Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Ahnert‐Hilger, Gudrun; Gratzl, Manfred

doi:10.1111/j.1471-4159.1987.tb00959.x

Cited by 32 publications

(9 citation statements)

References 35 publications

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“…The processes of membrane fusion during exocytosis have no immediate requirement for ATP (Cockcroft et al, 1987;Howell et al, 1989;Ahnert-Hilger and Gratzl, 1987;Holz et al, 1989;Vilmart-Seuwen et al, 1986;Baker and Whitaker, 1978;Moy et al, 1983;Sasaki and Epel, 1983;Crabb and Jackson, 1985). These observations accord with our finding that exocytosis was unaffected by the nonhydrolyzable ATP analogue AppNHp and suggest that protein phosphorylation per se is not one of the steps in the path to membrane fusion.…”

Section: How Might Thiophosphorylation Inhibit Exocytosis?supporting

confidence: 86%

Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

Whalley

Crossley

Whitaker

1991

The Journal of Cell Biology

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Abstract. We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATE ATPTS (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinase s, leading to irreversible protein thiophosphorylation (Gratecos, D., and E. H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP3,S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATPTS requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATPTS irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATPTS. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATPTS, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calciumstimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.

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Section: How Might Thiophosphorylation Inhibit Exocytosis?supporting

confidence: 86%

Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

Whalley

Crossley

Whitaker

1991

The Journal of Cell Biology

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“…Brain tissues express TRPC6 at high levels where it could contribute to excessive iron accumulation, leading to neurotoxicity. We show the relevance of this pathway using rat adrenal medullary tumour pheochromocytoma PC12 cells as a neuronal model, since they show higher NTBI uptake when differentiated into neuronal cells after treatment with NGF (nerve growth factor) [24][25][26][27]. We further support this model using HEK-293 (human embryonic kidney 293) cells transfected with TRPC6.…”

Section: Introductionmentioning

confidence: 53%

“…Differentiation of PC12 cells into neuronal cells after NGF treatment has been well-characterized [24,25,27]. PC12 cells can proliferate rapidly, but NGF treatment transforms them into a phenotype that exhibits neurite outgrowth and electrical properties of sympathetic neurons.…”

Section: Fe Uptake In Pc12 Cellsmentioning

confidence: 99%

Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells

Mwanjewe

Grover

2004

Biochemical Journal

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Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the a1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity.

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“…Investigation of stimulus-secretion coupling is facilitated by the use of permeabilized cells, whereby intracellular modulators can be introduced into the cell interior. For example, exocytotic release has been investigated in bovine adrenal chromaffin cells permeabilized by Staphylococcal a-toxin [1,2], in platelets permeabilized with high voltage discharge [3,4], and in rat mast cells permeabilized with streptolysin-O (SLO) [5].…”

Section: Introductionmentioning

confidence: 99%

Exocytosis in electropermeabilized neutrophils. Responsiveness to calcium and guanosine 5′-[γ-thio]triphosphate

Boonen

Steveninck

Dubbelman

et al. 1992

Biochemical Journal

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Electropermeabilized neutrophils were used to study the exocytotic response in rabbit neutrophils. Enzyme release from electropermeabilized neutrophils could be induced by elevating the Ca2+ concentration. Ca(2+)-induced secretion was significantly enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a concentration-dependent manner. The effect of GTP[S] could be blocked by guanosine 5'-[beta-thio]diphosphate (GDP[S]) and was not affected by pertussis toxin. GTP[S] did not induce enzyme release in the absence of Ca2+. Induction of an exocytotic response did not require addition of ATP. However, neutrophils permeabilized in the absence of ATP became refractory to stimulation due to a reduction in their affinity for Ca2+. Responsiveness to the effectors Ca2+ or Ca2+ + GTP[S] could be prolonged or restored by ATP. ATP was not the only agent that prolonged responsiveness; other nucleotides and inorganic phosphates were also effective. The protein kinase C inhibitors staurosporine and 1-O-hexadecyl-2-methyl-sn-glycerol did not inhibit exocytosis and had only a small effect on the prolongation and restoration of responsiveness by ATP. A hypothesis is presented suggesting that the loss of responsiveness is caused by dephosphorylation and that the restoration or prolongation of responsiveness is not mediated by protein kinase C. It is possible that an as yet unidentified Ca(2+)-binding protein is dephosphorylated, resulting in a decrease in Ca2+ affinity.

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Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Cited by 32 publications

References 35 publications

Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells

Exocytosis in electropermeabilized neutrophils. Responsiveness to calcium and guanosine 5′-[γ-thio]triphosphate

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