Activation of Tyrosine Hydroxylase in the Superior Cervical Ganglion by Nicotinic and Muscarinic Agonists

Both dimethylphenylpiperazinium (DMPP), a nicotinic agonist, and bethanechol, a muscarinic agonist, increase 3,4-dihydroxyphenylalanine (DOPA) synthesis in the superior cervical ganglion of the rat. DMPP causes approximately a fivefold increase in DOPA accumulation in intact ganglia whereas bethanechol causes about a two-fold increase in DOPA accumulation. These effects are additive with each other and with the increase in DOPA accumulation produced by 8-bromo cyclic AMP. The action of DMPP is dependent on extracellular Ca2+ while the actions of bethanechol and 8-bromo cyclic AMP are not dependent on extracellular Ca2+. Cholinergic agonists and cyclic nucleotides produce a stable activation of tyrosine hydroxylase (TH) in the ganglion. The activation of TH by nicotinic and muscarinic agonists can be detected after 5 min of incubation of the ganglia with these agents. The nicotinic response disappears after 30 min of incubation, whereas the muscarinic response persists for at least 30 min. The Ca2+ dependence of the TH activation produced by these agents is similar to the Ca2+ dependence of their effects on DOPA accumulation in intact ganglia. These data are consistent with the hypothesis that nicotinic agonists, muscarinic agonists, and cyclic AMP analogues increase TH activity by three distinct mechanisms. The activation of TH presumably underlies the increase in DOPA synthesis produced by these agents.     

Rapid Activation of Tyrosine Hydroxylase in Response to Nerve Growth Factor

Abstract: Nerve growth factor protein (NGF) was found to rapidly promote the activation of tyrosine hydroxylase in cultured rat PC 12 pheochromocytoma cells. PC 12 cultures were exposed to NGF for periods of less than 1 h and the soluble contents of homogenates prepared from the cells were assayed for tyrosine hydroxylase activity. Under these conditions, the specific enzymatic activity was increased by 60 ± 10% (n = 13) in comparison with that in untreated sister cultures. The increase was half maximal by 2–5 min of exposure and at NGF concentrations of about 10 ng/ml (0.36 n M ). Antiserum against NGF blocked the effect. Tyrosine hydroxylase activity could also be rapidly increased by NGF in cultures of PC12 cells that had been treated with the factor for several weeks in order to produce a neuron-like phenotype. This was achieved by withdrawing NGF for about 4 h and then readding it for 30 min. The NGF-induced increase of tyrosine hydroxylase activity in PC12 cultures was not affected by inhibition of protein synthesis and therefore appeared to be due to activation of the enzyme. Kinetic experiments revealed that NGF brought about no change in the apparent K_m of the enzyme for tyrosine or for co-factor (6-methyltetrahydropteridine), but that it did significantly increase the apparent maximum specific activity of the enzyme. These observations suggest that NGF (perhaps released by target organs) could promote a rapid and local enhancement of noradrenergic transmission in the sympathetic nervous system.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Phosphatidylcholine could be the source of 1,2-DAG which activates protein kinase C in EGF-stimulated colon carcinoma cells (HT29)

In our previous study (A. Balogh et al, Cell. Signalling 5 (6), 795-802, 1993.), we have shown that epidermal growth factor (EGF) increased protein kinase C (PKC) activities in colon carcinoma cell line (HT29), possibly through the increased 1,2-diacylglycerol (1,2-DAG) production via phosphatidylcholine (PC). Here we investigate the effect of the well-known PKC activator 12-O-tetradecanoyl-2 phorbol-13-acetate (TPA), on the levels of ³²P incorporation into EGF induced phosphatidylinositols (PI, PI4P, PI4, 5P₂) and different phospholipids (PC, PA, PS) as well as on induced tyrosine kinase activity. TPA significantly decreased the effects of EGF and it had the biggest inhibitory effect on EGF induced PC level. These data support our contention that PC plays an important role in the activation of PKC via 1,2-DAG production in the EGF stimulated pathway.     

Ebselen inhibits NO-induced apoptosis of differentiated PC12 cells via inhibition of ASK1-p38 MAPK-p53 and JNK signaling and activation of p44/42 MAPK and Bcl-2

Ebselen, a selenium-containing heterocyclic compound, prevents ischemia-induced cell death. However, the molecular mechanism through which ebselen exerts its cytoprotective effect remains to be elucidated. Using sodium nitroprusside (SNP) as a nitric oxide (NO) donor, we show here that ebselen potently inhibits NO-induced apoptosis of differentiated PC12 cells. This was associated with inhibition of NO-induced phosphatidyl Serine exposure, cytochrome c release, and caspase-3 activation by ebselen. Analysis of key apoptotic regulators during NO-induced apoptosis of differentiated PC12 cells showed that ebselen blocks the activation of the apoptosis signaling-regulating kinase 1 (ASK1), and inhibits phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal protein kinase (JNK). Moreover, ebselen inhibits NO-induced p53 phosphorylation at Ser15 and c-Jun phosphorylation at Ser63 and Ser73. It appears that inhibition of p38 MAPK and p53 phosphorylation by ebselen occurs via a thiol-redox-dependent mechanism. Interestingly, ebselen also activates p44/42 MAPK, and inhibits the downregulation of the antiapoptotic protein Bcl-2 in SNP-treated PC12 cells. Together, these findings suggest that ebselen protects neuronal cells from NO cytotoxicity by reciprocally regulating the apoptotic and antiapoptotic signaling cascades.     

