Role Played by Hypothalamic Nuclear Factor-B in Alcohol-Mediated Activation of the Rat Hypothalamic-Pituitary-Adrenal Axis
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内分泌学杂志 2005年第4期
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier@salk.edu.
Abstract
The DNA binding protein nuclear factor-B (NF-B) is a transcription factor translocated from the cytosol to the nucleus in response to stressors. Here we determined whether the known ability of alcohol to activate the hypothalamic-pituitary axis was mediated by NF-B, tested the hypothesis that this phenomenon was accompanied by increased hypothalamic NF-B transcripts, and investigated some of the mechanisms involved in this response. We found that alcohol-induced increase in plasma ACTH was blunted by the intracerebroventricular (icv) injection of a cell-permeable peptide that inhibits NF-B translocation. Alcohol also increased hypothalamic inhibitory factor B (IB) mRNA levels, a factor that regulates NF-B protein activation and the activity of NF-B DNA binding and whose expression is thought to reflect NF-B activity. This response, which was not accompanied by detectable changes in brain levels of proinflammatory cytokines, was partially retained in adrenalectomized/corticosterone-replaced rats. The icv injection of corticotropin-releasing factor (CRF), a hypothalamic peptide that is released by alcohol and mediates its influence on ACTH secretion, also stimulated hypothalamic IB transcription. We therefore determined whether brain CRF played a role in the influence of alcohol on NF-B signaling pathways. Indeed, the icv injection of the CRF antagonist -helCRF9–41 decreased alcohol-induced hypothalamic IB transcripts. Because this antagonist did not alter corticosterone levels, our data suggest that the role played by CRF was not modulated by this steroid. Collectively, our results provide evidence for a functional interaction between alcohol and NF-B-dependent pathway in stimulating the rat hypothalamic-pituitary axis activity that involves independent roles of corticosterone and CRF.
Introduction
THE DNA BINDING protein nuclear factor-B (NF-B) is a transcription factor that is present in the cytoplasm of most resting cells. NF-B forms a heterodimer that includes one 50-kDa (p50 or NF-B1) and one 65-kDa (p65 or RelA) protein (1, 2, 3). In the cytosol, NF-B dimers form a complex with one of the isoforms of the inhibitory protein IB, which becomes phosphorylated and degraded upon extracellular stimulation (4). This causes the release of free NF-B, which is translocated to the nucleus and binds to specific B sequences of the DNA to initiate the transcription of various genes, including IB (5). Commensurate with the role of NF-B as a central regulator for the expression of various genes involved in inflammation and infection, its brain levels are increased by proinflammatory cytokines, as well as bacterial and viral products (see Refs. 6 and 7). In addition, a vast array of categorically different stressors (for example, see Ref. 8) also up-regulate brain NF-B transcripts (see Refs. 9, 10, 11, 12, 13). Because stressors, in general (14, 15), and alcohol, in particular (16), are powerful activators of the hypothalamic-pituitary (HP) axis, we became interested in testing the hypothesis that there were functional connections between the expression of NF-B and the HP axis response to alcohol. An earlier report has indicated that acute, but not chronic, alcohol activated NF-B in the nuclear fraction of homogenized rat brains (17). In the present work, we first sought to determine whether our acute alcohol paradigm (the ip injection of a moderate dose of the drug) would alter NF-B levels in the endocrine hypothalamus, where the peptides that regulate the HP axis are synthesized (18, 19, 20), and whether manipulation of NF-B production by the intracerebroventricular (icv) injection of an inhibitor peptide (21) would alter the ACTH response to an acute alcohol injection. In view of the ability of glucocorticoids to interfere with NF-B signaling events (22), some of these experiments were done in both adrenal-intact and adrenalectomized (ADX) rats with replacement corticosterone pellets to determine the role played by this adrenal steroid in the responses induced by alcohol. The rationale for the second part of our work was that because corticotropin-releasing factor (CRF) stimulates ACTH synthesis and release (23, 24), we determined whether this peptide would increase tissue NF-B levels; and having observed that it did so, we investigated the role of endogenous CRF in the NF-B response to alcohol. Collectively, our results provide novel information regarding the role of NF-B, and its possible interaction with CRF, in mediating the hypothalamic-pituitary-adrenal (HPA) axis response to alcohol.
