Blockade by ifenprodil of high voltage‐activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N‐methyl‐D‐aspartate receptor antagonist actions

1 The block by ifenprodil of voltage‐activated Ca2+ channels was investigated on increases in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mm K+ (high‐[K+]o) in Fura‐2‐loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole‐cell voltage‐clamp. The effects of ifenprodil on voltage‐activated Ca2+ channels were compared with its antagonist actions on N‐methyl‐D‐aspartate‐ (NMDA) evoked responses in the same neuronal preparations. 2 Rises in [Ca2+]i evoked by transient exposure to high‐[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine‐sensitive Ca2+ channels, with smaller contributions from nifedipine‐resistant, ω‐conotoxin GVIA‐sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel‐web spider venom (Church et al., 1994). Ifenprodil (0.1 −200 μm) reversibly attenuated high‐[K+]o‐evoked rises in [Ca2+]i with an IC50 value of 17 ± 3 μm, compared with an IC50 value of 0.7 ± 0.1 μm for the reduction of rises in [Ca2+]i evoked by 20 μm NMDA. Tested in the presence of nifedipine 10 μm, ifenprodil (1–50 μm) produced a concentration‐dependent reduction of the dihydropyridine‐resistant high‐[K+]o‐evoked rise in [Ca2+]i with an IC50 value of 13 ± 4 μm. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage‐activated Ca2+ channels. 3 Application of the polyamine, spermine (0.25‐5 μm), produced a concentration‐dependent reduction of rises in [Ca2+]i evoked by high‐[K+]o. The antagonist effects of ifenprodil 20 μm on high‐[K+]o‐evoked rises in [Ca2+]i were attenuated by spermine 0.25 mm but not by putrescine 1 or 5 mm. In contrast, spermine 0.1 mm increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 μm) block of NMDA‐evoked rises in [Ca2+]i. 4 Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole‐cell voltage‐clamp. Ifenprodil attenuated both the peak and delayed whole‐cell IBa with an IC50 value of 18 ± 2 μ, whilst it attenuated steady‐state NMDA‐evoked currents with an IC50 of 0.8 ± 0.2 μm. Block of IBa by ifenprodil 10 μm was rapid in onset, fully reversible and occurred without change in the current‐voltage characteristics of IBa. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mm potentiated control NMDA‐evoked currents but attenuated IBa. In agreement with the microspectrofluorimetric studies, co‐application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 μm on NMDA‐evoked responses whereas the reduction of IBa by ifenprodil 10 μm in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive. 5 The results indicate that ifenprodil blocks high voltage‐activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.