BACKGROUND
the proliferation of astrocytes is stimulated by polypeptide growth factors such as pdgf, egf, bfgf and igf- <dig> acting on cellular signaling pathways which involve tyrosine kinases, protein kinase c, and the ras-raf-map kinase pathway  <cit> .
astroglial proliferation is also stimulated by neurotransmitters such as acetylcholine and glutamate  <cit> , by direct stimulation of protein kinase c with phorbol ester  <cit> , and by peptides such as endothelin and prolactin  <cit> .
astroglial proliferation is prominently inhibited by ethanol both in vivo and in vitro  <cit> , and this interference likely contributes to the development of the fetal alcohol syndrome  .
ethanol has been shown to potently antagonize proliferative effects of several individual astroglial mitogens including pdgf, igf- <dig>  acetylcholine and prolactin  <cit> .
the molecular target of ethanol's antimitogenic actions in astroyctes is not known with certainty, but inhibitory interactions of ethanol with lipid signaling pathways have been implicated  <cit> .
our group has recently reported strong evidence that the growth-inhibitory effect of ethanol in astrocytes is caused by the disruption of the phospholipase d  signaling pathway  <cit> .
under physiological conditions, pld catalyzes the hydrolysis of phosphatidylcholine  to yield phosphatidic acid  and choline.
in the presence of ethanol, however, pld forms phosphatidylethanol , a non-physiological phospholipid, at the expense of pa. this pld-specific phenomenon of transphosphatidylation is the reason why downstream events mediated by pld activation and pa formation are dose-dependently inhibited in the presence of ethanol .
in our previous work, we have found that astroglial pld is activated by mitogenic factors including fetal calf serum , pdgf, and phorbol ester, and we observed that ethanol reduced both astroglial proliferation and pa formation in a parallel manner.
1-butanol reduced pa formation and dna synthesis with the same potency while t-butanol was inactive for both effects  <cit> .
more recently, we demonstrated that exogenous pld as well as pa, when introduced into the cytosol by transient permeabilization, stimulated astroglial cell proliferation.
importantly, the action of pld was suppressed in the presence of ethanol  while the mitogenic effect of pa was not affected  <cit> .
thus, disruption of the pld signaling pathway by ethanol is sufficient to suppress astroglial cell proliferation.
recent findings from other groups are also compatible with a central role for the pld signaling pathway in ethanol toxicity in astrocytes.
first, several mitogenic factors including those that are known to be particularly sensitive to ethanol activate pld activity in astrocytes.
this holds true for pdgf  <cit> , acetylcholine  <cit> , glutamate  <cit> , phorbol esters  <cit> , endothelin  <cit> , and prolactin  <cit> .
in fact, disruption of pld signaling by ethanol was recently found to be responsible for ethanol's inhibitory effect on astroglial dna synthesis induced by muscarinic agonist  <cit> .
second, pld is activated via the mitogenic ras-ral pathway in many cell types  <cit> , and pa, the immediate product of pld activity, interacts with and activates proteins such as raf kinase, protein kinase cζ, and mtor which are known to be central to mitogenic signaling .
in addition, pa is a precursor of diacylglycerol , the endogenous activator of classical pkc's, and of lyso-pa, a potent mitogen in many cell types  <cit> .
taken together, current evidence suggests that intact pld signaling is a prerequisite for the proliferative effects of several mitogens, and that disruption of the pld pathway by ethanol may be a common theme in ethanol-induced inhibition of astroglial proliferation.
the present study in fetal astrocytes was motivated by recent reports that ethanol induces apoptosis in astrocytes, an effect that was accompanied by activation of the sphingomyelinase pathway and formation of ceramide  <cit> .
apoptosis denotes an active cellular program causing cellular death upon contact with toxicants.
apoptotic cell death in the cns has been under intensive study in recent years and involves several intracellular reaction cascades linked by the activation of caspases .
