Introduction

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Ethanol is toxic to cells in the developing and adult central nervous system (Charness et al., 1989). Although the molecular and cellular sites of action for ethanol are only partially characterized, recent data suggest that ethanol interacts directly with a subset of neuronal ion channels and protein kinases (Diamond and Gordon, 1997; Yamakura et al., 2001). Discrete ethanol binding sites may exist within hydrophobic pockets of selected proteins (Franks and Lieb, 1994; Dwyer and Bradley, 2000), and site-directed mutagenesis has identified critical amino acids that mediate particular physiological effects of alcohols and general anesthetics (Yamakura et al., 2001). The observation that ethanol acts within discrete protein domains suggests that it might be possible to identify antagonists for specific actions of ethanol.

We have proposed that interactions of ethanol with the immunoglobulin L1 cell adhesion molecule may be important for its teratogenic effects (Charness et al., 1994; Ramanathan et al., 1996). We noted that children with mutations in the gene for L1 have brain lesions that are similar to those of children with fetal alcohol syndrome (FAS). We therefore asked whether ethanol alters L1-mediated cell-cell adhesion (L1 adhesion). Ethanol did not affect the expression or cell surface localization of L1 (Charness et al., 1994). However, clinically relevant concentrations of ethanol inhibited L1 adhesion in NG108-15 neuroblastoma × glioma hybrid cells, in cerebellar granule cells, and in selected human L1-transfected murine fibroblasts (Charness et al., 1994; Ramanathan et al., 1996; Wilkemeyer and Charness, 1998). Bearer et al. (1999) demonstrated that comparably low concentrations of ethanol also inhibited L1-mediated neurite outgrowth in cerebellar granule cells.

L1 is a multifunctional, transmembrane protein that plays a critical role in nervous system development (Fransen et al., 1995, 1998; Demyanenko et al., 1999). L1 binds to other L1 molecules on adjacent cells and to selective proteins in the extracellular matrix, cell membrane, and cytoskeleton (Crossin and Krushel, 2000). L1 interactions trigger a series of signaling events that regulate growth cone motility, axon pathfinding, axon fasciculation, and neuronal migration (Crossin and Krushel, 2000; Schmid et al., 2000). Ethanol could alter these L1-dependent events by disrupting homophilic binding, heterophilic binding, or L1-mediated signal transduction.

To learn more about the interaction of ethanol with L1, we studied a series of straight, branched, and cyclic alcohols. These experiments revealed strict structural requirements for alcohol inhibition of L1 adhesion (Wilkemeyer et al., 2000). Interestingly, a subgroup of these alcohols had no effect on L1 adhesion, but blocked the effects of ethanol (Wilkemeyer et al., 2000, 2002). One ethanol antagonist, 1-octanol, also reduced the effects of ethanol on the morphology of dividing neural cells (Wilkemeyer et al., 2000) and prevented ethanol-induced apoptosis and dysmorphology in cultured mouse embryos (Chen et al., 2001). Thus, a molecule selected for its ability to antagonize ethanol's effects on L1 adhesion also prevented ethanol teratogenesis.

Recently, two neuroprotective peptides, SALLRSIPA (SAL) and NAPVSIPQ (NAP), were shown to prevent ethanol-induced fetal death and growth abnormalities in a mouse model of FAS (Spong et al., 2001). SAL, also known as activity-dependent neurotrophic factor (ADNF)-9, is an active fragment of ADNF, and NAP is an active fragment of activity-dependent neuroprotective protein. ADNF and activity-dependent neuroprotective protein are released by glial cells in response to vasoactive intestinal peptide (Brenneman and Gozes, 1996; Brenneman et al., 1998; Bassan et al., 1999). ADNF-9 has been shown to produce effects on transcriptional regulation that include an increase in nuclear factor-B DNA-binding activity in hippocampal neurons (Glazner et al., 2000) and the induction of neurite extension via enhanced cAMP response element-binding protein phosphorylation in dorsal root ganglia cultures (White et al., 2000). Femtomolar concentrations of NAP and SAL protect cultured neurons against a variety of toxins and both are neuroprotective in in vivo models of neurodegeneration and neural injury (Brenneman et al., 1998; Bassan et al., 1999; Gozes et al., 2000; Beni-Adani et al., 2001). The neuroprotective effects of NAP and SAL may be related to their ability to reduce oxidative injury, although other mechanisms of action may also be important. Because oxidative stress is one well established mechanism for ethanol-induced neurotoxicity (Kotch et al., 1995), it is conceivable that NAP and SAL prevent ethanol teratogenesis by blocking ethanol-induced oxidative stress. However, in view of the remarkably similar efficacy of NAP, SAL, and 1-octanol in preventing ethanol teratogenesis, we asked whether NAP and SAL also antagonize ethanol effects on L1.

