BRAIN NITRIC OXIDE SYNTHASE LEVELS INCREASE IN RESPONSE TO ANTENATAL ETHANOL EXPOSURE

Maria L. V. Dizon1, Lou Ann Brown3 and Stephen M. Black1,2,3,4,*

Departments of 1 Pediatrics and 2 Molecular Pharmacology, Northwestern University, Chicago, IL, 2Department of Pediatrics, Division of Neonatal–Perinatal Medicine, Emory University, Atlanta, GA, 3 Department of Biomedical and Pharmaceutical Sciences and the 4 International Heart Institute of Montana, University of Montana, Missoula, MT, USA

* Author to whom correspondence should be addressed at: International Heart Institute of Montana, St Patrick Hospital, 500 W Broadway, Missoula, MT 59802, USA. Tel.: +406 327 1673; Fax: +406 329 5880; E-mail: smblack{at}selway.umt.edu

(Received 24 March 2003; first review notified 25 July 2003; in revised form 10 September 2003; accepted 25 November 2003)


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aims: Our previous in vitro data have indicated that ethanol can increase nitric oxide synthase (NOS) expression. Thus, the Aims of this study were to determine whether ethanol produces the same effect in vivo. Methods: To accomplish this, we utilized the well-established prenatal ethanol (EtOH) exposure model in the guinea pig to examine the effect on brain NOS expression and activity. Results: Brain homogenates isolated from offspring of guinea pigs fed EtOH exhibited an increase in NOS protein expression and NOS activity compared to controls. Increased expression of neuronal NOS was observed only in soluble fractions of brain homogenates (P < 0.05 vs. control). Increased expression of a ~60 kDa band was detected in the soluble fraction that was immunoreactive against an antiserum raised against inducible NOS. In addition, an immunoreactive band of the correct predicted molecular weight for iNOS was found in the particulate fraction although the expression was unchanged between control and EtOH-treated animals. Endothelial NOS protein expression could not be detected in either soluble or particulate fractions from control or EtOH-treated animals. Conclusions: These results suggest that EtOH may exert its toxic effects antenatally via a mechanism of altered nitric oxide availability from NOS.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fetal alcohol syndrome (FAS) is the leading preventable cause of mental retardation in the United States (Abel and Sokol, 1986Go). Yet the mechanisms by which ethanol (EtOH) exerts its toxic effects are not well understood. Previous work conducted in vitro suggests that nitric oxide (NO) protects developing neurons against the toxic effects of EtOH. Thus, chronic alcohol exposure has been shown to increase nitric oxide (NO) levels in neuronal cultures (Chandler et al., 1997Go) and act in synergy with growth factors to increase nNOS expression (Phung and Black, 1999Go). In addition, studies in the rat have shown that nitric oxide synthase (NOS) inhibition will prevent the development of alcohol tolerance (Khanna et al., 1993Go). Inhibition of NOS is associated with the reversal of preference for alcohol versus water in alcohol-preferring rats (Rezvani et al., 1995Go) while nNOS null knock-out mice have increased vulnerability to alcohol-induced microencephaly and neuronal loss (Bonthius et al., 2002Go). However, although many in vivo studies have examined the effects of EtOH on NO signalling in the brain the results obtained have been contradictory. Both decreased NOS staining (Zima et al., 1998Go; Phillips et al., 2000Go; Zima et al., 2001Go) and increased (Xia et al., 1999Go) and decreased (Fataccioli et al., 1997Go) NOS activity have been observed. Thus, although it is likely that the generation of reactive nitrogen species is one of the mechanisms leading to brain abnormalities seen in FAS, including microencephaly (Archibald et al., 2001Go), a decrease in the absolute number of neurons (Maier et al., 1999aGo,bGo) and neuronal migrational disorders (Ozer et al., 2000Go), the mechanisms for these effects remain unresolved.

