Involvement of Extracellular Signal-Regulated Kinase (ERK) in Pardaxin-Induced Dopamine Release from PC12 Cells

  1. Eugenia Bloch-Shilderman1,
  2. Hao Jiang3,
  3. Saleh Abu-Raya1,
  4. Michal Linial1,2 and
  5. Philip Lazarovici2,1
  1. 1Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine (E.B.-S., S.A.-R., P.L.), and 2Department of Biological Chemistry, Life Sciences Institute (M.L.), The Hebrew University of Jerusalem, Jerusalem, Israel; 3William T. Gossett Neurology Laboratories, Henry Ford Health Sciences Center, and John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan (H.J.)

    Abstract

    Pardaxin (PX), an ionophore-peptide neurotoxin isolated from the fishPardachirus marmoratus, induces neurotransmitter release from neuronal preparations by both calcium-dependent and calcium-independent mechanisms. The aim of the present study was to investigate the role of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in pardaxin-induced dopamine (DA) release. The experiments were performed on variants of the PC12 cell line, an established cellular model for investigating DA release. Time course experiments indicated that PX, at nontoxic concentrations, stimulated ERK1 and ERK2 within 5 to 15 min, measured with a dual phospho-ERK antibody. PX stimulation of ERK activity was calcium (Ca2+)-dependent and followed by ERK translocation to the nucleus. This effect was temporally related to PX-induced exocytosis, and measured by [3H]dopamine release as well as by a vesicle fusion-based enzyme-linked immunosorbent assay. Blocking ERK activity with the specific mitogen-activated protein kinase kinase inhibitors PD98059 (50 μM for 45 min) and UO126 (30 μM for 30 min) inhibited PX-induced exocytosis in the presence but not in the absence of extracellular Ca2+. These results suggest the essential role of ERKs in PX-induced DA release under physiological conditions and support the hypothesis that ERKs are involved in regulating exocytosis.

    Footnotes

    • Send reprint requests to: Prof. Lazarovici Philip, Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, 91120, Israel. E-mail: lazph{at}md2.huji.ac.il

    • ↵1 M.L. is supported by the Ministry of Health and is a member of the Otto Levi Center for Molecular and Cellular Neurobiology.

    • ↵2 P.L. is affiliated with and supported by the David R. Bloom Center for Pharmacy at the Hebrew University.

    • Abbreviations:
      PX
      pardaxin
      DA
      dopamine
      MAPK
      mitogen-activated protein kinase
      ERK
      extracellular signal-regulated kinase
      MEK
      MAPK/ERK kinase
      MBP
      myelin basic protein
      NGF
      nerve growth factor
      DMEM
      Dulbecco's modified Eagle's medium
      PC12
      pheochromocytoma cell line
      M-M17-26
      a dominant-negative Ras PC12 variant cell line
      ELISA
      enzyme-linked immunosorbent assay
      PBS
      phosphate-buffered saline
      PAGE
      polyacrylamide gel electrophoresis
      CON
      control
      WT
      wild type
      • Received July 13, 2000.
      • Accepted November 11, 2000.
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    1. JPET March 1, 2001 vol. 296 no. 3 704-711
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