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.     

Phorbol esters inhibit agonist-induced [3H] inositol-1-phosphate accumulation in rat hippocampal slices

In rat hippocampal slices, carbachol and norepinephrine induce an accumulation of [3H]-inositol-1-phosphate which is markedly amplified in the presence of lithium. The tumor-promoting agents phorbol 12,13-dibutyrate (PDB) and 4 beta phorbol, 12 beta-myristate, 13 alpha-acetate (PMA) have no effect on [3H] inositol-1-phosphate accumulation alone, but when preincubated with hippocampal slices significantly inhibit the accumulation of [3H]-inositol-1-phosphate induced by carbachol and norepinephrine. The IC50 values for PDB and PMA are 0.2 microM and 25 microM respectively. In contrast, the weak tumor promoting agents 4-O-methylphorbol 12 myristate 13 acetate (MPMA) and phorbol 13,20-diacetate (P 13,20 DA) only slightly attenuate the agonist-induced response at concentrations less than or equal to 100 microM, whereas 4 alpha-phorbol (4 alpha-PHR), a biologically inactive phorbol, has no effect. These data suggest that phorbol ester receptor-mediated events may be negatively coupled to agonist-induced phosphatidylinositol hydrolysis.     

Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

The involvement of protein kinase C in the regulation of choline cotransport in Limulus.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.     

Protein Kinase C in Rat Brain Is Altered by Developmental Lead Exposure

The absence of learning-related redistribution of hippocampal protein kinase C (PKC) has been correlated with impairment of learning performance induced by developmental lead (Pb) exposure. This study was designed to examine whether the properties of brain PKC are altered by chronic Pb exposure during development. Two-tenth percent Pb acetate was administered to pregnant and lactating dams and then administered to weanlings in drinking water until postnatal day (PN) 56. Effects of Pb on translocation of PKC were studied in brain slices prepared from hippocampus. When the slices were treated with 0.33 M phorbol-12, 13-dibutyrate (PDBu) for 15 min, a significant increase in PKC activity was observed in the membrane fraction of hippocampal slices from Pb-exposed rats, suggesting that chronic Pb exposure potentiates PDBu-activated PKC translocation. Data obtained from saturation binding assays in the frontal cortices of Pb-exposed rats showed a decrease in the dissociation constant (K_D) in both membrane and cytosolic PKC. A decrease in the total binding sites (B_max) of [³H]PDBu binding was only observed in membrane PKC. Furthermore, developmental Pb exposure decreased PKC-, but not PKC-, -II, and - in the membrane fraction of the hippocampus and the frontal cortex. These results indicate that chronic Pb exposure during development increases phorbol ester binding affinity, enhances phorbol ester-induced translocation of PKC, and down-regulates membrane PKC, mainly PKC-.     

B-50/GAP-43 Phosphorylation and PKC Activity are Increased in Rat Hippocampal Synaptosomal Membranes After an Inhibitory Avoidance Training

Several lines of evidence indicate that protein kinase C (PKC) is involved in long-term potentiation (LTP) and in certain forms of learning. Recently, we found a learning-specific, time-dependent increase in [³H]phorbol dibutyrate binding to membrane-associated PKC in the hippocampus of rats subjected to an inhibitory avoidance task. Here we confirm and extend this observation, describing that a one trial inhibitory avoidance learning was associated with rapid and specific increases in B-50/GAP-43 phosphorylation in vitro and in PKC activity in hippocampal synaptosomal membranes. The increased phosphorylation of B-50/GAP-43 was seen at 30 min (+35% relative to naive or shocked control groups), but not at 10 or 60 min after training. This learning-associated increase in the phosphorylation of B-50/GAP-43 is mainly due to an increase in the activity of PKC. This is based on three different sets of data: 1) PKC activity increased by 24% in hippocampal synaptosomal membranes of rats sacrificed 30 min after training; 2) B-50/GAP-43 immunoblots revealed no changes in the amount of this protein among the different experimental groups; 3) phosphorylation assays, performed in the presence of bovine purified PKC or in the presence of the selective PKC inhibitor CGP 41231, exhibited no differences in B-50/GAP-43 phosphorylation between naive and trained animals. In conclusion, these results support the contention that hippocampal PKC participates in the early neural events of memory formation of an aversively-motivated learning task.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Flow threshold for enhanced phorbol ester binding in the ischemic gerbil brain

