NICOTINIC AND MUSCARINIC ACETYLCHOLINE RESPONSES IN DIFFERENTIATED PC12 CELLS

Nicotinic and muscarinic acetylcholine (ACh) responses were investigat ed in PC12 cells using the conventional whole-cell and nystatin perfor ated patch techniques. With the nystatin perforated patch, ACh induced three kinds of ionic currents: a rapid transient inward current, a su bsequent transient outward current and a long-lasting slow inward curr ent, whereas only a transient inward current was recorded by conventio nal whole-cell patch. The transient rapid inward current was mimicked by nicotine, but not by muscarine. On the contrary, the transient outw ard current and the long-lasting slow inward current were mimicked by muscarine but not by nicotine. Both nicotinic and muscarinic antagonis ts inhibited the transient inward current and the subsequent outward c urrent in a concentration-dependent manner. The current-voltage relati onship for the nicotine-induced transient current showed an inward rec tification and the reversal potential was close to the Na+ equilibrium potential. The ACh-, muscarine-, CCh- and oxotremorine-M induced outw ard currents increased in a sigmoidal fashion with an increase in the concentration. Neither McN-A-343, an M1 agonist, nor oxotremorine, an M2 agonist, mimicked the muscarinic response. The reversal potential o f the muscarinic response was close to the K+ equilibrium potential. T he muscarinic response was not affected by pre-treatment with pertussi s toxin but was enhanced by pre-treatment with Li+. In the cells perfu sed with Ca2+-free external solution, only the first application of AC h induced the muscarinic response. Calmodulin antagonists reversibly b locked the muscarinic response in a concentration-dependent manner. Ne ither protein kinase C inhibitor (H-7), protein kinase A inhibitor (H- 8), nor Ca-calmodulin dependent kinase II inhibitor (KN-62) affected t he muscarinic response. It was concluded that the ACh-induced rapid in ward current was passing through non-selective cation channels coupled with nicotinic ACh receptors. On the other hand, the muscarinic respo nse is mediated by the activation of M3 receptors coupled to IAP-insen sitive G-protein which stimulates the phosphatidylinositol pathway thr ough phospholipase C. Consequently, Ca2+ was released by the increase in IP3. Finally, Ca2+-calmodulin binding may lead to opening of the K channels.