	Materials and Methods

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Reagents. Ethanol was purchased from Fisher Scientific (Pittsburgh, PA); all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or as indicated. Peptides were purchased from Peptide Technologies Corporation (Gaithersburg, MD) and Sigma Genosys (Woodlands, TX). Purity (>95%) and identity were assessed by the company using high-performance liquid chromatography and mass spectrometry analyses. The peptides were dissolved in 10% dimethyl sulfoxide in phosphate-buffered saline (PBS; 0.13 M NaCl, 0.003 M KCl, 0.01 M Na₂HPO₄, and 0.002 M KH₂PO₄) and stored as 1 mM aliquots. SAL is only stable for several days at room temperature and 90% of SAL activity is lost after a single freeze-thaw cycle (Brenneman et al., 1998). We therefore used only freshly prepared stock solutions of SAL. NAP proved to be very stable in solution and could be aliquoted and frozen for later use without loss of activity (data not shown).

Cell Culture. NIH/3T3 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% normal calf serum (Intergen, Purchase, NY) and 400 µg/ml G418 (Invitrogen). Two subclones were used in these studies: 2A2-L1 and a vector-transfected cell line (Vec-1A5). The 2A2-L1 cell line is an ethanol-sensitive subclone derived from a stable transfection of NIH/3T3 cells with the human L1 cDNA, and Vec-1A5 is a subclone from a transfection with the empty expression vector (Wilkemeyer and Charness, 1998). NG108-15 cells (passages 21-30) were plated in serum-free, defined medium (Charness et al., 1986). Two days before the start of cell adhesion assays, serum-free medium containing bone morphogenetic protein-7 (BMP-7) (Creative BioMolecules, Hopkinton, MA) (10 ng/ml, final) was added daily to the NG108-15 cells. Both cell lines were cultured at 37°C, in an atmosphere of 90% air and 10% CO₂.

Cell Adhesion Assay. Cell-cell adhesion was measured using a modified short-term aggregation assay of subconfluent cells (Wilkemeyer and Charness, 1998; Wilkemeyer et al., 2000). Cells were detached by gentle agitation with calcium- and magnesium-free PBS supplemented with 0.1 mg/ml DNase, mechanically dissociated to obtain a single-cell suspension, and diluted to 330,000 cells/ml for the NIH/3T3 cells and 250,000 cells/ml for the NG108-15 cells. One milliliter of the cell suspension was added per well (4.5 cm²) to a 12-well plate. Peptides and ethanol were mixed before adding to the cells. After addition of ethanol or the ethanol/peptide mix, the cells were gently mixed for 30 min on ice. Cells were viewed at a final magnification of 200×, and each well was scored for single and adherent cells in five or six microscopic fields of view. We counted approximately 150 to 200 cells/field of view and 750 to 1000 cells/well. The percentage of adherent cells was calculated for each microscopic field of view and averaged. To ensure the reliability of the cell adhesion assays, most assays were scored without knowledge of the experimental conditions.

