Tyrosine Kinase-dependent Mechanism Research Articles

Mice with compromised 5-HTT function lack phosphotyrosine-mediated inhibitory control over prefrontal 5-HT responses.

The activity of the prefrontal cortex is essential for normal emotional processing and is strongly modulated by serotonin (5-HT). Yet, little is known about the regulatory mechanisms that control the activity of the prefrontal 5-HT receptors. Here, we found and characterized a deregulation of prefrontal 5-HT receptor electrophysiological signaling in mouse models of disrupted serotonin transporter (5-HTT) function, a risk factor for emotional and cognitive disturbances. We identified a novel tyrosine kinase-dependent mechanism that regulates 5-HT-mediated inhibition of prefrontal pyramidal neurons. We report that mice with compromised 5-HTT, resulting from either genetic deletion or brief treatment with selective serotonin reuptake inhibitors during development, have amplified 5-HT1A receptor-mediated currents in adulthood. These greater inhibitory effects of 5-HT are accompanied by enhanced downstream coupling to Kir3 channels. Notably, in normal wild-type mice, we found that these larger 5-HT1A responses can be mimicked through inhibition of Src family tyrosine kinases. By comparison, in our 5-HTT mouse models, the larger 5-HT1A responses were rapidly reduced through inhibition of tyrosine phosphatases. Our findings implicate tyrosine phosphorylation in regulating the electrophysiological effects of prefrontal 5-HT1A receptors with implications for neuropsychiatric diseases associated with emotional dysfunction, such as anxiety and depressive disorders.

Oxidized low-density lipoproteins induce rapid platelet activation and shape change through tyrosine kinase and Rho kinase–signaling pathways

AbstractOxidized low-density lipoproteins (oxLDL) generated in the hyperlipidemic state may contribute to unregulated platelet activation during thrombosis. Although the ability of oxLDL to activate platelets is established, the underlying signaling mechanisms remain obscure. We show that oxLDL stimulate platelet activation through phosphorylation of the regulatory light chains of the contractile protein myosin IIa (MLC). oxLDL, but not native LDL, induced shape change, spreading, and phosphorylation of MLC (serine 19) through a pathway that was ablated under conditions that blocked CD36 ligation or inhibited Src kinases, suggesting a tyrosine kinase–dependent mechanism. Consistent with this, oxLDL induced tyrosine phosphorylation of a number of proteins including Syk and phospholipase C γ2. Inhibition of Syk, Ca2+ mobilization, and MLC kinase (MLCK) only partially inhibited MLC phosphorylation, suggesting the presence of a second pathway. oxLDL activated RhoA and RhoA kinase (ROCK) to induce inhibitory phosphorylation of MLC phosphatase (MLCP). Moreover, inhibition of Src kinases prevented the activation of RhoA and ROCK, indicating that oxLDL regulates contractile signaling through a tyrosine kinase–dependent pathway that induces MLC phosphorylation through the dual activation of MLCK and inhibition of MLCP. These data reveal new signaling events downstream of CD36 that are critical in promoting platelet aggregation by oxLDL.

Nuclear-Cytoplasmic Trafficking of NTF2, the Nuclear Import Receptor for the RanGTPase, Is Subjected to Regulation

NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).

Amyloid-β oligomers stimulate microglia through a tyrosine kinase dependent mechanism

Alzheimer's disease (AD) has been well characterized by the presence of reactive microglia, often associated with β-amyloid (Aβ) plaque deposition. The oligomeric form of Aβ peptide (Aβo) has neurotoxic effects in the presence of microglia and is suggested to potentiate proinflammatory changes in microglia in AD. Primary murine microglia cultures stimulated with Aβo displayed increased protein phosphotyrosine and secreted tumor necrosis factor (TNF)-α levels which were attenuated by the Src/Abl inhibitor, dasatinib. Intracerebroventricular infusions of Aβo into C57BL6/J mice stimulated increased microgliosis and protein phosphotyrosine levels that were also attenuated by dasatinib administration. The rodent findings were validated in human AD brains versus age-matched controls demonstrating reactive microglial association with Aβo deposits and increased microglial protein phosphotyrosine and phospho-Src levels. These data suggest a role for Aβo in microglial activation through a tyrosine kinase-dependant pathway both in rodent models and human disease. Use of a selective nonreceptor tyrosine kinase inhibitor such as dasatinib to attenuate microglial-dependent proinflammatory changes may prove to be an important step toward developing anti-inflammatory treatments for AD.

