Gi- and Protein Kinase C-Mediated Heterologous Potentiation of Phospholipase C Signaling by G Protein-Coupled Receptors

  1. Martina Schmidt,
  2. Barbara Lohmann,
  3. Kerstin Hammer,
  4. Stephan Haupenthal,
  5. Matthias Voß,
  6. Christoph Nehls and
  7. Karl H. Jakobs
  1. Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany

    Abstract

    We recently reported that activation of the highly efficient phospholipase C (PLC) stimulatory m3 muscarinic acetylcholine receptor (mAChR) can induce a long-lasting Gi-mediated heterologous potentiation of PLC stimulation in human embryonic kidney (HEK) 293 cells, which was accompanied by an increased cellular level of the PLC substrate phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Here, we examined whether such a potentiated PLC response is also induced by the rather poorly PLC stimulatory m2 mAChR and the endogenously expressed purinergic and lysophosphatidic acid receptors. Pretreatment of m2 mAChR-expressing HEK 293 cells for 2 min with carbachol, followed by agonist washout and measurement of PLC activity ≥40 min later, caused a long-lasting (up to ∼90 min) heterologous potentiation of receptor- and G protein-mediated PLC stimulation. A similar heterologous potentiation of receptor-mediated PLC stimulation was induced by short term activation of lysophosphatidic acid and purinergic receptors. Either of the three receptor agonists increased the cellular level of PtdIns(4,5)P2 by ∼50%. The mAChR-induced PLC potentiation was fully prevented by either pertussis toxin or the protein kinase C (PKC) inhibitors staurosporine and Gö 6976, which did not affect acute PLC stimulation. On the other hand, the rise in PtdIns(4,5)P2 was prevented only by combined treatment of HEK 293 cells with pertussis toxin and PKC inhibitors. In conclusion, we demonstrated that activation of poorly PLC stimulatory receptors can also induce a long-lasting Gi-mediated heterologous potentiation of PLC signaling in HEK 293 cells and that this novel PLC regulatory process is under the control of PKC.

    Footnotes

    • Send reprint requests to: Dr. Martina Schmidt, Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. E-mail:martina.schmidt{at}uni-essen.de

    • This work was supported by the Deutsche Forschungsgemeinschaft and the IFORES program of the Universitätsklinikum Essen.

    • Abbreviations:
      GTPγS
      guanosine-5′-O-(3-thio)triphosphate
      HBSS
      Hanks’ balanced salt solution
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      Ins(1
      4,5)P3, inositol-1,4,5-trisphosphate
      LPA
      lysophosphatidic acid
      mAChR
      muscarinic acetylcholine receptor
      PKC
      protein kinase C
      PLC
      phospholipase C
      PMA
      phorbol-12-myristate-13-acetate
      PtdIns
      phosphatidylinositol
      PtdIns4P
      phosphatidylinositol-4-monophosphate
      PtdIns(4
      5)P2, phosphatidylinositol-4,5-bisphosphate
      PTX
      pertussis toxin
      HEK
      human embryonic kidney
      • Received September 16, 1997.
      • Accepted February 17, 1998.
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    1. Molecular Pharmacology June 1, 1998 vol. 53 no. 6 1139-1148
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