Western Blot Analysis. Protein extracts were prepared from 2A2-L1 and Vec-1A5 cells, with or without a 30-min exposure to 10⁷ M NAP or 10⁷ M SAL, to mimic the conditions of an adhesion assay. Cells were harvested in calcium/magnesium-free PBS, pelleted by centrifugation and resuspended in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 8.0, and 1.0% Nonidet P-40) containing protease inhibitors (100 µM phenylmethylsulfonyl fluoride, 100 µM leupeptin, and 50 µM pepstatin). The cells were homogenized by freeze thawing and vortexing, and insoluble material was removed by centrifugation at 10,000g for 20 min; 50 µg of protein extract was boiled for 5 min in the presence of 5× SDS-sample buffer (sodium dodecyl sulfate (10%), glycerol (50%), -mercaptoethanol (25%), Tris base pH 7.4 (100 mM), bromphenol blue (2 mg/100 ml); separated on a 4 to 15% polyacrylamide gel; and electrophoretically transferred to Immobilon P membranes (Millipore Corporation, Bedford, MA). The membranes were blocked with Tris-buffered saline (10 mM Tris-HCl pH 7.5 and 0.9% NaCl) containing 0.1% Tween 20, incubated for 2 h at room temperature with primary antibody SC1508 (Santa Cruz Biotechnology, Santa Cruz, CA) at a final concentration of 0.1 µg/ml. SC1508 is a goat polyclonal antibody raised against a peptide from the carboxy terminus of human L1. The membranes were incubated sequentially with biotinylated secondary antibody (sheep anti-goat IgG, 0.4 µg/ml; Santa Cruz Biotechnology) and avidin D-conjugated alkaline phosphatase (0.5 units/ml; Vector Laboratories, Burlingame, CA). The immunoreaction products were visualized with 5-bromo-4-chloro-3-indoyl phosphate/nitroblue tetrazolium (PerkinElmer Life Sciences, Boston, MA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Effects of NAP and SAL on L1 Expression and Adhesion. 1-Octanol and other antagonists had no effect on L1 adhesion, but blocked the effects of ethanol. Because NAP and SAL have complex pharmacological properties, we first explored whether either peptide modulates L1 expression or L1 adhesion. Cell adhesion assays were performed in 2A2-L1 cells in the absence and presence of a range of NAP and SAL concentrations. Neither peptide affected cell-cell adhesion at concentrations of up to 10⁵ M (Fig. 1A). Similarly, neither NAP nor SAL altered the morphology or viability of cells during the 30-min time course of the cell adhesion assay (data not shown). Treatment of 2A2-L1 cells for 30 min with 10⁷ M NAP or 10⁷ M SAL did not change levels of L1 expression or electrophoretic mobility, as determined by Western blot analysis (Fig. 1, B and C). These experiments indicate that brief treatment of L1-expressing NIH/3T3 cells with NAP or SAL does not alter L1 expression or L1 adhesion. Thus, NAP and SAL, like 1-octanol, have no agonist effects in this system.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of NAP and SAL on L1 adhesion and L1 expression. A, cell adhesion assays were performed with NIH/3T3 (2A2-L1) cells expressing human L1 in the absence and presence of either NAP or SAL. Shown are the means ± S.E.M. for the percentage of cell adhesion in the presence of each peptide at the indicated concentration (n = 3-4). The dashed line represents the mean percentage of cell-cell adhesion in the absence of peptide (42 ± 2%, n = 9). B, Western blot of protein extracts from two different passages of 2A2-L1 cells (lanes 1 and 2, P18; lanes 3 and 4, P19), using the polyclonal anti L1 antibody SC1508 (Santa Cruz Biotechnology). Protein extracts were prepared from untreated cells (lanes 1 and 3) or cells exposed to 107 M NAP for 30 min (lanes 2 and 4). The antibody recognizes two bands of approximate molecular weight 210 and 200 kDa, as described previously for L1 (Miura et al., 1992; Wilkemeyer and Charness, 1998). C, cells were treated as in B, but with 107 M SAL, rather than with NAP (lanes 1 and 2, P17; lanes 3 and 4, P9).

NAP and SAL Are Potent Antagonists of Ethanol Inhibition of L1-Mediated Cell-Cell Adhesion. We next asked whether NAP and SAL could antagonize ethanol inhibition of L1 adhesion. Cell adhesion assays were performed in the absence and presence of 100 mM ethanol using ethanol-sensitive, L1-expressing NIH/3T3 cells (2A2-L1). Ethanol reduced L1 adhesion by 52 ± 2%. As shown in Fig. 2, both NAP and SAL were potent antagonists of 100 mM ethanol. Antagonism by NAP was first apparent at concentrations of 10¹⁶ M and increased progressively over 8 log orders. The EC₅₀ value for NAP, based on linear regression analysis of the dose-response curve, was approximately 6 × 10¹⁴ M. The initial effect of SAL was first evident at concentrations of approximately 10¹³ M. Like NAP, SAL showed dose-dependent antagonism over many log orders. The approximate EC₅₀ value for SAL was 4 × 10¹¹ M. To verify that antagonist effects were specific and related to peptide structure, we evaluated two scrambled peptides derived from NAP. Neither PNIQVASP nor ASPNQPIV had any effect on L1 adhesion or on its inhibition by ethanol (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   NAP and SAL antagonism of ethanol inhibition of L1 adhesion. Cell adhesion assays were performed with NIH/3T3 cells expressing human L1 in the absence and presence of 100 mM ethanol and the indicated concentrations of either NAP or SAL. Shown are the means ± S.E.M. for the percentage of antagonist activity for each peptide (n = 4-16). Antagonist activity was calculated as [100 × (1  (% inhibition cell adhesion by ethanol plus peptide)/(% inhibition cell adhesion by ethanol alone))].