The guinea pig is one of the most widely used animal models used to investigate the effects of EtOH on the brain. Some of the most important investigations using this model have been performed by Kimura et al., utilizing a prenatal EtOH exposure model. However, these studies have focussed almost exclusively on the hippocampus where it was demonstrated that chronic maternal EtOH consumption suppressed NOS activity in hippocampus (Kimura et al., 1996Go) without changing NOS hippocampal expression (Kimura et al., 1999Go). However, no studies have as yet investigated the net effect of EtOH on NOS expression and activity within the brain as a whole nor have the effects on all the NOS isoforms been investigated in the same study. Thus, our studies have investigated the effects of antenatal EtOH exposure on the expression of the three NOS isoforms (endothelial, inducible and neuronal) as well as investigating the effects of antenatal EtOH exposure on NOS activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal treatment and sample preparation
Timed pregnant guinea pigs were purchased from Harlan Co and shipped at 30–35 days gestation to the Animal Resources Department of Emory University. Verification of pregnancy by palpation is difficult in the guinea pig and the vendor guaranteed pregnancy only in a range of 30–35 days gestation. The EtOH content of the drinking water was increased over 5 days to a final concentration of 4% (Abdollah and Brien, 1995Go). The EtOH content was incrementally increased to avoid decreased food intake and loss of pregnancy from inadequate caloric intake. Saccharin (8 mg/100 ml) was also included in the water to promote water intake. The only access to drinking water was the EtOH mixture. The dams received solid guinea pig chow ad libitum. For the controls, Maltose Dextrin 42 was added to the water in calories equivalent to the calories consumed in the water by the EtOH drinking dams. Saccharin was also included in the water of the controls. On days 55–60 of gestation (gestation = 71 days), pups were delivered by Caesarean section. Pups (n = 5, each from a different litter) were then decapitated and heads were snap frozen in liquid nitrogen. When required, heads were thawed on ice, brains removed and tissue homogenized (see below).

The diet or water consumed was not statistically different between the two groups. Blood alcohol of the dam and the pup were determined to be identical (0.079 ± 0.005%, n = 5 for both dams and pups) at the time of killing. Blood alcohol levels were determined using an established gas–liquid chromatographic procedure (Steenaart, 1985Go). At the time of delivery, the weight of the pups was not statistically different between the control and the EtOH-fed guinea pigs. The body weights of the pups were 47.2 ± 4.8 g and 42.1 ± 4.0 g for the control and EtOH groups, respectively (P > 0.05, n = 5 for both pair-fed and ethanol-fed groups). Head circumferences of the pups were 10.8 ± 0.2 and 9.8 ± 0.3 cm (P < 0.05) for the control and EtOH groups, respectively.

Whole brains were washed in 1 x phosphate-buffered saline. Homogenizing solution (20 mmol/l HEPES, pH 7.5 with 0.1 mmol/l EDTA, 1 mmol/l DTT and mammalian protease inhibitor cocktail) 2.5 ml per 0.5 g tissue was added to the samples. The tissue was then homogenized using a Tissue Tearor (Biospec Products). Homogenized tissue was transferred to 50 ml centrifuge tubes and centrifuged at 1000 g, 4°C for 20 min. Supernatant was decanted into fresh tubes and pellets were discarded. Then supernatant was centrifuged at 10 000 g, 4°C for 20 min. Supernatant was decanted and saved as soluble fractions. Pellets were resuspended in 2 ml additional homogenizing solution and centrifuged at 10 000 g, 4°C for 20 min. Supernatant was discarded. Pellets were resuspended in 1.5 ml homogenizing solution and saved as particulate fractions. All animal experimental protocols were approved by the Emory University Animal Care and Use Committee.

Western blot analysis
Homogenates were analysed for NOS protein expression using Western blot analysis. Protein was quantified using Coomassie protein assay reagent (Pierce Laboratories). Protein extracts (15 µg) were separated on 7.5% reducing SDS–polyacrylamide gels. Separated protein was electrophoretically transferred to nitrocellulose membranes. Membranes were first blocked in TBS/0.1% Tween containing 5% non-fat dry milk, then incubated with a primary antibody to neuronal- (nNOS), endothelial- (eNOS) or inducible- (iNOS) NOS. The anti-nNOS rabbit antibody was prepared as we have previously described (Sheehy et al., 1997Go). The anti-eNOS and anti-iNOS mouse antibodies were obtained from Transduction Laboratories. Membranes were then probed with secondary antibodies raised against the appropriate species (Pierce Laboratories). After washing with TBS/0.1% Tween, membranes were developed using a horseradish peroxidase chemiluminescent technique (SuperSignal West Femto Super Sensitive Substrate; Pierce Laboratories). Blots were imaged and results quantified using an Image Station 440CF (Kodak Digital Science).

