ELEVATION OF CYTOSOLIC CALCIUM BY CHOLINOCEPTOR AGONISTS IN SH-SY5Y HUMAN NEUROBLASTOMA-CELLS - ESTIMATION OF THE CONTRIBUTION OF VOLTAGE-DEPENDENT CURRENTS

1 Muscarinic but not nicotinic receptor stimulation in SH-SY5Y human neuroblastoma cells induces a concentration-dependent increase in [H-3]-inositol phosphate formation and a biphasic increase in [Ca2+]i. The latter involves release from both an intracellular store and Ca2+ entry across the plasma membrane. Here we examine the possibility that this agonist-stimulated Ca2+ entry occurs indirectly, as a consequence of depolarization. 2 Electrophysiological characterization, by whole cell patch-clamp techniques revealed that SH-SY5Y cells possess a tetrodotoxin-sensitive inward sodium current, a dihydropyridine-insensitive calcium current and an outward potassium current which was blocked by tetraethylammonium, 4-aminopyridine and intracellular caesium ions. The outward potassium current showed voltage-dependent activation and inactivation, similar to that seen for A-currents. 3 Application of nicotinic agonists evoked an inward current in cells voltage-clamped at negative holding potentials, but this current rectified, resulting in little or no outward current flow at positive potentials. The mean amplitude at a holding potential of - 60 mV was - 1.14 nA. Extrapolation of the current-voltage relation gave a reversal potential of + 8 mV, indicative of a non-specific cationic permeability. 4 Application of muscarinic agonists had no detectable effect in most of the cells tested. However, in one third of cells studied, a small slowly activating inward current was observed. The mean amplitude of this current at a holding potential of - 60 mV was - 8.3 pA. 5 This study confirms that SH-SY5Y cells possess voltage-dependent sodium, potassium and calcium currents. In addition, these cells are strongly depolarized by nicotinic agonists, which produce little change in [Ca2+]i. On the other hand, muscarinic agonists produce profound changes in [Ca2+]i with only a small inward current (depolarization). The contrasting effects of these two cholinoceptor agonists strongly implies that the Ca2+ entry after muscarinic receptor activation is not primarily due to activation of voltage-dependent calcium channels.