View this table: [in this window] [in a new window]   TABLE 1 Peptide antagonism of ethanol inhibition of L1 adhesion Cell adhesion assays were performed with NIH/3T3 cells expressing human L1. Antagonist activity was calculated as [100 × (1  (% inhibition cell adhesion by ethanol plus peptide)/(% inhibition cell adhesion by ethanol alone))]. Shown is the mean ± S.E.M. for the percentage of antagonist activity against 100 mM ethanol with 107 M of the indicated peptide (n = 4-10). Peptides are displayed by single letter code amino acid sequence, listed from amino to carboxy terminus.

NAP Is a Potent Antagonist in Neural Cells. Because we are modeling the effects of ethanol on the developing nervous system, we also conducted studies in a neural cell line. Treatment of neuroblastoma × glioma NG108-15 cells with BMP-7 increases cell-cell adhesion by inducing gene and protein expression for L1 and for the neural cell adhesion molecule (N-CAM) (Perides et al., 1992, 1993). Agonist and antagonist effects of alcohols are nearly identical in L1-transfected NIH/3T3 cells and in BMP-7-treated NG108-15 (Wilkemeyer et al., 2000). We therefore asked whether NAP could antagonize ethanol inhibition of cell-cell adhesion in NG108-15 cells. Cell adhesion assays were performed in the absence and presence of 100 mM ethanol in BMP-7-treated NG108-15 cells. As shown in Fig. 3A, NG108-15 cells grown in serum-free medium have low levels of cell-cell adhesion (17 ± 1%, n = 3). In contrast, NG108-15 cells incubated with 10 ng/ml BMP-7 for 48 h (Fig. 3B) exhibit increased cell-cell adhesion (42 ± 5%, n = 4), Exposure of NG108-15 cells to 100 mM ethanol significantly reduced cell-cell adhesion (Fig. 3C; cell adhesion, 28 ± 4%, n = 4). Ethanol inhibition of cell-cell adhesion was completely blocked by 10⁷ M NAP (Fig. 3D) (cell adhesion, 43 ± 3%, n = 4). The antagonist potency (EC₅₀ of 10¹² M) and efficacy (92 ± 5% antagonism with 10⁷ M) of NAP were similar in BMP-7-treated NG108-15 cells and in L1-transfected NIH/3T3 cells.

View larger version (130K): [in this window] [in a new window]   Fig. 3.   NAP antagonism of ethanol inhibition of cell-cell adhesion in neural cells. NG108-15 cells were cultured for 2 days in serum-free medium in the absence or presence of 10 ng/ml BMP-7. Cell adhesion assays were performed in the absence and presence of 100 mM ethanol and in the absence and presence of 107 M NAP. A to D, photomicrographs were obtained under phase contrast microscopy (200× magnification) from cells treated in the adhesion assay as follows: no BMP-7, no ethanol or peptide (A); 10 ng/ml BMP-7, no ethanol or peptide (B); 10 ng/ml BMP-7 and 100 mM ethanol (C); and 10 ng/ml BMP-7, 100 mM ethanol, and 107 M NAP (D). Scale bar, 100 µm.

Mechanisms and Kinetics of Antagonist Activity by NAP and SAL. We next asked whether NAP and SAL antagonism could be overcome by increasing concentrations of agonist. Both L1 and N-CAM contribute to the increased cell-cell adhesion in BMP-7-treated NG108-15 cells (Perides et al., 1992, 1993); hence, to focus on the interactions of ethanol and L1, we used L1-transfected NIH/3T3 cells for the remaining experiments. Adhesion assays were performed in the presence of increasing concentrations of ethanol and a fixed concentration near the EC₅₀ for NAP (10¹³ M) or SAL (10¹⁰ M).