NOS activity analysis
Homogenates were analysed for NOS activity utilizing the arginine-citrulline conversion assay originally described by Bush (Bush et al., 1992Go). Tissue samples (50 µg) were added to reaction tubes kept on ice. Specificity for NOS activity was demonstrated by pre-exposing brain homogenates to N{omega}-nitro-L-arginine methyl ester HCL (L-NAME), a non-specific NOS inhibitor. Then a reaction mixture containing L-arginine, FAD, BH4 and NADPH was added. MgCl2 was then added. Reactions were run both with and without CaCl2 and calmodulin (CaM). [3H]arginine was then added to each reaction tube and samples were incubated in a shaking water bath (37°C) for 1 h such that no more than 20% of the [3H]arginine was metabolized, to ensure that the substrate was not limiting. Final concentrations within the final reaction mixture were L-arginine (8 µmol/l), [3H]arginine (17 nmol/l), NADPH (1 mmol/l), FAD (5 µmol/l), BH4 (14 µmol/l), MgCl2 (1 mmol/l), CaCl2 (3 mmol/l) and calmodulin (25 units). Then the reaction was stopped with ice cold stop buffer (20 mmol/l Na citrate, pH 5.0 containing 1 mmol/l citrulline, 2 mmol/l EDTA 2 mmol/l and 0.2 mmol/l). Reactions mixtures were immediately poured through Dowex-50W columns, followed by 2 ml distilled H2O. Eluted fluid was collected in 15 ml scintillation vials, scintillation cocktail (ScintiVerse, Scintanalyzed; Fisher Scientific) 10 ml was added to each vial, and vials were counted for 3H using a LS6500 multipurpose scintillation counter (Beckman). NOS activity was estimated by the differences between counts in the presence and absence of L-NAME.

Statistical analysis
Quantitation of Western blots was performed using a Kodak image station 440CF and the KDS1D imaging software. The mean ± standard deviation of the densitometric analysis for each protein band was calculated. The mean ± standard deviation was also calculated for all NOS activities. In all cases comparisons between control and ethanol treated animals were made by ANOVA using the GB-STAT software program. A P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Soluble and particulate fractions of total brain homogenates were analysed separately for NOS isoform expression. The data obtained indicate that nNOS protein expression in the soluble fraction increased in animals exposed to EtOH in utero compared to controls (P < 0.05, Fig. 1A), but nNOS protein expression in the particulate fraction was unaffected (Fig. 1B). A protein band that cross-reacted with an antiserum specific to inducible NOS was also present in the soluble fraction, however the detected band had a mass of approximately 60 kDa rather than 130 kDa. The level of this protein within the soluble fraction was increased in EtOH-treated animals (P < 0.05 vs. control, Fig. 2A). Again iNOS protein expression in the particulate fraction was unchanged following antenatal EtOH exposure, although in the particulate fraction the detected band was present at the expected size of approximately 130 kDa (Fig. 2B). Endothelial NOS protein expression was absent in both the soluble and particulate fractions of controls and EtOH exposed animals (data not shown).




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Fig. 1. Western blot analysis of neuronal NOS (nNOS) expression in guinea pig brain homogenates: Control, and after antenatal exposure to ethanol (Ethanol). (A) Protein extracts (20 µg) prepared from the soluble fraction of total brain homogenates were separated on a 7.5% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analysed using a specific antiserum raised against nNOS. Figure is a representative blot. There is a significant increase in densitometric values for nNOS protein in the soluble fraction prepared from the ethanol fed guinea pig brains (n = 5). Values are mean ± standard deviation. *P < 0.05 vs. control. (B) Protein extracts (20 µg) prepared from the particulate fraction of total brain homogenates were separated on a 7.5% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analysed using a specific antiserum raised against nNOS. Figure is a representative blot. There is no significant difference in densitometric values for nNOS protein in the particulate fraction prepared from the control and ethanol-fed guinea pig brains (n = 5). Values are mean ± standard deviation.

 



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Fig. 2. Western blot analysis of inducible NOS (iNOS) expression in guinea pig brain homogenates: Control, and after antenatal exposure to ethanol (Ethanol). (A) Protein extracts (20 µg) prepared from the soluble fraction of total brain homogenates were separated on a 7.5% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analysed using a specific antiserum raised against iNOS. Figure is a representative blot. There is a significant increase in densitometric values for iNOS protein in the soluble fraction prepared from the ethanol fed guinea pig brains. However, the iNOS (iNOS*) detected is of a smaller molecular weight than the positive control (~130 kDa) (n = 5). Values are mean ± standard deviation. *P < 0.05 vs. control. (B) Protein extracts (20 µg) prepared from the particulate fraction of total brain homogenates were separated on a 7.5% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analysed using a specific antiserum raised against iNOS. Figure is a representative blot. There is no significant difference in densitometric values for iNOS protein in the particulate fraction prepared from the control and ethanol-fed guinea pig brains. However, the iNOS detected is of the predicted molecular weight (~130 kDa) (n = 5). Values are mean ± standard deviation.