apoptosis is almost universally accompanied by the formation of ceramide which may occur through de novo-synthesis, inhibition of ceramide breakdown, or activation of  sphingomyelinase , an enzyme which catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine .
ceramide has emerged as a second messenger for apoptotic pathways targeting kinases and phosphatases which are required for the execution of apoptotic cell death .
in cerebellar astrocytes and in glioma cells, ceramide levels were found to be reciprocally related to cell proliferation  <cit> .
for the present study, we developed the hypothesis that the formation of ceramide may be secondary to the inhibition of pld signaling which we had described earlier .
we now report that ethanol-induced ceramide formation in astrocytes is mimicked by 1-butanol, but not by t-butanol, and that pa, the product of pld activity, antagonizes ethanol-induced formation of ceramide.
we also found that ceramide is a potent inhibitor of stimulated pld activity.
thus, we obtained evidence of a cross-talk between pa and ceramide, two lipid messengers with opposite effects on cellular proliferation.
RESULTS
markers of apoptosis
when primary astrocyte cultures were exposed to ethanol , staining of the cells with hoechst  <dig>  a dye intercalating into dna, revealed condensation and fragmentation of the nuclei which was visible after  <dig> hrs; the maximum effect was observed after  <dig> hours .
higher magnification demonstrated the presence of "apoptotic bodies" in the nuclei .
a similar effect was observed after treatment of the cells with the well-known apoptogen, staurosporine , or with c2-ceramide  but not with t-butanol  .
in parallel experiments, ethanol caused fragmentation of nuclear dna in serum-starved astrocytes which is reflected by "dna laddering" on agarose electrophoresis .
serum withdrawal alone was not effective while incubation with c2-ceramide  mimicked the effect of ethanol.
ethanol at  <dig> %  was almost as effective as  <dig> % .
effects of ethanol on sphingomyelin hydrolysis
formation of -ceramide was measured after labeling sphingomyelin with serine.
under basal conditions, the ratio of -ceramide to -sphingomyelin  was approximately 1: <dig>  this ratio was not significantly changed during serum withdrawal .
the incubation of astrocytes with ethanol  in serum-free medium caused an increase of -ceramide  but did not significantly change the total labeling of the large pool of -sphingomyelin by -serine .
as the total incorporation of -serine into -sphingomyelin was somewhat variable between individual preparations, we used the c/s ratio to calculate ethanol-induced changes.
as shown in figs.
<dig> and  <dig>  ethanol caused a significant increase of the astroglial c/s ratio in a biphasic and dose-dependent manner.
the rapid and transient phase of ceramide formation occurred within  <dig> min and reached a maximum at  <dig> hour after addition of ethanol .
a second increase gradually developed after  <dig> hours and reached a maximum at  <dig> hours of ethanol exposure .
at this later time point, staurosporine  caused an increase of the c/s ratio to  <dig>  ±  <dig>  % .
inhibition of ceramide formation by pld activity and phosphatidic acid
to investigate whether ceramide formation is secondary to a disruption of pld signaling, we used the isomeric alcohols, 1-butanol and t-butanol.
1-butanol – but not t-butanol – is a substrate of pld for transphosphatidylation and leads to the formation of phosphatidyl-1-butanol at the expense of pa. the correlations between butanol exposures and disruption of pld signaling  in astrocytes were documented in detail in our previous work  <cit> .
in the present experiments we measured the differential effects of 1- and t-butanol on the formation of ceramide  in astrocytes.
as shown in fig.
