Extracellular ATP Stimulates Norepinephrine Uptake in PC12 Cells

Hardwick, Jean C.; Ehrlich, Yigal H.; Hendley, Edith D.

doi:10.1111/j.1471-4159.1989.tb08546.x

Cited by 32 publications

(17 citation statements)

References 29 publications

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“…Our previous studies have demonstrated that extracellular ATP can stimulate the highaffinity uptake of norepinephrine in PC12 cells (Hardwick et al, 1989). Norepinephrine uptake is the major means of transmitter inactivation at noradrenergic nerve terminals, requiring either Mgz+ or Ca2+ (Hendley, 1984).…”

Section: Discussionmentioning

confidence: 99%

“…The identification, localization, and characterization of specific endogenous protein substrates for phosphorylation activity are important for determining the physiological function of various protein kinases (Kennedy, 1983;Ehrlich, 1984Ehrlich, , 1987. Thus, the identification and characterization of the specific surface membrane proteins phosphorylated by extracellular ATP should provide insight into the mechanisms by which ecto-protein kinases are involved in the regulation of various neuronal functions (Ehrlich et al, 1986Ehrlich, 1987;Hendley et al, 1988;Muramoto et al, 1988;Hardwick et al, 1989).…”

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confidence: 99%

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Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Pawłowska

Hogan

Kornecki

et al. 1993

Journal of Neurochemistry

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Abstract:The phosphorylation of surface proteins by ectoprotein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC 12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ectoprotein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [y-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32P,. This activity WBS abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled P,. As also expected from ecto-protein kinase activity, PC 12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-a-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as e.wIusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca2+-dependent, high-affinity norepinephrine uptake in PC 12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra-and extracellular protein phosphorylation in intact PC 12 cells. Its hydrophilic analogue K-252b, had only minimal effects on intracellular protein phosphorylation but readily inhibited the phosphorylation of specific substrates of ecto-protein kinase in PC12 cells incubated with extracellular ATP, suggesting the involvement of ecto-protein kinase in the reported inhibition of NGF-induced neurite extension by K-252b. Preincubation of PC12 cells with 50 ng/ml of NGF for 5 min stimulated the activity of ecto-protein kinase toward all its endogenous substrates. Exposure of PC12 cells to the same NGF concentration for 3 days revealed another substrate of ecto-protein kinase, a 53K protein, whose surface phosphorylation is expressed only after NGF-induced neuronal differentiation. In the concentration range (10-100 p h f ) at which 6-thioguanine blocked NGF-promoted neurite outgrowth in PC12 cells, 6-thioguanine effectively inhibited the phosphorylation of specific proteins by ecto-protein kinase. This study provides the basis for continued investigation of the involvement of ecto-protein kinase and its surface protein substrates in neuronal differentiation, neuritogenesis, and synaptogenesis. Key Words: Protein phosphorylationEcto-protein kinase-Surface phosphoproteins-ExtracelMar ATP-Nerve growth factor...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Pawłowska

Hogan

Kornecki

et al. 1993

Journal of Neurochemistry

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“…For example, axoplasmic transport delivers key vesicular proteins from their site of synthesis in the cell body to the terminals; thus, vesicular uptake could also be reduced by structural or functional abnormalities of axonal trafficking that decrease the number of vesicles available. Alternatively, cell membrane and vesicular monoamine transport are energy-requiring processes (28,29), and intrinsic errors of mitochondrial function or metabolic insults posed by exogenous toxins could result in decreased neuronal uptake and vesicular sequestration. Moving forward, bases for the deficit in vesicular 18 F-DA uptake will need to be resolved.…”

Section: Discussionmentioning

confidence: 99%

Intra-neuronal vesicular uptake of catecholamines is decreased in patients with Lewy body diseases

