Departments of 1 Pediatrics and 2 Molecular Pharmacology, Northwestern University, Chicago, IL, 2Department of Pediatrics, Division of NeonatalPerinatal 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)
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ABSTRACT |
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INTRODUCTION |
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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., 1996) without changing NOS hippocampal expression (Kimura et al., 1999
). 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.
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MATERIALS AND METHODS |
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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 gasliquid chromatographic procedure (Steenaart, 1985). 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 SDSpolyacrylamide 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., 1997). 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., 1992). 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
-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.
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RESULTS |
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DISCUSSION |
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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, 2001
). 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., 1998
). 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, 1998
).
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., 2001).
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.
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ACKNOWLEDGEMENTS |
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