KCl stimulation increases norepinephrine transporter function in PC12 cells

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.     

Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects

Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.     

125I-[Tyr0,D-Trp8]somatostatin-14 binding sites in the locus coeruleus of the rat are located on both ascending and descending projecting noradrenergic cells

Radioautographic determinations of 125I-[Tyr0,D-Trp8]somatostatin-14 (125I-SRIF) binding sites were performed on frozen serial sections of the locus coeruleus (LC) of control rats and of rats subjected to either bilateral microinjections of 6 hydroxydopamine (6-OHDA) into the LC or unilateral microinjection into the ascending noradrenergic bundles. These experiments were performed in order to determine whether 125I-SRIF binding was localized to noradrenergic-containing cells and in which regions the cells which contain the binding sites are projecting. The extent of the lesions was assessed by measuring norepinephrine (NE) levels in the hippocampus (88% decrease as compared to sham-operated animals) for bilateral LC lesions and in the frontal cortex (87% reduction vs. contralateral side) for unilateral bundle lesions. In control rats, 125I-SRIF binding sites were restricted to the boundaries of the LC and followed closely the distribution of tyrosine hydroxylase-labeled cells. Three weeks after bilateral injections of 6-OHDA, 125I-SRIF binding decreased by 79% in all regions of the LC. In contrast, unilateral destruction of the ascending noradrenergic bundles resulted in a moderate decrease only in the middle part of the LC with a more important effect in the dorsal (55%) than in the ventral (24%) portion of the nucleus. These data demonstrate that: 1) most SRIF receptors in the LC are located in the vicinity of NE-containing cell bodies and 2) NE-containing cells bearing SRIF receptors project to the forebrain as well as to other terminal areas located more caudally in the brain. These data suggest a general role for SRIF in the control of the multiple functions of the LC.     

Control of the maturation and the survival of central noradrenergic neurons in culture.

Central noradrenergic neurons from the locus coeruleus express unique plastic properties. The aim of this study was to identify factors that specifically regulate the development and the survival of the noradrenergic cells. Primary dissociated cultures of embryonic locus coeruleus (LC) neurons were established. Norepinephrine (NE) uptake was used as an index of maturation of the noradrenergic neurons. The noradrenergic cells were identified and quantified following immunocytochemical staining for tyrosine hydroxylase antibody. We have examined the effect of hippocampal target tissue and of cyclic-AMP (cAMP) on the development of these cells. Coculturing LC cells with a low density of hippocampal target cells, resulted in a significant increase in NE uptake. However, when the amount of hippocampal target cells was doubled an enormous decrease in NE uptake occurred. The target stimulatory effect was mediated by both neurons and glia, whereas the inhibitory effect was mediated by direct contact between target glia and LC neurons and detected only in the presence of serum. In addition to target effect, we also tested the effect of elevated intracellular cAMP level on NE uptake versus GABA uptake. GABA uptake served as a developmental index of the non noradrenergic cells. Increasing the intracellular cAMP level, by application of the membrane permeable analog dibutyryl cyclic AMP (DbcAMP), resulted in a selective stimulation of NE uptake, due to enhanced survival of noradrenergic neurons. GABA uptake and the number of non-noradrenergic cells were not changed in the presence of DbcAMP. DbcAMP could maintain the survival of LC neurons in the absence of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase

We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100 mM) for 24 h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5′AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.     

A Further TWEAK to Multiple Sclerosis Pathophysiology

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF super family that controls many cellular activities including proliferation, migration, differentiation, apoptosis, and inflammation by binding to fibroblast growth factor-inducible 14 (Fn14), a highly inducible cell surface receptor. Recent studies have indicated that TWEAK–Fn14 axis signaling may contribute to chronic autoimmune diseases. TWEAK expression via microglia in cortical lesions, presence of TWEAK⁺ macrophages in inflamed leptomeninges, and absence of TWEAK/Fn14 expression in healthy brain implicates importance of this pathway in pathogenesis of multiple sclerosis lesions. TWEAK–Fn14 axis blockade has also shown promise in various multiple sclerosis animal models. Stimulation of the TWEAK/Fn14 pathway can result in activation of both canonical and noncanonical NF-κB signaling and could also stimulate mitogen-activated protein kinase (MAPK) signaling pathways. Here, we have reviewed evidence of the possible role of TWEAK–Fn14 axis in pathophysiology of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) via neuroinflammation, tissue remodeling, blood–brain barrier (BBB) disruption, neurodegeneration, and astrogliosis.     

Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

A growing number of studies have demonstrated that both macroautophagy/autophagy and apoptosis are important inner mechanisms of cell to maintain homeostasis and participate in the host response to pathogens. We have previously reported that a functional autophagy pathway is trigged by infection of classical swine fever virus (CSFV) and is required for viral replication and release in host cells. However, the interplay of autophagy and apoptosis in CSFV-infected cells has not been clarified. In the present study, we demonstrated that autophagy induction with rapamycin facilitates cellular proliferation after CSFV infection, and that autophagy inhibition by knockdown of essential autophagic proteins BECN1/Beclin 1 or MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) promotes apoptosis via fully activating both intrinsic and extrinsic mechanisms in CSFV-infected cells. We also found that RIG-I-like receptor (RLR) signaling was amplified in autophagy-deficient cells during CSFV infection, which was closely linked to the activation of the intrinsic apoptosis pathway. Moreover, we discovered that virus infection of autophagy-impaired cells results in an increase in copy number of mitochondrial DNA and in the production of reactive oxygen species (ROS), which plays a significant role in enhanced RLR signaling and the activated extrinsic apoptosis pathway in cultured cells. Collectively, these data indicate that CSFV-induced autophagy delays apoptosis by downregulating ROS-dependent RLR signaling and thus contributes to virus persistent infection in host cells.     

InsP3 signaling induces pulse-modulated Ca2+ signals in the nucleus of airway epithelial ciliated cells

The phenomenology of nuclear Ca(2+) dynamics has experienced important progress revealing the broad range of cellular processes that it regulates. Although several agonists can mobilize Ca(2+) from storage in the nuclear envelope (NE) to the intranuclear compartment (INC), the mechanisms of Ca(2+) signaling in the nucleus still remain uncertain. Here we report that the NE/INC complex can function as an inositol-1,4,5-trisphosphate (InsP(3))-controlled Ca(2+) oscillator. Thin optical sectioning combined with fluorescent labeling of Ca(2+) probes show in cultured airway epithelial ciliated cells that ATP can trigger periodic oscillations of Ca(2+) in the NE ([Ca(2+)](NE)) and corresponding pulses of Ca(2+) release to the INC. Identical results were obtained in InsP(3)-stimulated isolated nuclei of these cells. Our data show that [Ca(2+)](NE) oscillations and Ca(2+) release to the INC result from the interplay between the Ca(2+)/K(+) ion-exchange properties of the intralumenal polyanionic matrix of the NE and two Ca(2+)-sensitive ion channels-an InsP(3)-receptor-Ca(2+) channel and an apamin-sensitive K(+) channel. A similar Ca(2+) signaling system operating under the same functional protocol and molecular hardware controls Ca(2+) oscillations and release in/to the endoplasmic reticulum/cytosol and in/to the granule/cytosol complexes in airway and mast cells. These observations suggest that these intracellular organelles share a remarkably conserved mechanism of InsP(3)-controlled frequency-encoded Ca(2+) signaling.     

Angiotensin II type 1 receptor-modulated signaling pathways in neurons

Mammalian brain contains high densities of angiotensin II (Ang II) type 1 (AT1) receptors, localized mainly to specific nuclei within the hypothalamus and brainstem regions. Neuronal AT1 receptors within these areas mediate the stimulatory actions of central Ang II on blood pressure, water and sodium intake, and vasopressin secretion, effects that involve the modulation of brain noradrenergic pathways. This review focuses on the intracellular events that mediate the functional effects of Ang II in neurons, via AT1 receptors. The signaling pathways involved in short-term changes in neuronal activity, membrane ionic currents, norepinephrine (NE) release, and longer-term neuromodulatory actions of Ang II are discussed. It will be apparent from this discussion that the signaling pathways involved in these events are often distinct.     

Activated 3',5'-cyclic AMP-dependent protein kinase is sufficient to induce neuroendocrine-like differentiation of the LNCaP prostate tumor cell line

Neuroendocrine (NE) differentiation within prostate tumors is proposed to be a contributing factor in disease progression. However, the cellular origin and molecular mechanism controlling differentiation of prostatic NE cells are unresolved. The prostate tumor cell line, LNCaP, can reversibly acquire many NE characteristics in response to treatment with beta-adrenergic receptor agonists and activators of adenylate cyclase. In this study, we demonstrate that these treatments induce protein kinase A (PKA) activation in LNCaP cells and that ectopic expression of a constitutively activated form of the PKA catalytic subunit, CIalpha, results in acquisition of NE characteristics, including the extension of neuritic processes, cessation of mitotic activity, and production of neuron-specific enolase. Forskolin-, epinephrine-, and isoproterenol-dependent NE differentiation of LNCaP cells was significantly inhibited by expressing a dominant negative mutant of the PKA regulatory subunit, RIalpha. These results demonstrate that prostatic NE differentiation in response to these agents depends on PKA activation, and this signaling pathway may provide a therapeutic target for treating advanced forms of prostate cancer.     

Distribution of ectonucleotidases in the rodent brain revisited

Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5′-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5′-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain. This work was supported by the Deutsche Forschungsgemeinschaft (140/17-3).     

