Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: real-time analysis using fluorescent ligands and proteins.

The diacylglycerol signaling pathway, involving protein kinase C (PKC) and RasGRP, is a promising therapeutic target for both cancer and other indications. The phorbol esters, ultrapotent diacylglycerol analogues, bind to and activate PKC and RasGRP. Here, using fluorescent phorbol esters and complementary fluorescent PKC and RasGRP constructs, we determined the localization of the phorbol ester as a function of time after addition and how the resultant PKC or RasGRP3 translocation related to ligand localization. For these studies, we prepared fluorescently labeled phorbol esters of varying lipophilicities based on the BODIPY FL (green) or BODIPY 581/591 (red) fluorophores, and by using fusion constructs of green fluorescent protein or DsRed with PKC isoforms or RasGRP3 expressed in Chinese hamster ovary cells, we simultaneously compared the kinetics and pattern of localization of PKC or RasGRP3 with that of the fluorescent red or green phorbol esters. Binding assays showed that the fluorescent derivatives were potent ligands. Uptake followed a one-compartment pharmacokinetic model with a half-time of minutes to hours, depending on the ligand, and all of the fluorescent phorbol esters localized primarily to intracellular membranes, with little plasma membrane localization. The fluorescent phorbol esters induced translocation of and generally colocalized with PKCdelta or RasGRP3. However, PKCalpha and, initially, PKCdelta, translocated to the plasma membrane, in which little phorbol ester accumulated. The findings argue that the rate of uptake of phorbol esters influences the subsequent pattern of PKCdelta translocation, and that the specificity for PKCalpha translocation is dominated by factors other than the localization of the ligand.

An optimized protein kinase C activating diacylglycerol combining high binding affinity (Ki) with reduced lipophilicity (log P).

A small, focused combinatorial library encompassing all possible permutations of acyl branched alkyl chains-small and large, saturated and unsaturated-was generated from the active diacylglycerol enantiomer (S-DAG) to help identify the analogue with the highest binding affinity (lowest Ki) for protein kinase C (PK-C) combined with the minimum lipophilicity (log P). The selected ligand (3B) activated PK-C more effectively than sn-1,2-dioctanoylglycerol (diC8) despite being 1.4 log units more hydrophilic. Compound 3B indeed represents the most potent, hydrophilic DAG ligand to date. With the help of a green fluorescent protein (GFP)-tagged PK-Calpha, 3B was able to translocate the full length protein to the membrane with an optimal dose of 100 microM in CHO-K1 cells, while diC8 failed to achieve translocation even at doses 3-fold higher. Molecular modeling of 3B into an empty C1b domain of PK-Cdelta clearly showed the existence of a preferred binding orientation. In addition, molecular dynamic simulations suggest that binding discrimination could result from a favorable van der Waals (VDW) interaction between the large, branched sn-1 acyl group of 3B and the aromatic rings of Trp252 (PK-Cdelta) or Tyr252 (PK-Calpha). The DAG analogue of 3B in which the acyl groups are reversed (2C) showed a decrease in binding affinity reflecting the capacity of PK-C to effectively discriminate between alternative orientations of the acyl chains.

Structural determinants of phorbol ester binding in synaptosomes: pharmacokinetics and pharmacodynamics.

The present study used structurally distinct phorbol esters to investigate the relationship between their pharmacokinetics of binding to protein kinase C (PKC) in rat brain cortex synaptosomes, their affinity for PKC in synaptosomes and ability to enhance noradrenaline release from rat brain cortex. Affinity binding studies using [3deoxyphorbol 13-tetradecanoate (dPT)=PDB&z. Gt;12-deoxyphorbol 13-acetate (dPA)=phorbol 12,13-diacetate (PDA). In intact synaptosomes PDB, dPA and PDA rapidly displaced bound [3H]PDB whereas PMA and dPT were comparatively slow. However, the displacement rates for all the phorbol esters were equally rapid in synaptosomal membranes or synaptosomes permeabilised with Staphylococcus alpha-toxin. These results suggest that the lipophilic phorbol esters (dPT and PMA) are slower to displace [3H]PDB binding because they are hindered by the plasma membrane. In brain cortex slices it was found that the rate of displacement of [3H]PDB binding was closely correlated with the degree of elevation of transmitter noradrenaline release. Thus kinetic characteristics may determine biological responses and this may be particularly evident in events which occur rapidly or where there is fast counter-regulation.

