BLOCKADE BY IFENPRODIL OF HIGH VOLTAGE-ACTIVATED CA2+ CHANNELS IN RAT AND MOUSE CULTURED HIPPOCAMPAL PYRAMIDAL NEURONS - 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+](0)) in Fura-2-1oaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (I-Ba) 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, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 mu M) reversibly attenuated high-[K+](o)-evoked rises in [Ca2+](i) with an IC50 value of 17 +/- 3 mu M, compared with an IC50 value of 0.7 +/- 0.1 mu M for the reduction of rises in [Ca2+](i) evoked by 20 mu M NMDA. Tested in the presence of nifedipine 10 mu M, ifenprodil (1-50 mu M) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+](o)-evoked rise in [Ca2+](i) with an IC50 value of 13 +/- 4 mu M. 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 mM), produced a concentration-dependent reduction of rises in [Ca2+](i) evoked by high-[K+](o). The antagonist effects of ifenprodil 20 mu M 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 mu M) 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 I-Ba with an IC50 value of 18 + 2 mu M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 mu M. Block of I-Ba by ifenprodil 10 mu M was rapid in onset, fully reversible and occurred without change in the current-voltage characteristics of I-Ba The ifenprodil block of I-Ba 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 I-Ba. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 mu M on NMDA-evoked responses whereas the reduction of I-Ba by ifenprodil 10 mu M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on I-Ba 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.