorbol. V. On some ethers of phorbol].
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Chemical Phenomena↗
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[On the chemistry of phorbol. VI. Functional derivatives of phorbol-12-one and their circular dichroism].
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Acetates↗
Effects of phorbol and four diesters of phorbol on the incorporation of tritiated precursors into DNA, RNA, and protein in mouse epidermis.
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Acetates↗
Tumor-promoting activity of phorbol and four diesters of phorbol in mouse skin.
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Alcohols↗
Regulation of cell growth and the c-myc proto-oncogene expression by phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) in the androgen-independent human prostatic JCA-1 cells.
The proliferation of the human prostatic cancer JCA-1 cells showed a complex response to different concentrations of TPA. Whereas cells exposed for 24 h had growth reduction which was proportional to the concentration of TPA added to the culture media, they showed resistance to low (0.016 and 0.16 nM) but not high (> or = 1.6 nM) doses of TPA after 72 h. Growth-inhibited, treated cells also displayed a distinct cell morphology compared with the controls. Since c-myc expression has previously been shown to be correlated with cellular proliferation, we determined changes in its steady-state level in control and treated cells by Northern analysis. Following a 24h, 48h, and 72h treatment, with 16 nM TPA, c-myc mRNA was suppressed by 91%, 83%, and 78%, respectively, in good agreement with the extent of growth reduction observed. At the low dose of TPA (0.16 nM), however, the c-myc mRNA expression remained inhibited by 85% even though cell growth was only reduced by 10-14%. No difference in the total amount of c-myc protein could be detected between control and treated cells by Western analysis.
Androgens↗
Human growth hormone-stimulated growth of human cultured lymphocytes (IM-9) and its inhibition by phorbol diesters through down-regulation of the hormone receptors. Possible involvement of phosphorylation of a 55,000 molecular weight protein associated with the receptor in the down-regulation.
We report in this paper that human growth hormone (hGH) stimulates the growth of human cultured IM-9 lymphocytes in a low concentration (3%) of serum. The hormone-stimulated growth was inhibited with the phorbol diesters phorbol 12-myristate 13-acetate and phorbol 12,13-dibutylate (PDBu). The binding experiments of 125I-hGH to the phorbol diester-treated cells and to their detergent-solubilized receptors revealed that the phorbol diesters caused internalization of the hGH receptors from the cell surfaces but did not significantly affect their affinity (Ka = 8.5 x 10(9) M-1). About half of the receptors (1.4 x 10(3)/cell) were internalized in 30 min at 37 degrees C, and the half-effective doses of phorbol 12-myristate 13-acetate and PDBu were 5 and 35 nM, respectively. When culture was continued after washing with the culture medium, the phorbol diester-treated cells recovered their hGH-responsive growth, and the number of the surface hGH receptors was restored. The down-regulation of the hormone receptor was also induced with another phorbol diester, phorbol 12,13-didecanoate, but not with the phorbol or phorbol monoesters phorbol 12-myristate and phorbol 13-acetate. The synthetic activators of protein kinase C 1-oleoyl-2-acetyl-glycerol and N-(6-phenyl-hexyl)-5-chloro-1-naphthalenesulfonamide had an effect similar to that of the phorbol diesters. Staurosporine and sphingosine, inhibitors of protein kinase C, inhibited the phorbol diester-caused down-regulation with a half-inhibitory dose (IC50) of 8 nM and 130 microM, respectively. This suggests that protein kinase C was involved in the reaction. When 32Pi-loaded IM-9 cells were stimulated with PDBu at 37 degrees C, the phosphorylation of Mr 55,000, 88,000, and 114,000 proteins increased rapidly. The PDBu-stimulated phosphorylation of 55,000 protein was also inhibited by staurosporine at 10 nM, which was a comparable concentration to inhibit the phorbol diester-induced down-regulation of hGH receptors. Furthermore, among these proteins, the 55,000 protein was specifically coisolated with the hGH receptors by three different experiments: 1) immunoprecipitation by anti-hGH antibody; 2) immunoisolation using protein A-cellulose columns; and 3) affinity purification by hGH-fixed agarose gel. These results suggest that phorbol diesters reduce the hGH-stimulated growth of cultured IM-9 lymphocytes by the down-regulation of hGH receptors and that the receptor-associated 55,000 protein may be involved in this regulation through phosphorylation by protein kinase C.