Distribution of protein kinase C in the brain of Apteronotus leptorhynchus as revealed by phorbol ester binding.

An antibody to the mammalian protein kinase C alpha (PKCalpha) subunit and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 80 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in gymnotiform brain. The 80-kDa band was enriched in fish forebrain and cerebellum compared with hypothalamus and brainstem areas. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was used as a marker for the distribution of protein kinase C (PKC). [3H]PDBu binding was nearly completely displaced by excess cold PDBu; specific [3H]PDBu binding sites were heterogenously distributed with high densities in some gray matter regions and negligible densities in fiber tracts. A very high density of [3H]PDBu binding sites were found in the dorsal forebrain with far lower densities in most ventral forebrain nuclei. Low binding densities were observed in preoptic and hypothalamic areas with the exception of the nucleus diffusus and nucleus tuberis anterior. The thalamus and midbrain also had only low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was negligible. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal molecular layer, which contains cerebellar parallel fibers; the other layers of the ELL had far lower binding densities.

Conformationally constrained analogues of diacylglycerol having a perhydrofuro[3,4-c]furan-1,4-dione bis-gamma-butyrolactone skeleton.

Bis-gamma-lactones (1, 2) having a perhydrofuro[3,4-c]furan-1,4-dione skeleton were designed as conformationally constrained diacylglycerol analogues. They were synthesized from D-apiose in 11 steps, and evaluated as PKC-alpha ligands by measuring their ability to displace bound [3H]-PDBU from the enzyme. The compounds showed moderate binding affinities with Ki values of 13.89 (+/- 5.67) microM and 11.47 (+/- 0.89) microM, respectively. Their similar binding affinities indicate that these two bicyclic compounds were not effectively discriminated by PKC-alpha in terms of the direction of the side chain as other ligands built on similar bis-gamma-lactones.

Protein kinase C in focal ischemic rat brain: dual autoradiographic analysis of [14C]iodoantipyrine (IAP) and [3H]phorbol-12,13-dibutyrate (PDBu).

Protein kinase C (PKC) activity was measured in rat brain with 2 h of middle cerebral artery (MCA) and common carotid artery (CCA) occlusion, using dual autoradiography of [14C]iodoantipyrine (IAP) and [3H]phorbol-12,13-dibutyrate (PDBu). In the ischemic brain, it required more than 120 min of incubation to obtain a plateau in PDBu binding. In contrast, the binding of PDBu in non-ischemic brain reached a plateau with incubation for 60 min. This delay of PDBu binding in the ischemic brain suggests that the affinity of this ligand is reduced due to a change in structure of the cell membrane caused by ischemia. PDBu binding in the ischemic brain increased significantly compared to the non-ischemic brain. This finding provides further evidence that excessive activation of PKC in the ischemic brain may play an important role in ischemic neuronal damage.

Changes in protein kinase C and adenylate cyclase in the temporal lobe from subjects with schizophrenia.

Changes in G-protein linked neurotransmitter receptors have been reported in a number of regions of the brain of schizophrenic subjects. These changes, if functional, could cause a change in proteins such as protein kinase C (PKC) and adenylate cyclase (AC) which are important components of the G-protein linked second messenger cascades. We therefore used autoradiography to measure the distribution and density of [3H]phorbol ester binding to PKC and [3H]forskolin binding to AC in tissue obtained at autopsy from schizophrenic and non-schizophrenic subjects (Controls). There were significant decreases in the density of PKC in the parahippocampal gyrus (687 +/- 60 vs. 885 +/- 51 fmol/mg TE; mean +/- SEM; p < 0.01) and in AC in the dentate gyrus (75 +/- 4.9 vs. 92 +/- 6.5, p < 0.05) from the schizophrenic subjects. These data could indicate that changes in neurotransmitter receptors in the hippocampus from subjects with schizophrenia could have resulted in a change in their associated second messenger systems.