Effects of chlordiazepoxide on footshock- and corticotropin-releasing factor-induced increases in cortical and hypothalamic norepinephrine secretion in rats

Noradrenergic and corticotropin-releasing factor (CRF) neuronal systems within the brain have been implicated in stress and anxiety. Synaptic release of cerebral norepinephrine (NE) is increased during stress, and following intracerebral CRF administration. Benzodiazepines are commonly used anxiolytic drugs but information on their effects on the stress- and CRF-related release of NE is limited. We have used in vivo microdialysis to test the effects of the benzodiazepine, chlordiazepoxide (CDP) on the noradrenergic responses to footshock and intracerebroventricular CRF in the medial hypothalamus and the medial prefrontal cortex (PFM) of freely moving rats. Footshock (60 x 0.1-0.2 mA shocks in 20 min) significantly increased microdialysate concentrations of NE in the first sample collected after initiating the footshock. In the hypothalamus, microdialysate NE was augmented 64% above baseline. A second footshock session (100 min after the first footshock) increased microdialysate NE to 313% of the baseline. Thus the noradrenergic responses to footshock were enhanced by preceding footshocks. CRF (100 ng) administered into the locus coeruleus (LC) almost tripled microdialysate concentrations of NE in the PFM. CDP (5mg/kg, i.p.) had no statistically significant effects on the basal dialysate concentrations of NE, but it significantly attenuated both footshock- and CRF-induced increases in dialysate NE. CDP may exert a direct inhibitory effect on the noradrenergic neurons, alter the input to LC noradrenergic neurons, or alter the ability of CRF to activate the LC noradrenergic system.     

IGF-I mediated survival pathways in normal and malignant cells

The type-I and -II insulin-like growth factors (IGF-I, II) are now established as survival- or proliferation-factors in many in vitro systems. Of note IGFs provide trophic support for multiple cell types or organ cultures explanted from various species, and delay the onset of programmed cell death (apoptosis) through the mitochondrial (intrinsic pathway) or by antagonizing activation of cytotoxic cytokine signaling (extrinsic pathway). In some instances, IGFs protect against other forms of death such as necrosis or autophagy. The effect of IGFs on cell survival appears to be context specific, being determined both by the cell origin (tissue specific) and the cellular stress that induces loss of cellular viability. In many human cancers, there is a strong association with dysregulated IGF signaling, and this association has been extensively reviewed recently. IGF-regulation is also disrupted in childhood cancers as a consequence of chromosomal translocations. IGFs are implicated also in acute renal failure, traumatic injury to brain tissue, and cardiac disease. This article focuses on the role of IGFs and their cellular signaling pathways that provide survival signals in stressed cells.     

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.     

Interaction with dopamine D2 receptor enhances expression of transient receptor potential channel 1 at the cell surface

Receptor signaling is mediated by direct protein interaction with various types of cytoskeletal, adapter, effector, and additional receptor molecules. In brain tissue and in cultured neurons, activation of dopamine D2 receptors (D2Rs) has been found to impact cellular calcium signaling. Using a yeast two-hybrid approach, we have uncovered a direct physical interaction between the D2R and the transient receptor potential channel (TRPC) subtypes 1, 4 and 5. The TRPC/D2R interaction was further validated by GST-pulldown assays and coimmunoprecipitation from mammalian brain. Ultrastructural analysis of TRPC1 and D2R expression indicates colocalization of the two proteins within the cell body and dendrites of cortical neurons. In cultured cells, expression of D2Rs was found to increase expression of TRPC1 at the cell surface by 50%. These findings shed new light on the constituents of the D2R signalplex, and support the involvement of D2Rs in cellular calcium signaling pathways via a novel link to TRPC channels.     

Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function.     

Amphetamine Stimulates Endocytosis of the Norepinephrine and Neuronal Glutamate Transporters in Cultured Locus Coeruleus Neurons

Amphetamines and amphetamine-derivatives elevate neurotransmitter concentrations by competing with endogenous biogenic amines for reuptake. In addition, AMPHs have been shown to activate endocytosis of the dopamine transporter (DAT) which further elevates extracellular dopamine (DA). We previously found that the biochemical cascade leading to this cellular process involves entry of AMPH into the cell through the DAT, stimulation of an intracellular trace amine-associated receptor, TAAR1, and activation of the small GTPase, RhoA. We also showed that the neuronal glutamate transporter, EAAT3, undergoes endocytosis via the same cascade in DA neurons, leading to potentiation of glutamatergic inputs. Since AMPH is a transported inhibitor of both DAT and the norepinephrine transporter (NET), and EAAT3 is also expressed in norepinephrine (NE) neurons, we explored the possibility that this signaling cascade occurs in NE neurons. We found that AMPH can cause endocytosis of NET as well as EAAT3 in NE neurons. NET endocytosis is dependent on TAAR1, RhoA, intracellular calcium and CaMKII activation, similar to DAT. However, EAAT3 endocytosis is similar in all regards except its dependence upon CaMKII activation. RhoA activation is dependent on calcium, but not CaMKII, explaining a divergence in AMPH-mediated endocytosis of DAT and NET from that of EAAT3. These data indicate that AMPHs and other TAAR1 agonists can affect glutamate signaling through internalization of EAAT3 in NE as well as DA neurons.

     

The conserved Nup107-160 complex is critical for nuclear pore complex assembly

Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex.