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European Biophysics Journal

, Volume 33, Issue 3, pp 255–264 | Cite as

Phospholipid metabolism is required for M1 muscarinic inhibition of N-type calcium current in sympathetic neurons

  • Liwang Liu
  • Mandy L. Roberts
  • Ann R. RittenhouseEmail author
Article

Abstract

The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca2+ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein Gq and identified phospholipase C (PLC), phospholipase A2 (PLA2), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M2/M4 muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M1 muscarinic receptors (M1Rs), Gqα, PLC, PLA2, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA2 cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M1Rs, Gqα, PLC, PIP2, PLA2, and AA for N-current inhibition.

Keywords

Arachidonic acid Muscarinic receptors N-current inhibition Oxotremorine-M Superior cervical ganglion neurons 

Abbreviations

AA

arachidonic acid

BAPTA

1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

BSA

bovine serum albumin

DAG

diacylglycerol

DEDA

7,7-dimethyleicosadienoic acid

ETYA

5,8,11,14-eicosatetraynoic acid

FPL

FPL 64176

IP3

inositol-1,4,5-trisphosphate

L-channel

L-type calcium channel

L-current

L-type calcium current

LPA

lysophosphatidic acid

M1R

M1 muscarinic receptor

N-channel

N-type calcium channel

N-current

N-type calcium current

NMN

nimodipine

OAG

1-(cis-9-octadecenoyl)-2-acetyl-sn-glycerol

OPC

oleoyloxyethyl phosphorylcholine

Oxo-M

oxotremorine methiodide

PIP2

phosphatidylinositol-4,5-bisphosphate

PLC

phospholipase C

PLA2

phospholipase A2

PTX

pertussis toxin

SCG

superior cervical ganglion

Notes

Acknowledgements

Robert Carraway helped with developing the protocol for Western blot analysis. We thank Claire Baldwin, John F. Heneghan, and Maggie Lee for critically reading various versions of the manuscript and helping with the editing. This project was funded by an Established Investigator Award from the American Heart Association (ARR) and by a grant from NINDS (NS34195).

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Copyright information

© EBSA 2004

Authors and Affiliations

  • Liwang Liu
    • 1
  • Mandy L. Roberts
    • 1
  • Ann R. Rittenhouse
    • 1
    Email author
  1. 1.Department of PhysiologyUniversity of Massachusetts Medical SchoolWorcesterUSA

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