Alterations of local cerebral blood flow, phorbol 12,13-dibutyrate binding activity, and histological damage during acute focal ischaemia in rat brain. A pathophysiology of acute focal ischaemia: Part 1.

The alterations of the local cerebral blood flow (LCBF), 3H-phorbol 12,13-dibutyrate (PDBu) binding activity were measured, and histological findings were also examined during the closed time course (0, 1, 3, 5, 7 hour) after middle cerebral artery occlusion (MCAO) in rat brain to assess the complex pathophysiology of acute focal ischaemia. From 1 to 3 hours after the start of MCAO, significant (p < 0.01) hyperreactivity of the second messenger system involving PDBu binding may be present, despite low perfusion of LCBF, and severe damage in the striatum whereas sparing almost completely the cortex on histological examination. At 5 hours, the PDBu binding activity increased slightly but not significantly but is reduced markedly at 7 hours after MCAO compared with the control group. The measurement of PDBu binding activity, additionally to measuring the LCBF and observation of the histological change might be a useful indicator in determining the threshold and duration of ischaemia which cause functionally irreversible cell damage in the brain.

Characterization by [3H]resiniferatoxin binding of a human vanilloid (capsaicin) receptor in post-mortem spinal cord.

Membranes obtained from post-mortem human spinal cord specimens bound [3H]resiniferatoxin (RTX) with an affinity of 11 nM in a non-cooperative fashion. This binding behaviour contrasted with the high affinity [3H]RTX binding (Kd = 24 pM) to rat spinal cord membranes which displayed apparent positive cooperativity (cooperativity index = 1.8) but was in accord with the low affinity (Kd = 5 nM) non-cooperative RTX binding to guinea pig spinal cord preparations. We conclude that the [3H]RTX binding assay utilizing post-mortem human spinal cord membranes affords a novel biochemical approach to explore structure-activity relations at human vanilloid receptors.

Heterologous sensitization of adenylate cyclase activity by serotonin in the rat cerebral cortex.

In vitro exposure of rat cerebrocortical slices to microM concentrations of serotonin (5HT) results in an increased response of adenylate cyclase to isoproterenol (ISO). No change in the affinity of the beta-adrenoceptor toward the agonist was found after 5HT exposure when measuring ISO displacement of [3H]CGP 12177 binding. A similar increase of adenylate cyclase response was also found when using VIP as a stimulatory agent. The dose-response curve of adenylate cyclase to the GTP analogue, GppNHp, was modified by 5HT, which promotes a significantly higher maximal response without altering the potency of GppNHp. Forskolin-stimulated adenylate cyclase activity was not affected by 5HT. Serotonergic 5HT2 receptors are involved in the sensitization of adenylate cyclase to GppNHp, since the selective 5HT2 antagonist ketanserin inhibits the effect of 5HT, whereas the 5HT2 agonist DOI mimics 5HT. The involvement of 5HT2 receptor-coupled activation of protein kinase C is also demonstrated: direct protein kinase C activators such as phorbol esters and s,n-dioctanoylglycerol behave in the same manner as 5HT, while the protein kinase C inhibitor CGP 41251 prevents 5HT from increasing adenylate cyclase responsiveness to GppNHp. Moreover, in vitro exposure of cortical slices to 5HT results in reduced inhibition of adenylate cyclase by somatostatin. Since no change was observed at the receptor level and in the direct stimulation of the catalytic subunit of the enzyme, we propose that 5HT might accomplish the sensitization of adenylate cyclase through protein kinase C by inactivating the inhibitory coupling protein Gi and facilitating the interaction of the exogenous GppNHp with the stimulatory coupling protein Gs.

Environmental stimulation promotes changes in the distribution of phorbol ester receptors.

