Transthoracic vertebrectomy for metastatic spinal tumors

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC alpha along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC epsilon. In contrast, thrombin had a smaller effect on nuclear translocation of PKC alpha and did not influence PKC epsilon, but instead induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC alpha and epsilon within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms, alpha, delta, epsilon and zeta in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC alpha and epsilon to the perinuclear region and into the nucleus, while PKC delta and zeta showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC alpha and epsilon was reduced to or below control values. At 8 h, increased nuclear expression of isoforms alpha, epsilon and zeta was observed, while isoform delta was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.     

The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications

Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.     

The influence of oral glucose intake on binding and degradation of 125I-insulin by receptors on erythrocytes as well as on insulin and C-peptide serum levels in patients after myocardial infarction and healthy individuals

In this study, we investigated the influence of glucose administration on binding and degradation of 125I-insulin by receptors on erythrocytes as well as on insulin and C-peptide serum levels in 15 patients after myocardial infarction and in 15 age-matched healthy persons. Venous blood samples were taken directly before and at 30, 60 and 120 minutes after oral administration of 75 g of glucose. In the collected blood samples serum glucose, insulin and C-peptide levels were determined. Binding and degradation of 125I-insulin by specific receptors on red blood cells were evaluated using the method described by Gambhir and modified by the authors. Serum insulin and C-peptide levels were significantly higher while binding of 125I-insulin to erythrocytes was decreased in patients after myocardial infarction. These results seem to support the hypothesis that insulin resistance and hyperinsulinism play a role in the pathogenesis of ischaemic heart disease. Impaired degradation of 125I-insulin during the oral glucose tolerance test in the patients after myocardial infarction indicates that insulin resistance is located at the receptor level.     

Cytosolic and nuclear mitogen-activated protein kinases are regulated by distinct mechanisms

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPPK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.     

Chronic effects of glucose on insulin signaling in A-10 vascular smooth muscle cells

To examine the effects of hyperglycemia on insulin signaling in A-10 vascular smooth muscle cells, cells were treated with extracellular D-glucose and effects of insulin were studied on the diacylglycerol-protein kinase C signaling system. A-10 cells specifically bound 125I-insulin, and insulin-like growth factor-I did not displace the label. 125I-insulin binding was unaltered under hyperglycemic conditions. To determine if insulin receptors were coupled to other insulin-regulated processes, diacylglycerol, protein kinase C, and glucose transport were evaluated. Insulin increased cellular diacylglycerol (DAG) levels which were also increased following glucose treatment and not further stimulated by insulin. The uptake of 2-[3H]deoxy-D-glucose (2-DOG) was stimulated by insulin and 12-O-tetradecanoyl phorbol 13-acetate (TPA). Insulin- and TPA-stimulated 2-[3H]DOG uptake was inhibited by a protein kinase inhibitor, staurosporine. Preincubation of cells with 500 nM TPA overnight resulted in the inhibition of insulin- and TPA-stimulated 2-[3H]DOG uptake. Protein kinase C activity was translocated from cytosolic to membrane fractions following insulin treatment. Overnight glucose (25 mM) treatment resulted in a 50% decrease in protein kinase C enzyme activity and > 90% decrease in protein kinase C beta immunoreactive levels. Protein kinase C activity and levels were not affected by osmotic control media containing mannitol. A-10 cells express GLUT4-type glucose transporters. Neither insulin-regulatable glucose transporter (GLUT4) mRNA nor GLUT4 protein levels were diminished by glucose. Significant decreases in insulin- and TPA-stimulated 2-[3H]DOG uptake occurred, however, with glucose. The down-regulation of protein kinase C beta and resultant inhibition of 2-[3H]DOG uptake by chronic glucose suggests a biochemical link between hyperglycemia and DAG-protein kinase C signaling in vascular smooth muscle cells.     

Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation

Excessive cerebral accumulation of the 42-residue amyloid beta-protein (Abeta) is an early and invariant step in the pathogenesis of Alzheimer's disease. Many studies have examined the cellular production of Abeta from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how Abeta is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted Abeta under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic Abeta at picomolar to nanomolar concentrations was completely inhibited by the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody removed its Abeta-degrading activity. IDE was present in BV-2 cytosol, as expected, but was also released into the medium by intact, healthy cells. To confirm the extracellular occurrence of IDE in vivo, we identified intact IDE in human cerebrospinal fluid of both normal and Alzheimer subjects. In addition to its ability to degrade Abeta, IDE activity was unexpectedly found be associated with a time-dependent oligomerization of synthetic Abeta at physiological levels in the conditioned media of cultured cells; this process, which may be initiated by IDE-generated proteolytic fragments of Abeta, was prevented by three different IDE inhibitors. We conclude that a principal protease capable of down-regulating the levels of secreted Abeta extracellularly is IDE.     

Polar redistribution of 125I-labelled insulin on the plasma membrane of cultured human lymphocytes

Cultured human lymphocytes of the IM-9 line have specific binding sites for 125I-insulin that have been characterized in detail. When the hormone--cell interaction was studied by quantitative electron microscopic (EM) autoradiography, it was shown that initial binding was to the plasma membrane and that at 37 degrees C a small fraction of the ligand was internalized by the cell. The internalized ligand was found preferentially localized in lysosomes in the Golgi region of the cell. It is well known that di- or multivalent ligands redistribute in the plane of the membrane before internalization. We now report that following initial binding the univalent ligand insulin also undergoes a polar redistribution ('capping') before internalization.     

Mitogen receptors in chick embryo fibroblasts. Kinetics, specificity, unmasking, and synthesis of 125I-insulin binding sites

Insulin, a mitogen for cultured chick embryo fibroblasts (Temin, H.M. (1968) Cancer 3, 771-787), has been employed to characterize the effects of mitogen/cell membrane interactions as it relates to growth. The specific binding of 125I-insulin to substratum-attached cells is time- and temperature dependent and is optimum at a pH of 7.0. Fetal calf and chicken sera, somatomedin "A/C mixed," and desalanine or native porcine insulin compete with 125I-insulin for membrane-binding sites. Proinsulin, although competing less effectively than native insulin for binding, is more effective than desoctapeptide insulin. Unrelated polypeptide hormones do not compete for 125I-insulin binding. The lowest concentration of insulin at which specific binding is detected is 0.1 nM. Scatchard plot analysis of the binding data indicates that there are two types of binding sites in confluent cultures of fibroblasts: one of high affinity (K1 = 2 to 6 X 10(8) M-1) and low capacity, the other of low affinity (K2 = 0.8 to 3.0 X 10(7) M-1) and high capacity. Approximately 1.9 and 7.1 X 10(3) molecules of insulin are bound at each site, respectively. A 10-min incubation at 24 degrees of the fibroblasts with 10 mug/ml of trypsin causes a 2-fold stimulation of specific 125I-insulin binding and a similar 2-fold increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation. Neuraminidase treatment also produces a 37% increase in specific 125I-insulin binding but treatment with alpha-chymotrypsin or phospholipase C are without significant effect. The results of this and additional experiments support the hypothesis that trypsin treatment of chick embryo fibroblasts leads to an unmasking of 125I-insulin binding sites. Serum starvation of fibroblasts for 12 or 24 h produces a 2.5- to 5-fold increase in specific 125I-insulin binding. This increase is the result of an increase in the number of hormone-binding sites from 9 X 10(3) to 6 X 10(4) per cell which are predominantly of the low affinity type. There is no change in the affinity constants. The presence of camptothecin, or cordycepin, or cycloheximide in the incubation medium completely blocks the increase in number of 125I-insulin-binding sites resulting from serum starvation. The addition of native insulin to the medium of serum-starved cultures also blocks this increase. The magnitude of insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation correlates with the levels of occupancy of the low affinity 125I-insulin-binding sites in untreated fibroblasts. In fibroblasts cultured in the absence of serum, the marked increase in insulin-stimulated 2-deoxy-D-glucose uptake and thymidine incorporation parallels the increase in number of mitogen receptors. The concentration of insulin that produces a half-maximum stimulation of thymidine incorporation is calculated to be 5 X 10(-8) M. At this concentration of insulin, 42% of the receptor sites are occupied.     