Alkaloids↗
Expression and phorbol ester-induced down-regulation of protein kinase C isozymes in osteoblasts.
The protein kinase C (PKC) enzyme family consists of at least 11 isozymes in three classes, with characteristic tissue distributions. Phorbol esters activate and ultimately down-regulate phorbol-sensitive isozymes. PKC is a signal transducer in bone, and phorbol esters influence bone resorption. Little is known about specific PKC isozymes in this tissue, however. We describe here the expression and phorbol ester-induced down-regulation of PKC isozymes in osteoblasts. Normal mouse osteoblasts and seven osteoblastic cell lines (rat UMR-106, ROS 17/2.8, ROS 24/1, and human MG-63, G-292, SaOS-2, HOS-TE85) were screened for isozyme expression by Western immunoblotting using isozyme-specific anti-PKC antibodies. The conventional alpha and beta I isozymes, but not gamma, were present in each of the osteoblasts examined; PKC-beta II was detectable in all but the ROS 24/1 line. PKC-epsilon was expressed in all osteoblasts screened, but other novel PKCs, delta, eta, and theta, were detectable only in select lines. The atypical zeta and iota/lambda PKCs were in all osteoblasts examined. To determine the sensitivity of the isozymes to prolonged phorbol ester treatment, normal osteoblasts and the UMR-106 cell line were treated with vehicle or 1 microM phorbol 12, 13-dibutyrate (PDB) for 1, 3, 6, 12, 24, or 48 h, and Western blot analysis was performed. Normal and UMR-106 cells showed similar phorbol sensitivities; conventional (alpha, beta I) and novel (delta, epsilon, eta) isozymes were down-regulated by prolonged phorbol treatment but atypical isozymes were not. Down-regulation of all sensitive PKCs was detectable within 6 h of phorbol treatment; the novel delta and epsilon isozymes, however, showed more rapid and dramatic down-regulation than conventional isozymes. The observed down-regulation was dose-dependent (0.3-3 microM) and specific; 48 h treatment with the inactive phorbol, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), failed to down-regulate PDB-sensitive isozymes. The phorbol-induced down-regulation was also reversible; 24 h after withdrawing PDB, all phorbol-sensitive isozymes, except PKC-eta, had recovered at least partially. These studies, the first to characterize thoroughly PKC isozyme expression in osteoblastic cells from several species, demonstrate that osteoblasts have a characteristic PKC isozyme profile, including both phorbol ester-sensitive and -insensitive isozymes. The time course of down-regulation and the presence of phorbol-insensitive PKCs must be considered in interpreting the effects of phorbol esters on bone remodeling.
Animals↗
Relationship between biological responsiveness to phorbol esters and receptor levels in GH4C1 rat pituitary cells.
During a 24-hr incubation of GH4C1 cells with phorbol esters or thyrotropin-releasing hormone, there is a decrease in the number of phorbol esters receptors (down-modulation). The purpose of this study was to investigate whether this decrease in receptor number attenuated cellular responsiveness to subsequent challenge with phorbol esters. Accordingly, cellular sensitivity to a phorbol ester-mediated biological response, namely the decrease in binding of epidermal growth factor, was compared in control and down-modulated cells. This phorbol ester-mediated event is closely associated with phorbol ester receptor occupancy, and it is therefore an effect for which altered dose-response characteristics correlating with alterations in the number of phorbol ester receptors could be anticipated. In fact, during a 48-hr exposure to phorbol esters, GH4C1 cells become refractory to the effect on epidermal growth factor binding. This time course is similar to that for the loss of phorbol ester receptors. However, when cells are down modulated by pretreatment with phorbol ester or thyrotropin-releasing hormone and then (re)challenged with phorbol ester, no differences in dose-response characteristics were observed between control and down-modulated cells. We therefore conclude that phorbol ester receptor down-modulation does not affect cellular responsiveness to phorbol esters, at least when decreased epidermal growth factor binding is used as the marker for the phorbol ester-mediated event.
Animals↗
Effect of a chemical carcinogen and phorbol esters on sterol metabolism of mouse skin.
