Elevation of cytosolic calcium by cholinoceptor agonists in SH‐SY5Y human neuroblastoma cells: estimation of the contribution of voltage‐dependent currents

Muscarinic but not nicotinic receptor stimulation in SH‐SY5Y human neuroblastoma cells induces a concentration‐dependent increase in [³H]‐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. 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. 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 +8mV, indicative of a non‐specific cationic permeability. 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. 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. 1992 British Pharmacological Society