<dig>  there was a tendency for an increased level of the c/s ratio after addition of 1-butanol at  <dig> % while highly significant increases were observed with  <dig> %.
in contrast, t-butanol  had no effect.
this pattern was identical at the  <dig> hour and  <dig> hours time points .
this pattern suggested a change of the c/s ratio that was secondary to the disruption of the pld pathway.
to obtain more direct evidence for this hypothesis, we tested the influence of exogenous pa, the product of pld, on the c/s ratio.
for this purpose, we used a permeabilization procedure which makes use of an oxygen-insensitive mutant  of streptolysin-o  to introduce the membrane-impermeable pa into the astroglial cytosol  <cit> .
in preliminary experiments, we found that transient permeabilization with sl-o for  <dig> min by itself did not affect -ceramide levels .
basal ceramide levels were also unchanged if exogenous pa was added to the astrocytes in the absence  or presence of sl-o .
however, the ceramide formation evoked by ethanol  was significantly reduced in the presence of pa .
at the  <dig> h timepoint, the ethanol-induced effect  was reduced by pa pretreatment to  <dig>  ±  <dig>  %, a relative reduction by  <dig> percent .
at the  <dig> h timepoint, pa pretreatment reduced ethanol-induced ceramide formation by  <dig> percent .
inhibition of phospholipase d activity by ceramide
as the previous experiments indicated an inhibitory effect of the pld pathway on ceramide formation, the following experiment tested a possible effect of ceramide on pld activity measured by the transphosphatidylation assay.
in these experiments, the membrane-permeable c2-ceramide  slightly but non-significantly reduced basal pld activity by 30% .
however, when pld activity was stimulated by addition of serum  or by pdb, a phorbol ester and stimulator of protein kinase c , c2-ceramide at both  <dig> and  <dig> μm strongly and significantly reduced pld activity.
interestingly, serum-induced pld stimulation was more sensitive to ceramide than pdb-induced pld activity; the relative inhibitions for  <dig> and  <dig> μm c2-ceramide were   <dig> and  <dig> % and   <dig> and  <dig> %, respectively.
discussion
we have previously documented that ethanol suppresses signaling through the mitogenic phospholipase d  pathway, and we and others have provided evidence that this effect may be responsible for the antiproliferative actions of ethanol in astrocytes  <cit> .
the present study was motivated by findings that ethanol induces astroglial apoptosis via activation of the sphingomyelinase pathway  <cit> .
we confirm these earlier reports by showing the induction of apoptotic markers and ceramide formation in ethanol-treated astrocytes.
the novel finding of our study is that ethanol-induced formation of ceramide is reciprocally regulated by phosphatidic acid  and the phospholipase d pathway which is itself inhibited by ceramide.
we used three different approaches to demonstrate that apoptotic cell death in astrocytes can be induced by exposure to ethanol.
first, we report that ethanol can induce nuclear condensation and degradation .
second, application of ethanol to serum-starved astroglial cultures caused "dna laddering", a typical hallmark of apoptotic degradation of nuclear dna .
the effect of ethanol was mimicked by a cell-permeable ceramide, c2-ceramide , and by staurosporine .
third, ethanol induced an increase of ceramide in astroglial cultures .
our findings clearly confirm that ethanol can induce apoptosis and ceramide formation in astrocytes, a finding which is in agreement with some  <cit>  but not all  <cit>  previous studies.
we performed a time course of -ceramide formation after ethanol exposure and found that it was maximal at  <dig> and  <dig> hours.
biphasic formations of ceramide such as those observed here have been described previously in a range of peripheral cell types although their significance is unclear; they may reflect different modes of ceramide formation, or different pools of ceramide  <cit> .
at both time points of maximum ceramide formation , we observed the same ethanol-evoked enhancement of ceramide formation.
importantly, apoptotic cell death and ceramide formation were induced by ethanol levels as low as  <dig> mm which corresponds to blood alcohol levels  which are found in heavy drinkers.
it should be noted that the present experiments do not unequivocally identify the mechanism of ceramide formation.
we used -serine to pre-label sphingomyelin for  <dig> hours, removed the precursor, and measured formation of ceramide as an increase of the -ceramide/ -sphingomyelin ratio during incubations with ethanol.
this ratio most likely reflects the activity of sphingomyelinase, and sphingomyelinase activity was actually shown to be responsible for ethanol-induced ceramide formation in a recent study  <cit> .
