Changes of phorbol ester binding sites in rat brain following intracerebroventricular administration of thyrotropin-releasing hormone (TRH): An in vitro macroautoradiographic investigation

We examined the influence of the intracerebroventricular (icv) administration of thyrotropin-releasing hormone (THH) on protein kinase C (PKC) activities in various rat forebrain regions in order to cast light on the mechanism of extra-pituitary non-endocrine physiological actions of TRH in the central nervous system. An in vitro macroautoradiographic method, with [³H]phorbol 12, 13-dibutyrate (PDBu) as the radioactive ligand, was used to investigate quantitative alterations of PKC activities. The optical densities for PDBu binding sites in the striatum and hippocampal formation were significantly increased after the icv administration of TRH, while those in the frontal cortex and septum were unchanged. These findings suggest that TRH may exert some of its non-endocrine functions through striatal and hippocampal neurons which used PKC in their second messenger systems.     

Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the and isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH₃ somatomammotrophic cell line. AtT-20/D16-V corticotrophic and T3-1 gonadotrophic murine cell lines contained no PKC-. The or isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca²⁺-independent PKCs in T3-1 and GH₃ cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca²⁺-dependent PKC activities were of similar sensitivity to PDBu in GH₃, T3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca²⁺-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca²⁺-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)     

Phospholipid and Ca++ dependency of phorbol ester receptors

The phospholipid and Ca++ dependency of a partially purified phorbol ester apo-receptor from the soluble fraction of mouse brain homogenates was studied. This apo-receptor is believed to be identical with the Ca++ and phospholipid-dependent protein kinase C. Binding of phorbol esters to the receptor/kinase C was shown to be entirely dependent on phospholipids. The negatively charged phospholipids phosphatidylserine, phosphatidylinositol, and phosphatidic acid all fully reconstituted binding. The neutral phospholipids were inactive. Among active phospholipids and mixtures of phospholipids, substantial differences (greater than 100-fold) were observed in the amounts required to achieve reconstitution. Although Ca++ was not required for reconstitution of binding activity, it dramatically (up to 100-fold) increased the potency of phospholipids for reconstitution. The phospholipids not only permitted reconstitution of the apo-receptor but also played a major role in determining the binding characteristics of the complex. The KD values of [3H]phorbol 12,13-dibutyrate were in the range of 0.8 nM for the complex with phosphatidylserine to 30 nM for the complex with dioleoyl-phosphatidic acid. Like the binding affinity, the stimulation of protein kinase C activity by phorbol esters was dependent on the phospholipid into which the receptor/kinase C was reconstituted. The importance of the lipid domain for controlling the receptor/kinase C activity and for modulation of cellular sensitivity to phorbol esters is discussed.     

Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study

Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer’s disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin – the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn²⁺-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.     

Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen

Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from beta-amyloid peptide (Abeta) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (alpha, betaI, betaII), novel (delta, epsilon, theta) and atypical (zeta, iota/lambda) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against Abeta toxicity. In addition, peptide inhibitors of conventional, betaI, or betaII PKC isoforms significantly reduce protection afforded by PMA or 17beta-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by Abeta toxicity.     

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.     

Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester-treated Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane-bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.     

Fluorescent tetradecanoylphorbol acetate: A novel probe of phorbol ester binding domains

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N′-di(2-hydroxyethyl)amino)-7-nitrobenz-2-oxa-1,3-diazole-aminododecanoate (N-C₁₂-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C₁₂-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C₁₂-Ac(13) at 25°C, P₃HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C₁₂-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [³H]-phorbol-12,13-dibutyrate ([³H]-PBu₂) from rat brain cytosol with N-C₁₂-Ac(13) gave an apparent dissociation constant (K_d) of 11 nM. N-C₁₂-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C₁₂-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P₃HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C₁₂-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C₁₂-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.     

