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 [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. 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 [Ca2t]1. On the other hand, muscarinic agonists produce profound changes in [Ca2+1J 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.
Forsythe
I D
ID
Department of Physiology, University of Leicester.
Lambert
D G
DG
Nahorski
S R
SR
Lindsdell
P
P
D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004553:Electric Conductivity; D004594:Electrophysiology; D005731:Ganglionic Stimulants / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D015640:Ion Channel Gating; D009116:Muscarine / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009538:Nicotine / Q000494:pharmacology; D010277:Parasympathomimetics / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005731:Ganglionic Stimulants; D007295:Inositol Phosphates; D010277:Parasympathomimetics; D009538:Nicotine; D009116:Muscarine; D002217:Carbachol; D011188:Potassium; D002118:Calcium