Gardenamide A attenuated cell apoptosis induced by serum deprivation insult via the ERK1/2 and PI3K/AKT signaling pathways.

Gardenamide A (GA) is a stable genipin derivative with neuroprotective properties. It rescued pheochromocytoma cell (PC12) sympathetic cultures and retinal neuronal cells from apoptosis insult induced by serum deprivation. GA attenuated the accumulation of intracellular reactive oxygen species (ROS) and the loss of mitochondrial membrane potential. Western blotting with specific phospho-antibodies indicated that GA increased the phosphorylation of both the protein kinase B (Akt) and the extracellular signal-regulated kinase (ERK1/2) in PC12 cells. The GA neuroprotective effect was inhibited by either the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or the mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059. These results propose that the neuroprotective effect of GA on PC12 neuronal cell cultures was mediated through both the PI3K/Akt and ERK1/2 signaling pathways. Therefore, GA may serve as a pharmacological tool to investigate neuroprotective mechanisms of neurons afflicted by different insults.

Interferon-gamma-induced neuronal differentiation of human umbilical cord blood-derived progenitors.

Human umbilical cord blood (HUCB) provides a source of progenitors for cell therapy. We isolated and characterized an HUCB-derived population of progenitors (HUCBNP), differentiated toward neuronal phenotype by human neuroblastoma-conditioning medium (CM) and nerve growth factor (NGF), which have been found to confer neuroprotection toward hypoxia-mediated neuronal injury. This study investigated whether interferon-gamma (IFN-gamma) contributes to HUCBNP differentiation. IFN-gamma was detected in the CM used for the induction of differentiation of HUCBNP and a neutralizing antibody of IFN-gamma significantly inhibited either IFN-gamma or CM-induced differentiation. Transcriptome analysis of CM-differentiated HUCBNP, identified 86 genes as highly upregulated, among them 25 were IFN-induced (such as 2',5'-oligoadenylate synthetase 1 and 2, IFN-induced protein and transmembrane proteins, STAT1 (IFN-gamma-receptor signal transducer and activator of transcription) and chemokine C-X-C motif ligand 5). Treatment of HUCBNP with human recombinant IFN-gamma, inhibited cell proliferation in a dose-dependent manner. IFN-gamma (1-100 ng/ml) enhanced neuronal differentiation, expressed by neurite outgrowths and increased expression of the neuronal markers beta-tubulin III, microtubule-associated protein 2, neuronal nuclei, neurofilament M and neuronal-specific enolase. IFN-gamma additively cooperated with NGF to induce the differentiation of HUCBNP. These data indicate that IFN-gamma promotes neuronal differentiation of HUCB-derived progenitors, proposing its use in future protocols towards cell therapy.

31P magnetic resonance spectroscopy of endothelial cells grown in three-dimensional matrigel construct as an enabling platform technology: I. The effect of glial cells and valproic acid on phosphometabolite levels.

Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.

31P magnetic resonance spectroscopy of endothelial cells grown in three-dimensional matrigel constructs as an enabling platform technology: II. The effect of anti-inflammatory drugs on phosphometabolite levels.

In the accompanying study, the authors presented phosphometabolite patterns of endothelial cells grown under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). Here the authors describe the effect of nonsteroidal anti-inflammatory drugs (NSAIDs), using this enabling platform technology, which is relevant for evaluating drug effects in tissue-engineered endothelial constructs. Treatment with indomethacin significantly changed the phosphometabolite fingerprint in this endothelial model, by, respectively, increasing (81%) and decreasing (42%) glycerophosphocholine (GPC) and phosphomonoesters (PM). Furthermore, a safer approach using a NSAID prodrug was also demonstrated in this study with a indomethacin phospholipid-derived prodrug (DP-155). Like the parental drug, DP-155 increased and decreased the levels of GPC and PM by 100% and 20%, respectively. These changes represent useful biomarkers to monitor NSAID effects on endothelized tissue-engineered constructs for the purpose of controlling endothelial cell survival and inflammation upon implantation.

Signaling pathways for PC12 cell differentiation: making the right connections.

