Nitric oxide modulates agonist-evoked Ca2+ release and influx responses in PC12-64 cells

Nitric oxide (NO) is a signalling molecule involved in events crucial to neuronal cell function such as neurotransmitter release gene transcription, and neurotoxicity. In these, as well as in many other neuronal processes, a key role may be played by the increases of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) occuring in response to activation of plasma membrane receptors coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis. Such a [Ca²⁺]_i increases are sustained by release of the cation from intracellular stores and stimulation of influx through specific Ca²⁺ channels. We have investigated the role of NO in modulating the two above Ca²⁺ processes occuring subsequently to muscarinic receptor activation in a selected clone (PC12-64) of PC12 cells, a neurosecretory/neuronal cell model. Analysis of [Ca²⁺]_i variations in fura-2-loaded cells, exposed to different NO synthase inhibitors or NO donors, showed that Ca²⁺ release from intracellular stores was moderately inhibited and stimulated by these two groups of drugs, respectively, while Ca²⁺ influx through the channels directly coupled to muscarinic receptors was found to be insensitive to NO action. In contrast, Ca²⁺ influx activated by muscarinic receptor-induced store depletion (investigated also by Mn²⁺ quenching of the fura-2 signal) was increased by NO generation and inhibited by NO synthase blockade. Incubation of the cells with 8-bromo cGMP did not mimick the action of NO, suggesting that the effect of the messenger on Ca²⁺ influx is exerted through a signalling pathway different from cGMP generation.