1 We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells.
2 The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau(1) (1.1+/-0.21s) and tau(2) (8.9+/-1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents.
3 Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness.
4 Inclusion of the protein kinase inhibitor, staurosporine (1 mu M) or of okadaic acid (1 mu M), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity.
5 Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau(1) (1.4+/-0.28s) and tau(2) (11.3+/-1.7s). The complex, however, prevented the loss of 5-HT3 receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 mu M).
6 The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 mu M) showed a half-life time (tau 1/2 of 2.6+/-0.12 min in control cells which was reduced to 1.6+/-0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex.
7 We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.
|Number of pages||5|
|Journal||British Journal of Pharmacology|
|Publication status||Published - Aug-1996|
- 5-HT3 receptors
- NG108-15 cells
- receptor desensitization
- DEPENDENT PROTEIN-KINASE
- ROOT GANGLION NEURONS