however, our present data do not exclude alternative pathways of increased ceramide formation such as de novo-synthesis of ceramide or inhibition of ceramidase.
the important findings of this study relate to the interaction between lipid second messenger pathways.
it was known from previous work  that pa, the product of phospholipase d , mediates mitogenic stimulation in astrocytes whereas formation of ceramide by sphingomyelinase activation accompanies apoptosis.
we now tested the hypothesis that ethanol causes astroglial apoptosis by inhibiting pld and, as a consequence, stimulates the sphingomyelinase pathway.
the results shown in figs.
<dig> and  <dig> are evidence of a direct inhibitory influence of the pld pathway on ceramide formation.
first, we observed that the addition of 1-butanol, a primary alcohol which suppresses pld signaling, caused an increase of ceramide levels .
this effect was not seen with the inactive isomer t-butanol which does not interfere with pld signaling in astrocytes .
second, we used transient permeabilization of astrocytes by streptolysin-o to introduce pa, the product of pld, into the astroglial cytosol  <cit> .
we found that exogenous pa almost completely prevented the ethanol-induced increase of ceramide at early  and delayed  phases of ceramide formation .
the fact that ethanol and 1-butanol, but not t-butanol increase ceramide formation, whereas pa antagonized this effect, gives strong evidence that pld-mediated formation of pa keeps ceramide levels low under basal conditions , and that pld activity antagonizes ceramide formation under the influence of toxicants .
unfortunately, we could not determine the effect of exogenous pa on astroglial apoptosis because the permeabilization procedure was found to induce a delayed apopototic response in astrocytes .
we also probed the reciprocal effects of ceramide signaling on pld activity.
the results shown in fig.
<dig> demonstrate that c2-ceramide inhibits pld activity at a concentration as low as  <dig> μm.
basal pld activity was only slightly inhibited, but the increases of pld activity induced by addition of serum or phorbol ester were strongly antagonized.
this finding in astrocytes corroborates previous reports that ceramide can inhibit pld signaling in peripheral cell types  <cit> .
it remains a matter of speculation why serum-induced pld was somewhat more susceptible to inhibition by ceramide than phorbol ester-stimulated activity.
growth factors in serum and phorbol ester may affect different signaling pathways leading to pld activation, and we previously presented inhibition data with bacterial toxins which supported this idea for astroglial pld  <cit> .
interestingly, ethanol was also observed to inhibit serum- and growth factor-mediated astroglial proliferation more effectively than phorbol ester-induced proliferation  <cit> .
at this time, we cannot distinguish which isoform of pld is responsible for pa formation; previous attempts to selectively down-regulate astroglial plds failed due to the long biological half-lives of the proteins  <cit> .
the molecular target of ceramide for inhibiting pld also remains to be identified; previous work has implicated direct inhibition of pld by ceramide as well as upstream molecules such as protein kinase c which activate pld  <cit> .
CONCLUSIONS
in summary, the present results give evidence of a cross-talk between lipid-signaling pathways in astrocytes such that the product of pld, namely pa, inhibits ceramide formation whereas ceramide inhibits pld activation .
the experimental evidence suggests that the ratio "pa:ceramide" contributes to the decision whether astrocytes proliferate or undergo apoptosis.
our data suggest that ethanol induces astroglial apoptosis during brain development by disrupting pld signaling, thereby reducing pa and increasing ceramide formation.
this effect likely contributes to the microencephaly and delay of brain development observed in fetal alcohol syndrome.

