TY - JOUR
T1 - Melittin stimulates fatty acid release through non-phospholipase-mediated mechanisms and interacts with the dopamine transporter and other membrane-spanning proteins
AU - Keith, Dove J.
AU - Eshleman, Amy J.
AU - Janowsky, Aaron
N1 - Funding Information:
We thank Dr. Kim Neve for providing the dopamine D 2 receptor cDNA. This project was supported by NRSA grant 5F31DA016499-02 and Tartar Trust Fellowship (DJK), and NIH/VA Interagency Agreement Y1-DA5007, the VA Merit Review , and VA Research Career Scientist Programs (AJ).
PY - 2011/1/15
Y1 - 2011/1/15
N2 - Phospholipase A2 releases the fatty acid arachidonic acid from membrane phospholipids. We used the purported phospholipase A2 stimulator, melittin, to examine the effects of endogenous arachidonic acid signaling on dopamine transporter function and trafficking. In HEK-293 cells stably transfected with the dopamine transporter, melittin reduced uptake of [3H]dopamine. Additionally, measurements of fatty acid content demonstrated a melittin-induced release of membrane-incorporated arachidonic acid, but inhibitors of phospholipase C, phospholipase D, and phospholipase A2 did not prevent the release. Subsequent experiments measuring [125I]RTI-55 binding to the dopamine transporter demonstrated a direct interaction of melittin, or a melittin-activated endogenous compound, with the transporter to inhibit antagonist binding. This effect was not specific to the dopamine transporter, as [3H]spiperone binding to the recombinant dopamine D2 receptor was also inhibited by melittin treatment. Finally, melittin stimulated an increase in internalization of the dopamine transporter, and this effect was blocked by pretreatment with cocaine. Thus, melittin acts through multiple mechanisms to regulate cellular activity, including release of membrane-incorporated fatty acids and interaction with the dopamine transporter.
AB - Phospholipase A2 releases the fatty acid arachidonic acid from membrane phospholipids. We used the purported phospholipase A2 stimulator, melittin, to examine the effects of endogenous arachidonic acid signaling on dopamine transporter function and trafficking. In HEK-293 cells stably transfected with the dopamine transporter, melittin reduced uptake of [3H]dopamine. Additionally, measurements of fatty acid content demonstrated a melittin-induced release of membrane-incorporated arachidonic acid, but inhibitors of phospholipase C, phospholipase D, and phospholipase A2 did not prevent the release. Subsequent experiments measuring [125I]RTI-55 binding to the dopamine transporter demonstrated a direct interaction of melittin, or a melittin-activated endogenous compound, with the transporter to inhibit antagonist binding. This effect was not specific to the dopamine transporter, as [3H]spiperone binding to the recombinant dopamine D2 receptor was also inhibited by melittin treatment. Finally, melittin stimulated an increase in internalization of the dopamine transporter, and this effect was blocked by pretreatment with cocaine. Thus, melittin acts through multiple mechanisms to regulate cellular activity, including release of membrane-incorporated fatty acids and interaction with the dopamine transporter.
KW - Arachidonic acid
KW - Dopamine receptor
KW - Dopamine transporter
KW - Endocytosis
KW - Phospholipase
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U2 - 10.1016/j.ejphar.2010.10.023
DO - 10.1016/j.ejphar.2010.10.023
M3 - Article
C2 - 20969853
AN - SCOPUS:78651085635
SN - 0014-2999
VL - 650
SP - 501
EP - 510
JO - European Journal of Pharmacology
JF - European Journal of Pharmacology
IS - 2-3
ER -