Insulin-like growth factors cross the blood-brain barrier

Although evidence exists that insulin may cross the blood-brain barrier, little is known about the ability of insulin-like growth factors (IGF-I and -II) to cross this barrier. In the present studies, equimolar concentrations of equal specific activity 125I-labeled IGF-I, IGF-II, or insulin were infused into the carotid artery of anesthetized adult rats. The perfusions were carried out for 3 min in the presence or absence of excess unlabeled ligand or insulin, with three or more animals in each group. Immediately after the perfusion, brains were frozen and sectioned for autoradiography. All ligands were detected in choroid plexus, median eminence, and blood vessels, but [125I]IGF-I and -II were also prominently localized in brain parenchyma. Densitometric analysis of film autoradiographs (28-day exposure for all ligands) revealed that radiolabeled IGFs, especially IGF-I, were significantly more abundant throughout the forebrain than [125I]insulin, especially in the paraventricular nucleus, where [125I]IGF-I was 10-fold and [125I]IGF-II was 5-fold more abundant than [125I]insulin. The difference in [125I]IGF-I vs. [125I]insulin accumulation was confirmed by parallel measurements of radioactivity in anatomically matched brain sections using a gamma-spectrometer. The uptake of radiolabeled IGF-I, IGF-II, and insulin by brain parenchyma and vasculature was completely inhibited by excess (1,000-fold) unlabeled ligand; however, insulin (10,000-fold excess) did not completely abolish [125I]IGF-I and -II accumulation. Microscopic evaluation of nuclear emulsion-coated brain sections revealed that radioactivity associated with [125I]IGF-I and -II perfusions was selectively concentrated in capillaries and medium-sized parenchymal cells in the paraventricular nucleus and, to a lesser extent, the supraoptic nucleus and anterior nucleus of the thalamus, whereas in other brain regions the radioligands were mostly bound to capillaries. These results suggest that radiolabeled IGF-I and -II bind to brain capillaries and cross the blood-brain barrier into brain parenchyma more readily than radiolabeled insulin.     

A cytosolic activity distinct from crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.     

Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris

Exogenous angiogenin undergoes rapid nuclear translocation in cultured human umbilical artery endothelial cells at 37 degrees C but not at 4 degrees C. Treatment of cells with colchicine, nocodazole and taxol, which disrupt the microtubule system, does not affect the nuclear translocation process of angiogenin, suggesting that cells transport internalized angiogenin in a microtubule independent fashion. Lysosomal inhibitors, chloroquine and leupeptin, neither inhibit nor enhance the nuclear translocation of angiogenin, indicating that lysosomal targeting and processing are not required for, and do not compete with, the nuclear translocation. Moreover, treatment of cells with a tyrosine kinase antagonist, genistein, does not change the ability of the cells to translocate angiogenin into the nucleus. We suggest that exogenous angiogenin is translocated to the nucleus by a mechanism that does not require activation of tyrosine kinase, but includes receptor-mediated endocytosis, microtubule and lysosome independent transport across the cytoplasm, and nuclear localization sequence-assisted nuclear import.     

Distinct nuclear localization and activity of tissue transglutaminase

Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.     

Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence

BACKGROUND: The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus. RESULTS: We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coli beta-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells. CONCLUSIONS: The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.     

Activation of caspase-1 in the nucleus requires nuclear translocation of pro-caspase-1 mediated by its prodomain

The interleukin-1beta-converting enzyme-like protease precursor, pro-caspase-1, has an N-terminal prodomain that is removed during cleavage activation of the protease. Here we show that tumor necrosis factor treatment of HeLa cells induced apoptosis without detectable proteolytic activation of caspase-1 in the cytosol. Instead, tumor necrosis factor induced the translocation of pro-caspase-1 to the nucleus where it was proteolytically activated, releasing the intact prodomain. We identified a nuclear localization signal in the prodomain, which was required for translocation of both pro-caspase-1 as well as its prodomain to the nucleus. Surprisingly, transfected MCF-7 carcinoma or embryonic kidney 293T cells expressing the prodomain alone underwent apoptosis. These results show that death signal-induced nuclear targeting is a novel activity of a caspase prodomain and indicate that caspase-1 and its prodomain may have hitherto unsuspected nuclear functions in apoptosis.     

