Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms

Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high-K⁺ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K⁺ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K⁺ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.     

Activation of Mitogen-Activated Protein Kinase by Epidermal Growth Factor in Hippocampal Neurons and Neuronal Cell Lines

Abstract: Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.     

丝裂原活化蛋白激酶(MAPK)信号通路的研究进展

丝裂原活化蛋白激酶（MAPK）信号通路是广泛存在于各种细胞中的一条信号转导途径,由一组级联活化的丝／苏氨酸蛋白激酶组成,对于细胞周期的运行和基因表达具有重要调控作用。MAPK包括多个成员,活化后向核内迁移,磷酸化包括转录因子在内的核蛋白和膜受体,实现对基因转录和其他事件的调节。MAPK激酶（MAPKK）是MAPK的上游激活分子,催化MAPK的Tyr和Thr残基双特异性磷酸化。Mos是脊椎动物生殖细胞中特有的MAPKK,通过MAPKK／MAPK途径活化成熟促进因子,启动卵母细胞成熟发育并维持中期阻滞。MAPK的下游分子包括MAPK活化的蛋白激酶（MAPKAPK）、核转录因子、热休克蛋白和细胞质磷脂酶A2等,执行由MAPK所介导的细胞生命活动调节功能。     

Rat Brain Protein Kinase C: Purification, Antibody Production, and Quantification in Discrete Regions of Hippocampus

Protein kinase C (PKC), a calcium- and phospholipid-dependent kinase, is highly enriched in rat brain, where it may function in signal transduction processes. We purified rat brain PKC to homogeneity by a three-column procedure of diethylaminoethyl-cellulose, phenyl-Sepharose, and protamine-agarose with a yield of 16% and a final specific activity of 9,600 pmol of [3H]phorbol-12,13-dibutyrate bound/mg of protein. The pure protein consisted of a doublet of 80 and 78 kilodaltons. Rabbit antibodies prepared against a beta-type PKC synthetic peptide sequence (RAKIGQGTKAPEEKTANTISK) showed high specificity and sensitivity for PKC and recognized only the 78-kilodalton form of PKC. Micropunches (300 microns in diameter) of rat hippocampal subregions were solubilized in sodium dodecyl sulfate (SDS) sample buffer, electrophoresed on SDS-10% polyacrylamide gels, and transferred to nitrocellulose. PKC was visualized by 125I-protein A autoradiography and quantified by densitometry. The highest concentrations of PKC were found in the CA1 pyramidal cell layer (0.43 +/- 0.04 OD), with the lowest amounts in the CA3 and CA4 pyramidal cell layers (0.11 +/- 0.02 and 0.085 +/- 0.006 OD, respectively). These results demonstrate a simple way of preparing antibodies against domains of PKC. We also describe a procedure for quantifying the relative amounts of PKC in discrete brain regions.     

Evidence for Protein Kinase C Involvement in the Short-Term Activation by Prolactin of Tyrosine Hydroxylase in Tuberoinfundibular Dopaminergic Neurons

Abstract: The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a K _1(DA) value of 29.92 ± 0.49 μ M , the other being × 15-fold more sensitive to DA inhibition with a K _1(DA) of 1.96 ± 0.09 μ M , likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low K _1(DA) remained unaffected, whereas the K _1(DA) of the purported active form of TH increased to 62.6 ± 0.8 μ M , suggesting an increase in the enzyme phosphorylation. This increase in the K _I(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12- O -tetradecan-oylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.     

Identification of an Ectokinase Activity in Cerebellar Granule Primary Neuronal Cultures

Abstract: Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 n M [γ-³²P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for ∼20 min. Unlike what was seen with [γ-³²P]ATP, in the presence of [³²P] orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80–90% in the presence of 1 µ M unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 m M PO₄³⁻. Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 m M KCl or 100 µ M glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl₂ but inhibited in the presence of MnCl₂ or NaF and in the absence of CaCl₂. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80–90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.     

细胞因子受体的组成,结构功能及信号传导机制

细胞因子受体种类繁多,分属于不同受体超家族。活化受体的功能可分为：ＰＴＫ型受体或其结合蛋白具有ＰＴＫ活性、丝／苏氨酸蛋白激酶型受体以及与Ｇ蛋白耦联的受体等。不同受体与其配体结合后,通过对受体后信号传导成分的可逆的磷酸化反应传递信号,最终通过对其终端成分,如酶活力、基因表达、细胞骨架蛋白的功能、膜通透性等的调节,导致细胞的生物效应。     

Neurotrophins Rescue Cerebellar Granule Neurons from Oxidative Stress-Mediated Apoptotic Death: Selective Involvement of Phosphatidylinositol 3-Kinase and the Mitogen-Activated Protein Kinase Pathway

Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K⁺ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K⁺. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K⁺ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K⁺/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K⁺ to low-K⁺/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K⁺ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1–100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K⁺-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K⁺ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K⁺ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.     

Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high-affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen-activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.     

Protein Kinase C Activity in Rat Brain Cortex

The procedure used to obtain cerebral tissue for analysis of protein kinase C (PKC) activity may affect the subcellular distribution of the enzyme. We compared different methods of tissue preparation and found that the proportion of PKC activity associated with the particulate fraction of the cerebral cortex was only 30% when the brain was frozen in situ while the animal was on life support or after decapitation followed by delayed freezing. Other methods of obtaining cerebral tissue resulted in 49-56% of the PKC activity in the particulate fraction. Freezing per se had no apparent effect on the activity or subcellular distribution of PKC. In addition, whenever the particulate PKC activity was high (greater than 48%), there was also a significant increase in the proportion of particulate protein (from 51 to approximately 63%, p less than 0.05).     