 
Soluble and particulate fractions of brain homogenates were analysed separately for NOS activity. For soluble fractions, in utero EtOH exposure increased Ca2+/CaM-independent NOS activity by 2.79-fold as compared to untreated animals (6.95 ± 0.393 pmol/min/mg protein vs. 2.49 ± 0.309 pmol/min/mg protein, P < 0.05, Fig. 3A). For soluble fractions, in utero EtOH exposure similarly increased Ca2+/CaM-dependent activity by 3.22-fold (7.37 ± 1.40 pmol/min/mg protein vs. 2.29 ± 0.251 pmol/min/mg protein, P < 0.05, Fig. 3A). For the particulate fractions, in utero EtOH exposure increased Ca2+/CaM-independent NOS activity by 5.38-fold as compared to untreated animals (9.42 ± 2.02 pmol/min/mg protein vs. 1.75 ± 0.164 pmol/min/mg protein, P < 0.05, Fig. 3B). For particulate fractions, in utero EtOH exposure increased Ca2+/CaM-dependent NOS activity by 4.12-fold as compared to untreated animals (10.3 ± 2.70 pmol/min/mg protein vs. 2.49 ± 0.217 pmol/min/mg protein, P < 0.01, Fig. 3B).



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Fig. 3. NOS activity in guinea pig brain homogenates: Control, and after antenatal exposure to ethanol (Ethanol). NOS activity was estimated in protein extracts (50 µg) prepared from either the soluble (A) or particulate (B) fractions of total brain homogenates using [3H]arginine to [3H]citrulline conversion. Activities are presented as pmols/min/mg protein in the presence and absence of calcium calmodulin activation. *P < 0.05 vs. control.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have demonstrated for the first time the in utero effects of antenatal EtOH exposure on multiple isoforms of nitric oxide synthase within guinea pig brain homogenates. The effects of EtOH on NOS protein expression and activity were not uniform across isoforms. EtOH appears to alter nNOS expression. Neuronal NOS was both present in cytosolic and membrane fractions of both treated and untreated animals. However, nNOS protein was increased in response to EtOH treatment only within the cytosolic fraction. In contrast, eNOS expression was absent from both cytosolic and membrane fractions and EtOH treatment did not induce eNOS protein expression.

In the particulate fraction the iNOS protein was found at the predicted molecular weight of ~130 kDa. However, within the cytosol, a protein that cross-reacted with the iNOS antiserum was found to be present with an approximate molecular weight of 60 kDa. This was detected in the cytosol of both the EtOH-treated and control animals and was significantly increased in EtOH-fed animals. Whether this protein is actually a truncated form of iNOS is unclear. However, a potential trypsin cleavage site is present within the CaM region of NOS linking the heme and the reductase domains that, if cleaved at this location, would yield a fragment of approximately 60 kDa. Thus, one possible explanation for the finding of a smaller molecular weight band is that the iNOS protein is degraded in the cytosolic fraction perhaps via the proteasomal degradation pathway as previously described (Musial and Eissa, 2001Go). Alternatively, it is possible that we are observing a splice variant of iNOS as has been previously described in the fetal lung (Rairigh et al., 1998Go). It is unclear whether this shorter form of iNOS has catalytic activity. However, if this is a cleaved fragment of iNOS it is possible that it still retains catalytic activity as it has been previously demonstrated that incubation of the oxygenase and reductase domains of eNOS together in vitro, can yield catalytic activity (Nishida and Ortiz de Montellano, 1998Go).

Our results also indicate that antenatal EtOH exposure exerts an affect on NOS activity. Both Ca2+/CaM-dependent NOS activity, corresponding to the neuronal and endothelial isoforms, and Ca2+/CaM-independent activity, corresponding to the inducible isoform, increased to varying degrees in response to EtOH, and to differing degrees depending on the fraction assayed. Both Ca2+/CaM-dependent and Ca2+/CaM-independent NOS activities were greater in particulate fractions compared to soluble fractions although no changes in NOS expression were detected in this fraction. As alterations in protein expression and activity are not well correlated this suggests that there may be regulation of the NOS isoforms at a post-translational level. However, further studies will be required to elucidate the mechanisms for these effects.