A key issue in signal transduction is how signaling pathways common to many systems-so-called canonical signaling cassettes-integrate signals from molecules having a wide spectrum of activities, such as hormones and neurotrophins, to deliver distinct biological outcomes. The neuroendocrine cell line PC12, derived from rat pheochromocytoma, provides an example of how one canonical signaling cassette-the Raf --> mitogen-activated protein kinase kinase (MEK) --> extracellular signal-regulated kinase (ERK) pathway-can promote distinct outcomes, which in this case include neuritogenesis, gene induction, and proliferation. Two growth hormones, epidermal growth factor (EGF) and nerve growth factor (NGF), use the same pathway to cause PC12 proliferation and differentiation, respectively. In addition, pituitary adenylate cyclase-activating polypeptide (PACAP), a neurotransmitter that also causes differentiation, uses the same canonical cassette as NGF but in a different way. The Connections Map for PC12 Cell Differentiation brings into focus the complex array of specific cellular responses that rely on canonical signal transduction systems.

Expression levels of the nerve growth factor receptors TrkA and p75 in effusions and solid tumors of serous ovarian carcinoma patients.

PURPOSE: The purpose of this study was to analyze the expression of the high- and low-affinity nerve growth factor (NGF) receptors TrkA and p75 in effusions and in primary and metastatic tumors of serous ovarian carcinoma patients, as well as to evaluate their association with clinicopathological parameters and disease outcome. EXPERIMENTAL DESIGN: Sections from 77 malignant effusions and 78 primary and metastatic lesions were evaluated for protein expression of TrkA and p75 using immunohistochemistry (IHC). Expression of the phosphorylated form of TrkA (p-TrkA) was evaluated in 75 effusions using IHC. TrkA and p75 mRNA expression was studied in 44 effusions using reverse transcription-PCR (RT-PCR). RESULTS: TrkA protein membrane expression was detected in carcinoma cells in 30 of 77 (39%) effusions and 64 of 78 (82%) solid tumors. The decrease in TrkA expression in effusions approached, but did not reach, statistical significance when only corresponding lesions were analyzed (P = 0.06 in the comparison of effusions and primary tumors, P = 0.09 for effusions and metastases). Conversely, p75 protein membrane expression was more common in effusions, which was detected in 16 of 77 (21%) effusions as compared with 6 of 78 (8%) solid tumors (P > 0.05 in analysis of corresponding lesions). Expression of p-TrkA in carcinoma cells was limited to 5 of 75 effusions. Interestingly, 11 of 16 p75-positive effusions were also immunoreactive for the antibody against TrkA (P = 0.001), suggesting NGF activation using two signaling pathways. TrkA and p75 protein expression in tumor cells was similar in pleural and peritoneal effusions (P > 0.05). Using reverse transcription-PCR, TrkA mRNA was detected in 2 of 45 effusions, whereas p75 mRNA was present in 3 of 45 specimens. TrkA and p75 showed no association with tumor grade, Fédération Internationale des Gynaecologistes et Obstetristes stage, chemotherapy status, the extent of residual disease, or survival (P > 0.05). CONCLUSIONS: TrkA and p75 are both expressed in advanced-stage ovarian carcinoma, but whereas p75 expression is elevated in effusions, TrkA shows an opposite trend. The different expression of NGF receptors in effusions may relate to the different microenvironment and growth factor availability in body cavities, as also supported by the infrequent activation of TrkA in effusions. The similar expression of TrkA and p75 in carcinoma cells in pleural and peritoneal effusions provides further evidence for our hypothesis that there are few, if any, phenotypic differences between ovarian carcinoma cells at these two sites. TrkA and p75 expression in effusions does not appear to be a predictor of disease outcome in advanced-stage serous ovarian carcinoma.

Neuroprotective effects of novel cholinesterase inhibitors derived from rasagiline as potential anti-Alzheimer drugs.

