dizocilpine-maleate and benzamil

During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na+ channels opened) induces an increase in neuronal survival. In the present work we show that 3 microM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 microM amiloride (an inhibitor of Ca2+ channels), 25 microM benzamil (an inhibitor of Na+ channels), 30 microM dantrolene and 7.5 microM caffeine (both inhibitors of Ca2+ release from the endoplasmatic reticulum) and 10 microM BAPTA-AM (an intracellular Ca2+ chelator). However, 5 microM nifedipine (a selective inhibitor of voltage-dependent L-type Ca2+ channels) and 100 microM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 microM genistein (an inhibitor of tyrosine kinase enzymes), 20 microM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 microM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na+ influx, intracellular Ca2+ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.

Topics: Amiloride; Animals; Caffeine; Calcium Channel Blockers; Calcium Channels, T-Type; Cell Death; Cell Survival; Cells, Cultured; Chelating Agents; Dantrolene; Diuretics; Dizocilpine Maleate; Egtazic Acid; Excitatory Amino Acid Antagonists; In Vitro Techniques; Membrane Potentials; Muscle Relaxants, Central; Nifedipine; Phosphodiesterase Inhibitors; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Retinal Ganglion Cells; Veratridine

Five minutes of oxygen and glucose deprivation (termed "in vitro ischemia") causes long-term synaptic transmission failure (LTF) in the CA1 region of the rat hippocampal slice. Dependence of LTF on cell calcium was tested by generating graded reductions in cell Ca. There was a strong correlation between the average level of exchangeable cell Ca in CA1 during ischemia, and the extent of LTF. In standard buffer, exchangeable cell Ca in CA1 increased by 35% after 3 min of ischemia and remained elevated for the entire 5 min of ischemia. Unidirectional Ca influx increased by 35% during the first 2.5 min of ischemia and remained at that level for the next 2.5 min. There were no changes in unidirectional Ca efflux during this period. Thus, the accumulation results from increased influx of Ca. Ca influx during the first 2.5 min of ischemia depended entirely on NMDA channels; it was completely blocked by the noncompetitive NMDA receptor antagonist MK-801. However MK-801 had no effect during the second 2.5 min. This inactivation of NMDA-mediated influx during ischemia appears to result from dephosphorylation. Okadaic acid increased Ca influx during the second 2.5 min of ischemia and this increase was blocked by MK-801. The ischemia-induced Ca influx during the second 2.5 min of ischemia was attenuated 25% by nifedipine (50 microM) and an additional 35% by the Na/Ca exchange inhibitor benzamil (100 microM). The AMPA/kainate antagonist DNQX had no effect on the Ca influx. Antagonists were used to relate Ca influx to LTF. Blockade of enhanced Ca entry during ischemia in standard buffer (2.4 mM Ca) had no effect on LTF, consistent with total cell Ca prior to ischemia being adequate to cause complete LTF. However, MK-801 strongly protected against LTF when the buffer contained 1.2 mM Ca, a more physiological level. MK-801 combined with DNQX prevented transmission damage in standard buffer. Thus, AMPA/kainate receptor activation contributes to ischemic damage, although not by enhancing Ca entry.

Topics: Amiloride; Animals; Calcium; Dizocilpine Maleate; Ethers, Cyclic; Guanidines; Hippocampus; In Vitro Techniques; Ischemic Attack, Transient; Kinetics; Male; Microscopy, Electron; Okadaic Acid; Pyramidal Tracts; Quinoxalines; Rats; Rats, Sprague-Dawley; Time Factors