Three-dimensional distribution of Ca2+ through quail fibroblasts transfected with nicotinic aceylcholine receptors after stimulation with the nicotinic agonist suberyldicholine.

Cross K.M.L., Jane S.D., Wild A.E., Foreman R.C, Manzotti,P.S. and Chad J.E (1995b)

World Wide Web: URL (Uniform Resource Location) -http://www.neuro.soton.ac.uk/Big/Papers/SDC/sdc.html

Abstract

  1. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1, was used to measure intracellular calcium ion (Ca2+i) activity in quail fibroblasts (QF18 cells) stably transfected with constructs encoding muscle-type nicotinic acetylcholine receptors (AChRs).
  2. Application of the nicotinic 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 164ñ22nM (mean ñ s.e.mean; n=40 cells) rising to 600ñ81nM on addition of SDC (10æM; n=15 cells), whereas no increase in Ca2+i was seen in nontransfected control QT6 fibroblasts (before: 128ñ9nM, n=40; after: 113ñ13nM, n=15).
  3. The increases in Ca2+i caused by application of SDC were dose dependent, with an EC50 value of 12.7ñ5.9æM (n=14).
  4. The responses to SDC in QF18 cells were blocked by prior application of à- bungarotoxin (200nM), by the addition of Cd2+ (100æM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and -conotoxin, which act with IC50 values of 100nM and 100pM 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.
Keywords; nicotinic acetylcholine receptors; confocal laser scanning microscopy; suberyldicholine; voltage-sensitive calcium channels; quail fibroblasts; Indo-1.

Reference.

K.M.L. Cross, S.D. Jane, A.E. Wild1, R.C. Foreman & J.E. Chad, (1995) Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts increases intracellular calcium. Brit. J.Pharm. submitted.

Supplemental Images referenced in the Paper.

Following on from the Abstract of the paper.
  1. The first group (54Kb) show three dimensional pictures of the calcium levels in various Z-planes of quail fibroblasts transfected with foetal nicotinic acetylcholine receptors. Calcium levels are colour coded blue to red, red being highest. Click on the first image to see the sequence in detail

  2. The second group (59Kb) show the calcium levels in the lowest Z-plane from the above cells over time. The calcium levels are colour coded from blue to red, and are shown as a 3-dimensional peak, with the higher the peak, the higher the calcium levels.