The translocation of protein kinase C (PKC) from the cytosol to the membrane might be functionally involved in learning and memory. Using [3H]-phorbol 12,13-dibutyrate (3H-PDBu) binding three pools of binding sites could be distinguished in tissue preparations: Pool a comprised the soluble receptors which bound phorbol ester with low affinity in the absence of calcium. Pool b was composed of high-affinity phorbol ester binding sites identified in the soluble fraction upon addition of calcium. Pool c represented stably membrane-bound receptors binding phorbol ester independently of calcium. 3H-PDBu binding was then measured in the cortices and hippocampi of rats trained in an eight-arm radial maze. A progressive training-dependent increase of membrane-bound binding activity with a concomitant decrease in the soluble fraction was detected independent of learning the maze task. These results suggest that it is the experience of an enriched environment by the repeated behavioral stimulation in a maze rather than the acquisition of a memory task that leads to enhanced incorporation of phorbol ester receptors (PKC) into the cell membrane.

Immobilization stress induces alterations of second-messenger systems in the gerbil brain.

The effects of immobilization stress on the cerebral second messenger (adenylate cyclase and phosphoinositide) were investigated autoradiographically in mongolian gerbils. After 10 min (10-min stress group, n = 7), or after 6 h (6-h stress group, n = 7) of fixation on a flat board while supine, in vitro autoradiography was performed using [3H]forskolin (3H-FK) and [3H]phorbol-12,13-dibutyrate (3H-PDBu) as specific ligands to identify the distribution of adenylate cyclase and protein kinase C, respectively. In another group of 7 gerbils (control group), the same autoradiographic procedure was performed immediately after the animals were removed from the cage. In the 10-min stress group, FK binding was significantly decreased in the hypothalamus and amygdala, but significantly increased in the basal ganglia including the caudate-putamen and globus pallidus. FK binding in the 6-h stress group tended to increase throughout the brain, rising significantly in the basal ganglia. PDBu binding in either stress group did not change significantly compared to the control group in any region except the hippocampal CA3 region of the 6-h stress group. Under immobilization stress, the adenylate cyclase system may undergo time-dependent and regionally specific changes, while the phosphoinositide system remains relatively stable.

Second messenger systems in brains of patients with Parkinson's or Huntington's disease.

Alterations in protein kinase C (PKC) and myo-inositol 1,4,5-trisphosphate (IP3) receptors were studied in the autopsied human striata from 21 patients with Parkinson's disease (PD) (Yahr III, IV, and V), 8 patients with Huntington's disease (HD), and 23 age-matched and postmortem time-matched nonneurological controls. The concentrations of PKC and IP3 receptors were determined using [3H]4 beta-phorbol 12,13-dibutyrate (PDBu) and [3H]IP3 as respective ligands. Both the specific [3H]-PDBu and [3H]IP3 bindings were significantly reduced in the striata of Yahr V patients with dementia (PDD) and in that of HD patients, as compared to findings in the controls. These bindings were unchanged when all the PD patients without dementia, Yahr (III plus IV) patients, or Yahr V patients without dementia were compared with evidence from the controls. Immunoquantification of four PKC subspecies (alpha, beta I, beta II, and gamma) in the HD putamen revealed a selective reduction in the beta II-PKC immunoreactions. These results are supported by immunohistochemical findings in the rat brain that beta II-PKC is expressed in the striatal gabaergic efferent pathway, while the alpha-PKC is present in the nigrostriatal dopaminergic neurons. The neurochemical pathophysiology of PD differs between patients with and without dementia.

Cholinergic denervation alters [3H]phorbol-12,13-dibutyrate binding to rat hippocampal membranes.

Cholinergic denervation of the rat hippocampus caused by electrolytic lesions of the medial septum (MS) results in a time-bound ingrowth of peripheral sympathetic noradrenergic fibers from the superior cervical ganglion to the dentate gyrus and CA3 region of the hippocampus. To determine the functional significance of hippocampal sympathetic ingrowth (HSI), [3H]phorbol-12,13-dibutyrate (PDBu) binding was assessed 4 weeks after MS lesions. In control animals, affinity for [3H]PDBu binding was found to be greater in the dorsal compared to ventral hippocampus, while the number of binding sites (Bmax) was similar between regions. Regardless of the presence of HSI, MS lesions resulted in increased affinity in the dorsal hippocampus, while the Bmax was found to 'normalize' in the ventral hippocampus by HSI. These results suggest that HSI is functional and can alter important cellular events.

The role of the phorbol ester receptor/protein kinase C in the sensitivity of leukemic cells to anthracyclines.