Antenatal EtOH exposure increased the concentrations of both nNOS and iNOS within the cytosol, a location where both isoforms are known to exist while in the active state. Given the absence of eNOS within the soluble fraction, we attribute the Ca2+/CaM-dependent activity within the cytosol to nNOS. Ca2+/CaM-independent NOS activity within the cytosol is, at first glance, easily attributable to iNOS. On the other hand, the lack of any large difference between Ca2+/CaM-dependent and -independent activity despite an increase in both nNOS and iNOS protein, suggests that nNOS may be partly responsible for the increase in Ca2+/CaM-independent activity. Perhaps EtOH exposure alters the association between calmodulin and nNOS resulting in a loss of Ca2+ dependence. Although this is speculative at the present time, studies from Song et al. have demonstrated that HSP90 can stabilize the Ca2+/CaM binding to nNOS which increases activity independent of changes in protein levels (Song et al., 2001Go).

NOS activity increased in the membrane fraction in response to EtOH, even though eNOS was absent in that fraction. Neuronal NOS protein concentrations did not increase significantly in this fraction even with EtOH treatment and so an increase in protein does not explain an increase in NOS activity. Likewise, Ca2+/CaM-independent activity increased at the membrane in response to EtOH, despite the fact that iNOS protein expression did not increase significantly. Together this suggests that EtOH causes nNOS activity and/or iNOS activity to increase at an ectopic location. Either the enzyme activity of iNOS is increased or perhaps nNOS has a decreased dependence on Ca2+. In other words, NOS activity may be increased at an ectopic location in response to EtOH exposure, and it is this activation, rather than an increase in protein production, that accounts for the increased NOS activity.

In summary, our data demonstrate that antenatal EtOH exposure increases nNOS and iNOS protein levels and both Ca2+/CaM-dependent and -independent NOS activity within the guinea pig brain. Furthermore, we have shown that NOS activity can be increased independent of changes in protein levels. Thus, these changes in NOS expression and activity may contribute to the mechanism by which EtOH exerts its effect on neuronal development. EtOH may perturb neural cell differentiation in vivo by increasing the availability of NO, thereby altering the local oxidative milieu, perhaps augmenting differentiation in some cases and heightening cell death in others. We propose that further studies are warranted to investigate the roles played by iNOS and nNOS in FAS.


    ACKNOWLEDGEMENTS
 
This research was supported in part by grants HL60190, HL67841 and HD398110 from the National Institutes of Health, and FY00-98 from the March of Dimes, all to S.M.B.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abdollah, S. and Brien, J. F. (1995) Effect of chronic maternal ethanol administration on glutamate and N-methyl-D-aspartate binding sites in the hippocampus of the near-term fetal guinea pig. Alcohol 12, 377–382.[CrossRef][ISI][Medline]

Abel, E. L. and Sokol, S. J. (1986) Fetal alcohol syndrome is now leading cause of mental retardation. Lancet 2, 1222.

Archibald, S. L., Fennema-Notestine, C., Gamst, A., Riley, E. P., Mattson, S. N. and Jernigan, T. L. (2001) Brain dysmorphology in individuals with severe prenatal alcohol exposure. Developmental Medicine and Child Neurology 43, 148–154.[ISI][Medline]

Bonthius, D. J., Tzouras, G., Karacay, B., Mahoney, J., Hutton, A., McKim, R. and Pantazis, N. J. (2002) Deficiency of neuronal nitric oxide synthase (nNOS) worsens alcohol-induced microencephaly and neuronal loss in developing mice. Brain Research: Developmental Brain Research 138, 45–59.[ISI][Medline]

Bush, P. A., Gonzalez, N. E., Griscavage, J. M. and Ignarro, L. J. (1992) Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine. Biochemical and Biophysical Research Communications 185, 960–966.[ISI][Medline]

Chandler, L. J., Sutton, G., Norwood, D., Sumners, C. and Crews, F. T. (1997) Chronic ethanol increases N-methyl-D-aspartate-stimulated nitric oxide formation but not receptor density in cultured cortical neurons. Molecular Pharmacology 51, 733–740.[Abstract/Free Full Text]

