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
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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
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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.
- The American Society for Pharmacology and Experimental Therapeutics
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