1 We have recently shown that the alkaloid methyl-laudanosine blocks SK channel-mediated afterhyperpolarizations (AHPs) in midbrain dopaminergic neurones. However, the relative potency of the compound on the SK channel subtypes and its ability to block AHPs of other neurones were unknown. 2 Using whole-cell patch-clamp experiments in transfected cell lines, we found that the compound blocks SK1, SK2 and SK3 currents with equal potency: its mean IC 50 s were 1.2, 0.8 and 1.8 mM, respectively. IK currents were unaffected. In rat brain slices, methyl-laudanosine blocked apaminsensitive AHPs in serotonergic neurones of the dorsal raphe and noradrenergic neurones of the locus coeruleus with IC 50 s of 21 and 19 mM, as compared to 15 mM in dopaminergic neurones. However, at 100 mM, methyl-laudanosine elicited a constant hyperpolarization of serotonergic neurones of about 9 mV, which was inconsistently (i.e. not in a reproducible manner) antagonized by atropine and hence partly due to the activation of muscarinic receptors. 3 While exploring the pharmacology of related compounds, we found that methyl-noscapine also blocked SK channels. In cell lines, methyl-noscapine blocked SK1, SK2 and SK3 currents with mean IC 50 s of 5.9, 5.6 and 3.9 mM, respectively. It also did not block IK currents. Methyl-noscapine was slightly less potent than methyl-laudanosine in blocking AHPs in brain slices, its IC 50 s being 42, 37 and 29 mM in dopaminergic, serotonergic and noradrenergic neurones, respectively. Interestingly, no significant non-SK effects were observed with methyl-noscapine in slices. At a concentration of 300 mM, methyl-noscapine elicited the same changes in excitability in the three neuronal types than did a supramaximal concentration of apamin (300 nM). 4 Methyl-laudanosine and methyl-noscapine produced a rapidly reversible blockade of SK channels as compared with apamin. The difference between the IC 50 s of apamin (0.45 nM) and methyllaudanosine (1.8 mM) in SK3 cells was essentially due to a major difference in their k À1 (0.028 s À1 for apamin and X20 s À1 for methyl-laudanosine). 5 These experiments demonstrate that both methyl-laudanosine and methyl-noscapine are medium potency, quickly dissociating, SK channel blockers with a similar potency on the three SK subtypes. Methyl-noscapine may be superior in terms of specificity for the SK channels.

Activity-dependent neurotrophic factor (ADNF) and a 14-amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect neurons from death associated with an array of toxic conditions, including amyloid ␤-peptide, N-methyl-D-aspartate, tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report that an even smaller, nine-amino acid fragment (ADNF9) with the sequence SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons from oxidative injury and neuronal apoptosis induced by FeSO 4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species. Key Words: Activity-dependent neurotrophic factor-Neurons-Trophic factor withdrawalReactive oxygen species-Apoptosis. J. Neurochem. 73, 2341-2347 (1999).A novel, glial-derived neuroprotective polypeptide (14 kDa; pI 8.3) was recently isolated by sequential chromatographic methods and termed activity-dependent neurotrophic factor (ADNF) (Brenneman and Gozes, 1996). ADNF is regulated by vasoactive intestinal peptide, which is released by neurons upon depolarization, binds neighboring astrocytes on high-affinity vasoactive intestinal peptide receptors, and mediates the release of ADNF. The name ADNF comes from the fact that is was first shown to protect neurons from death associated with electrical blockade by tetrodotoxin. Further analysis determined that a 14-amino acid fragment of ADNF, termed ADNF14, was sufficient to provide neuroprotection equivalent to that seen with full-length ADNF. This peptide (VLGGGSALLRSIPA) is very similar, although not identical, to a section of heat shock protein 60 (VLGGGCALLRCIPA), differing only by the replacement of cysteines in the heat shock protein 60 peptide with serines in ADNF14 (Brenneman and Gozes, 1996;Brenneman et al., 1998). Both ADNF and ADNF14 protect cultured central neurons from death associated with several insults, including the neurotoxic HIV envelope glycoprotein gp120, NMDA, amyloid ␤-peptide (A␤) 25-35, and tetrodotoxin. Perhaps the most striking feature of the neuroprotective effect of ADNF is the potency. ADNF and ADNF14 peptide confer maximal in vitro protection at concentrations as low as 1 fM. In vivo studies have shown that ADNF can protect cortical cells from death after NMDA injection . The range of insults that are alleviated by ADNF indicates a general rather than a specific mode of protection.The importance of endogenous ADNF is shown by a study in which an antiserum against ADNF14 kills cultured embryonic rat cortical neurons . ADNF shares with more extensively studied neurotrophic factors the ability to stimulate mitotic activity, accelerating embryonic development in cultured whole mouse embryos (Glazner et al., 1999). These studies implicate ADNF as a critical endogenous regulator of nervous system development and maintenance. Extensive peptide analysis has shown that the smallest active form of ADNF is a nine-amino acid fra...

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This study was designed to assess the effect of 50 Hz electromagnetic fields (EMFs) on hippocampal cell cultures in the presence or absence of either sodium nitroprusside (SNP, a NO donor) or Fe2+ induced oxidative stress. One week old cultured rat hippocampal cells were exposed to either intermittent EMFs (IEMFs, 50 Hz, 0-5 mT, 1 min ON/OFF cycles, repeated 10 times every 2 h, 6 times/day during 48 h) or continuous EMFs (CEMFs, 50 Hz, 0-5 mT for 48 h). In a second set of experiments, the effect on such EMFs applied in combination with oxidative stress induced by 0.5 microM Fe2+ or SNP was estimated. At the end of both sets of experiments, cell mortality was assessed by lactate dehydrogenase measurements (LDH). Neither type of exposure to EMFs was observed to modify the basal rate of cell mortality. The exposure to CEMFs in presence of either NO or Fe2+ did not induce any significant increase in cell death. However, when cells were exposed to EMFs in the presence of NO, we observed a significant increase in cell death of 11 and 23% (P<0.001) at 2.5 and 5 mT, respectively. This effect had some specificity because IEMFs did not modify the effect of Fe2+ on cell mortality. Although the effects of IEMFs reported in this study were only observed at very high intensities, our model may prove valuable in trying to identify one cellular target of EMFs.

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