Factor XIII supports platelet activation and enhances thrombus formation by matrix proteins under flow conditions

Activated coagulation factor XIII (FXIIIa) is a transglutaminase that crosslinks fibrin at sites of vascular injury. FXIIIa also associates with blood platelets, although its role in platelet function is unclear and requires clarification. To evaluate the ability of FXIIIa to support platelet adhesion and spreading under conditions of physiologic flow, and to identify the underpinning receptors and signaling events. Platelet adhesion to immobilized FXIIIa was measured by fluorescence microscopy, and signaling events were characterized by immunoblotting. Immobilized FXIIIa supported platelet adhesion and spreading under static conditions through mechanisms that were dually and differentially dependent on integrins α(IIb)β(3) and α(v)β(3). Platelet adhesion was independent of FXIIIa transglutaminase or protein disulfide isomerase activity. Moreover, adhesion was abolished by antibodies that prevented interaction with FXIIIa, but maintained when potential interactions with fibrinogen were blocked. Platelet adhesion to FXIIIa was reduced significantly by either the specific α(IIb)β(3) antagonist tirofiban or the selective α(v)β(3)-blocking antibody LM609, and abolished when they were used in combination. Importantly, platelet adhesion was preserved under venous and arterial flow conditions in which both integrins played essential roles. In contrast, FXIIIa stimulated the formation of filopodia and lamellipodia in adherent platelets that was mediated exclusively by α(IIb)β(3) and eliminated by the Src-family inhibitor 4-amino-5-(4-methylphenyl-7-(t-butyl)pyrazolo(3,4-d)pyrimidine, indicating a tyrosine kinase-dependent mechanism. Crucially, under conditions of arterial shear, FXIIIa accentuated platelet recruitment by von Willebrand factor and collagen. Our data demonstrate a potential role for FXIIIa in supporting platelet adhesion at sites of vascular damage, particularly in association with other thrombogenic matrix proteins.

Rotenone reduces Mg 2+-dependent block of NMDA currents in substantia nigra dopamine neurons

Rotenone is a pesticide that has been successfully used to produce a rodent model of Parkinson's disease. We reported previously that rotenone potently augmented N-methyl- d-aspartate (NMDA)-evoked currents in rat dopamine neurons via a tyrosine kinase-dependent mechanism. In this study, we investigated the effect of rotenone on the current–voltage relationship of NMDA-induced currents in substantia nigra zona compacta neurons recorded with whole-cell patch pipettes in slices of rat brain. In a physiologic concentration of extracellular Mg 2+ (1.2 mM), a 30 min perfusion with rotenone (100 nM) produced a marked increase in current evoked by bath application of NMDA (30 μM), especially when measured at relatively hyperpolarized currents. In the presence of rotenone, NMDA currents lost the characteristic region of negative-slope conductance that is normally produced by voltage-dependent block by Mg 2+. The voltage-dependent effect of rotenone on NMDA currents was mimicked by a low extracellular concentration of Mg 2+ (0.2 mM) and was antagonized by a high level of Mg 2+ (6.0 mM). Moreover, we report that the tyrosine kinase inhibitor genistein blocked the ability of rotenone to augment NMDA receptor currents. These results suggest that rotenone potentiates NMDA currents by a tyrosine kinase-dependent process that attenuates voltage-dependent Mg 2+ block of NMDA-gated channels. These results support the hypothesis that an excitotoxic mechanism might participate in rotenone-induced toxicity of midbrain dopamine neurons.

Globular adiponectin increases cGMP formation in blood platelets independently of nitric oxide.

Platelet-derived nitric oxide (NO) has been shown to play conflicting roles in platelet function, although it is accepted that NO mediates its actions through soluble guanylyl cyclase (sGC). This confusion concerning the roles of platelet NO may have arisen because of an uncharacterized mechanism for activation of sGC. To examine the ability of the novel platelet agonist globular adiponectin (gAd) to stimulate the NO-independent cGMP-protein kinase G (PKG) signaling cascade. We used three independent markers of NO signaling, [(3)H]l-citrulline production, cGMP accrual, and immunoblotting of vasodilator-stimulated phosphoprotein (VASP), to examine the NO signaling cascade in response to gAd. gAd increased platelet cGMP formation, resulting in a dose- and time-dependent increase in phospho-VASP(157/239). Phosphorylation of VASP in response to gAd was mediated by both protein kinase A and PKG. Importantly, cGMP formation occurred in the absence of NO synthase (NOS) activation and in the presence of NOS inhibitors. Indeed, inhibition of the NOS signaling cascade had no influence on gAd-mediated platelet aggregation. Exploration of the mechanism demonstrated that NO-independent cGMP formation, phosphorylation of VASP and association of sGCalpha(1) with heat shock protein-90 induced by gAd were blocked under conditions that inhibited Src kinases, implying a tyrosine kinase-dependent mechanism. Indeed, sGCalpha1 was reversibly tyrosine phosphorylated in response to gAd, collagen, and collagen-related peptide, an effect that required Src kinases and downstream Ca(2+) mobilization. These data demonstrate activation of the platelet cGMP signaling cascade by a novel tyrosine kinase-dependent mechanism in the absence of NO.

