TPA and resiniferatoxin-mediated activation of NADPH-oxidase. A possible role for Rx-kinase augmentation of PKC.

The non-tumour promoting irritant, resiniferatoxin, was capable of activating the NADPH-oxidase respiratory burst of starch-elicited, but not resident mouse peritoneal macrophages in vitro. Unlike TPA, the response was synergised by incubation with zymosan. The Rx-stimulated NADPH-oxidase activity in a cell-free assay was selectively enhanced in the presence of exogenous Rx-kinase rather than PKC and in the absence of Ca2+. Since resiniferatoxin is a poor activator of PKC, it is probable that the Ca2(+)-independent Rx-kinase plays a role in activation of the macrophage respiratory burst following stimulation by zymosan.

Activation of the PKC-isotypes alpha, beta 1, gamma, delta and epsilon by phorbol esters of different biological activities.

Phorbol esters, tetradecanoylphorbolacetate, sapintoxin-A, 12-deoxyphorbol-phenylacetate, 12-deoxyphorbol-phenylacetate-20-acetate, thymeleatoxin and resiniferatoxin were investigated for their abilities to activate the PKC-isotypes alpha, beta 1, gamma, delta and epsilon. PKC-isotypes were grouped into two classes on the basis of Ca2+ requirements for activation by phorbol esters; alpha, beta 1, and gamma being Ca(2+)-dependent forms and delta and epsilon being Ca(2+)-independent. PKC-isotype selective activation by phorbol esters was observed in that SAPA failed to activate PKC-delta up to a concentration of 1000 ng.ml-1 and DOPPA only activated PKC-beta 1 over the same range of concentrations.

A phorbol ester and a daphnane ester stimulate a calcium-independent kinase activity from human mononuclear cells.

TPA and a non-promoting, pro-inflammatory ester RX were used to stimulate the forms of PKC isolated from human mononuclear cells. Three peaks of kinase activity corresponding to gamma, beta and alpha PKC were stimulated by TPA in the presence and absence of calcium and/or phosphatidylserine (PS) but were not activated by RX. A fourth peak eluted at high phosphate concentration was activated by TPA and RX in the presence of PS and the absence of calcium. Activity in this fraction was labile to freezing and thawing and was inhibited by staurosporine.

Molecular modelling of tumour-promoting and non-promoting diterpene esters and molecular co-ordinates of the potent irritant resiniferatoxin.

The lowest energy conformer of seventeen diterpenes, representing five different phorbol and daphnane diterpene nuclei, has been generated. TPA possessed the highest minimum free energy of these compounds; all other compounds possessed a lower minimum free energy. Compounds based on the resiniferonol nucleus possessed the lowest minimum free energy (9.4-16.6% of that of TPA). The molecular co-ordinates of the non-promoting but potent irritant resiniferatoxin (Rx) are also reported. These studies may be important in elucidation of the biochemical mechanisms of action of diterpene esters, including an understanding of the interactions of diterpene esters with the phorbol ester binding domain of the protein kinase C isoform family.

PKC in rat cortical synaptosomes: effects of depolarization.

PKC isotypes were studied in rat cortical synaptosomes. Resting synaptosomes, subfractionated into cytosolic and particulate (Triton X100-soluble and insoluble) fractions, containing PKC beta 1, gamma, delta and epsilon isotypes but very little PKC beta 2 or alpha. PKC delta and epsilon were evenly distributed in the cytosolic and particulate fractions; PKC beta 1 was mainly in the cytosol (70%) and PKC gamma was primarily particulate (80%). Triton X-100 extracted most of the PKC delta and epsilon from the particulate fraction but only half of the PKC gamma, indicating PKC gamma association with the cytoskeleton. Following KC1 treatment (30 mM), both the classical PKC isotypes beta 1 and gamma shifted from the cytosolic to the particulate fraction, with PKC beta 1 moving specifically to the detergent insoluble fraction whereas PKC gamma appeared to move to both. It is concluded that PKC beta 1, gamma, delta, and epsilon isotypes can occur presynaptically and suggested that PKC beta 1 and gamma are directly involved in synaptic transmission. Apparent mol. wt changes in translocating forms may be related to phosphorylation and subsynaptosomal location.