Phorbol Ester Differentiates the Levels of [<Superscript>3</Superscript>H]MK-801 Binding in Rats Lines Selected for Differential Sensitivity to the Hypnotic Effects of Ethanol

These studies addressed the possible involvement between sensitivity to the hypnotic action of ethanol and function of the NMDA receptor. The studies were carried out using high-alcohol sensitive (HAS) and low-alcohol sensitive (LAS) rats, two rats having differential sensitivity to the acute hypnotic action of ethanol. The animal models were developed by a selective breeding experiment. Using a quantitative autoradiograph technique, it was demonstrated that [³H]MK-801 binding to the NMDA receptor was highest in hippocampus in both HAS and LAS rats, but significant [³H]MK-801 binding was also detected in cortex, caudate-putamen, and thalamus of HAS and LAS rats. The density of [³H]MK-801 binding was lower only in cerebellar granule layers of untreated HAS rats as compared to the same brain area in untreated LAS rats. Activation of protein kinase C (PKC) by 100 nM PDBu, increased [³H]MK-801 binding in cortex, caudate-putamen, thalamus, central gray, and cerebellum of HAS rats but activation of PKC did not influence [³H]MK-801 binding in LAS rats. These activation of PKC differentiates between [³H]MK-801 binding of HAS and LAS rats in frontal cortex (layer II-IV and cingulate), caudate-putamen, and ventral lateral thalamic nuclei. The basal level of PKC- mRNA was higher in HAS rats than that of LAS rats. These results suggest that the activation of PKC potentiates NMDA receptor function of the rat line which is more sensitive to alcohol (HAS) but does not affect [³H]MK-801 binding of alcohol resistant (LAS) rats.     

Receptors for phorbol esters are primarily localized in neurons: Comparison of neuronal and glial cultures

Binding of [³H]PDB has been measured in the present study to determine the levels of protein kinase C in the neuronal and astrocytic glial cells in culture from rat brain. Binding of [³H]PDB to homogenates of cultured neuronal cells from the brains of normotensive and hypertensive rats was time-dependent and specific. The relative potency for competition by various phorbol esters to [³H]PDB binding was TPA > -PDD > POE > -PDD 4phorbol. Scatchard analysis showed that neuronal cultures from normotensive rat brains contained 2–3 fold more phorbol ester receptors compared with the glial cultures from the same brains. No differences in theK _d andB _max were observed between neuronal cultures from normotensive and spontaneously hypertensive rat brains. These studies suggest that the phorbol ester receptors are primarily localized in neuronal cells.     

Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.     

A combined analysis of regional energy metabolism and immunohistochemical ischemic damage in the gerbil brain

By combining immunohistochemical technique with microassay methods, we analyzed regional energy metabolism in vulnerable and tolerant areas of gerbil brains during evolution of neuronal damage after bilateral common carotid artery occlusion for 10 min with subsequent reperfusion. Four animals were used for each reperfusion period. Based on the information from the immunohistochemical examination, we dissected out vulnerable and tolerant subregions of the hippocampus, cerebral cortex, and thalamus from freeze-dried 20-microm-thick sections, and measured the levels of creatine phosphate (P-Cr), adenine nucleotides, guanine nucleotides, and purine bodies by HPLC, and the levels of glucose, glycogen, and lactate by an enzyme-immobilized column method. There were no significant differences in the levels of metabolites between vulnerable and tolerant subregions of control brains. After reperfusion, both vulnerable and tolerant subregions recovered preischemic metabolic profiles by 2 days. Although the regional differences between vulnerable and tolerant subregions were minimal at each reperfusion period, there were delays in the recovery of P-Cr, ATP, and/or total adenine nucleotides in all vulnerable subregions. A decline of P-Cr, ATP, and GTP levels without change in %ATP, AMP, or purine bodies occurred after reperfusion for 3 days, coinciding with the development of immunohistochemical damage by the immunoreaction for microtubule-associated protein 1A. The results supported the notion that subtle but sustained impairment of energy metabolism caused by mitochondrial dysfunction in the early reperfusion period might trigger delayed neuronal death in vulnerable subregions.     