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View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of increasing agonist concentration on peptide antagonist activity. Cell adhesion assays were performed with NIH/3T3 cells expressing human L1 in the presence of increasing concentrations of ethanol and either NAP or SAL. A, mean ± S.E.M. for the percentage of inhibition of cell-cell adhesion by ethanol alone () by ethanol plus 1010 M SAL (). Ethanol (100 mM) produces near maximum inhibition of cell adhesion. Exposure of NIH/3T3 cells to >400 mM ethanol lead to disruptions in membrane integrity and cell death. Note that increasing concentrations of ethanol eliminate SAL antagonist activity. B, mean ± S.E.M. for the percentage of inhibition of cell-cell adhesion by ethanol alone () or with ethanol and 1013 M NAP (). Ethanol (100 mM) produces near maximum inhibition of cell adhesion. Note that in contrast to SAL, increasing concentrations of ethanol do not overcome NAP antagonist activity.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Reversibility of ethanol antagonism by SAL and NAP. 2A2-L1 cells were exposed to 1013 M NAP or 1010 M SAL in cell culture medium (pretreat) or medium alone (control). After a 30-min exposure at 37°C, the medium was removed and the flasks were washed three times, for 10 min each, with excess cell culture medium. Cell adhesion assays were then performed in the absence or presence of 100 mM ethanol. Control flasks were not pretreated with peptide. A, mean ± S.E.M. percentage of cell-cell adhesion in control cells () and cells pretreated with the indicated peptide (). Pretreatment with NAP or SAL had no effect on levels of cell adhesion. B, mean ± S.E.M. for the percentage of inhibition of cell-cell adhesion by ethanol for control cells or cells pretreated with the indicated peptide (n = 4-6). Note that ethanol inhibition of L1 adhesion is only reduced slightly in cells pretreated with SAL, relative to control cells. However, pretreating the cells with NAP prevents ethanol inhibition of L1 adhesion.

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	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The major finding of this study is that the neuroprotective peptides NAP and SAL are extraordinarily potent and effective antagonists of ethanol inhibition of L1 adhesion. NAP was significantly more potent than SAL, but both peptides showed half-maximal effects in the femtomolar (NAP) to picomolar (SAL) range. We used 100 mM ethanol in our experiments, because this very high concentration is observed in alcoholics (Charness et al., 1989) and reliably produces teratogenic effects in several models of FAS (Kotch et al., 1995; Chen et al., 2001). At micromolar concentrations, both peptides virtually abolished the effects of 100 mM ethanol. Thus, NAP and SAL antagonize a specific cellular action of ethanol, even at the highest clinically attainable ethanol concentrations.

Most of our experiments were carried out in NIH/3T3 cells transfected with human L1. We have shown that anti-L1 Fab fragments reduce cell-cell adhesion in L1-transfected cells to levels observed in NIH/3T3 cells transfected with an empty vector (Wilkemeyer and Charness, 1998). Moreover, ethanol does not reduce levels of adhesion in vector-transfected NIH/3T3 cells or in L1-transfected NIH/3T3 cells that have been pretreated with anti-L1 Fab fragments (Ramanathan et al., 1996; Wilkemeyer and Charness, 1998). Therefore, it is reasonable to assume that L1 mediates the component of cell adhesion that is inhibited by ethanol. It is not yet clear whether ethanol, NAP, or SAL produce their effects in L1-transfected NIH/3T3 cells by interacting directly with L1 or by affecting the interaction of L1 with other cellular constituents. L1-adhesion could be modulated through phosphorylation of L1 (Garver et al., 1997; Tuvia et al., 1997; Long et al., 2001) or through interactions of L1 with multiple heterophilic binding partners (Crossin and Krushel, 2000). Ethanol and its antagonists might disrupt any of these L1 interactions.

NAP and SAL might antagonize ethanol inhibition of L1 adhesion through an indirect mechanism, by increasing the cell-cell adhesion of L1-transfected NIH/3T3 cells. However, treatment with NAP or SAL neither increased L1 adhesion nor altered the expression of L1. More likely, NAP and SAL are interacting with the same target sites as ethanol to block its effects. Amino acid sequence, rather than amino acid composition, seems to be important for NAP and SAL antagonist activity. Two scrambled NAP peptides were completely inactive. Brenneman et al. (1998) have shown that the neuroprotective activity of SAL is sensitive to deletions and single amino acid substitutions. Further structure-activity relation analysis may reveal unique motifs within these small peptides that are necessary for ethanol antagonist activity, providing additional clues about their target sites.