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

The Cholinergic Stimulating Effects of Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor Are Mediated by Protein Kinase C

Abstract: The intracellular mechanisms through which two trophic factors, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), regulate cholinergic development were examined in sympathetic neuron cultures. Treatment with CNTF or LIF increased levels of choline acetyltransferase (ChAT) activity by 375 and 350%, respectively. However, in neuronal cultures depleted of protein kinase C (PKC) activity by chronic phorbol ester treatment, neither CNTF nor LIF elevated ChAT activity. Further, the stimulation of ChAT due to increased cell density was not observed in PKC-depleted sympathetic neurons. The inhibition of CNTF-stimulated ChAT by phorbol ester occurred in a dose-dependent manner and chronic phorbol ester treatments did not alter the levels of the catecholamine biosynthetic enzyme tyrosine hydroxylase. Moreover, increased levels of diacylglycerol, an endogenous activator of PKC, were observed in sympathetic neurons treated with CNTF. However, neither CNTF nor LIF stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate. These observations suggest that a common PKC-dependent pathway, which is independent of phosphatidylinositol 4,5-bisphosphate hydrolysis, mediates the cholinergic stimulating effects of CNTF, LIF, and cell-cell contact in cultured sympathetic neurons.     

Activation of Protein Kinase C Augments Peptide Release from Rat Sensory Neurons

Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 n M phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4α-PDBu analogue at 100 n M had no effect on release. Furthermore, 100 n M bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 n M or 1-oleoyl-2-acetyl- sn -glycerol at 50 µ M did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 n M staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12- O -tetradecanoylphorbol 13-acetate or sn -1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 ± 1 h) in these cells.     

Protein Kinase C Activation Attenuates N-Methyl-d-Aspartate-Induced Increases in Intracellular Calcium in Cerebellar Granule Cells

Abstract: Activation of the N-methyl-d -aspartate (NMDA) subtype of glutamate receptor increases levels of intracellular calcium and can lead to stimulation of protein kinase C activity. Several reports have demonstrated that stimulation of protein kinase C can, in turn, increase electrophysiological responses to NMDA in certain cells or in oocytes expressing certain NMDA receptor subunits. In the present study, the effects of protein kinase C activation on NMDA receptor-mediated increases in intracellular Ca²⁺ levels were investigated in primary cultures of rat cerebellar granule cells using fura-2 fluorescence spectroscopy. Pretreatment of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not the inactive analogue 4α-phorbol 12-myristate 13-acetate, inhibited NMDA-induced increases in intracellular Ca²⁺ levels. Coincubation of cells with PMA and the kinase inhibitor staurosporine or calphostin C blocked the PMA effect. The potency of NMDA was reduced twofold, and the potency of the NMDA receptor coagonist, glycine, to enhance the response to NMDA was decreased fourfold by pretreatment of cells with PMA. The effect on glycine was mimicked by pretreatment with okadaic acid, a protein phosphatase inhibitor. PMA treatment did not significantly alter Mg²⁺ inhibition of the NMDA response but decreased the potency of the competitive antagonist CGS-19755. These data suggest that, in cerebellar granule cells, the function of the NMDA receptor may be subject to feedback inhibition by protein kinase C stimulation. Under physiological conditions, this inhibition may result from a decreased effectiveness of the endogenous coagonists, glutamate and glycine.     

Novel Chimeric Peptide Inhibits Protein Kinase C and Induces Apoptosis in Human Immune Cells

Protein kinase C (PKC) participates in a myriad of cellular processes. Protein kinase C isoforms play different roles based on their cellular expression balance and activation. The activity of classical PKC isoforms has been shown to be crucial for immune cell population homeostasis, playing a positive role in survival and proliferation. Protein kinase C inhibitors have been used for conditions where up-regulated PKC results in a pathological state. The most commonly investigated PKC inhibitors are highly effective in inhibiting PKC function but they are relatively unspecific, some of them even inhibiting other kinase families. Protein kinase C pseudosubstrates are auto-inhibitory domains which have been used to inhibit more specifically PKC in vitro but they do not freely penetrate cells. This could be resolved by using cell-permeable PKC pseudosubstrates which would more accurately modulate cellular PKC activity and PKC-related functions in intact cells. Here we show the development of a chimeric peptide inhibitor of classical PKC isoforms, consisting of a cell permeable sequence and a pseudosubstrate sequence which was able to translocate into cells, inhibiting PKC kinase activity and PKC T-cell-specific substrate phosphorylation. We also demonstrate a dramatic reduction in T-cell proliferation at high chimeric peptide concentration; this was attributed to apoptosis induction, as demonstrated by cell shrinking, phosphatidylserine exposure and DNA fragmentation. As expected, the control peptide (pseudosubstrate) did not penetrate cells, affect cell proliferation or survival. We also show that a neoplastic T-cell line which expresses higher levels of PKC is more resistant to chimeric peptide-mediated cell death than normal cells, corroborating a PKC role in apoptosis resistance. This chimeric peptide could be useful for the specific modulation of the PKC signalling pathway in pathological conditions.