TV3326, (N-propargyl-(3R)-aminoindan-5-yl-ethyl,methyl carbamate) was prepared in order to combine the neuroprotective effects of rasagiline, a selective inhibitor of monoamine oxidase (MAO)-B with the cholinesterase (ChE) inhibitory activity of rivastigmine as a potential treatment for Alzheimer's disease. The study reported here examined the neuroprotective effects of TV3326 against various insults in vitro and in vivo. TV3326 caused a dose related (10-500 microM) reduction in death induced in NGF differentiated rat pheochromocytoma (PC12) cells by 3-4 hour exposure to oxygen-glucose deprivation. A single s.c. injection of TV3326 given five minutes after closed head injury in mice significantly reduced the cerebral edema, and accelerated the recovery of motor function and spatial memory several days later. Unilateral icv injection of streptozotocin (STZ) 1.5 mg in rats, caused specific damage to myelinated neurones in the fornix and corpus callosum accompanied by microgliosis. Three bilateral injections of STZ, 0.25 mg each, caused more widespread damage, and a marked impairment in spatial memory. Chronic oral treatment with TV3326 (75 mumols/kg) reduced the neuronal damage and microgliosis and almost completely prevented the memory impairment. The neuroprotective effect in PC12 cells may be due to a combination of ChE inhibition and antiapoptotic activity. The latter does not result from ChE inhibition. It is associated with the presence of the propargyl group, since it occurs with other propargylamines that do not inhibit MAO, but not with drugs that inhibit only ChE.

Tamoxifen inhibits nerve growth factor-induced proliferation of the human breast cancerous cell line MCF-7.

An array of polypeptide growth factors contribute to the development of breast cancer, the most common tumor-related cause of death in women of Western countries. Therefore, breast cancer therapy should be aimed at inhibition of growth factor-dependent breast cancerous cell proliferation. However, the relative contribution of each individual factor in the development and maintenance of the transformed phenotype is largely unknown. Here we report for the first time that the proliferative effects of nerve growth factor, (NGF) a typical neurotrophin, are similar to those of epidermal growth factor (EGF) and insulin-like growth factor II, and are enhanced by 17beta-estradiol in the human breast cancer cell line MCF-7. The effect of NGF appeared to be mediated by its trkA receptors (trkA(NGFR)), as suggested by the potent inhibition of both MCF-7 cell proliferation and trkA(NGFR) phosphorylation occurring upon treatment of cultures with the selective trkA(NGFR) inhibitor K252a. Surprisingly, the antiestrogen drug tamoxifen (TAM) inhibited NGF-induced MCF-7 cell proliferation and trkA(NGFR) phosphorylation in a concentration-related fashion. The effect of TAM seemed to be estrogen receptor-independent, because the pure estrogen receptor antagonist ICI 182.780 was unable to block NGF-induced trkA(NGFR) phosphorylation. Our data underline the new emerging role of trkA(NGFR) in breast tumor growth, and suggest a related novel therapeutic use of TAM in breast cancer.

Involvement of extracellular signal-regulated kinase (ERK) in pardaxin-induced dopamine release from PC12 cells.

Pardaxin (PX), an ionophore-peptide neurotoxin isolated from the fish Pardachirus marmoratus, induces neurotransmitter release from neuronal preparations by both calcium-dependent and calcium-independent mechanisms. The aim of the present study was to investigate the role of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in pardaxin-induced dopamine (DA) release. The experiments were performed on variants of the PC12 cell line, an established cellular model for investigating DA release. Time course experiments indicated that PX, at nontoxic concentrations, stimulated ERK1 and ERK2 within 5 to 15 min, measured with a dual phospho-ERK antibody. PX stimulation of ERK activity was calcium (Ca(2+))-dependent and followed by ERK translocation to the nucleus. This effect was temporally related to PX-induced exocytosis, and measured by [(3)H]dopamine release as well as by a vesicle fusion-based enzyme-linked immunosorbent assay. Blocking ERK activity with the specific mitogen-activated protein kinase kinase inhibitors PD98059 (50 microM for 45 min) and UO126 (30 microM for 30 min) inhibited PX-induced exocytosis in the presence but not in the absence of extracellular Ca(2+). These results suggest the essential role of ERKs in PX-induced DA release under physiological conditions and support the hypothesis that ERKs are involved in regulating exocytosis.

A double cysteine trkA mutant exhibiting reduced NGF binding and delayed Erk signaling.