NAP was also a potent ethanol antagonist in neural cells. The NG108-15 cell line is a neuroblastoma × glioma hybrid that expresses a strongly neuronal phenotype (Hamprecht, 1977). Treatment of NG108-15 cells with BMP-7 increases cell adhesion by inducing gene and protein expression for both L1 and N-CAM (Perides et al., 1992, 1993). Ethanol inhibits cell-cell adhesion in NG108-15 cells (Charness et al., 1994), but the target molecules are less certain than in L1-transfected NIH/3T3 cells, because at least two cell adhesion molecules contribute to the increased adhesion of NG108-15 cells. N-CAM is not likely to be a major target of ethanol in NG108-15 cells, because ethanol does not inhibit cell-cell adhesion in NIH/3T3 cells transfected with the 140-kDa isoform of human N-CAM (Ramanathan et al., 1996). Moreover, the pharmacology of various alcohols for inhibition of cell-cell adhesion is identical in BMP-7-treated NG108-15 cells and in L1-transfected NIH/3T3 cells (Wilkemeyer et al., 2000). Hence, it is likely that ethanol inhibits cell-cell adhesion in BMP-7-treated NG108-15 cells by interacting with L1. Our data indicate that NAP antagonizes the effects of ethanol on L1 adhesion both in neural cells as well as in fibroblasts.

The kinetics for antagonism of ethanol by SAL and NAP showed important differences. SAL antagonism was overcome completely by increasing the concentration of ethanol, whereas NAP antagonism was unaffected. Likewise, the antagonist effects of SAL were largely reversible, whereas those of NAP were not. Surprisingly, even 1 min of exposure at 4°C to 10¹³ M NAP abolished ethanol inhibition of L1 adhesion for up to 2 h. It should be noted that although the cells were only exposed to NAP for 1 min, approximately 1 h passes from the time cell harvesting begins until the cell adhesion assay is complete. During this time, signaling events triggered by NAP could prevent ethanol inhibition of L1 adhesion. These data suggest that SAL is a reversible and possibly competitive antagonist of ethanol inhibition of L1 adhesion. NAP, on the other hand, seems to be an irreversible antagonist, at least over the time parameters of these studies.

We have also characterized a group of alcohols that antagonize the effects of ethanol on L1 adhesion (Wilkemeyer et al., 2000, 2002). These alcohols showed striking structural specificity for their antagonist effects. Antagonist potency for 1-alcohols increased progressively from 1-pentanol to 1-dodecanol and then declined gradually from 1-tridecanol to 1-tetradecanol. This cutoff effect suggested that, like the agonist target site, the antagonist target site has important size limitations. As observed for SAL, increasing the concentration of agonist overcame the antagonism of 3-buten-1-ol, benzyl alcohol, cyclopentanol, and 3-pentanol. As observed for NAP, increasing the concentration of agonist did not overcome the antagonist effects of 4-methyl-1-pentanol, 2-methyl-2-pentanol, 1-pentanol, 2-pentanol, 1-octanol, and 2,6-di-isopropylphenol. These findings suggest that NAP, SAL, and selective straight, branched, and cyclic alcohols act at multiple, discrete sites to antagonize the actions of ethanol on L1 adhesion.

The pharmacokinetic differences between NAP and SAL may arise from differences in the binding affinities for their targets. If the binding affinity of NAP were similar to its EC₅₀ for ethanol antagonism (6 × 10¹⁴ M) then the dissociation half-time for NAP would be in the order of days. This very slow rate of dissociation might account for the inability to overcome NAP antagonism with increasing concentrations of agonist as well as the apparent irreversibility of NAP activity. The inability to reduce NAP antagonism with increasing concentrations of agonist could also result from the interaction of NAP with an allosteric site that regulates ethanol inhibition of L1 adhesion (Wilkemeyer et al., 2000). This may be the case for 1-octanol, which was less potent than NAP or SAL, but which was also a noncompetitive, fully reversible antagonist (Wilkemeyer et al., 2000). Finally, NAP and SAL may activate different signaling pathways that differ in their latency for inducing enduring effects.

