Alcohol and Alcoholism

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Alcohol and Alcoholism Vol. 34, No. 4, pp. 506-510, 1999
© 1999 Medical Council on Alcoholism

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Rapid Communication

THE SYNERGISTIC ACTION OF ETHANOL AND NERVE GROWTH FACTOR IN THE INDUCTION OF NEURONAL NITRIC OXIDE SYNTHASE

Yume T. Phung and Stephen M. Black^*

Department of Pediatrics, University of California at San Francisco, San Francisco, CA 94143-0106, USA

Received 26 January 1999; accepted 16 March 1999

	ABSTRACT

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Ethanol alone had no effect on neuronal nitric oxide synthase (nNOS) expression in PC12 cells. However, in the presence of nerve growth factor (NGF), nNOS expression was amplified (threefold, P < 0.05), compared to NGF alone. This increase was eliminated with pretreatment of PC12 cells with staurosporine, suggesting that the effects of ethanol on nNOS expression are mediated by a protein kinase C-dependent pathway.

	INTRODUCTION

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Many studies have identified the brain as a major target organ for the harmful effects of alcohol. Alcohol exposure has been shown to alter both the normal development of the brain (Kotkoskie and Norton, 1988) and affect neuromorphogenesis (Miller, 1986). Alcohol appears to interfere with the formation of normal neural processes. Thus, exposure to ethanol inhibits dendrite growth and differentiation in the pyramidal cells of the developing rat neocortex and hippocampus, and decreases dendritic branching and spine density in CA1 hippocampal neurons and Purkinje cells in adult rats and mice (Hammer, 1986). Chronic alcohol exposure has been shown to increase nitric oxide (NO) levels in neuronal cultures (Chandler et al., 1994) and may be responsible for the acquisition of alcohol tolerance. Studies in the rat have shown that nitric oxide synthase (NOS) inhibition will prevent the development of alcohol tolerance (Khanna et al., 1993). NOS activity also appears to be linked to the development of alcohol addiction (Beauge et al., 1994). Inhibition of NOS is associated with the reversal of preference for alcohol vs water in alcohol-preferring rats (Beauge et al., 1994; Rezvani et al., 1995). Additionally when PC12 cells are exposed to methanol, ethanol, or propanol in the presence of suboptimal concentrations of nerve growth factor (NGF) (30 ng/ml), each produces rapid morphological differentiation and extensive neurite outgrowth (Wooten and Ewald, 1991). Since we had previously demonstrated that NGF increases the expression of neuronal NO synthase (nNOS) in PC12 cells, we undertook a study to determine if ethanol could also exert an effect on nNOS expression in PC12 cells. The results obtained indicate that, although ethanol by itself is incapable of affecting nNOS gene expression, it acts synergistically with NGF to ‘super-induce’ nNOS gene expression in a protein kinase C (PKC)-dependent manner.

	MATERIALS AND METHODS

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Cell culture and treatments
PC12 cells were cultured and passaged as previously described (Sheehy et al., 1997). For differentiation experiments, cells were plated onto collagen IV (BioCoat)-coated 60-mm dishes (for protein studies) or 24-well plates (for neurite outgrowth determination) in defined medium, as previously described (Ferriero et al., 1994; Sheehy et al., 1997). Cells were allowed to attach overnight before being treated. Differentiation was induced in the presence of NGF (1–40 ng/ml) for 5 days. For experiments with ethanol, the dishes were sealed using paraffin to prevent evaporation and the medium changed daily. To inhibit PKC activity, PC12 cells were incubated with staurosporine (1 nM).

Western blot analysis
PC12 cells were harvested into ice-cold phosphate buffered saline (PBS), centrifuged, resuspended in PBS, and sonicated prior to protein quantification (Bradford reagent, Bio-Rad). Protein extracts (50 µg) were separated on 7.5% reducing SDS– polyacrylamide gels. The gel was electrophoretically transferred to nitrocellulose, blocked in PBS/0.1% Tween containing 5% non-fat dry milk and incubated with a primary antiserum that recognized the haem domain of nNOS (Sheehy et al., 1997). After washing to remove excess secondary antibody, membranes were developed using chemiluminescent techniques (Pierce Labs). Quantification of autoradiographic results was performed by scanning (Hewlett Packard SCA Jet IICX) the bands of interest into an image editing software program (Adobe Photoshop). Band intensities were analysed densitometrically on a Macintosh computer (model 7100/66) using the public domain NIH Image program. ANOVA was used to determine any significant changes in densitometric values. A P < 0.05 was considered statistically significant.

