ς2-Receptor Regulation of Dopamine Transporter via Activation of Protein Kinase C
- Dr. Linda L. Werling, Department of Pharmacology, The George Washington University Medical Center, 2300 Eye St. NW, Washington, DC 20037. E-mail: phmllw{at}gwumc.edu
Abstract
The elucidation of the mechanisms underlying ς2-receptor activation and signal transduction is crucial to the understanding of ς2-receptor function. Previous studies in our laboratory have demonstrated ς2-receptor-mediated regulation of the dopamine transporter (DAT) as measured by amphetamine-stimulated release of [3H]dopamine (DA) from both rat striatal slices and PC12 cells. The regulation of the DAT in the PC12 cell model was dependent upon activation of Ca2+/calmodulin-dependent kinase II. We have now studied the second messenger systems involved in ς2-receptor-mediated regulation of amphetamine-stimulated [3H]DA release in rat striatal slices, including Ca2+/calmodulin-dependent kinase II, protein kinase C, and sources of calcium required for the enhancement of release produced by ς2-receptor activation. The Ca2+/calmodulin-dependent kinase II inhibitors 1-[N,O-bis-(5-isoquionolinesulfonyl)]-N-methyl-l-tyrosyl-4-phenylpiperazine andN-[2-[[[3-(4′-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxy-benzenesulfonamide phosphate did not significantly affect the (+)-pentazocine-mediated enhancement of amphetamine-stimulated [3H]DA release. However, we found that an inhibitor of protein kinase C, 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-1H-pyrrole-2,5-dione, blocks the (+)-pentazocine-mediated enhancement in rat striatal slices. The protein kinase C activator phorbol 12-myristate 13-acetate, but not the inactive isophorbol 4α,9α,12α,13α,20-pentahydroxytiglia-1,6-dien-3-one, enhanced the amphetamine-stimulated [3H]DA release comparable to the enhancement seen by (+)-pentazocine alone. Additionally, the L-type voltage-dependent calcium channel inhibitor nitrendipine or prior treatment with thapsigargin, but not the N-type voltage-dependent calcium channel ω-conotoxin MVIIA, attenuated the (+)-pentazocine-mediated enhancement. Together, these data suggest that activation of ς2-receptors results in the regulation of DAT activity via a calcium- and protein kinase C-dependent signaling mechanism.
Footnotes
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This study was supported by National Institute on Drug Abuse Grant DA 06667 (to L.L.W.) and National Institute on Drug Abuse Predoctoral Fellowship DA 06002 (to A.E.D.). A.E.D. is a predoctoral student in the Department of Pharmacology, The George Washington Institute for Biomedical Sciences. This work will be part of a dissertation presented to the above-mentioned department in partial fulfillment of the requirements for the Ph.D. degree. The findings herein were reported in preliminary form in Derbez AE, Mody RM, Weatherspoon JK, and Werling LL (1999) Mechanisms by which sigma2 (ς2) receptors may regulate dopamine release ([3H]DA release) from slices of brain tissue and cells in culture. Soc Neurosci Abstr25:1475 and Derbez AE and Werling LL (2000) Regulation of dopamine transporter via sigma-2 receptor activation of calcium-dependent second messengers.FASEB Abstr14:1372.
- Abbreviations:
- SKF10,047
- N-allylnormetazocine
- ER
- endoplasmic reticulum
- AMPH
- amphetamine
- DA
- dopamine
- Ca2+/CaM kinase II
- Ca2+/calmodulin-dependent protein kinase II
- DAT
- dopamine transporter
- PKC
- protein kinase C
- SUP
- supernatant
- MKB
- modified Krebs-HEPES buffer
- ANOVA
- analysis of variance
- TTX
- tetrodotoxin
- S1
- first ampthetamine stimulus
- ISI
- interstimulus interval
- S2
- second amphetamine stimulus
- VDCC
- voltage-dependent calcium channel
- GF109,203X
- 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-1H-pyrrole-2,5-dione
- KN-62
- 1-[N,O-bis-(5-isoquionolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine
- KN-92
- 2-[N-(4′-methoxybenzenesulfonyl)]amino-N-(4′chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate
- KN-93
- N-[2-[[[3-(4′-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxy-benzenesulfonamide phosphate
- PMA
- phorbol-12-myristate-13-acetate
- NIT
- nitrendipine
- BIMU-8
- endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-(1-methyl)ethyl-2-oxo-1H-bezimidazole-1-carboxamide hydrochloride
- 4α-phorbol
- isophorbol (4α,9α,12α,13α,20-pentahydroxytiglia-1,6-dien-3-one)
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- Received August 24, 2001.
- Accepted December 13, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