The correlation between regional phorbol ester binding and cerebral blood flow (CBF) was evaluated in the gerbil brain after 2-hour unilateral common carotid artery occlusion [³H]phorbol 12, 13-dibutyrate (PDBu) was used as a specific ligand for estimating the translocation of protein kinase C (PKC), and CBF was determined by the [¹⁴C]iodoantipyrine method. A quantitative autoradiographic method permitted concurrent measurement of these two parameters in the same brain. In the ischemia group of the animals, statistically significant, inverse correlations were noted between the CBF and PDBu binding in the hippocampus (CA₁ and CA₃ regions and dentate gyrus), the caudate-putamen and lateral nuclei of the thalamus. In these regions, the PDBu binding increased progressively as CBF fell below 35–40 ml/100 g/min. On the other hand, the PDBu binding in the cerebral cortices did not show any significant changes even when CBF was decreased to below 35 ml/100 g/min. The above data suggest that (1) the translocation of PKC to the cell membrane may be regionally specific in response to ischmia and may remain in the regions particularly vulnerable to ischemia such as the hippocampus, caudate-putamen and lateral nuclei of the thalamus in the early ischemic phase; (2) the threshold of CBF below which PKC begins to translocate to the cell membrane in the above regions, may be 35–40 ml/100 g/min in 2-hour ischemia.     

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.     

Synthesis and Tumor-promoting Activities of 12-Epi-phorbol-12,13-dibutyrate

12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O₂ ^-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the β-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.     

Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum

Abstract: The effects of lithium on muscarinic cholinoceptor-stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [³H]inositol or [³H]cytidine prelabelling and inositol 1,4,5-trisphosphate [lns(1,4,5)P₃] and inositol 1,3,4,5-tetrakisphosphate [lns(1,3,4,5)P₄] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P₃ and lns(1,3,4,5)P₄ mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30-min incubation period. Carbachol-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations were inhibited by lithium in all brain regions studied in a time-and concentration-dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC₅₀ values for inhibition of agonist-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [³H]CMP-phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo-inositol to reverse these effects, as well as lithium-suppressed lns(1,4,5)P₃ accumulation, implicates myo-inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [³H]inositol monophosphate and [³H]CMP-phosphatidate levels and inhibitions of lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations. However, the inability of striatal slices to sustain carbachol-stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo-inositol may indicate differences in phosphoinositide signalling in this brain region.     

Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Effect of Brain Ischemia on Protein Kinase C

We examined the influence of brain ischemia on the activity and subcellular distribution of protein kinase C (PKC). Two different models of ischemic brain injury were used: postdecapitative ischemia in rat forebrain and transient (6-min) cerebral ischemia in gerbil hippocampus. In the rat forebrain model, at 5 and 15 min postdecapitation there was a steady decrease of total PKC activity to 60% of control values. This decrease occurred without changes in the proportion of the particulate to the soluble enzyme pools. Isolated rat brain membranes also exhibited a concomitant decrease of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding with an apparent increase of the ligand affinity to the postischemic membranes. On the other hand, the ischemic gerbil hippocampus model displayed a 40% decrease of total PKC activity, which was accompanied by a relative increase of PKC activity in its membrane-bound form. This resulted in an increase in the membrane/total activity ratio, indicating a possible enzyme translocation from cytosol to the membranes after ischemia. Moreover, after 1 day of recovery, a statistically significant enhancement of membrane-bound PKC activity resulted in a further increase of its relative activity up to 162% of control values. In vitro experiments using a synaptoneurosomal particulate fraction were performed to clarify the mechanism of the rapid PKC inhibition observed in cerebral tissue after ischemia. These experiments showed a progressive, Ca(2+)-dependent, antiprotease-insensitive down-regulation of PKC during incubation. This down-regulation was significantly enhanced by prior phorbol (PDBu) treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester receptors in cerebral cortex of cats with GM1 gangliosidosis