Fataccioli, V., Gentil, M., Nordmann, R. and Rouach, H. (1997) Inactivation of cerebellar nitric oxide synthase by ethanol in vitro. Alcohol and Alcoholism 32, 683–691.[Abstract]

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.[CrossRef][ISI][Medline]

Kimura, K. A., Parr, A. M. and Brien, J. F. (1996) Effect of chronic maternal ethanol administration on nitric oxide synthase activity in the hippocampus of the mature fetal guinea pig. Alcoholism: Clinical and Experimental Research 20, 948–953.[ISI][Medline]

Kimura, K. A., Reynolds, J. N. and Brien, J. F. (1999) Ontogeny of nitric oxide synthase I and III protein expression and enzymatic activity in the guinea pig hippocampus. Brain Research: Developmental Brain Research 116, 211–216.[ISI][Medline]

Maier, S. E., Cramer, J. A., West, J. R. and Sohrabji, F. (1999a) Alcohol exposure during the first two trimesters equivalent alters granule cell number and neurotrophin expression in the developing rat olfactory bulb. Journal of Neurobiology 41, 414–423.[CrossRef][ISI][Medline]

Maier, S. E., Miller, J. A., Blackwell, J. M. and West, J. R. (1999b) Fetal alcohol exposure and temporal vulnerability: regional differences in cell loss as a function of the timing of binge-like alcohol exposure during brain development. Alcoholism: Clinical and Experimental Research 23, 726–734.[ISI][Medline]

Musial, A. and Eissa, N. T. (2001) Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway. Journal of Biological Chemistry 276, 24268–24273.[Abstract/Free Full Text]

Nishida, C. R. and Ortiz de Montellano, P. R. (1998) Electron transfer and catalytic activity of nitric oxide synthases. Chimeric constructs of the neuronal, inducible, and endothelial isoforms. Journal of Biological Chemistry 273, 5566–5571.[Abstract/Free Full Text]

Ozer, E., Sarioglu, S. and Gure, A. (2000) Effects of prenatal ethanol exposure on neuronal migration, neuronogenesis and brain myelination in the mice brain. Clinical Neuropathology 19, 21–25.[ISI][Medline]

Phillips, D. E., Cummings, J. D. and Wall, K. A. (2000) Prenatal alcohol exposure decreases the number of nitric oxide synthase positive neurons in rat superior colliculus and periaqueductal gray. Alcohol 22, 75–84.[CrossRef][ISI][Medline]

Phung, Y. T. and Black, S. M. (1999) The synergistic action of ethanol and nerve growth factor in the induction of neuronal nitric oxide synthase. Alcohol and Alcoholism 34, 506–510.[Abstract/Free Full Text]

Rairigh, R. L., Le Cras, T. D., Ivy, D. D., Kinsella, J. P., Richter, G., Horan, M. P., Fan, I. D. and Abman, S. H. (1998) Role of inducible nitric oxide synthase in regulation of pulmonary vascular tone in the late gestation ovine fetus. Journal of Clinical Investigation 101, 15–21.[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.[CrossRef][ISI][Medline]

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

Song, Y., Zweier, J. L. and Xia, Y. (2001) Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy. American Journal of Physiology: Cell Physiology 281, C1819–C1824.[ISI][Medline]

Steenaart, N. A. E., Clarke, D. W. and Brien, J. F. (1985) Gas–liquid chromatography analysis of ethanol and acetaldehyde in blood with minimal artifactual acetaldehyde formation. Journal of Pharmacological Methods 14, 199–212.[CrossRef][ISI][Medline]

Xia, J., Simonyi, A. and Sun, G. Y. (1999) Chronic ethanol and iron administration on iron content, neuronal nitric oxide synthase, and superoxide dismutase in rat cerebellum. Alcoholism: Clinical and Experimental Research 23, 702–707.[ISI][Medline]

Zima, T., Druga, R. and Stipek, S. (1998) The influence of chronic moderate ethanol administration on NADPH-diaphorase (nitric oxide synthase) activity in rat brain. Alcohol and Alcoholism 33, 341–346.[Abstract]

Zima, T., Fialova, L., Mestek, O., Janebova, M., Crkovska, J., Malbohan, I., Stipek, S., Mikulikova, L. and Popov, P. (2001) Oxidative stress, metabolism of ethanol and alcohol-related diseases. Journal of Biomedical Science 8, 59–70.[CrossRef][ISI][Medline]





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