Abstract
The role of protein kinase C and intracellular Ca2+ on amphetamine-mediated dopamine release through the norepinephrine plasmalemmal transporter in undifferentiated PC12 cells was investigated. The selective protein kinase C inhibitor chelerythrine completely inhibited endogenous dopamine release elicited by 1 μM amphetamine. Direct activation of protein kinase C increased dopamine release in a Ca2+-insensitive, imipramine-sensitive manner and the release was not additive with amphetamine. Exocytosis was not involved since these events were not altered by either deletion of extracellular Ca2+ or reserpine pretreatment. Down-regulation of protein kinase C activity by long-term phorbol ester treatment resulted in a dramatic decrease in amphetamine-mediated dopamine release with no apparent effect on [3H]dopamine uptake. To more completely examine a role for Ca2+, intracellular Ca2+ was chelated in the cells. Depletion of intracellular Ca2+ considerably decreased dopamine release in response to 1 μM amphetamine compared with vehicle-treated cells, but had no effect on the [3H]dopamine uptake. Thus, our results suggest that amphetamine-mediated dopamine release through the plasmalemmal norepinephrine transporter is highly dependent on protein kinase C activity and intracellular but not extracellular Ca2+. Furthermore, protein kinase C and intracellular Ca2+ appear to regulate [3H]dopamine inward transport and amphetamine-mediated outward transport of dopamine independently in PC12 cells.
Footnotes
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Send reprint requests to: Margaret E. Gnegy, Department of Pharmacology, 2220 MSRB III, University of Michigan School of Medicine, Ann Arbor, MI 48109-0632. E-mail: pgnegy{at}umich.edu
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This work was supported by National Research Service Award Grant 5F32DA05912 (to L.K.), Basic Science Research Partnership Grant, University of Michigan Medical School, and Grant DA11697 from the National Institutes of Health.
- Abbreviations:
- AMPH
- amphetamine
- NE
- norepinephrine
- DA
- dopamine
- NET
- norepinephrine transporter
- DAT
- dopamine transporter
- PKC
- protein kinase C
- TPA
- 12-O-tetradecanoylphorbol 13-acetate
- BAPTA-AM
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester
- KRB
- Krebs-Ringer buffer
- HPLC
- high-performance liquid chromatography
- PAGE
- polyacrylamide gel electrophoresis
- VMAT
- vesicular monoamine transporter
- ANOVA
- analysis of variance
- Received December 1, 2000.
- Accepted January 31, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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