Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts: effects on intracellular calcium.
1. The aim of these experiments was to determine the ability of the muscle-type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1 was used. 2. Application of the nicotine agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n = 40 cells) rising to 600 +/- 81 nM on addition of SDC (10 microM; n = 15 cells), whereas no increase in [Ca2+]i was seen in non-transfected control QT6 fibroblasts (before: 128 +/- 9 nM, n = 40; after; 113 +/- 13 nM, n = 15). 3. The increase in [Ca2+]i caused by application of SDC was dose-dependent, with an EC50 value of 12.7 +/- 5.9 microM (n = 14). 4. The responses to SDC in QF18 cells were blocked by prior application of alpha-bungarotoxin (200 nM), by the addition of Ca2+ (100 microM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5. We conclude that activation of the nicotinic AChRs leads to a Na(+)-dependent depolarization and hence activation of endogenous voltage-sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.
Cross
K M
KM
Department of Physiology & Pharmacology, University of Southampton.
Jane
S D
SD
Wild
A E
AE
Foreman
R C
RC
Chad
J E
JE
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002794:Choline / Q000031:analogs & derivatives / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D018722:Nicotinic Agonists / Q000494:pharmacology*; D009543:Nifedipine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011784:Quail; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection; D020866:omega-Conotoxin GVIA
D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D018722:Nicotinic Agonists; D010455:Peptides; C009335:subecholine; D020866:omega-Conotoxin GVIA; D009543:Nifedipine; D002794:Choline; D002118:Calcium