Modulation of α7 Nicotinic Receptor-Mediated Calcium Influx by Nicotinic Agonists

Abstract

Our previous work had demonstrated stable expression of rat α7 α-bungarotoxin (α-BGT) binding sites by GH₄C₁ rat pituitary cells, a clonal line that does not endogenously express nicotinic receptors. The stably expressed α7 sites had similar binding affinities, pharmacological profiles, kinetic properties, and molecular size as rat brain α-BGT receptors, suggesting that they represent a good model system for studying receptor function. The present data show that nicotinic receptor agonists increase intracellular calcium levels ([Ca²⁺]_i), as assessed using Fura-2, in α7/GH₄C₁ cells in a dose-dependent manner with EC₅₀ values that correlate well with the affinity of these ligands for α7/GH₄C₁ α-BGT receptors. Nicotinic receptor antagonists inhibited agonist-induced increases in [Ca²⁺]_i, with IC₅₀ values in the nanomolar to micromolar range. The nicotinic agonist-induced increase in [Ca²⁺]_i required extracellular calcium and did not occur in the presence of CdCl₂, suggesting that agonist-induced increases in [Ca²⁺]_i are due to an influx of extracellular calcium through voltage-gated calcium channels. Preexposure of the α7/GH₄C₁ cells to 8-bromo cAMP resulted in an enhanced [Ca²⁺]_i in response to agonist, suggesting that phosphorylation by adenylate cyclase may regulate receptor responsiveness. Interestingly, short-term preexposure (40–60 sec) of the cells to subthreshold concentrations of nicotinic agonist-enhanced receptor-stimulated calcium influx (up to 55%) while activating agonist concentrations completely blocked receptor-mediated responses. Long-term exposure of α7/GH₄C₁cells to K⁺ resulted in about a 2-fold increase in α-BGT receptors and in agonist-evoked calcium influx. The sensitivity of these up-regulated receptors were modulated by subthreshold and activating concentrations of agonist in a manner similar to control receptors. The present results, demonstrating a biphasic regulation of α7 receptor-mediated calcium influx by nicotinic agonists, suggest that these receptors may play an important role in neuronal function under control and depolarizing conditions.