Globular adiponectin induces platelet activation through the collagen receptor GPVI-Fc receptor gamma chain complex.

The adipocyte-derived cytokine, adiponectin (Ad), exerts potent vascular effects, although the direct effects of Ad on blood platelets are unclear. The influence of globular Ad (gAd) on blood platelet function was investigated. We measured platelet aggregation and tyrosine phosphorylation signaling events in human and mouse platelets. The ability of gAd to activate Glycoprotein VI (GPVI) activity was determined with a NFAT luciferase reporter assay. gAd, but not full length Ad, induced rapid aggregation and granule secretion of human and mouse platelets through a pathway that is ablated under conditions of Src kinase inhibition, indicating a tyrosine kinase-dependent mechanism. Consistent with this, gAd stimulates rapid tyrosine phosphorylation of several proteins in human and mouse platelets. The pattern of increase in tyrosine phosphorylation was similar to that induced by collagen, with the tyrosine kinase Syk and PLCgamma2 being identified among the list of tyrosine phosphorylated proteins. As collagen activates platelet through the GPVI-Fc receptor gamma-chain (FcRgamma) complex, we used FcRgamma null platelets (which also lack GPVI) to explore the mechanism by which gAd stimulates platelets. Stimulation of tyrosine phosphorylation and platelet aggregation by gAd was abolished in FcRgamma null platelets and markedly reduced in the absence of PLCgamma2. Further, GPVI was confirmed as a collagen receptor for gAd by increased luciferase activity in Jurkat T-cells transfected with GPVI. We identify gAd as a novel ligand for GPVI that stimulates tyrosine kinase-dependent platelet aggregation. Our data raise the possibility that gAd may promote unwanted platelet activation at sites of vascular injury.

Genistein inhibits voltage-gated sodium currents in SCG neurons through protein tyrosine kinase-dependent and kinase-independent mechanisms

Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na(+) currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC(50)) at 9.1 +/- 0.9 microM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na(+) current. The inactive genistein analog daidzein also inhibited Na(+) currents, but was less effective than genistein. The IC(50) for daidzein-induced inhibition was 20.7 +/- 0.1 microM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na(+) currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na(+) currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na(+) currents. These results suggest that genistein inhibits Na(+) currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na(+) channel.

Acetaldehyde dissociates the PTP1B–E-cadherin–β-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

Interactions between E-cadherin, beta-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell-cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, beta-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and beta-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and beta-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and beta-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of beta-catenin on tyrosine residues, and abolished the interaction of beta-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of beta-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and beta-catenin was reduced by tyrosine phosphorylation of beta-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)-PTP1B. The pairwise binding study showed that GST-E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of beta-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, beta-catenin and PTP1B by a phosphorylation-dependent mechanism.

The Decreased Susceptibility of Bcr/Abl Targets to NK Cell-Mediated Lysis in Response to Imatinib Mesylate Involves Modulation of NKG2D Ligands, GM1 Expression, and Synapse Formation

Chronic myeloid leukemia is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein, a constitutively active tyrosine kinase. In previous studies, we have provided evidence that Bcr/Abl overexpression in leukemic cells increased their susceptibility to NK-mediated lysis by different mechanisms. In the present study, using UT-7/9 cells, a high level Bcr/Abl transfectant of UT-7 cells, we show that the treatment of Bcr/Abl target by imatinib mesylate (IM), a specific Abl tyrosine kinase inhibitor, hampers the formation of the NK/target immunological synapse. The main effect of IM involves an induction of surface GM1 ganglioside on Bcr/Abl transfectants that prevents the redistribution of MHC-related Ag molecules in lipid rafts upon interaction with NK cells. IM also affects cell surface glycosylation of targets, as assessed by binding of specific lectins resulting in the subsequent modulation of their binding to lectin type NK receptor, particularly NKG2D. In addition, we demonstrate that the tyrosine kinase activity repression results in a decrease of MHC-related Ags-A/B and UL-16-binding protein expression on Bcr/Abl transfectants UT-7/9. We show that NKG2D controls the NK-mediated lysis of UT-7/9 cells, and IM treatment inhibits this activating pathway. Taken together, our results show that the high expression of Bcr/Abl in leukemic cells controls the expression of NKG2D receptor ligands and membrane GM1 via a tyrosine kinase-dependent mechanism and that the modulation of these molecules by IM interferes with NK cell recognition and cytolysis of the transfectants.