Goldstein¹,

Holmes²,

Kopin³

et al. 2011

J. Clin. Invest.

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Several neurodegenerative disorders, including Parkinson disease (PD), are characterized by the presence of Lewy bodies -cytoplasmic inclusions containing α-synuclein protein aggregates -in the affected neurons. A poorly understood feature of Lewy body diseases is loss of sympathetic nerves in the heart and other organs, manifesting as orthostatic hypotension (OH; also known as postural hypotension). We asked whether sympathetic denervation is associated with decreased uptake of catecholamines, such as dopamine and norepinephrine, into storage vesicles within sympathetic neurons. We used 6-[ 18 F]-dopamine ( 18 F-DA) to track myocardial uptake and retention of catecholamines. Concurrently, the fate of intra-neuronal 18 F-DA was followed by assessment of arterial plasma levels of the 18 F-DA metabolite 18 F-dihydroxyphenylacetic acid ( 18 F-DOPAC). The ratio of myocardial 18 F-DA to arterial 18 F-DOPAC provided an index of vesicular uptake. Tracer concentrations were measured in patients with PD with or without orthostatic hypotension (PD+OH, PD-No-OH); in patients with pure autonomic failure (PAF, a Lewy body disease without parkinsonism); in patients with multiple system atrophy (MSA, a non-Lewy body synucleinopathy); and in normal controls. Patients with PD+OH or PAF had decreased vesicular 18 F-DA uptake and accelerated 18 F-DA loss, compared with MSA and control subjects. PD-No-OH patients could be subtyped into one of these categories based on their initial 18 F-DA uptake. We conclude that sympathetic denervation in Lewy body diseases is associated with decreased vesicular uptake of neuronal catecholamines, suggesting that vesicular monoamine transport is impaired. Vesicular uptake may constitute a novel target for diagnosis, treatment, and prevention.

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“…7). Ectoprotein kinases use extracellular ATP as a phosphate donor to phosphorylate endogenous cell surface proteins as well as soluble proteins and have been implicated in a number of biological phenomena including cell adhesion (52), Ca 2ϩ influx (53), neurotransmitter uptake (54,55), synaptogenesis (41), neurite outgrowth (45,56), synaptic plasticity, and long term potentiation (47). We reported previously that the developmental change in the localization of NGC on the Purkinje cells correlates well with synaptogenesis of the climbing fiber system on Purkinje cells (16), suggesting that NGC is involved in the selective synaptogenesis in the cerebellum.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Neuroglycan C, a Brain-specific Transmembrane Chondroitin Sulfate Proteoglycan, and Its Localization in the Lipid Rafts

Yamauchi¹,

Tokita²,

Aono³

et al. 2002

Journal of Biological Chemistry

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Neuroglycan C (NGC) is a brain-specific transmembrane chondroitin sulfate proteoglycan. In the present study, we examined whether NGC could be phosphorylated in neural cells. On metabolic labeling of cultured cerebral cortical cells from the rat fetus with 32 P i , serine residues in NGC were radiolabeled. Some NGC became detectable in the raft fraction from the rat cerebrum, a signaling microdomain of the plasma membrane, with cerebral development. NGC from the non-raft fraction, not the raft fraction, could be phosphorylated by an in vitro kinase reaction. The phosphorylation of NGC was inhibited by adding to the reaction mixture a recombinant peptide representing the ectodomain of NGC, but not by adding a peptide representing its cytoplasmic domain. NGC could be labeled by an in vitro kinase reaction using [␥-32 P]GTP as well as [␥-32 P]ATP, and this kinase activity was partially inhibited by 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole, a selective inhibitor of casein kinase II. In addition to the intracellular phosphorylation, NGC was also phosphorylated at the cell surface by an ectoprotein kinase. This is the first report to demonstrate that NGC can be phosphorylated both intracellularly and pericellularly, and our findings suggest that a kinase with a specificity similar to that of casein kinase II is responsible for the NGC ectodomain phosphorylation.

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Extracellular ATP Stimulates Norepinephrine Uptake in PC12 Cells

Cited by 32 publications

References 29 publications

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Ecto‐Protein Kinase and Surface Protein Phosphorylation in PC12 Cells: Interactions with Nerve Growth Factor

Intra-neuronal vesicular uptake of catecholamines is decreased in patients with Lewy body diseases

Phosphorylation of Neuroglycan C, a Brain-specific Transmembrane Chondroitin Sulfate Proteoglycan, and Its Localization in the Lipid Rafts

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