The NGF receptor trkA is a tyrosine kinase receptor comprising an extracellular domain with a ligand-binding site, a transmembrane-spanning domain (TMD), and an intracellular domain composed of a juxtamembrane region (JMR), a tyrosine kinase domain, and a short carboxy-terminal tail. Nerve growth factor (NGF) binds and activates this receptor, leading to phosphorylation of signaling substrates involved in neuronal proliferation, differentiation, and survival. Human trkA contains one cysteine residue in the TMD (C423) and another, separated by 12 residues, in the JMR (C436). We hypothesized that the removal of one or both of the cysteines would affect NGF-induced signaling of the trkA receptor. Here we show that NGF induces rapid receptor autophosphorylation in a wild-type, trkA-expressing clone (WT11), in a single cysteine trkA mutants (C423T or C436A), but lower autophosphorylation activity in a double-cysteine trkA mutant (C423T/C436A). WT11 and SM cells had similar binding affinity, but that of DM cells was lower, according to the NGF radioreceptor assay. NGF-induced Erk phosphorylation was rapid in WT11 and C423T cells, but delayed in C436A and C423T/C436A cells. NGF induced [3H]thymidine incorporation into WT11 and SM cells, but had no effect on DM cells. However, basic fibroblast growth factor (bFGF) induced rapid phosphorylation of Erk1/2, and [3H]thymidine incorporation in NIH3T3, WT11, single mutant (SM), and double mutant (DM) cells, suggesting that the impaired NGF-induced Erk phosphorylation and thymidine incorporation observed in DM cells are due to the double-cysteine mutations in the trkA receptor. Cumulatively, our findings support a model in which Cys436 of the trkA is responsible for the rapid transfer of the transmembrane occupancy signal to the SHC adaptor protein for activation of the Ras-Erk pathway and DNA synthesis.

Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity.

Neurotrophins (NTs) promote neuronal survival and maintenance during development and after injury. However, their role in the communication between the nervous system and the immune system is not yet clear. We observed recently that passively transferred activated T cells of various antigen specificities home to the injured central nervous system (CNS), yet only autoimmune T cells specific to a CNS antigen, myelin basic protein (MBP), protect neurons from secondary degeneration after crush injury of the rat optic nerve. Here we examined the involvement of NTs in T-cell-mediated neuroprotection, and the possible significance of the antigen specificity of the T cells in this activity. Analysis of cytokine and NT expression in various rat T cell lines showed that the T cells express mRNA for cytokines of Th1, Th2, and Th3 phenotypes. In addition, the T cells express mRNA and protein specific to nerve growth factor, brain-derived neurotrophic factor, NT-3, and NT-4/5. Antigen activation significantly increased NT secretion. Thus, reactivation of CNS autoimmune T cells by locally presented antigens to which they are specific can lead to enhanced secretion of NTs and possibly also of other factors in injured optic nerves. mRNA for TrkA, TrkB and p75 receptors was expressed in the injured nerve, suggesting that these specific receptors can mediate the effects of the T-cell-derived NTs. The neuroprotective effect of the passively transferred autoimmune anti-MBP T cells in injured optic nerves was significantly decreased after local applicaiton of a tyrosine kinase inhibitor known to be associated with NT-receptor activity. These results suggest that the neuroprotective effect of autoimmune T cells involves the secretion of factors such as NTs by the T cells reactivated by their specific antigen in the injured CNS. T cell intervention in the injured CNS might prove to be a useful means of promoting post-injury CNS maintenance and recovery, possibly via supply of NTs and other factors.

Ca2+-activated K+ channels in human leukemic Jurkat T cells. Molecular cloning, biochemical and functional characterization.

Previous studies have demonstrated the presence of apamin-sensitive, small-conductance Ca(2+)-activated K(+) currents in human leukemic Jurkat T cells. Using a combined cDNA and reverse transcriptase-polymerase chain reaction cloning strategy, we have isolated from Jurkat T cells a 2.5-kilobase cDNA, hSK2, encoding the human isoform of SK2 channels. Northern blot analysis reveals the presence of a 2.5-kilobase hSK2 transcript in Jurkat T cells. While present in various human tissues, including brain, heart, skeletal muscle, kidney, and liver, no hSK2 mRNA could be detected in resting and activated normal human T cells. The hSK2 gene is encoded by 8 exons and could be assigned to chromosome 5 (q21.2-q22.1). The protein encoded by hSK2 is 579 amino acids long and exhibits 97% identity with its rat counterpart rSK2. When expressed in Chinese hamster ovary cells, hSK2 produces Ca(2+)-activated K(+) currents with a unitary conductance of 9.5 pS and a K(0.5) for calcium of 0.7 microm; hSK2 currents are inhibited by apamin, scyllatoxin, and d-tubocurarine. Overexpression of the Src family tyrosine kinase p56(lck) in Jurkat cells, up-regulates SK2 currents by 3-fold. While IKCa channels are transcriptionally induced upon activation of normal human T cells, our results show that in Jurkat cells SK2 channels are constitutively expressed and down-regulated following mitogenic stimulation.

NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor.

The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway.

Heterologous upregulation of nerve growth factor-TrkA receptors in PC12 cells by pituitary adenylate cyclase-activating polypeptide (PACAP).

The capacity for the neurotrophic factor PACAP38 to regulate expression of nerve growth factor (NGF)-trkA receptors in PC12 cells has been examined. Treatment of PC12 cells with 5 nM PACAP38 for 48 h elicited a 2.5-fold increase in 125I-NGF binding sites. FACS and Western analysis of trkA receptor protein indicate an abundance of receptors. The PACAP38-selective antagonist PACAP 6-38 blocked trkA receptor upregulation elicited by PACAP38. The expression of epidermal growth factor receptors was not affected by PACAP38 suggesting that upregulation of trkA represents a selective effect of this neurotrophic peptide. Similarly, expression of the pan-neurotrophin binding receptor p75 was not altered by PACAP38 treatment. In addition to effects on trkA observed in wild-type PC12 cells, PACAP38 stimulated an increase in the level of expressed human trkA receptors stably transfected into PC12 cells. PACAP38 provoked an increase in basal and NGF-stimulated phosphorylation of trkA. Enhanced phosphorylation of trkA was detected as early as 6 h following addition of PACAP38 and was maximal at 48 h. Increased incorporation of phosphate occurs on both serine and tyrosine residues of trkA. These results suggest that PACAP38 is able to promote upregulation of trkA receptors, an event associated with elevated serine/tyrosine phosphorylation of trkA.

A disulfide conjugate between anti-tetanus antibodies and HIV (37-72)Tat neutralizes tetanus toxin inside chromaffin cells.

Conjugates between anti-tetanus F(ab')2 fragments and the (37-72) fragment of the HIV Tat protein were taken up by chromaffin cells, NG108-15 neurohybridoma cells and Rev-2-T-6 lymphoma cells. The uptake could not be inhibited by competition with (37-72)Tat, but was reduced in the presence of metabolic inhibitors or at low temperature. The disulfide as well as the thioether conjugate were translocated to the cytoplasmic space, but only the disulfide conjugate moderately restored the stimulated transmitter release inhibited by tetanus toxin. Therefore, disulfide conjugates are more promising than thioethers for the neutralization of intracellular antigens. These conjugates provide new tools to study neuroprotection against bacterial neurotoxins.

Rasagiline, a monoamine oxidase-B inhibitor, protects NGF-differentiated PC12 cells against oxygen-glucose deprivation.

In our in vitro model, rasagiline a selective irreversible monoamine oxidase-B (MAO-B) inhibitor, protected nerve growth factor (NGF)-differentiated PC12 cells from cell death under oxygen and glucose deprivation (OGD). The severity of the OGD insult, as expressed by cell death, was time-dependent. Exposure of the cells to OGD for 3 hr followed by 18 hr of reoxygenation caused about 30-40% cell death. Under these conditions, the neuroprotective effect of rasagiline was dose-dependent: rasagiline reducing OGD-induced cell death by 68% and 80% at 100 nM and 1 microM, respectively. The neuroprotective effect of rasagiline was also observed when added after the OGD insult (55% reduction in cell death). Under rasagiline treatment, there was a lesser decrease in ATP content in cultures exposed to OGD compared with that in untreated cultures. OGD followed by reoxygenation resulted in a several fold increase in PGE(2) release into the extracellular medium. Rasagiline (100 nM-1 microM) markedly inhibited OGD-induced PGE(2) release. Clorgyline, a monoamine oxidase-A (MAO-A) inhibitor, did not protect NGF-differentiated PC12 cells against OGD-induced cell death. As NGF-differentiated PC12 cells contain exclusively MAO type A, these data suggest that the neuroprotective effect of rasagiline under OGD conditions is independent of MAO inhibition.

Nerve growth factor (NGF)-induced calcium influx and intracellular calcium mobilization in 3T3 cells expressing NGF receptors.