Tyrosine phosphorylation of the N-methyl- d-aspartate receptor is enhanced in synaptic membrane fractions of the adult rat hippocampus

Hippocampal N-methyl- d-aspartate receptors (NMDARs) contribute to the expression of certain types of synaptic plasticity, such as long-term potentiation (LTP). It is well documented that tyrosine kinases increase NMDAR phosphorylation and potentiate NMDAR function. However, it is unclear how these phosphorylation changes result in enhanced NMDAR activity. We previously reported that NMDAR surface expression can be increased by LTP-inducing stimulation via tyrosine kinase-dependent mechanisms in the adult hippocampus [D.R. Grosshans, D.A. Clayton, S.J. Coultrap, M.D. Browning, Nat. Neurosci., 5 (2002) 27–33]. We therefore hypothesized that tyrosine phosphorylation of the NMDAR may enhance the trafficking of the receptor to the synaptic membrane. Here, we show that the stoichiometry of NR2A and NR2B tyrosine phosphorylation is significantly higher in synaptosomal membranes than intracellular microsomal/light membranes. Interestingly, NR2B tyrosine-1472, but not NR1 serine-896 or -897, phosphorylation is significantly higher in synaptosomal membranes than intracellular microsomal/light membranes. Furthermore, treatment of hippocampal slices with either a tyrosine phosphatase inhibitor or a tyrosine kinase inhibitor alters NMDAR tyrosine phosphorylation and produces a corresponding change in the concentration of NMDARs in the synaptosomal membrane fraction. Taken together, these data support the hypothesis that tyrosine phosphorylation may enhance NMDAR activity by increasing the number of NMDARs at the synaptic membrane.

Inhibition of glycogen synthase kinase 3β suppresses coxsackievirus-induced cytopathic effect and apoptosis via stabilization of β-catenin

Coxsackievirus B3 (CVB3), a common human pathogen for viral myocarditis, induces a direct cytopathic effect (CPE) and apoptosis on infected cells. To elucidate the mechanisms that contribute to these processes, we studied the role of glycogen synthase kinase 3beta (GSK3beta). GSK3beta activity was significantly increased after CVB3 infection and addition of tyrosine kinase inhibitors blocked CVB3-triggered GSK3beta activation. Inhibition of caspase activity had no inhibitory effect on CVB3-induced CPE; however, blockage of GSK3beta activation attenuated both CVB3-induced CPE and apoptosis. We further showed that CVB3 infection resulted in reduced beta-catenin protein expression, and GSK3beta inhibition led to the accumulation and nuclear translocation of beta-catenin. Finally, we found that CVB3-induced CPE and apoptosis were significantly reduced in cells stably overexpressing beta-catenin. Taken together, our results demonstrate that CVB3 infection stimulates GSK3beta activity via a tyrosine kinase-dependent mechanism, which contributes to CVB3-induced CPE and apoptosis through dysregulation of beta-catenin.

Differential Roles of Vascular Endothelial Growth Factor Receptors 1 and 2 in Dendritic Cell Differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.

Regulation of cardiac Na–Ca exchange activity by selective tyrosine kinase inhibition

British Journal of Pharmacology (2004) 143, 929–930. doi:10.1038/sj.bjp.0706009

Fibroblast growth factor-1 improves cardiac functional recovery and enhances cell survival after ischemia and reperfusion: A fibroblast growth factor receptor, protein kinase c, and tyrosine kinase-dependent mechanism

We sought to investigate the role of fibroblast growth factor (FGF)-1 during acute myocardial ischemia and reperfusion. The FGFs display cardioprotective effects during ischemia and reperfusion. We investigated FGF-1-induced cardioprotection during ischemia and reperfusion and the intracellular signaling pathways responsible for these effects in an ex vivo murine setup of myocardial ischemia and reperfusion. Cardiac-specific human FGF-1 overexpression was associated with enhanced post-ischemic hemodynamic recovery and decreased lactate dehydrogenase release during reperfusion. Inhibition of the FGF receptor, protein kinase C (PKC), and tyrosine kinase (TK) resulted in blockade of FGF-1-induced protective effects on cardiac functional recovery and cell death. The overexpression of FGF-1 induces cardioprotection through a pathway that involves the FGF receptor, PKC, and TK.

