Opposing contributions of NR1 and NR2 to protein kinase C modulation of NMDA receptors

N-Methyl-D-aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor-mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK-293). Phorbol 12-myristate 13-acetate (PMA) pretreatment of HEK-293 cells enhanced or suppressed NMDA receptor-mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1(011), conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium-45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor-mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C-terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.     

Phosphorylation and modulation of brain glutamate transporters by protein kinase C

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.     

Independent and exclusive modulation of cardiac delayed rectifying K+ current by protein kinase C and protein kinase A

We have recently established that local exposure to a 929.2 MHz electromagnetic near-field, used for cellular phones, does not promote rat liver carcinogenesis in a medium-term bioassay system. In the present study, a 1.439 GHz electromagnetic near-field (EMF), another microwave band employed for cellular phones in Japan, was similarly investigated. Time division multiple access (TDMA) signals for the Personal Digital Cellular (PDC) Japanese cellular telephone standard system were directed to rats through a quarter-wavelength monopole antenna. Numerical dosimetry showed that the peak SARs within the liver were 1.91-0.937 W/kg, while the whole-body average specific absorption rates (SARs) were 0.680-0.453 W/kg, when the time-averaged antenna radiation power was 0.33 W. Exposure was for 90 min a day, 5 days a week, over 6 weeks, to male F344 rats given a single dose of diethylnitrosamine (200 mg/kg, i.p.) 2 weeks previously. At week 3, all rats were subjected to a two-thirds partial hepatectomy. At week 8, the experiment was terminated and the animals were killed. Carcinogenic potential was scored by comparing the numbers and areas of the induced glutathione S-transferase placental form (GST-P)-positive foci in the livers of exposed (48) and sham-exposed rats (48). Despite increased serum levels of corticosterone, adrenocorticotropic hormone (ACTH) and melatonin, the numbers and the areas of GST-P-positive foci were not significantly altered by the exposure. These findings clearly indicated that local body exposure to a 1.439 GHz EMF, as in the case of a 929.2 MHz field, has no promoting effect on rat liver carcinogenesis in the present model.     

Differential effects of protein kinase A on Ras effector pathways

Ras mutants with the ability to interact with different effectors have played a critical role in the identification of Ras-dependent signaling pathways. We used two mutants, RasS35 and RasG37, which differ in their ability to bind Raf-1, to examine Ras-dependent signaling in thyroid epithelial cells. Wistar rat thyroid cells are dependent upon thyrotropin (TSH) for growth. Although TSH-stimulated mitogenesis requires Ras, TSH activates protein kinase A (PKA) and downregulates signaling through Raf and the mitogen-activated protein kinase (MAPK) cascade. Cells expressing RasS35, a mutant which binds Raf, or RasG37, a mutant which binds RalGDS, exhibited TSH-independent proliferation. RasS35 stimulated morphological transformation and anchorage-independent growth. RasG37 stimulated proliferation but not transformation as measured by these indices. TSH exerted markedly different effects on the Ras mutants and transiently repressed MAPK phosphorylation in RasS35-expressing cells. In contrast, TSH stimulated MAPK phosphorylation and growth in cells expressing RasG37. The Ras mutants, in turn, exerted differential effects on TSH signaling. RasS35 abolished TSH-stimulated changes in cell morphology and thyroglobulin expression, while RasG37 had no effect on these activities. Together, the data indicate that cross talk between Ras and PKA discriminates between distinct Ras effector pathways.     

The potential for isoenzyme-selective modulation of protein kinase C

Protein kinase C (PKC) is a phospholipid-dependent serine/threonine kinase family consisting of at least 11 closely related isoenzymes. The different PKC isoenzymes play important roles in signal transduction pathways. The exact significance of each isoenzyme is not known at present; therefore, the elucidation of the roles of the various PKC isoenzymes is important. To explain the function of distinct PKC isoenzymes, the availability of isoenzyme-specific inhibibitors or activators would be an advantage. PKC inhibitors have been known for some time, but these compounds are not isoenzyme-specific and also inhibit other kinases. Recently, an inhibitor selective for PKC alpha and another one selective for PKCbetaI and betaII were described. Both compounds compete with the ATP binding sites that exhibit high homologies among the different PKC isoenzymes. Among others, the phosporyl transfer region, the pseudosubstrate domain, the phorbolester binding sequences, and the phosphorylation sites may also be targets for modulation of isoenzyme-specific PKC activity. The question is whether the differences in these domains and the substrate specificity of the PKC isoenzymes will allow isoenzyme-specific inhibition. In this review the human sequences of these sites, isoenzyme-specific substrates, inhibitory compounds, and inhibitory peptides are summarized.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.     