 the proliferation of astrocytes is stimulated by polypeptide growth factors such as pdgf, egf, bfgf and igf- acting on cellular signaling pathways which involve tyrosine kinases, protein kinase c, and the ras-raf-map kinase pathway. astroglial proliferation is also stimulated by neurotransmitters such as acetylcholine and glutamate, by direct stimulation of protein kinase c with phorbol ester, and by peptides such as endothelin and prolactin. astroglial proliferation is prominently inhibited by ethanol both in vivo and in vitro, and this interference likely contributes to the development of the fetal alcohol syndrome. ethanol has been shown to potently antagonize proliferative effects of several individual astroglial mitogens including pdgf, igf- acetylcholine and prolactin. the molecular target of ethanol's antimitogenic actions in astroyctes is not known with certainty, but inhibitory interactions of ethanol with lipid signaling pathways have been implicated. our group has recently reported strong evidence that the growth-inhibitory effect of ethanol in astrocytes is caused by the disruption of the phospholipase d signaling pathway. under physiological conditions, pld catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. in the presence of ethanol, however, pld forms phosphatidylethanol, a non-physiological phospholipid, at the expense of pa. this pld-specific phenomenon of transphosphatidylation is the reason why downstream events mediated by pld activation and pa formation are dose-dependently inhibited in the presence of ethanol. in our previous work, we have found that astroglial pld is activated by mitogenic factors including fetal calf serum, pdgf, and phorbol ester, and we observed that ethanol reduced both astroglial proliferation and pa formation in a parallel manner. 1-butanol reduced pa formation and dna synthesis with the same potency while t-butanol was inactive for both effects. more recently, we demonstrated that exogenous pld as well as pa, when introduced into the cytosol by transient permeabilization, stimulated astroglial cell proliferation. importantly, the action of pld was suppressed in the presence of ethanol while the mitogenic effect of pa was not affected. thus, disruption of the pld signaling pathway by ethanol is sufficient to suppress astroglial cell proliferation. recent findings from other groups are also compatible with a central role for the pld signaling pathway in ethanol toxicity in astrocytes. first, several mitogenic factors including those that are known to be particularly sensitive to ethanol activate pld activity in astrocytes. this holds true for pdgf, acetylcholine, glutamate, phorbol esters, endothelin, and prolactin. in fact, disruption of pld signaling by ethanol was recently found to be responsible for ethanol's inhibitory effect on astroglial dna synthesis induced by muscarinic agonist. second, pld is activated via the mitogenic ras-ral pathway in many cell types, and pa, the immediate product of pld activity, interacts with and activates proteins such as raf kinase, protein kinase cζ, and mtor which are known to be central to mitogenic signaling. in addition, pa is a precursor of diacylglycerol, the endogenous activator of classical pkc's, and of lyso-pa, a potent mitogen in many cell types. taken together, current evidence suggests that intact pld signaling is a prerequisite for the proliferative effects of several mitogens, and that disruption of the pld pathway by ethanol may be a common theme in ethanol-induced inhibition of astroglial proliferation. the present study in fetal astrocytes was motivated by recent reports that ethanol induces apoptosis in astrocytes, an effect that was accompanied by activation of the sphingomyelinase pathway and formation of ceramide. apoptosis denotes an active cellular program causing cellular death upon contact with toxicants. apoptotic cell death in the cns has been under intensive study in recent years and involves several intracellular reaction cascades linked by the activation of caspases. apoptosis is almost universally accompanied by the formation of ceramide which may occur through de novo-synthesis, inhibition of ceramide breakdown, or activation of sphingomyelinase, an enzyme which catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine. ceramide has emerged as a second messenger for apoptotic pathways targeting kinases and phosphatases which are required for the execution of apoptotic cell death. in cerebellar astrocytes and in glioma cells, ceramide levels were found to be reciprocally related to cell proliferation. for the present study, we developed the hypothesis that the formation of ceramide may be secondary to the inhibition of pld signaling which we had described earlier. we now report that ethanol-induced ceramide formation in astrocytes is mimicked by 1-butanol, but not by t-butanol, and that pa, the product of pld activity, antagonizes ethanol-induced formation of ceramide. we also found that ceramide is a potent inhibitor of stimulated pld activity. thus, we obtained evidence of a cross-talk between pa and ceramide, two lipid messengers with opposite effects on cellular proliferation.  