Analysis and purification of phorbol esters using normal phase HPLC and photodiode-array detection.

For the first time a normal-phase HPLC method using photodiode-array detection is described for the analysis and purification of phorbol esters. The use of the method is demonstrated with examples of 10 different tigliane and daphnane esters (TPA, DOPP, DOPPA, Sap A, Sap B, Sap C, Sap D, Thy A, Ro and Rx). Both analytical and semipreparative techniques were developed. The method has been used in the final purification of DOPP and Rx from plant extracts. The method can be employed in the areas of phytochemistry, biochemistry and pharmacology/toxicology, where small samples of the toxic materials are required for research.

Properties of a resiniferatoxin-stimulated, calcium inhibited but phosphatidylserine-dependent kinase, which is distinct from protein kinase C isotypes alpha, beta 1, gamma, delta, epsilon and eta.

We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca2+ the Ka for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nM. Using a phosphate gradient of 20-500 mM, peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nM phosphate and resiniferatoxin kinase recovered in 500 mM phosphate. At the optimum concentration of 160 nM, the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 microM failed to activate the kinase. A Scatchard analysis of [3H] phorbol dibutyrate binding produced a linear plot (Kd 41.6 nM; Bmax 11.6 fmol unit-1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabelled resiniferatoxin binding assay was also used to demonstrate specific binding of [3H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes alpha, beta 1, gamma, delta and epsilon by means of immunological analysis and from the eta isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca2+ (Ki 0.1-0.5 nM free Ca2+). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70-90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.

Effects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, glycogen synthase kinase-3beta inhibition, and viral replication: a screening approach for new bipolar disorder drugs derived from the valproic acid core structure.

Inositol-1,4,5-trisphosphate (InsP3) depletion has been implicated in the therapeutic action of bipolar disorder drugs, including valproic acid (VPA). It is not currently known whether the effect of VPA on InsP3 depletion is related to the deleterious effects of teratogenicity or elevated viral replication, or if it occurs via putative inhibitory effects on glycogen synthase kinase-3beta (GSK-3beta). In addition, the structural requirements of VPA-related compounds to cause InsP3 depletion are unknown. In the current study, we selected a set of 10 VPA congeners to examine their effects on InsP3 depletion, in vivo teratogenic potency, HIV replication, and GSK-3beta activity in vitro. We found four compounds that function to deplete InsP3 in the model eukaryote Dictyostelium discoideum, and these drugs all cause growth-cone enlargement in mammalian primary neurons, consistent with the effect of InsP3 depletion. No relationship was found between InsP3 depletion and teratogenic or elevated viral replication effects, and none of the VPA congeners were found to affect GSK-3beta activity. Structural requirements of VPA congers to maintain InsP3 depletion efficacy greater than that of lithium are a carboxylic-acid function without dependence on side-chain length, branching, or saturation. Noteworthy is the enantiomeric differentiation if a chiral center exists, suggesting that InsP3 depletion is mediated by a stereoselective mode of action. Thus, the effect of InsP3 depletion can be separated from that of teratogenic potency and elevated viral replication effect. We have used this to identify two VPA derivatives that share the common InsP3-depleting action of VPA, lithium and carbamazepine, but do not show the side effects of VPA, thus providing promising novel candidates for bipolar disorder treatment.

Lithium inhibits glycogen synthase kinase-3 by competition for magnesium.

The mechanism by which lithium (Li(+)) inhibits the protein kinase glycogen synthase kinase-3 (GSK-3) is unknown. Here, we demonstrate that Li(+) is a competitive inhibitor of GSK-3 with respect to magnesium (Mg(2+)), but not to substrate or ATP. This mode of inhibition is conserved between mammalian and Dictyostelium GSK-3 isoforms, and is not experienced with other group I metal ions. As a consequence, the potency of Li(+) inhibition is dependent on Mg(2+) concentration. We also found that GSK-3 is sensitive to chelation of free Mg(2+) by ATP and is progressively inhibited when ATP concentrations exceed that of Mg(2+). Given the cellular concentrations of ATP and Mg(2+), our results indicate that Li(+) will have a greater effect on GSK-3 activity in vivo than expected from in vitro studies and this may be a factor relevant to its use in the treatment of depression.