The pathogenesis of neuronal dysfunction in the gangliosidoses is poorly understood. Studies of the feline gangliosidoses and in vitro experiments implicate ganglioside inhibition of protein kinase C (PKC) in the pathogenesis of these neurological diseases. Therefore, in the present study, the binding of [³H]phorbol-12, 13 dibutyrate was measured to determine the levels of PKC in cerebral cortex of cats with GM₁ gangliosidosis (mutant) and age matched normal siblings. This binding of ([³H]PDB) to cerebral cortex homogenates in both normal and mutant cats was highly specific. The specificity of receptors was ascertained also from displacement studies using nonradioactive phorbol ester analogues to displace [³H]PDB bound to its receptors. In both mutant and normal cat brain, phorbol 12, 13-dibutyrate (PDB), 4--phorbol 12,13-didecanoate (-PDA) and 4--phorbol 12,13-dibenzoate (-PDBz) were highly potent (approximately to same degree) and effective in displacing [³H]PDB. On the other hand, 4- phorbol 12,13-diacetate (-PDA) was a weak displacer and 4--phorbol did not displace the bound [³H]PDB in either normal or mutant brain. Scatchard analysis of the binding data indicated a homogenous single class of binding sites in normal and mutant brain (Normal: K_d=1.42×10^–7 M, B_max=8.40 pmoles/mg protein. Mutant: K_d=1.60×10^–7 M, B_max=10.00 pmoles/mg protein). Sphingosine inhibited the binding to approximately the same extent in normal and mutant cortex. These studies demonstrate the presence of highly specific, homogenous, single type phorbol ester receptors in cerebral cortex of cats with GM₁ gangliosidosis which are qualitatively and quantitatively similar to normal cat brain.     

Regulation of the Na+-Dependent Glutamate/Aspartate Transporter in Rodent Cerebellar Astrocytes

The regulation of the Na⁺-dependent glutamate/aspartate transporter system GLAST expressed in rat and mouse cerebellar and cortical astrocytic cultures was examined. Pretreatment of the cerebellar cells with l-glutamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known Ca²⁺/ diacylglicerol-dependent protein kinase (PKC) activator, produced a decrease in [³H]-d-aspartate uptake. This reduction was dose- and time-dependent and sensitive to PKC inhibitors. Furthermore, the l-glutamate–dependent [³H]-d-aspartate uptake decrease is a non-receptor dependent process, because neither of the agonists or antagonists were effective in mimicking or reverting the effect. Interestingly, transportable substrates could reproduce the l-glutamate effect. In sharp contrast, in cortical astrocytes, both l-glutamate and TPA pre-exposure result in an augmentation of the [³H]-d-aspartate uptake. These findings suggest that the Na⁺-dependent glutamate uptake GLAST undergoes a region-specific regulation.     

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 phosphatidyl-serine (PS) or the synthetic diacylglycerol, DiC8, 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 reduced the epidermal growth factor binding of MCF-7 cells to an extent similar to that found with DiC8 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 (muramyl-tripeptide) 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.     

Changes in [3H]Forskolin Binding to Adenylate Cyclase and [3H]Phorbol Dibutyrate Binding to Protein Kinase C in Pentobarbital Tolerant/Dependent Rats

These studies were designed to examine the effect of chronic administration of pentobarbital on activity of adenylate cyclase (AC) and protein kinase C (PKC) in the rat brain by autoradiography. Recently, it has been suggested that the phosphorylation of specific proteins may be involved in the development of physical dependence. An experimental model of barbiturate tolerance and dependence was developed using i.c.v. infusion of pentobarbital (300 g/ 10 l/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [³H]forskolin binding were elevated (28–67%) in cortex, thalamus, dentate gyrus, hippocampal CA3 and cerebellum of the pentobarbital withdrawal animals, while these changes were not observed in tolerant rats. The levels of [³H]phorbol dibutyrate binding were highly elevated (38–65%) in the region of cortex, caudate putamen, septum, thalamus, dentate gyrus, and cerebellum of rats withdrawal from pentobarbital. These results show that the levels of AC and PKC were significantly elevated in pentobarbital withdrawal rats, and suggest that the levels of AC and PKC are altered in a region-specific manner during pentobarbital withdrawal.