markers of apoptosis when primary astrocyte cultures were exposed to ethanol, staining of the cells with hoechst a dye intercalating into dna, revealed condensation and fragmentation of the nuclei which was visible after hrs; the maximum effect was observed after hours. higher magnification demonstrated the presence of "apoptotic bodies" in the nuclei. a similar effect was observed after treatment of the cells with the well-known apoptogen, staurosporine, or with c2-ceramide but not with t-butanol. in parallel experiments, ethanol caused fragmentation of nuclear dna in serum-starved astrocytes which is reflected by "dna laddering" on agarose electrophoresis. serum withdrawal alone was not effective while incubation with c2-ceramide mimicked the effect of ethanol. ethanol at % was almost as effective as %. effects of ethanol on sphingomyelin hydrolysis formation of -ceramide was measured after labeling sphingomyelin with serine. under basal conditions, the ratio of -ceramide to -sphingomyelin was approximately 1: this ratio was not significantly changed during serum withdrawal. the incubation of astrocytes with ethanol in serum-free medium caused an increase of -ceramide but did not significantly change the total labeling of the large pool of -sphingomyelin by -serine. as the total incorporation of -serine into -sphingomyelin was somewhat variable between individual preparations, we used the c/s ratio to calculate ethanol-induced changes. as shown in figs. and ethanol caused a significant increase of the astroglial c/s ratio in a biphasic and dose-dependent manner. the rapid and transient phase of ceramide formation occurred within min and reached a maximum at hour after addition of ethanol. a second increase gradually developed after hours and reached a maximum at hours of ethanol exposure. at this later time point, staurosporine caused an increase of the c/s ratio to ± %. inhibition of ceramide formation by pld activity and phosphatidic acid to investigate whether ceramide formation is secondary to a disruption of pld signaling, we used the isomeric alcohols, 1-butanol and t-butanol. 1-butanol – but not t-butanol – is a substrate of pld for transphosphatidylation and leads to the formation of phosphatidyl-1-butanol at the expense of pa. the correlations between butanol exposures and disruption of pld signaling in astrocytes were documented in detail in our previous work. in the present experiments we measured the differential effects of 1- and t-butanol on the formation of ceramide in astrocytes. as shown in fig. there was a tendency for an increased level of the c/s ratio after addition of 1-butanol at % while highly significant increases were observed with %. in contrast, t-butanol had no effect. this pattern was identical at the hour and hours time points. this pattern suggested a change of the c/s ratio that was secondary to the disruption of the pld pathway. to obtain more direct evidence for this hypothesis, we tested the influence of exogenous pa, the product of pld, on the c/s ratio. for this purpose, we used a permeabilization procedure which makes use of an oxygen-insensitive mutant of streptolysin-o to introduce the membrane-impermeable pa into the astroglial cytosol. in preliminary experiments, we found that transient permeabilization with sl-o for min by itself did not affect -ceramide levels. basal ceramide levels were also unchanged if exogenous pa was added to the astrocytes in the absence or presence of sl-o. however, the ceramide formation evoked by ethanol was significantly reduced in the presence of pa. at the h timepoint, the ethanol-induced effect was reduced by pa pretreatment to ± %, a relative reduction by percent. at the h timepoint, pa pretreatment reduced ethanol-induced ceramide formation by percent. inhibition of phospholipase d activity by ceramide as the previous experiments indicated an inhibitory effect of the pld pathway on ceramide formation, the following experiment tested a possible effect of ceramide on pld activity measured by the transphosphatidylation assay. in these experiments, the membrane-permeable c2-ceramide slightly but non-significantly reduced basal pld activity by 30%. however, when pld activity