Muscarinic m3 receptors and dynamics of intracellular Ca²⁺ cerebellar granule neurons

Activation of muscarinic receptors with carbachol has no effect on intracellular Ca²⁺ concentration in cerebellar granule cell cultures. Only after elevating intracellular Ca²⁺ concentrations using either 40 mM KCl or activating glutamatergic receptors was carbachol able to increase intracellular Ca²⁺. The response lasted about 10 s, and the median increase in intracellular Ca²⁺ with either 100 μM or 300 μM carbachol was about 85 nM. Carbachol at 30 μM elicited an increase in intracellular calcium that was half maximal. After a 16 min or 32 min delay following cell depolarization, responses to carbachol were only found in about 20% of the cells studied and the median increase in intracellular Ca²⁺ was about 14 nM. (-)-Quinuclidinylxanthene-9-carboxylate hemioxalate (QNX) at 10 nM (a muscarinic m3 antagonist), but not methoctramine at 5 μM (a muscarinic m2 antagonist), attenuated the action of 100 μM carbachol. This carbachol-mediated response was elicited in the absence of extracellular Ca²⁺ and in the presence of 10 μM dantrolene, but was blocked by 1 μM thapsigargin. Lastly, treatments of cerebellar granule cell cultures with carbachol (100 μM) for up to 12 h results in a desensitization of the carbachol-mediated release of stored Ca²⁺. Thus, carbachol acts on muscarinic m3 receptors to induce the release of Ca²⁺ from IP₃-sensitive Ca²⁺ stores that must be filled by a prior period of high intracellular Ca²⁺.