Both NAP and SAL showed a progressive, dose-dependent increase in ethanol antagonist effect over 8 log orders of peptide concentration. This unusually broad dose-response curve is consistent with a complex mechanism of action that might involve negative cooperativity, multiple binding sites, or activation of multiple signaling pathways. The dose-response curve for antagonism of ethanol had interesting similarities and differences with those observed in models of neuroprotection. NAP is also more potent than SAL in most neuroprotection experiments (Bassan et al., 1999; Gozes et al., 2000; Spong et al., 2001). However, the shape of the dose-response curve varies, depending on the choice of neurotoxin; in most instances, both peptides show their neuroprotective effects over less than 6 log orders. Protection by NAP against glutathione depletion in neuroblastoma cells (Offen et al., 2000) and against -amyloid, N-methyl-D-aspartate, glycoprotein-120, and tetrodotoxin in neurons shows a biphasic curve, with decreasing effect above certain peptide concentrations (Bassan et al., 1999). In contrast, SAL protection of hippocampal neurons against FeSO₄ toxicity is dose-dependent and does not show a drop-off at higher concentrations (Glazner et al., 1999).

The mechanism of neuroprotection by NAP and SAL seems to be complex. Pretreatment of pregnant mice with NAP alone significantly decreased ethanol-induced fetal death, whereas pretreatment with SAL alone did not (Spong et al., 2001). The neuroprotective potency of SAL is approximately 10,000-fold less in pure neuronal cultures than in mixed neuronal-glial cultures (Brenneman et al., 1998), suggesting an important role for neuronal-glial interactions. Several cellular actions of these peptides may contribute to neuroprotection, including induction of nuclear factor-B activity and heat-shock protein 60, increased levels of cGMP, inhibition of oxidative stress, reduction in reactive oxygen species, release of neurotrophic factor-3, and enhancement of basal transport of glucose and glutamate in synaptosomes (Glazner et al., 1999; Zamostiano et al., 1999; Blondel et al., 2000; Glazner et al., 2000; Guo and Mattson, 2000; Ashur-Fabian et al., 2001). It is unclear which of these many actions of NAP and SAL are responsible for their protective effects against ethanol teratogenesis.

Oxidative stress is a well established mechanism of ethanol-induced cellular injury and seems to play a central role in ethanol teratogenesis (Kotch et al., 1995; Chen and Sulik, 1996). Antioxidants, such as vitamin E, catalase, and superoxide dismutase, reduce ethanol-induced injury in cultured neurons and in whole embryo culture (Kotch et al., 1995; Chen and Sulik, 1996; Mitchell et al., 1999; Chen and Sulik, 2000). Interestingly, Spong et al. (2001) found that NAP and SAL protection of mouse embryos from ethanol toxicity was associated with a decrease in reduced glutathione. This observation suggests that NAP and SAL block the induction by ethanol of reactive oxygen species. We have proposed that loss of L1-mediated cell-cell adhesion may also be linked to oxidative injury through a process known as anoikis, or the induction of apoptotic cell death triggered by loss of cell-cell or cell-substrate contact (Chen et al., 2001). Anoikis leads to cell death through activation of oxidative injury. Conceivably NAP and SAL antagonize the effects of ethanol in two ways: by acting upstream to prevent loss of L1 adhesion and by acting downstream of the many convergent apoptotic pathways that trigger oxidative injury.

We undertook these experiments, because research in our two laboratories showed that 1-octanol, NAP, and SAL were remarkably effective in preventing ethanol teratogenesis. 1-Octanol was first tested for its ability to block ethanol teratogenesis, because it potently antagonized ethanol inhibition of L1 adhesion. The present experiments now demonstrate that two peptides, identified for their ability to protect against ethanol teratogenesis and neurotoxicity, are extremely potent antagonists of ethanol inhibition of L1 adhesion. Thus, two structurally unrelated groups of compounds, alcohols and polypeptides, share common actions: antagonism of ethanol inhibition of L1 adhesion and prevention of ethanol teratogenesis. These findings support the hypothesis that ethanol effects on L1 contribute to its teratogenic actions. These highly potent ethanol antagonists may be valuable tools for identifying the target sites through which ethanol disrupts central nervous system development and for designing drugs to prevent FAS.