was stimulated by addition of serum or by pdb, a phorbol ester and stimulator of protein kinase c, c2-ceramide at both and μm strongly and significantly reduced pld activity. interestingly, serum-induced pld stimulation was more sensitive to ceramide than pdb-induced pld activity; the relative inhibitions for and μm c2-ceramide were and % and and %, respectively. discussion we have previously documented that ethanol suppresses signaling through the mitogenic phospholipase d pathway, and we and others have provided evidence that this effect may be responsible for the antiproliferative actions of ethanol in astrocytes. the present study was motivated by findings that ethanol induces astroglial apoptosis via activation of the sphingomyelinase pathway. we confirm these earlier reports by showing the induction of apoptotic markers and ceramide formation in ethanol-treated astrocytes. the novel finding of our study is that ethanol-induced formation of ceramide is reciprocally regulated by phosphatidic acid and the phospholipase d pathway which is itself inhibited by ceramide. we used three different approaches to demonstrate that apoptotic cell death in astrocytes can be induced by exposure to ethanol. first, we report that ethanol can induce nuclear condensation and degradation. second, application of ethanol to serum-starved astroglial cultures caused "dna laddering", a typical hallmark of apoptotic degradation of nuclear dna. the effect of ethanol was mimicked by a cell-permeable ceramide, c2-ceramide, and by staurosporine. third, ethanol induced an increase of ceramide in astroglial cultures. our findings clearly confirm that ethanol can induce apoptosis and ceramide formation in astrocytes, a finding which is in agreement with some but not all previous studies. we performed a time course of -ceramide formation after ethanol exposure and found that it was maximal at and hours. biphasic formations of ceramide such as those observed here have been described previously in a range of peripheral cell types although their significance is unclear; they may reflect different modes of ceramide formation, or different pools of ceramide. at both time points of maximum ceramide formation, we observed the same ethanol-evoked enhancement of ceramide formation. importantly, apoptotic cell death and ceramide formation were induced by ethanol levels as low as mm which corresponds to blood alcohol levels which are found in heavy drinkers. it should be noted that the present experiments do not unequivocally identify the mechanism of ceramide formation. we used -serine to pre-label sphingomyelin for hours, removed the precursor, and measured formation of ceramide as an increase of the -ceramide/ -sphingomyelin ratio during incubations with ethanol. this ratio most likely reflects the activity of sphingomyelinase, and sphingomyelinase activity was actually shown to be responsible for ethanol-induced ceramide formation in a recent study. however, our present data do not exclude alternative pathways of increased ceramide formation such as de novo-synthesis of ceramide or inhibition of ceramidase. the important findings of this study relate to the interaction between lipid second messenger pathways. it was known from previous work that pa, the product of phospholipase d, mediates mitogenic stimulation in astrocytes whereas formation of ceramide by sphingomyelinase activation accompanies apoptosis. we now tested the hypothesis that ethanol causes astroglial apoptosis by inhibiting pld and, as a consequence, stimulates the sphingomyelinase pathway. the results shown in figs. and are evidence of a direct inhibitory influence of the pld pathway on ceramide formation. first, we observed that the addition of 1-butanol, a primary alcohol which suppresses pld signaling, caused an increase of ceramide levels. this effect was not seen with the inactive isomer t-butanol which does not interfere with pld signaling in astrocytes. second, we used transient permeabilization of astrocytes by streptolysin-o to introduce pa, the product of pld, into the astroglial cytosol. we found that exogenous pa almost completely prevented the ethanol-induced increase of ceramide at early and delayed phases of ceramide formation. the fact that ethanol and 1-butanol, but not t-butanol increase ceramide formation, whereas pa antagonized this effect, gives strong evidence that pld-mediated formation of pa keeps ceramide levels low under basal conditions, and that pld activity antagonizes ceramide formation under the influence of toxicants. unfortunately, we could not determine the effect of exogenous pa on astroglial apoptosis because the permeabilization procedure was found to induce