Activity correlations in the phorbol ester series

Activity correlations in the phorbol ester series. The production of inflammation by phorbol esters on mammalian skin correlates on a structural basis with in vitro measurements of lymphocyte mitogenesis and mobilization of prostaglandins. All of the pro-inflammatory phorbol esters tested in our laboratory have been shown to activate the enzyme protein kinase C, and such an interaction could in large part explain the induction of an inflammatory response in vivo. Certain of these compounds additionally induce aggregation of human and rabbit blood platelets. This activity does not structurally correlate with the induction of inflammation but may correlate with the known tumour-promoting actions of phorbol derivatives. Compounds which induce platelet aggregation stimulate the secretion of a biologically active substance which we have termed 'Factor W'. The production of Factor W occurs into human plasma following platelet stimulation by phorbol tumour-promoting agents. It is an unstable substance, distinct in its aggregating properties from phorbol esters, ADP, 5-hydroxytryptamine, thrombin, platelet aggregating factor and the products of arachidonate oxidative metabolism.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Phorbol ester induces CYP2E1 in astrocytes,through a protein kinase C- and tyrosine kinase-dependent mechanism

Cytochrome P450 2E1 (CYP2E1) is highly inducible in a subset of astrocytes in vivo following ischemic or mechanical injury and in vitro by lipopolysaccharide or interleukin-1beta. In the present study, phorbol-12,13-dibutyrate (PDBu) was found to induce catalytically active CYP2E1 more than fourfold in cortical glial cultures. Little induction was seen up to 12 h, and full effects only at 21-24 h of PDBu treatment. CYP2E1 expression in PDBu-treated cells was enriched in a subset of astrocytes. The protein kinase C inhibitors, staurosporine and calphostin C, and the tyrosine kinase inhibitor genistein, but not its inactive analogue daidzein, prevented the induction of CYP2E1 by PDBu. It is suggested that CYP2E1, together with interleukin-6 and ciliary neurotrophic factor, is part of a response of astrocytes to cellular stress elicited by, e.g. cerebral injury, cytokines or phorbol ester, and mediated in part through protein kinase C.     

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca²⁺ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C , ₁, , , and / were all present in rat midbrain cytosolic extract, PKC , ₁, , and / were present in spleen cytosol, and PKC and / were present in COS 7 cell cytosol. The predominance of and activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay.The PKC activity assay was configured such that the Ca²⁺ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca²⁺-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca²⁺, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca²⁺ whereas in the presence of Ca²⁺, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca²⁺. Maximum values for Ca²⁺-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca²⁺-dependent PKC isoform activity in a Ca²⁺ free environment. The greater potency of mezerein on PKC was confirmed using PKC and further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca²⁺ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca²⁺-dependent isoforms in Ca²⁺ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca²⁺. The concentration-dependence of DOG was examined on PKC and PKC further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC . However, [³H]PDBu binding data using PKC purified from several sources gave very different IC₅₀ values when DOG was used as a displacer, and in general these values correlated with the EC₅₀ values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor.     

Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle.

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.     

The effect of acute and chronic electroconvulsive shock on [3H]phorbol-dibutyrate binding to rat brain membranes

The present study investigated the effect of single and repeated electroconvulsive shock (ECS) on proteinkinase C in rat cerebral cortex, cerebellum, hippocampus and striatum using [3H]Phorbol-12, 13-butyrate binding. In the postictal period and 24 hr after a single ECS there was no alteration in any brain region. Twenty four hr after 10 once-daily ECS there was a significant decrease the number of binding sites in cerebral cortex (30%) and in cerebellum (20%) without a change in the affinity constant. These findings are discussed with regard to earlier reports on phosphoinositide turnover following chemically and electrically induced seizures.