Insulin degradation: progress and potential

Insulin degradation is a regulated process that plays a role in controlling insulin action by removing and inactivating the hormone. Abnormalities in insulin clearance and degradation are present in various pathological conditions including type 2 diabetes and obesity and may be important in producing clinical problems. The uptake, processing, and degradation of insulin by cells is a complex process with multiple intracellular pathways. Most evidence supports IDE as the primary degradative mechanism, but other systems (PDI, lysosomes, and other enzymes) undoubtedly contribute to insulin metabolism. Recent studies support a multifunctional role for IDE, as an intracellular binding, regulatory, and degradative protein. IDE increases proteasome and steroid hormone receptor activity, and this activation is reversed by insulin. This raises the possibility of a direct intracellular interaction of insulin with IDE that could modulate protein and fat metabolism. The recent findings would place intracellular insulin-IDE interaction into the insulin signal transduction pathway for mediating the intermediate effects of insulin on fat and protein turnover.     

Insulin binding and fluid-phase endocytosis stimulation in the mouse neuroblastoma cell line 41A3

As well as many other hormones and growth factors, insulin is known to influence several processes in the CNS; its specific effects, however, are still poorly understood. Neuroblastoma cell lines represent a useful experimental system for the analysis of the insulin-specific effect on neurons, in the absence of possible regulatory mechanisms elicited by other neuronal/glial cells and/or soluble factors. The expression and the binding properties of insulin receptors, as well as the insulin effects on both membrane fluidity and cell surface architecture, have been investigated in 41A3 mouse neuroblastoma cells, by radioligand-binding fluorescence spectroscopy and scanning electron microscopy, respectively the same cells, insulin-induced modifications on cytoskeletal organisation also have been studied. Binding studies were performed using 125I-insulin, while the cationic fluorescent probe trimethylammonium 1,6-diphenyl-1,3,5-hexatriene was used for biophysical investigations. The results presented in this paper provide evidence that insulin interacts with 41A3 neuroblastoma cells through a receptor-mediated mechanism and that, in these cells, insulin binding modifies the cell surface morphology and stimulates endocytosis.     

Decreased leukotriene C4 synthesis accompanies adherence-dependent nuclear import of 5-lipoxygenase in human blood eosinophils

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [3H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types.     

Differential modulation of protein kinase C isozymes in rat parotid acinar cells. Relation to amylase secretion

We investigated the expression, distribution, and activation parameters of protein kinase C (PKC) isozymes in isolated rat parotid acinar cells. By analyzing cellular extracts by western blot analysis and for isozyme-specific RNA, the Ca(2+)-independent PKC-delta, -epsilon, and -zeta were detected in the cytosolic, particulate (plasma membrane), and nuclear fractions of unstimulated cells, whereas the Ca(2+)-dependent PKC-alpha was confined to the cytosolic and particulate fractions. The expressed isozymes showed distinct responses to phorbol 12-myristate 13-acetate (PMA), thymeleatoxin, and cell surface receptor agonists with respect to translocation from cytosol to particulate fraction and nucleus, as well as sensitivity to down-regulation caused by prolonged exposure to PMA (3-20 hr). The marked susceptibility to down-regulation displayed by PKC-alpha and -delta was accompanied by an enhanced secretory response to norepinephrine as compared with control cells. Further, the selective PKC inhibitors Ro 31-8220 and CGP 41,251 also produced a concentration-dependent enhancement of norepinephrine-induced amylase secretion. Our findings suggest that PKC-alpha or -delta plays a negative modulatory role, rather than an obligatory role, in amylase secretion. Also, the localization and redistribution of PKC-epsilon and -delta to the nucleus by PKC activators imply that one or both of these isozymes may regulate such processes as cellular proliferation and/or differentiation.