a delayed apopototic response in astrocytes. we also probed the reciprocal effects of ceramide signaling on pld activity. the results shown in fig. demonstrate that c2-ceramide inhibits pld activity at a concentration as low as μm. basal pld activity was only slightly inhibited, but the increases of pld activity induced by addition of serum or phorbol ester were strongly antagonized. this finding in astrocytes corroborates previous reports that ceramide can inhibit pld signaling in peripheral cell types. it remains a matter of speculation why serum-induced pld was somewhat more susceptible to inhibition by ceramide than phorbol ester-stimulated activity. growth factors in serum and phorbol ester may affect different signaling pathways leading to pld activation, and we previously presented inhibition data with bacterial toxins which supported this idea for astroglial pld. interestingly, ethanol was also observed to inhibit serum- and growth factor-mediated astroglial proliferation more effectively than phorbol ester-induced proliferation. at this time, we cannot distinguish which isoform of pld is responsible for pa formation; previous attempts to selectively down-regulate astroglial plds failed due to the long biological half-lives of the proteins. the molecular target of ceramide for inhibiting pld also remains to be identified; previous work has implicated direct inhibition of pld by ceramide as well as upstream molecules such as protein kinase c which activate pld.  in summary, the present results give evidence of a cross-talk between lipid-signaling pathways in astrocytes such that the product of pld, namely pa, inhibits ceramide formation whereas ceramide inhibits pld activation. the experimental evidence suggests that the ratio "pa:ceramide" contributes to the decision whether astrocytes proliferate or undergo apoptosis. our data suggest that ethanol induces astroglial apoptosis during brain development by disrupting pld signaling, thereby reducing pa and increasing ceramide formation. this effect likely contributes to the microencephaly and delay of brain development observed in fetal alcohol syndrome. 
moreover, exogenous pa, added to transiently permeabilized astrocytes, suppressed ethanol-induced -ceramide formation. concomitantly, in cells prelabeled with -serine, ethanol caused a dose-dependent, biphasic increase of the -ceramide/ -sphingomyelin ratio after and hours of incubation. as primary alcohols such as ethanol and 1-butanol were shown to inhibit the phospholipase d -mediated formation of pa, a mitogenic lipid messenger, we tested their effects on ceramide formation. vice versa, addition of c2-ceramide to astrocytes inhibited pld activity induced by serum or phorbol ester. ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid formation by phospholipase d. we propose that the formation of ceramide in ethanol-exposed astrocytes is secondary to the disruption of phospholipase d signaling. ethanol reduces the pa:ceramide ratio in fetal astrocytes, a mechanism which likely participates in ethanol-induced glial apoptosis during brain development. evidence is presented that the formation of pa and ceramide may be reciprocally linked during ethanol exposure.
 ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid formation by phospholipase d. the present study investigates ethanol's effect on the induction of apoptosis in astrocytes and the formation of ceramide, an apoptotic signal. evidence is presented that the formation of pa and ceramide may be reciprocally linked during ethanol exposure.  in cultured rat cortical astrocytes, ethanol induced nuclear fragmentation and dna laddering indicative of apoptosis. concomitantly, in cells prelabeled with -serine, ethanol caused a dose-dependent, biphasic increase of the -ceramide/ -sphingomyelin ratio after and hours of incubation. as primary alcohols such as ethanol and 1-butanol were shown to inhibit the phospholipase d -mediated formation of pa, a mitogenic lipid messenger, we tested their effects on ceramide formation. in astrocytes prelabeled with -serine, ethanol and 1-butanol, in contrast to t-butanol, significantly increased the formation of -ceramide. moreover, exogenous pa, added to transiently permeabilized astrocytes, suppressed ethanol-induced -ceramide formation. vice versa, addition of c2-ceramide to astrocytes inhibited pld activity induced by serum or phorbol ester.  we propose that the formation of ceramide in ethanol-exposed astrocytes is secondary to the disruption of phospholipase d signaling. ethanol reduces the pa:ceramide ratio in fetal astrocytes, a mechanism which likely participates in ethanol-induced glial apoptosis during brain development. 