The effect of 20-methylcholanthrene and phorbol esters on sterol metabolism of mouse skin was studied. When 4 beta-phorbol esters were administered to mice that were previously painted once with 20-methylcholanthrene, a depression of some sterols in skin occurred, of which that of lathosterol was most marked. This effect was not observed when the order of application was reversed. Using a metabolic inhibitor, diazacholesterol, it was shown that sterols which reduce in mouse skin by administration of carcinogen and promoters were similar to those which reduce by administration of carcinogen only and are the members of one of the two cholesterol-biosynthetic pathways, i.e., a pathway which proceeds through intermediates with a saturated side chain. The intensity of the lathosterol-depressing effect of phorbol esters depends on the order of application of 20-methylcholanthrene and promoters, the amount of promoters, molecular species of alcoholic moiety of esters, and configuration at C-4 of phorbol moiety. Of the phorbol esters tested, 4 beta-phorbol-12-myristate-13-acetate revealed the highest activity, which was followed by 4 beta-phorbol-12,13-didecanoate, 4 beta-phorbol-12,13-dibutyrate, 4 beta-phorbol-12,13-dibenzoate, 4 beta-phorbol-12,13-diacetate, 4 alpha-phorbol-12,13-didecanoate, and 4 alpha-phorbol. 4 alpha-Phorbol was practically inactive. When beta-naphthoflavone was substituted for 20-methylcholanthrene, little effect was observed except in TPA, which revealed a rather marked lathosterol-depressing activity. Phorbol esters themselves did show some activity of lathosterol depression without prior application of 20-methylcholanthrene, but the effects were much weaker. When anthralin was applied to mouse skin after the painting of 20-methylcholanthrene, a low but definite lathosterol-depressing effect was observed.
Administration, Topical↗
Specific binding of phorbol ester tumor promoters.
[20-(3)H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a K(d) of 25 nM; this value is very close to the 50% effective dose (ED(50)), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-(3)H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 x 10(4) molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (K(i) = 2 nM), mezerein (K(i) = 180 nM), phorbol 12,13-dibenzoate (K(i) = 180 nM), phorbol 12,13-diacetate (K(i) = 1.7 muM), phorbol 12,13,20-triacetate (K(i) = 39 muM), and phorbol 13-acetate (K(i) = 120 muM). The measured K(i) values are all within a factor of 3.5 of the ED(50) values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 mug/ml) and 4alpha-phorbol 12,13-didecanoate (10 mug/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 muM), phenol (1 mM), iodoacetic acid (1.7 muM), and cantharidin (75 muM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 muM), retinoic acid (10 muM), or prostaglandin E(2) (1 muM). These agents thus appear to act at a target distinct from that of the phorbol esters.
Animals↗
Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine.
A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.
Animals↗
Phorbol esters induce interleukin 2 mRNA in sensitive but not in resistant EL4 cells.
Phorbol ester sensitive EL4 cells become growth-inhibited and produce interleukin 2 when treated with phorbol-12, 13-dibutyrate. Resistant cells lack both responses. To determine whether the defect in phorbol ester-resistant EL4 cells occurs pre- or post-transcriptionally, a hybridization assay for interleukin 2 mRNA was developed using two synthetic oligonucleotides complementary to mouse interleukin 2 mRNA as probes. Both probes hybridized to a 1-kilobase band in RNA from phorbol ester-treated sensitive cells. This RNA was detectable within 3 h of phorbol ester administration, and accumulation peaked by 12 h. The 1-kilobase band was induced in a concentration-dependent manner by 4-beta-phorbol-12, 13-dibutyrate but not by the inactive analog, 4-alpha-phorbol-12, 13-dibutyrate. No bands hybridizing with the interleukin 2 probe were detected in RNA isolated from unstimulated cells or from phorbol ester-resistant EL4 cells at any time up to 24 h following phorbol ester stimulation. The accumulation of the RNA in sensitive cells was blocked when the protein synthesis inhibitors, cycloheximide (75 microM) or puromycin (90 microM) were added within 1 h of the addition of phorbol ester. If cycloheximide was added 2 or more h after phorbol ester treatment, superinduction of the 1-kilobase band was observed. These results indicate that the failure of phorbol ester-resistant EL4 cells to produce interleukin 2 is due to a defect proximal to interleukin 2 transcription and that the accumulation of interleukin 2 mRNA in phorbol ester-sensitive EL4 cells requires protein synthesis.
Animals↗
Scanning mutagenesis studies reveal multiple distinct regions within the human protein kinase C alpha regulatory domain important for phorbol ester-dependent activation of the enzyme.