The phorbol ester receptor (PER), which is believed to be identical to protein kinase C (PKC), has been implicated in the control of the sensitivities of tumor cells to certain forms of cancer chemotherapy. We evaluated the effects of regulating the expression of PER/PKC on the sensitivity of the R1B6 subclone of HL-60 human promyelocytic leukemic cells to anthracyclines. This cell line is resistant to the effects of phorbol esters on cellular differentiation by virtue of a down-regulated PER. R1B6 cells were maintained in phorbol 12, 13 dibutyric acid (PDBu). Twenty four to thirty six hours after removal of PDBu from the medium there was a 3-fold increase in the number of receptors compared to baseline values measured by [3H]-PDBu binding and a marked increase in the activity of PKC as measured by calcium-phospholipid dependent incorporation of [32P] into histone. Despite these changes in the number of PER and in the activity of PKC there was no difference in the cellular accumulation of [3H]-daunomycin or in the sensitivity of cells to the toxic effects of doxorubicin. Furthermore, there was no difference between the R1B6 cells and the parental HL-60 line in their intrinsic sensitivities to doxorubicin. These studies demonstrate that alterations in the expression of PER and the activity of PKC alone are not sufficient to influence anthracycline accumulation or toxicity in R1B6 human leukemic cells in contrast to recently reported results using other cell lines.

Bicarbonate dependence of cAMP accumulation induced by phorbol esters in hamster spermatozoa.

Phorbol esters stimulate cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in hamster spermatozoa under conditions for in vitro capacitation. The 20-50-fold elevation of cAMP levels induced by 1 microM phorbol 12-myristate 13-acetate (PMA) in spermatozoa depends on the presence of sodium bicarbonate in the medium (ED50: 15 mM) and it is independent of extracellular pH. Sodium bicarbonate stimulates adenylate cyclase activity in membrane preparations by 4-fold (ED50: 40 mM). After solubilization, the bicarbonate-sensitive moiety elutes as a single peak of 55 kDa in a gel filtration column. Blockers of bicarbonate chloride antiporters diisothiocyanate stilbene 2,2'-disulfonic acid (DIDS) or acetamido 4'-isothiocyanate stilbene 2,2'-disulfonic acid (SITS) inhibit the bicarbonate dependent PMA effect on cAMP in living spermatozoa (ED50: 100 microM). Maximal (85%) inhibition in cAMP accumulation is observed at 1 mM. Motility is inhibited only at high concentrations of the blockers. Pretreatment of living cells with 1 mM DIDS does not affect membrane adenylate cyclase activity which remains responsive to bicarbonate. These results suggest that controlled transport of bicarbonate through the sperm plasma membrane could be associated to the regulation of cAMP synthesis.

Protein kinase C is localized in focal contacts of normal but not transformed fibroblasts.

Transformed cells differ from normal cells in that they fail to respond to normal signals for regulation of growth and differentiation. This disordered signal transduction probably contributes to maintenance of the transformed phenotype. Several lines of evidence suggest that changes in the Ca2(+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), may be important for transformation. To determine the role of PKC in transformation, we compared the levels and subcellular distribution of total phorbol ester receptors and PKC in normal and SV40-transformed rat embryo fibroblasts (REF52 cells). We also used our alpha-PKC (Type 3)-specific monoclonal antibodies to compare alpha-PKC content and regulation. We found no differences in quantity or subcellular distribution of PKC in 100,000 x g soluble and pelletable fractions. Downmodulation, which represents a feedback loop for limiting PKC activity, occurs to the same extent in both cell types. A major difference between the normal and transformed cells was revealed by immunofluorescence of alpha-PKC. In normal cels, alpha-PKC is tightly associated with the cytoskeleton and appears to be organized into focal contacts because it colocalizes with talin. In contrast, in SV40-REF52 cells, alpha-PKC is not tightly associated with the cytoskeleton and does not colocalize with talin. The difference in subcellular localizations correlates with a loss of two alpha-PKC-binding proteins in the transformed cells. These results indicate that inappropriate subcellular location of alpha-PKC may contribute to maintenance of the transformed phenotype.