Loperamide blocks high-voltage-activated calcium channels and N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurons

The effects of the antidiarrheal agent loperamide on high-voltage-activated (HVA) calcium channel activity and excitatory amino acid-evoked responses in two preparations of cultured hippocampal pyramidal neurons were examined. In rat hippocampal neurons loaded with the calcium-sensitive dye fura-2, rises in intracellular free calcium concentration ([Ca²⁺]_i) evoked by transient exposure to 50 mM K⁺-containing medium [high extracellular potassium concentration ([K⁺]_o)] were mediated by Ca²⁺ flux largely through nifedipine-sensitive Ca²⁺ channels, with smaller contributions from ω-conotoxin GVIA (ω-CgTx)-sensitive Ca²⁺ channels and channels insensitive to both nifedipine and ω-CgTx. Loperamide reversibly blocked rises in [Ca²⁺]_i evoked by high [K⁺]_o in a concentration-dependent manner, with an IC₅₀ of 0.9 ± 0.2 μM. At the highest concentration tested (50 μM), loperamide eliminated rises in [Ca²⁺]: evoked by high [K⁺]_o, a result otherwise achieved only in Ca²⁺-free medium or by the combined application of nifedipine, ω-CgTx, and funnel web spider venom to Ca²⁺-containing medium. The action of loperamide was neither naloxone sensitive nor mimicked by morphine and was seen at concentrations substantially less than those required to block influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor-operated ionophore. Similar results were obtained in cultured mouse hippocampal pyramidal neurons under whole-cell voltage clamp. Voltage-activated Ca²⁺ channel currents carried by barium ions (I_Ba) could be discriminated pharmacologically into nifedipine-sensitive (L-type) and nifedipine-resistant, ω-CgTx-sensitive (N-type) components. Loperamide (0.1-50 μM) produced a concentration-dependent reduction of the peak I_Ba with an IC₅₀ value of 2.5 ± 0.4 μM and, at the highest concentration tested, could fully block I_Ba in the absence of any other pharmacological agent. The loperamide-induced block was rapid in onset and offset, was fully reversible, and did not appear to be related to the known calmodulin antagonist actions of loperamide. The current-voltage characteristics of the whole-cell I_Ba were unaffected by loperamide and the block was not voltage dependent. Loperamide also attenuated NMDA-evoked currents recorded at a membrane potential of -60 mV, with an IC₅₀ of 73 ± 7 μM. The block of NMDA-evoked currents was not competitive in nature, was not reversed by elevation of the extracellular glycine or spermine concentration, and was not affected by changes in the membrane holding potential. Steady state currents evoked by kainate and DL-α-amino-3-hydroxy-5-methylisoxazolepropionic acid were, in contrast, relatively unaffected by 100 μM loperamide. We conclude that loperamide, applied at low micromolar concentrations, is a broad-spectrum blocker of neuronal HVA Ca²⁺ channels. At higher concentrations, it reduces Ca²⁺ flux through NMDA receptor-operated channels. Loperamide may prove to be a useful tool in experiments in which a general and reversible suppression of neuronal HVA Ca²⁺ channel activity is required.