While phorbol ester-binding sites within protein kinase C alpha (PKCalpha) have been identified and characterized utilizing fragments of the enzyme, it remains unclear whether additional regions within the enzyme may play an important role in its ability to be activated by phorbol ester. To examine this hypothesis, we generated 20 glutathione-S-transferase-tagged, V1-deficient, human PKCalpha holoenzyme constructs in which tandem six or 12 amino acid residue stretches along the full regulatory domain were changed to alanine residues. Each protein was assessed for its ability to bind phorbol ester and to induce growth repression when its catalytic activity was activated by phorbol ester upon expression in yeast cells. Mutagenesis of residues 99-158 potently reduced phorbol binding, consistent with previously published findings on the importance of the C1b region in phorbol binding. In addition, we identified a number of regions within the PKC regulatory domain that, when mutagenized, blocked the activation of PKC-mediated growth repression by phorbol ester while actually enhancing phorbol ester binding in vitro (residues 33-62, and 75-86). This study thus helps distinguish regions important for phorbol binding from regions important for the ability of phorbol ester to activate the enzyme. Our findings also suggest that multiple regions within C2 are necessary for full activation of the enzyme by phorbol ester, in particular residues 231-254. Finally, three regions, when mutagenized, completely, blocked catalytic domain activity in vivo (residues 33-62, 75-86, and 123-146), underscoring the important role of regulatory domain sequences in influencing catalytic domain function, even in the absence of the V1 region containing the pseudosubstrate sequence. This is the first tandem mutagenesis study for PKC that assesses the importance of regions for both phorbol binding and for phorbol-dependent activation in the context of the entire holoenzyme.
Alanine↗
Possible mechanism of phorbol diester-induced maturation of human promyelocytic leukemia cells.
The phorbol diesters are the most potent inducers of differentiation of the promyelocytic leukemia cell line, HL-60. Soluble phorbol diester receptors from HL-60 cells were obtained from the cytosolic fraction and from the particulate fraction by either divalent ion chelation or detergent extraction. The partially purified soluble phorbol diester receptors required exogenous Ca2+ and phospholipid for maximal binding and displayed a dissociation constant (KD) of 8.1 nM for [3H]phorbol 12,13-dibutyrate. Phorbol diester analogues inhibited [3H]phorbol 12,13-dibutyrate binding in a stereospecific manner consistent with their biologic potency. The soluble phorbol diester receptors prepared by all three methods copurified in a constant ratio with the Ca2+/phospholipid-dependent protein kinase C through ammonium sulfate precipitation, DEAE ion exchange, and gel filtration chromatography. Partially purified protein kinase C was directly activated by the phorbol diesters even in the absence of exogenous Ca2+. The ability of a series of phorbol analogues to activate the kinase correlated with their known activity as inducers of cell differentiation. In addition, phorbol diester stimulation altered the phosphate acceptor substrate profile of protein kinase C, at least in part, by alteration of the Michaelis constant (Km). These data suggest that protein kinase C is the phorbol diester receptor and that phorbol diester-induced macrophage maturation of HL-60 cells may be mediated by activation of intracellular protein kinase C.
Caenorhabditis elegans Proteins↗
Phorbol esters and d-tubocurarine up-regulate alpha-bungarotoxin sites in chromaffin cells in culture via distinct mechanisms.