Testosterone enhances calcium reabsorption by the kidney

The kidney is a target tissue for androgens, but the role of these hormones in the regulation of calcium (Ca 2+) reabsorption remains unclear. The present study examines the effects of testosterone on Ca 2+ transport by the luminal membranes of proximal and distal nephrons of the rabbit kidney. Tubule suspensions were pre-incubated in the presence or absence of the hormone, and 45 Ca 2+ uptake by the luminal membranes was measured using the rapid filtration technique. In the proximal tubules, testosterone did not influence Ca 2+ uptake. In the distal tubules, a 5 min incubation with the hormone increased this uptake with a maximal response at 10 −10 M. Ca 2+ transport by the distal membranes shows a dual kinetics. Testosterone enhanced the V max value of the low affinity component. In an attempt to identify the underlying mechanisms involved in this action, several messenger inhibitors were introduced in the tubule suspension. PD 98059 and U0 126 as well as AG 99 and genistein interfered with the hormone action suggesting the implication of a MEK kinase and a tyrosine kinase. To determine the type of the channels involved in this effect, Ca 2+ uptake was measured in the presence of diltiazem, ω-conotoxin MVIIC and mibefradil, i.e. selective inhibitors of the L-type, P/Q type and T-type channels. An inhibition of Ca 2+ transport was observed exclusively with mibefradil. These results indicate that testosterone enhances Ca 2+ transport by opening a T-type Ca 2+ channel in the distal luminal membrane, via MEK kinase and tyrosine kinase dependent mechanisms.

1002-100 Tyrosine kinase-dependent mechanism occurs downstream of protein kinase C activation in the pharmacological preconditioning by alpha-1 adrenoceptor stimulation

1002-100 Tyrosine kinase-dependent mechanism occurs downstream of protein kinase C activation in the pharmacological preconditioning by alpha-1 adrenoceptor stimulation

Depolarization-induced signaling to Ras, Rap1 and MAPKs in cortical neurons

In neurons, membrane depolarization triggers pleiotropic signaling which includes the activation of the small GTPases, Ras and Rap1, and the mitogen-activated protein kinases (MAPKs) Erk1/2. We have studied the intracellular signaling mechanisms which regulate these events in mouse-cultured cortical neurons. We show that depolarization induces activation of both Ras and Rap1, although with different kinetics: Ras activation is strong and fast while Rap1 activation is slower and weaker. Blockade of calmodulin affects the GTP-loading of Ras and Rap1 and prevents the MAPK response. Moreover, protein kinase A (PKA) activity is required for depolarization-induced Rap1 activation and full Erk stimulation, but is not involved in that of Ras. This PKA-dependent Rap1 activation does not require Src family kinases, but, in contrast to Ras, is sensitive to genistein, indicating the involvement of a tyrosine kinase-dependent mechanism. Our data provide new insights into the regulation of Ras and Rap1 activation in neurons.

NADPH oxidase activity in preeclampsia with immortalized lymphoblasts used as models.

Upon activation, neutrophils release reactive oxygen species that are believed to contribute to the widespread manifestation of preeclampsia. Neutrophils have an NADPH oxidase enzyme that catalyzes the production of reactive oxygen species. Little is known about the manifestations of the activated response and the upstream signaling pathways that regulate this process in preeclampsia. It is hypothesized that genetic factors may contribute to the release of reactive oxygen species and consequently the pathophysiology of the disease. We used Epstein-Barr virus-immortalized lymphoblasts from third-trimester, preeclamptic, postpartum preeclamptic women and their respective control subjects to assess NADPH oxidase-mediated reactive oxygen species production by using luminol-derived chemiluminescence and dihydrorhodamine-123 fluorescence. There was no effect of pregnancy status on the lymphoblast phorbol ester-stimulated luminol chemiluminescence area under the curve. However, lymphoblasts from preeclamptic patients had significant elevation of the lymphoblast phorbol ester-stimulated luminol area under the curve (F statistic 10.922, P<0.002). Similar findings were evident with dihydrorhodamine-123. No differences were revealed between preeclamptic and control cells when measuring the abundance of the phox proteins using Western blotting. Studies with genistein and tyrphostin implicated tyrosine kinase-dependent mechanisms in the control of NADPH oxidase-associated increased reactive oxygen species production in preeclampsia. These data show that preeclampsia is associated with a predisposition to increased agonist-stimulated NADPH oxidase-mediated reactive oxygen species production. The enhancement of reactive oxygen species generation may be important in mediating the endothelial dysfunction seen in preeclampsia.