Characterization of phorbol ester binding to protein kinase C isotypes.

A mixed micellar assay was used to study the in vitro binding of [3H]phorbol-12, 13-dibutyrate ([3H]PDBu) to pure recombinant protein kinase C (PKC)-alpha, -beta 1, -beta 2, -gamma, -delta, -epsilon, and -zeta isotypes expressed in the baculovirus/insect cell system. Scatchard analysis revealed that all isotypes except PKC-zeta were able to specifically bind PDBu, with Kd values ranging from 1.6 to 18 nM in the presence of calcium. In the absence of calcium PKC-alpha, -beta 1, -beta 2, and -delta were observed to have a 2-3-fold drop in affinity, although Bmax values remained unchanged, at a stoichiometry of 1.4-2.8 mol of PDBu/mol of enzyme. Competition with specific [3H]PDBu binding was assessed for the phorbol esters PDBu, 12-tetradecanoylphorbol-13-O-acetate, 12-deoxyphorbol-13-O-phenylacetate, 12-deoxyphorbol-13-O-phenylacetate-20-acetate, thymeleatoxin, resiniferatoxin, and sapintoxin A. Resiniferatoxin and 12-deoxyphorbol-13-O-phenylacetate-20-acetate were found to compete effectively only with PDBu bound to the PKC-beta 1 and -beta 2 isotypes and were the least potent of the phorbol esters tested (IC50, > 5 microM). The phorbol esters sapintoxin A, 12-deoxyphorbol-13-O-phenylacetate, 12-tetradecanoylphorbol-13-O-acetate, and PDBu (in order of potency) competed for binding to all isotypes (IC50 values ranging from 2 to 70 nM), with unchanged or slightly decreased potency when calcium was replaced by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thymeleatoxin, which was similar in other respects to these potent phorbol esters, was found to be less able to compete with binding to PKC-alpha and -epsilon isotypes (IC50, 3-5 microM). It appears that, whereas the binding of phorbol esters to PKC depends primarily on the C20 substituent, other areas of the molecule have an influence on this interaction and the PKC isotypes themselves display heterogeneity in their phorbol ester-binding characteristics.

HL-60 cell differentiation induced by phorbol- and 12-deoxyphorbol-esters.

The human promyelocytic leukaemia cell (HL-60) undergoes differentiation into a macrophage-like form when exposed to both tumour promoting- and non-promoting phorbol esters. We have investigated the effect of the two non-promoting phorbol esters, 12-deoxyphorbol-13-O-phenylacetate (Dopp) and 12-deoxyphorbol-13-O-phenylacetate-20-acetate (Doppa) on HL-60 cultures, and compared them with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All phorbol esters tested were found to be able to stop HL-60 proliferation and induce cell adherence and morphological changes characteristic of differentiation. TPA, fully differentiating at 1 nM, was more potent than Dopp and Doppa, which required 100 nM for full differentiation effects within the 4 day study. Doppa initially appeared weaker than Dopp at inhibiting incorporation of thymidine, the earliest effect studied, but we were able to detect rapid C-20 deacylation of Doppa, converting it to Dopp, using an HPLC protocol presented here. A detailed study of this thymidine incorporation inhibition showed that both TPA (10 nM or greater) and Dopp (500 nM or greater) have very similar time courses, with 50% inhibition occurring at approximately 12 h, in contrast to Doppa which had a significantly delayed time course at all doses tested. Exposure tests indicated that Dopp and Doppa could be washed from the cells much more easily than TPA. The data presented here strongly support the notion that the metabolic conversion of Doppa to Dopp by HL-60 cells was necessary to mediate its differentiating effects. Since protein kinase C (PKC)-beta 1, present in HL-60 cells, has been found to be the only PKC isotype activated so far in vitro by Doppa, our results suggest that activation of this isotype is not sufficient to drive HL-60 differentiation in vivo.