Previous work had shown that nicotinic antagonists resulted in a marked up-regulation of alpha-bungarotoxin sites in chromaffin cells in culture. The present experiments were done to determine the intracellular mechanism(s) whereby nicotinic antagonists might mediate their effects on these receptors. Chromaffin cells were cultured for three days with various concentrations of 4 beta-phorbol 12-myristate 13-acetate, an agent which affects protein kinase C by mimicking the actions of diacylglycerol. The phorbol ester resulted in a dose-dependent increase in alpha-bungarotoxin binding which was maximal with 100 nM 4 beta-phorbol 12-myristate 13-acetate. This increase in binding appeared to be due to an increase in the maximal number of alpha-bungarotoxin sites. Time dependence studies showed that the effect of the phorbol was undetectable with incubations of 24 h or less and appeared to plateau by 72-96 h. A similar increase in toxin binding was also observed with 4 beta-phorbol 12,13-dibutyrate. On the other hand, an inactive analog of 4 beta-phorbol 12-myristate 13-acetate had no significant effect on binding. D-Sphingosine, an inhibitor of protein kinase C, was able to partially block the phorbol ester-induced increase in toxin binding while polymyxin B, another protein kinase C inhibitor, completely prevented the up-regulation of the alpha-bungarotoxin sites. Carbachol and nicotine prevented this enhancement of toxin binding in the presence of 4 beta-phorbol 12-myristate 13-acetate. Although the phorbol ester resulted in an increase in toxin binding, acetylcholine-evoked catecholamine secretion from chromaffin cells in culture was decreased, indicating a dissociation between the functional nicotinic acetylcholine receptor population and the alpha-bungarotoxin sites. To determine whether agents which affect protein kinase C can alter the up-regulation of alpha-bungarotoxin sites by d-tubocurarine, 4 beta-phorbol 12-myristate 13-acetate was added to the cells in combination with the nicotinic antagonist. The up-regulation of toxin binding sites induced by d-tubocurarine was additive with that induced by the phorbol and was not affected by polymyxin B. Thus, the results would suggest that there are at least two mechanisms by which alpha-bungarotoxin binding sites can be regulated. One is mediated via an interaction at nicotinic receptors, while the other occurs in response to phorbol esters and thus may be mediated by protein kinase C. Interestingly, although the molecular mechanisms resulting in alpha-bungarotoxin receptor up-regulation differ, both the d-tubocurarine- and the phorbol ester-induced increases were prevented by nicotinic receptor ligands.
Adrenal Medulla↗
The structural requirements for phorbol esters to enhance serotonin and acetylcholine release from rat brain cortex.
The effects of various phorbol-based protein kinase C (PKC) activators on the electrical stimulation-induced (S-I) release of serotonin and acetylcholine was studied in rat brain cortical slices pre-incubated with [3H]-serotonin or [3H]-choline to investigate possible structure-activity relationships. 4beta-phorbol 12,13-dibutyrate (4betaPDB, 0.1-3.0 microM), enhanced S-I release of serotonin in a concentration-dependent manner whereas the structurally related inactive isomer 4alpha-phorbol 12, 13-dibutyrate (4alphaPDB) and phorbol 13-acetate (PA) were without effect. Another group of phorbol esters containing a common 13-ester substituent (phorbol 12,13-diacetate, PDA; phorbol 12-myristate 13-acetate, PMA; phorbol 12-methylaminobenzoate 13-acetate, PMBA) also enhanced S-I serotonin release with PMA being least potent. The deoxyphorbol monoesters, 12-deoxyphorbol 13-acetate (dPA), 12-deoxyphorbol 13-angelate (dPAng), 12-deoxyphorbol 13-phenylacetate (dPPhen) and 12-deoxyphorbol 13-isobutyrate (dPiB) enhanced S-I serotonin release but 12-deoxyphorbol 13-tetradecanoate (dPT) was without effect. The 20-acetate derivatives of dPPhen and dPAng were less effective in enhancing S-I serotonin release compared to the parent compounds. With acetylcholine release all phorbol esters tested had a far lesser effect when compared to their facilitatory action on serotonin release with only 4betaPDB, PDA, dPA, dPAng and dPiB having significant effects. The effects of the phorbol esters on serotonin release were not correlated with their reported in vitro affinity and isozyme selectivity for PKC. A comparison across three transmitter systems (noradrenaline, dopamine, serotonin) suggests basic similarities in the structural requirements of phorbol esters to enhance transmitter release with short chain substituted mono- and diesters of phorbol being more potent facilitators of release than the long chain esters. Some compounds notably PDA, PMBA, dPPhen, dPPhenA had different potencies across noradrenaline, dopamine and serotonin.
Acetylcholine↗
Excess K+ and phorbol ester activate protein kinase C and support the survival of chick sympathetic neurons in culture.
The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester-supported sympathetic neurons possessed desipramine-sensitive [3H]-norepinephrine uptake mechanism, and therefore were noradrenegic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF-supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol ester-supported neurons the activity in the particulate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in less than 1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 microgram for 15 min), there was no change in the enzyme activity. Phorbol 13-acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+) Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. In NGF-supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration- and time-dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.
Animals↗
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 4 beta-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 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-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 Ca2+-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.
Animals↗