	RESULTS

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We initially treated PC12 cells with ethanol (200 mM) in the presence or absence of NGF (40 ng/ml) and determined the effect on neurite outgrowth (Fig. 1 A) or nNOS protein expression (Fig. 1 B). In the absence of NGF, ethanol had no obvious effects on either neurite outgrowth or nNOS expression. However, in the presence of NGF, the expression of nNOS was induced significantly (threefold, P < 0.05) over that achieved with NGF alone (Fig. 1 C). This increase in nNOS expression was paralleled by an increase in neurite formation (Fig. 1 A).

View larger version (107K): [in this window] [in a new window]   Fig. 1. Effects of ethanol, nerve growth factor (NGF) or both on neurite production and neuronal nitric oxide synthase (nNOS) gene expression in PC12 cells in culture. Ethanol alone did not induce nNOS. PC12 cells were grown for 5 days on collagen IV-coated plates in the presence of ethanol (200 mM) and/or NGF (40 ng/ml). Neurite production was then visualized (A). Protein extracts (50 µg) were also prepared from these cells and separated on a 7.5% reducing SDS–polyacrylamide gel and analysed by Western blotting (B). nNOS protein expression was determined using densitometry and values expressed relative to that obtained with NGF (40 ng/ml) alone (C). Values are means ± SEM from six experiments. *P < 0.05 vs NGF-treated cells. Only in the presence of NGF does ethanol stimulate neurite outgrowth and nNOS expression.

 
This synergistic effect of ethanol and NGF on nNOS expression was found with ethanol concentrations as low as 25 mM (Fig. 2 A, C). Similarly, at high ethanol concentrations (200 mM), NGF levels as low as 10 ng/ml could increase nNOS expression (Fig. 2 B, C).

View larger version (63K): [in this window] [in a new window]   Fig. 2. The effect of ethanol and nerve growth factor (NGF) on neuronal nitric oxide synthase (nNOS) expression in PC12 cells. PC12 cells were grown for 5 days on collagen IV-coated plates in the presence of NGF (0–40 ng/ml) and 100 mM ethanol (A) or in the presence of ethanol (0–200 mM) and 40 ng/ml NGF (B). Protein extracts (50 µg) were prepared from each treatment and separated on a 7.5% reducing SDS–polyacrylamide gel and analysed by Western blotting. nNOS expression was determined using densitometry and values expressed relative to that obtained with NGF (40 ng/ml) alone (C). Values are means ± SEM from three experiments. *P < 0.05 vs 40 ng/ml NGF alone; P < 0.05 vs 40 ng/ml NGF alone.

 
This synergistic induction of nNOS expression could be blocked by the PKC inhibitor, staurosporine (Fig. 3 A, B).

View larger version (54K): [in this window] [in a new window]   Fig. 3. Protein kinase C (PKC) inhibition attenuates neuronal nitric oxide synthase (nNOS) induction by ethanol. PC12 cells were grown for 5 days on collagen IV-coated plates in the presence of NGF (40 ng/ml) and 100 mM ethanol and in the presence or absence of the PKC inhibitor, staurosporine (1 nM). Protein extracts (50 µg) were prepared from each treatment and separated on a 7.5% reducing SDS–polyacrylamide gel and analysed by Western blotting. The densitometric values for nNOS protein for each treatment were calculated from three different experiments (B). Values are means ± SEM. *P < 0.05 vs NGF-treated cells. The presence of staurosporine significantly reduces the ethanol induced over-expression of nNOS.

 

	DISCUSSION

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
PC12 cells exposed to ethanol in the presence of NGF increase their rate of neurite production even in the presence of suboptimal concentrations of NGF (Wooten and Ewald, 1991). However, the effector molecule in this process is unknown at the present time. Since we have previously demonstrated that NO is required for NGF-induced PC12 cell differentiation, we felt that it was reasonable to hypothesize that ethanol may stimulate NO production by increasing the expression of the nNOS gene. Treatment of PC12 cells indicated that, in the absence of NGF, ethanol had no obvious effects on either neurite outgrowth or nNOS expression. However, in the presence of NGF, the expression of nNOS was significantly induced over that achieved with NGF alone. There was also a parallel increase in neurite formation. Induction of nNOS expression could be achieved at levels of NGF as low as 10 ng/ml, suggesting that ethanol sensitizes PC12 cells such that they will react to growth factor levels that would not normally elicit a morphological response. This ‘super-induction’ (or permissive effect) of nNOS expression was blocked by the PKC inhibitor, staurosporine, indicating that the induction of nNOS was dependent on a functional PKC pathway. This result fits well with those from Messing et al. (1991) and Messing (1993), who have also shown that ethanol increases the expression PKC and PKC and that PKC appears to mediate the enhanced NGF-induced neurite outgrowth (Hundle et al., 1997).