Loss of a prolyl oligopeptidase confers resistance to lithium by elevation of inositol (1,4,5) trisphosphate.

The therapeutic properties of lithium ions (Li+) are well known; however, the mechanism of their action remains unclear. To investigate this problem, we have isolated Li+-resistant mutants from Dictyostelium. Here, we describe the analysis of one of these mutants. This mutant lacks the Dictyostelium prolyl oligopeptidase gene (dpoA). We have examined the relationship between dpoA and the two major biological targets of lithium: glycogen synthase kinase 3 (GSK-3) and signal transduction via inositol (1,4,5) trisphosphate (IP3). We find no evidence for an interaction with GSK-3, but instead find that loss of dpoA causes an increased concentration of IP3. The same increase in IP3 is induced in wild-type cells by a prolyl oligopeptidase (POase) inhibitor. IP3 concentrations increase via an unconventional mechanism that involves enhanced dephosphorylation of inositol (1,3,4,5,6) pentakisphosphate. Loss of DpoA activity therefore counteracts the reduction in IP3 concentration caused by Li+ treatment. Abnormal POase activity is associated with both unipolar and bipolar depression; however, the function of POase in these conditions is unclear. Our results offer a novel mechanism that links POase activity to IP3 signalling and provides further clues for the action of Li+ in the treatment of depression.

Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein.

Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the DICTYOSTELIUM: STAT protein Dd-STATa. Here we show that it also induces serine phosphorylation by GskA, a homologue of glycogen synthase kinase-3 (GSK-3). Tyrosine phosphorylation occurs within 10 s of stimulation, whereas serine phosphorylation takes 5 min, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes.

A molecular cell biology of lithium.

Lithium (Li(+)), a mood stabilizer, has profound effects on cultured neurons, offering an opportunity to investigate its cellular biological effects. Here we consider the effect of Li(+) and other psychotropic drugs on growth cone morphology and chemotaxis. Li(+) inhibits GSK-3 (glycogen synthase kinase-3) at a therapeutically relevant concentration. Treated cells show a number of features that arise due to GSK-3 inhibition, such as altered microtubule dynamics, axonal branching and loss of semaphorin 3A-mediated growth cone collapse. Li(+) also causes growth cones to spread; however, a similar effect is seen with two other mood stabilizers, valproic acid and carbamazepine, but without changes in microtubules or axon branching. This common effect of mood stabilizers is mediated by changes in inositol phosphate signalling, not GSK-3 activity. Given the presence of neurogenesis in the adult brain, we speculate that changes in growth cone behaviour could also occur during treatment of mental disorders.

12-Deoxyphorbol-13-O-phenylacetate-20-acetate is not protein kinase C-beta isozyme-selective in vivo.

The phorbol ester, 12-deoxyphorbol-13-O-phenylacetate-20-acetate (DOPPA) has been shown to activate specifically the protein kinase C (PKC)-beta 1 isozyme in vitro (1). We have investigated the potential of DOPPA as a PKC-beta 1/2 isozyme-specific agonist in intact cells, employing U937 cells, which express beta 1/2, epsilon and zeta PKC and in Swiss 3T3 cells which lack PKC-beta 1/2 but express alpha, delta, epsilon and zeta PKC. Immunoblot analysis with isozyme-specific antibodies indicated that DOPPA can mediate the subcellular redistribution and down-modulation of all endogenous PKC isozymes (except PKC-zeta) in both U937 and Swiss 3T3 cells. Prolonged treatment (> 6 h) of cultures in down-modulation studies is complicated by the metabolism of DOPPA to 12-deoxyphorbol-13-phenylacetate (DOPP), a compound which activates all PKC isozymes tested in vitro (Ryves, W. J., et al. (1991) FEBS Lett., 288, 5-9). Nevertheless, because DOPPA induced rapid and dose-dependent phosphorylation of p80 in cells which do not express PCK-beta, p80 phosphorylation in Swiss 3T3 cells indicates that DOPPA can activate a non-beta PKC in vivo. The data suggest that DOPPA cannot be used as a PKC-beta-selective agonist in intact cell studies.