The neurotrophins have been implicated in the acute regulation of synaptic plasticity. Neurotrophin-stimulated presynaptic calcium uptake appears to play a key role in this process. To understand the mechanism of neurotrophin-stimulated calcium uptake, the regulation of calcium uptake and intracellular mobilization by nerve growth factor (NGF) was investigated using NIH 3T3 cells stably transfected with either the high affinity NGF receptor p140(trk) (3T3-Trk) or the low affinity NGF receptor p75(NGFR) (3T3-p75). In 3T3-Trk cells, NGF increased both calcium uptake and intracellular calcium mobilization. In 3T3-p75 cells, NGF increased calcium uptake but not intracellular calcium mobilization. K-252a alone increased intracellular calcium in 3T3-Trk cells but not in 3T3-p75 cells. Nifedipine, an inhibitor of calcium uptake through L-type calcium channels, inhibited the action of NGF on both 3T3-Trk cells and 3T3-p75 cells, indicating that both p140(trk) and p75(NGFR) receptors are linked to nifedipine-sensitive L-type calcium channels. These studies show that either NGF receptor will support increases in intracellular calcium but that p140(trk) does so by increasing both uptake and mobilization, whereas p75(NGFR) does so by increasing uptake only.

Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells.

In this study, we examined the role of specific protein kinase C (PKC) isoforms in the differentiation of PC12 cells in response to nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells express PKC-alpha, -beta, -gamma, -delta, -epsilon, -mu, and -zeta. For PKC-delta, -epsilon, and -zeta, NGF and EGF exerted differential effects on translocation. Unlike overexpression of PKC-alpha and -delta, overexpression of PKC-epsilon caused enhanced neurite outgrowth in response to NGF. In the PKC-epsilon-overexpressing cells, EGF also dramatically induced neurite outgrowth, arrested cell proliferation, and induced a sustained phosphorylation of mitogen-activated protein kinase (MAPK), in contrast to its mitogenic effects on control cells or cells overexpressing PKC-alpha and -delta. The induction of neurite outgrowth by EGF was inhibited by the MAPK kinase inhibitor PD95098. In cells overexpressing a PKC-epsilon dominant negative mutant, NGF induced reduced neurite outgrowth and a more transient phosphorylation of MAPK than in controls. Our results suggest an important role for PKC-epsilon in neurite outgrowth in PC12 cells, probably via activation of the MAPK pathway.

Characterization of pardaxin-induced dopamine release from pheochromocytoma cells: role of calcium and eicosanoids.

Pardaxin, an excitatory neurotoxin, induced dopamine release from pheochromocytoma (PC12) cells both in the presence and absence of extracellular calcium ([Ca]o). In the presence of extracellular calcium, nifedipine, an L-type calcium channel blocker, did not affect dopamine release, whereas 1,2-bis (2-aminophenoxy) ethane N,N, N'N'-tetra-acetic acid (BAPTA), a chelator of cytosolic calcium, and dantrolene, a blocker of calcium release from intracellular stores, inhibited only partially (30-40%) pardaxin-induced dopamine release. In the absence of [Ca]o, BAPTA and dantrolene were ineffective. Pardaxin stimulated the arachidonic acid (AA) cascade in PC12 cells independently of [Ca]o. The phospholipase inhibitors mepacrine and bromophenacyl bromide inhibited both pardaxin-induced AA release and pardaxin-induced dopamine release. Dopamine release induced by pardaxin also was blocked by the lipoxygenase inhibitors nordihydroguaiaretic acid, esculetin, and 2-(12-hydroxydodeca-5, 10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone. Under these conditions, a parallel reduction in 5-hydroxyeicosatetranoic acid release also was observed. Suppression of pardaxin-induced dopamine release by inhibitors of phospholipase A2 and lipoxygenase was more pronounced in calcium-free medium. These results indicate the involvement of the lipoxygenase pathway in pardaxin-induced dopamine release and suggest the use of this toxin as a novel pharmacological tool for investigating the mechanism of calcium-independent neurotransmitter release.

Pardaxin, a new pharmacological tool to stimulate the arachidonic acid cascade in PC12 cells.

The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A2 (PLA2) activity (measured by [3H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA2 activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the alpha-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA2 was markedly inhibited by the nonselective PLA2 inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA2 and the calcium-independent PLA2 and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran -2- one], a highly specific inhibitor of calcium-independent PLA2. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE2 release. The release of other eicosanoids, such as 6-keto-PGF1alpha and thromboxane B2, was also augmented. Pardaxin-induced PGE2 release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE2 release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA2 plays an important role in Pardaxin stimulation of the AA cascade.