Characterization of the interaction of influenza virus NS1 with Akt

Avian influenza viruses belong to the genus influenza A virus of the family Orthomyxoviridae. The influenza virus consists of eight segmented minus stranded RNA that encode 11 known proteins. Among the 11 viral proteins, NS1 (non-structural protein 1, encoded on segment 8) has been implicated in the regulation of several important intra-cellular functions.In this report, we investigated the functional interaction of NS1 with serine threonine kinase Akt, a core intra-cellular survival regulator. In co-immunoprecipitation assays and GST pull-down assays, NS1 directly interacted with Akt. The interaction was mediated primarily through the Akt-PH (Pleckstrin Homology) domain and the RNA-binding domain of NS1. NS1 preferentially interacted with phosphorylated Akt, but not with non-phosphorylated Akt. Functionally, the NS1-Akt interaction enhanced Akt activity both in the intra-cellular context and in in vitro Akt kinase assays. Confocal microscopic analysis revealed that phosphorylated Akt interacted with NS1 during the interphase of the cell cycle predominantly within the nucleus. Finally, mass spectrometric analysis demonstrated the position at Thr215 of NS1 protein is primary phosphorylation target site through Akt activation. The results together supported the functional importance of influenza virus NS1 with Akt, a core intra-cellular survival regulator.     

Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells

The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal-regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c-fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c-fos stability and activation in an ERK-dependent manner in PC12 cells. This is achieved through ERK-dependent phosphorylation of c-fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c-fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF-dependent transactivation of c-fos. In addition, a consensus ERK binding site (DEF domain) is also required for c-fos transactivation. However, stability is controlled by ERK-dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post-translational regulation of c-fos following NGF treatment of PC12 cells. Because these ERK-dependent phosphorylations are required for proper c-fos function, and occur sequentially, we propose that c-fos is a sensor for ERK signaling duration in the neuronal-like cell line PC12.     

Rat mesenchymal stem cells increase tyrosine hydroxylase expression and dopamine content in ventral mesencephalic cells in vitro

Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. It has been shown that MSCs secrete neurotrophic factors involving nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Also, these neurotrophic factors can upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells and neural stem cells. Here, we investigated the effect of co-culturing rat E13.5 ventral mesencephalic cells (VMCs) with MSCs from rat bone marrow on TH expression and dopamine (DA) content. The study consisted of 3 groups: MSC, VMC and a combined MSC+VMC group. All groups were cultured in serum-free neuro-basal medium for 3 days. Thereafter, each group was analyzed by RT-PCR, western blotting, and HPLC. The co-culture group showed a higher expression at TH and DA than the VMC group. However, TH and DA were not present in the MSC group. These observations suggest that MSCs could be an alternative source for treating neurodegenerative diseases such as Parkinson's disease (PD).     

Nilotinib Induces Autophagy in Hepatocellular Carcinoma through AMPK Activation

Hepatocellular carcinoma (HCC) is the most common liver cancer and the third-leading cause of cancer death worldwide. Nilotinib is an orally available receptor tyrosine kinase inhibitor approved for chronic myelogenous leukemia. This study investigated the effect of nilotinib on HCC. Nilotinib did not induce cellular apoptosis. Instead, staining with acridine orange and microtubule-associated protein 1 light chain 3 revealed that nilotinib induced autophagy in a dose- and time-dependent manner in HCC cell lines, including PLC5, Huh-7, and Hep3B. Moreover, nilotinib up-regulated the phosphryaltion of AMP-activated kinase (AMPK) and protein phosphatase PP2A inactivation were detected after nilotinib treatment. Up-regulating PP2A activity suppressed nilotinib-induced AMPK phosphorylation and autophagy, suggesting that PP2A mediates the effect of nilotinib on AMPK phosphorylation and autophagy. Our data indicate that nilotinib-induced AMPK activation is mediated by PP2A, and AMPK activation and subsequent autophagy might be a major mechanism of action of nilotinib. Growth of PLC5 tumor xenografts in BALB/c nude mice was inhibited by daily oral treatment with nilotinib. Western blot analysis showed both increased phospho-AMPK expression and decreased PP2A activity in vivo. Together, our results reveal that nilotinib induces autophagy, but not apoptosis in HCC, and that the autophagy-inducing activity is associated with PP2A-regulated AMPK phosphorylation.     

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

Abstract

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr⁴¹⁶) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.