Previously we have demonstrated that a functional nNOS is required for NGF-induced differentiation in PC12 cells with NO being necessary, but not sufficient, to induce differentiation. Together, the results presented here along with our previous studies strongly suggest that nNOS, via NO release, plays a major role in transducing the effects of ethanol into neuronal differentiation. This enhanced neurite outgrowth could be detrimental in vivo to the developing brain. For example, the increased dendrite length caused by this induction of neurite outgrowth could place synapses at greater distances from the cell soma at risk, decreasing electrical conduction along the dendrite to the soma. In addition, increased neurite connections could interfere with the normal cycles of neural process elimination and subsequent synaptic connection rearrangements that are an integral part of brain development. Furthermore, process elongation may lead to disruptions in neural network output by causing an imbalance in inhibitory and excitatory signals. Further analysis of how ethanol stimulates the expression of nNOS is thus warranted.

	ACKNOWLEDGEMENTS

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This work was supported in part by NIH grants HL60190 and HD28825, a James A. Shannon Director's award also from the NIH, and by a grant from the UCSF Academic Senate Committee on Research.

	FOOTNOTES

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
^* Author to whom correspondence should be addressed at: Division of Neonatology, Northwestern University Medical School, Ward 13-317 (MS# W-140), 303 E. Chicago Avenue, Chicago, IL 60611-3008, USA

	REFERENCES

TOP FOOTNOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Beauge, F. J., Seibre, N. and Vesperini, D. (1994) Involvement of nitric oxide (NO) in ethanol intake and preference in rats. International Society for Biomedical Research on Alcoholism, Gold Coast, Australia, Abstract 614.

Chandler, L. J., Sumners, C. and Crews, F. T. (1994) Effects of chronic ethanol exposure on nitric oxide formation in primary neuronal and astroglial cultures. Research Society on Alcoholism, Maui, HI Abstract 154.

Ferriero, D. M., Sheldon, R. A. and Messing, R. O. (1994) Somatostatin enhances nerve growth factorinduced neurite outgrowth in PC12 cells. Brain Research, Developmental Brain Research 80, 13–18.[Medline]

Hammer, R. P., Jr (1986) Alcohol effects on developing neuronal structure. In Alcohol and Brain Development, West, J. R. ed., pp. 184–203. Oxford University Press, New York.

Hundle, B., McMahon, T., Dadgar, J. C., Mochly-Rosen, D. and Messing, R. (1997) An inhibitory fragment derived from protein kinase C epsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters. Journal of Biological Chemistry 272, 15028–15035.[Abstract/Free Full Text]

Khanna, J. M., Morato, G. S., Shah, G., Chau, A. and Kalant, H. (1993) Inhibition of nitric oxide synthesis impairs rapid tolerance to ethanol. Brain Research Bulletin 32, 43–47.[Web of Science][Medline]

Kotkoskie, L. A. and Norton, S. (1988) Prenatal brain malformations following acute ethanol exposure in the rat. Alcoholism: Clinical and Experimental Research 12, 579–587.

Messing, R. O. (1993) Ethanol as an enhancer of neural differentiation. Alcohol and Alcoholism 28 (Suppl. 2), 289–293.

Messing, R. O., Henteleff, M. and Park, J. J. (1991) Ethanol enhances growth factor-induced neurite formation in PC12 cells. Brain Research 565, 301–311.[Web of Science][Medline]

Miller, M. W. (1986) Effects of alcohol on the generation and migration of cerebral cortical neurons. Science 233, 1308–13011.[Abstract/Free Full Text]

Rezvani, A. H., Grady, D. R., Peek, A. E. and Pucilowski, O. (1995) Inhibition of nitric oxide synthesis attenuates alcohol consumption in two strains of alcohol-preferring rats. Pharmacology, Biochemistry and Behavior 50, 265–270.[Web of Science][Medline]

Sheehy, A. M., Phung, Y. T., Riemer, R. K. and Black, S. M. (1997) Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells. Molecular Brain Research 52, 71–77.[Medline]

Wooten, M. W. and Ewald, S. J. (1991) Alcohols synergize with NGF to induce early differentiation of PC12 cells. Brain Research 550, 333–339.[Web of Science][Medline]

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This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (5) Request Permissions Google Scholar Articles by Phung, Y. T. Articles by Black, S. M. Search for Related Content PubMed PubMed Citation Articles by Phung, Y. T. Articles by Black, S. M. Social Bookmarking          What's this?

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