Melittin stimulates fatty acid release through non-phospholipase-mediated mechanisms and interacts with the dopamine transporter and other membrane-spanning proteins

Dove J. Keith, Amy J. Eshleman, Aaron Janowsky

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)501-510
Number of pages10
JournalEuropean Journal of Pharmacology
Volume650
Issue number2-3
DOIs
StatePublished - Jan 15 2011

Fingerprint

Melitten
Dopamine Plasma Membrane Transport Proteins
Membrane Proteins
Fatty Acids
Phospholipases A2
Arachidonic Acid
Membranes
Spiperone
Phospholipase D
Dopamine Agents
Dopamine D2 Receptors
HEK293 Cells
Type C Phospholipases
Cocaine
Dopamine
Phospholipids

Keywords

  • Arachidonic acid
  • Dopamine receptor
  • Dopamine transporter
  • Endocytosis
  • Phospholipase

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Melittin stimulates fatty acid release through non-phospholipase-mediated mechanisms and interacts with the dopamine transporter and other membrane-spanning proteins",
abstract = "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.",
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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

PY - 2011/1/15

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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.

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