Modulation by calcineurin of 5-HT3 receptor function in NG108-15 neuroblastoma x glioma cells

Br J Pharmacol. 1996 Aug;118(7):1836-40. doi: 10.1111/j.1476-5381.1996.tb15611.x.

Abstract

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 microM) or of okadaic acid (1 microM), 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 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) 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.

MeSH terms

  • 1-Methyl-3-isobutylxanthine / pharmacology
  • Amino Acid Sequence
  • Animals
  • Brain Neoplasms / enzymology
  • Brain Neoplasms / metabolism*
  • Calcineurin
  • Calmodulin-Binding Proteins / antagonists & inhibitors
  • Calmodulin-Binding Proteins / pharmacology*
  • Chelating Agents / pharmacology
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Enzyme Inhibitors / pharmacology
  • Glioma / enzymology
  • Glioma / metabolism*
  • Half-Life
  • Molecular Sequence Data
  • Neuroblastoma / enzymology
  • Neuroblastoma / metabolism*
  • Okadaic Acid / pharmacology
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphoprotein Phosphatases / metabolism
  • Phosphoprotein Phosphatases / pharmacology*
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism
  • Rats
  • Receptors, Serotonin / drug effects
  • Receptors, Serotonin / metabolism*
  • Staurosporine / pharmacology
  • Tumor Cells, Cultured

Substances

  • Calmodulin-Binding Proteins
  • Chelating Agents
  • Enzyme Inhibitors
  • Receptors, Serotonin
  • Okadaic Acid
  • Egtazic Acid
  • Protein-Serine-Threonine Kinases
  • Calcineurin
  • Phosphoprotein Phosphatases
  • Staurosporine
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • 1-Methyl-3-isobutylxanthine