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.     

Receptor-operated calcium influx mediated by protein tyrosine kinase pathways

Calcium influx from the extracellular space elicited by activation of heterotrimeric G protein-coupled and heptahelical receptors plays a critical role in transmembrane signal transduction in a wide variety of cell systems. In nonexcitable cells, the precise voltage-independent mechanism by which calcium enters the cell remains unknown. Multiple mechanisms appear to be operating in different cell types (1-3): 1. G protein-operated calcium influx, 2. Second messenger-operated calcium influx, 3. Capacitative calcium influx, and 4. Phosphorylation of calcium channels. Receptor-operated calcium channels have a fundamental role in stimulus-secretion coupling in many different cells, but these channels remain to be purified and cloned. This review proposes that receptor-operated calcium influx is mediated by protein tyrosine kinase pathways. The function of protein tyrosine kinase pathways and their interactions with other receptor-operated calcium influx mechanisms are described.     

Involvement of protein kinase C during activation of the mitogen-activated protein kinase cascade by leukemia inhibitory factor. Evidence for participation of multiple signaling pathways

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulates the activation of mitogen-activated protein kinase kinase (MAPKK), mitogen-activated protein kinase (MAPK), and S6 protein kinase (S6K) activities both in a time- and dose-dependent manner. A single peak of MAPKK activity, four peaks of activity against the S6 synthetic peptide, RRLSSLRA (S6 peptide), and three distinct peaks toward myelin basic protein (MBP) were observed after Mono-Q chromatography of LIF-stimulated cell extracts. Two of the MBP kinase activities correlated with the stimulation of extracellular signal-regulated kinases 1 and 2. Interestingly, down-regulation of protein kinase C (PKC) by chronic treatment of 3T3-L1 cells with phorbol ester was found to attenuate, but not block, the LIF-mediated stimulation of MAPKK, MAPK, and S6K activities in 3T3-L1 cells. Treatment of 3T3-L1 cells with epidermal growth factor increased MAPKK, MAPK, and S6K activities to a similar extent as LIF, but this activation was not attenuated by down-regulation of PKC. Our results suggest that the full activation of the MAPK cascade by LIF may require inputs from multiple signaling pathways, one of which is dependent upon the presence of functional PKC.     

The synergistic effects of vitamin D metabolites and transforming growth factor-beta on costochondral chondrocytes are mediated by increases in protein kinase C activity involving two separate pathways

Transforming growth factor-beta (TGFbeta), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFbeta effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFbeta on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC. This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFbeta showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFbeta was added to GC cells or when 1,25 plus TGFbeta were added to GC or RC cells. The addition of 1,25 plus TGFbeta and 24,25 plus TGFbeta to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFbeta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFbeta for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFbeta may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFbeta only affected PKC activity through G protein. The results of the present study indicate that vitamin D metabolites and TGFbeta produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFbeta was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFbeta through G protein activation.     

Astrocytic dye coupling in rat hippocampus: topography, developmental onset, and modulation by protein kinase C

Previous studies have shown that astrocytes constitute a functional syncytium whereas the cytoplasmata of individual cells are connected via gap junctions. Many studies have used cultured astrocytes and have examined electrical coupling with the help of double electrode techniques. Another approach has been the immunohistochemical detection of gap junction proteins in sections of brain tissue. From the results of these experiments it is difficult to infer the extent of astrocytic coupling in situ. To get an impression of the distribution of coupled astrocytes we took advantage of the hippocampal slice preparation which leaves the topography of neurons and astrocytes intact. We performed injections of low molecular weight dyes into single electrophysiologically identified astrocytes. As these dyes can pass through gap junctions this leads to the staining of all connected cells in a certain area, limited by the diffusional spread of the dye. The results show that there is virtually no border to astrocytic coupling between the diverse hippocampal subdivisions. This widespread coupling could already be detected at postnatal day 4, the earliest age tested. Activation of protein kinase C with phorbol esters showed that it is possible to reduce gap junctional communication by some extent. We conclude that although no compartmentalization was seen with respect to astrocytic coupling, the intercellular communication of these glial cells has the capability of being regulated for example by neuronal signals which activate the phospholipase C pathway in astrocytes.     

Irreversible inactivation of protein kinase C by glutathione

The tripeptide glutathione (GSH) is the predominant low molecular weight thiol reductant in mammalian cells. In this report, we show that at concentrations at which GSH is typically present in the intracellular milieu, GSH and the oxidized GSH derivatives GSH disulfide (GSSG) and glutathione sulfonate each irreversibly inactivate up to 100% of the activity of purified Ca2+- and phosphatidylserine (PS)-dependent protein kinase C (PKC) isozymes in a concentration-dependent manner by a novel nonredox mechanism that requires neither glutathiolation of PKC nor the reduction, formation, or isomerization of disulfide bridges within PKC. Our evidence for a nonredox mechanism of PKC inactivation can be summarized as follows. GSSG antagonized the Ca2+- and PS-dependent activity of purified rat brain PKC with the same efficacy (IC50 = 3 mM) whether or not the reductant dithiothreitol was present. Glutathione sulfonate, which is distinguished from GSSG and GSH by its inability to undergo disulfide/thiol exchange reactions, was as effective as GSSG in antagonizing Ca2+- and PS-dependent PKC catalysis. The irreversibility of the inactivation mechanism was indicated by the stability of the inactivated form of PKC to dilution and extensive dialysis. The inactivation mechanism did not involve the nonspecific phenomena of denaturation and aggregation of PKC because it obeyed pseudo-first order kinetics and because the hinge region of PKC-alpha remained a preferential target of tryptic attack following GSH inactivation. The selectivity of GSH in the inactivation of PKC was also indicated by the lack of effect of the tripeptides Tyr-Gly-Gly and Gly-Ala-Gly on the activity of PKC. Furthermore, GSH antagonism of the Ser/Thr kinase casein kinase 2 was by comparison weak (<25%). Inactivation of PKC-alpha was not accompanied by covalent modification of the isozyme by GSH or other irreversible binding interactions between PKC-alpha and the tripeptide, but it was associated with an increase in the susceptibility of PKC-alpha to trypsinolysis. Treatment of cultured rat fibroblast and human breast cancer cell lines with N-acetylcysteine resulted in a substantial loss of Ca2+- and PS- dependent PKC activity in the cells within 30 min. These results suggest that GSH exerts negative regulation over cellular PKC isozymes that may be lost when oxidative stress depletes the cellular GSH pool.     

Distinct effects of fatty acids on translocation of gamma- and epsilon-subspecies of protein kinase C

Effects of fatty acids on translocation of the gamma- and epsilon-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). gamma-PKC-GFP and epsilon-PKC-GFP predominated in the cytoplasm, but only a small amount of gamma-PKC-GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of gamma-PKC-GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on gamma-PKC-GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of epsilon-PKC-GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of epsilon-PKC-GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of epsilon-PKC-GFP than that of gamma-PKC-GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of gamma-PKC-GFP. Simultaneous application of arachidonic acid (25 MicroM) and DiC8 (10 microM) elicited a slow, irreversible translocation of gamma-PKC- GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of gamma- and epsilon-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.     

Developmental change in the modulation of acetylcholine receptor channel by protein kinase C activation in Xenopus embryonic muscle cells

Protein phosphorylation is important in synaptic transmission and plasticity. We report here that phorbol 12-myristate 13-acetate (TPA), a protein kinase C (PKC) activator, enhances the postsynaptic response at developing neuromuscular junctions by increasing the open time of embryonic acetylcholine (ACh) channels at earlier stages of cultured myocytes. Compared with day-1 cultures, the effects of TPA declined or disappeared on day-3 cultures. Adenosine 5'-triphosphate (ATP) which is co-stored and co-released with ACh at motor nerve terminals and is reported to enhance spontaneous synaptic currents by the activation of PKC, also shows similar developmental changes in the modulation of embryonic ACh channels in Xenopus embryonic myocytes.     

Differential roles of protein kinase C and pertussis toxin-sensitive G-binding proteins in modulation of melanoma cell proliferation and motility by thrombospondin 1

Thrombospondin 1 (TSP1) is an angiogenesis inhibitor that decreases tumor growth. We now report that TSP1 directly inhibits the proliferation of human melanoma cells. TSP1, peptides, and a recombinant fragment from the type I repeats, but not peptides that bind CD36 or CD47, inhibit the proliferation of A2058 melanoma cells. In contrast, chemotaxis is mediated by peptides or recombinant fragments from the procollagen, type I, type II, and cell-binding domains. The antiproliferative activity of TSP1 is mediated by a different signal transduction pathway than those mediating motility responses to the same protein. Activators of protein kinase A and protein kinase C inhibit chemotaxis but not the antiproliferative activity of TSP1, whereas the antiproliferative activity is reversed by inhibiting the tyrosine kinase or phosphatase activities. TSP1-mediated chemotaxis is partially dependent on a pertussis toxin (PT)-sensitive G-binding protein, whereas haptotaxis is not. Chemotaxis stimulated by the procollagen domain and the CD47-binding sequences from the COOH-terminal domain are also sensitive to PT, but responses to the type I and type III domains are not sensitive to PT. Residual chemotaxis to TSP1 in the presence of PT may therefore be mediated by the activities of the type I or type III repeats. Thus, TSP1 elicits several intracellular signals in melanoma cells that result from interactions with several domains of this protein and differentially affect growth and motility.     

Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C

PURPOSE OF STUDY: Interleukin-2 (IL-2) is a potent activator of lymphocytes, but its effectiveness as an anti-cancer agent is compromised by several adverse side effects including pulmonary edema. One explanation for the pulmonary toxicity of IL-2 is that activated lymphocytes directly induce the pulmonary vascular endothelium to become more leaky. METHODS: To test this hypothesis the number of total lymphocytes, gamma delta T cells, and CD2-positive cells (alpha beta T cells and natural killer cells) in peripheral blood and lung lymph of sheep were compared before and after IL-2 infusion. Hemodynamic and lymph dynamic changes were also evaluated. RESULTS: IL-2 decreased mean aortic pressure, increased cardiac output, lowered systemic vascular resistance, and doubled lung lymph flow (P < or = 0.05), but had no effect on plasma or lymph oncotic pressure. The lymph protein concentration and the lymph-to-plasma protein concentration ratio were not different after IL-2 infusion. IL-2 had no effect on the number of total lymphocytes, gamma delta T cells, or CD2-positive cells in the peripheral blood. In contrast, the number of total lymphocytes, gamma delta T cells, and CD2-positive cells in lung lymph decreased significantly (P < or = 0.05). CONCLUSIONS: The lymphocyte populations decreased more than could be explained by the increase in lymph flow, demonstrating that lung lymphocytes were not reduced simply by dilution. These results imply that the pulmonary edema associated with IL-2 is not caused by activated lymphocytes.     

TGF-beta- and angiotensin-II-induced mesangial matrix protein secretion is mediated by protein kinase C

BACKGROUND: Glomerulonephritis is characterized by the accumulation of extracellular matrix protein within the glomerulus. This process, when allowed to proceed unimpeded, leads to glomerulosclerosis and eventually to cessation of glomerular filtration. There is evidence that protein kinase C (PKC) activation plays an important role in mediating at least some of the effects of TGF-beta in vascular smooth-muscle cells. The current study was undertaken to determine whether PKC activity is required for both TGF-beta and angiotensin II (Ang II) to induce mesangial cell matrix protein secretion. METHODS: PKC was inhibited by two separate methods, and [3H]thymidine incorporation was assessed in both the presence and the absence of PKC inhibition. Conditioned medium from cells stimulated with TGF-beta or Ang II was collected and analysed for secreted matrix proteins and sulphated proteins by SDS-polyacrylamide gel electrophoresis and western blotting. RESULTS: Twenty-four-hour incubation of rat mesangial cells with phorbol-12-myristate-13-acetate (PMA) reduced total PKC activity to basal levels. Both TGF-beta and Ang II were mitogenic in mesangial cells, and chronic PMA pre-incubation inhibited this DNA synthesis. TGF-beta-and Ang-II-induced sulphated protein secretion into conditioned medium was markedly attenuated in PKC-downregulated cells. Secretion of the specific matrix proteins laminin and fibronectin by mesangial cells stimulated with either TGF-beta or Ang II was also diminished in PKC-downregulated cells and in cells pre-incubated with the specific PKC inhibitor, chelerythrine. There was no evidence of generalized cell toxicity or decreased non-specific protein synthesis caused by these PKC inhibitors. CONCLUSIONS: PKC is a key intermediary in the process by which TGF-beta and Ang II cause DNA synthesis and mesangial cell matrix protein production. Thus, PKC inhibitors deserve further study as potential therapeutic agents for a variety of glomerular diseases.     

Signal transduction by protein kinase C in mammalian cells

1. The past two decades have witnessed great advances in our understanding of the role of protein kinase C (PKC) in signal transduction. The Ca(2+)-activated, phospholipid-dependent protein kinase discovered by Nishizuka's group in 1977 is now a family of at least 11 isoforms. Protein kinase C isoforms exist in different proportions in a host of mammalian cells and each isoform has a characteristic subcellular distribution in each cell type. 2. Stimulation of a specific PKC isoform often causes redistribution of the isoform from one subcellular compartment to another compartments where it complexes with and phosphorylates a specific protein substrate. 3. The interaction of a specific PKC isoform with its protein substrate may directly activate a specific function of the cell or may trigger a cascade of protein kinases that ultimately stimulates a specific response in differentiated cells or regulates growth and proliferation in undifferentiated cells.