Materials and Methods
Animals
Adult male Sprague Dawley rats (200–220 g) were kept under standard light (lights on, 0630–1830 h) and feeding (rat chow and water ad libitum) regimens. Surgeries included the stereotaxic placement, 8–10 d before the assay, of an icv cannula that allowed delivery of treatment to the right lateral brain ventricle (25); aseptic insertion of an ip or intragastric (ig) cannula, 7–10 d before the assay; and a right jugular iv catheter, 2–3 d before testing (26). The iv, ip, and ig surgeries were performed under isoflurane anesthesia, whereas icv cannulae were implanted under sc administration of ketamine (100 mg/kg)/acepromazine (4 mg/kg)·xylazine (10 mg/kg). Adrenalectomy was performed under isoflurane anesthesia via the lumbar approach, and the rats were implanted at that time with corticosterone pellets (35 mg/3 wk; Innovative Research of America, Sarasota, FL) that maintained steroid levels at 25.2 ± 2.9 ng/ml. All animals were singly housed to prevent chewing of the cannulae. All protocols were approved by the Salk Institute Institutional Animal Care and Use Committee.
Experimental protocols
On the day of the experiment, the animals were removed to a soundproof room, singly housed in opaque buckets, and left undisturbed for 3 h. All experiments were conducted in nonhandled, freely moving rats that were not exposed to nonspecific stressors. When appropriate, blood samples (0.3 ml) were taken through the iv cannula and immediately replaced with an equivalent volume of apyrogenic isotonic saline. Alcohol was administered through indwelling cannulae (see below) to otherwise undisturbed, unhandled rats. It is important to note that although alcohol activates the HPA axis (16) and can thus be classified as a stressor, the procedure of injecting this drug is not itself stressful because delivery is carried out through the indwelling cannula. Blood was drawn into tubes that contained EDTA (10 μl of a 60 mg/ml solution) and placed on ice. They were centrifuged at 4 C, and plasma was stored at –20 C until assayed. When tissues were collected at the end of an experiment, the animals were either rapidly decapitated and tissues were removed for Northern blot analysis and ribonuclease (RNase) protection assay or they were anesthetized for perfusion and tissue collection for in situ hybridization (see below).
Reagents
Alcohol was diluted with saline so that its final injected concentration was 18% (vol/vol). Alcohol was injected ip at a dose (3.0 g/kg) that is commonly used by us (16) and others (for example, see Refs. 27, 28, 29, 30). It induces a moderate degree of intoxication (31, 32) and significantly stimulates ACTH release (16) as well as paraventricular nucleus (PVN) CRF neuronal activity (23). Several experiments were repeated with injection of the drug through an indwelling permanent ig cannula to verify that results were not specific to one mode of administration. In all cases, results were comparable between the ip and ig routes. However, because not all experiments were performed in both models, here we only illustrate data obtained in ip-injected rats. For studies that investigated the role played by NF-B, a survey of the published literature indicated the availability of several well-defined cell-permeable peptides that inhibit NF-B translocation by competing with the NF-B complexes for the cellular machinery responsible for nuclear translocation of NF-B (for example, see Refs. 21 and 33, 34, 35). We chose the well-known SN50 NF-B antagonist (SN50 ANT) with the amino acid sequence AAVALLPAVLLALLAPVQRKQKLMP because it has already been tested and validated in rats (21), as opposed to only being used in isolated cells. This antagonist was originally shown to inhibit nuclear induction of the NF-B proteins (36), although at high doses, it may also compete for proteins generally involved in nuclear import (37). The route and regimen of administration described by these authors (21) were used as a basis for our preliminary studies and were later modified as needed (see Results). The active peptide antagonist (molecular weight, 2781.5) and its corresponding inactive control scrambled peptide were purchased from Calbiochem (La Jolla, CA) and dissolved in sterile apyrogenic water. Rat/human (r/h)CRF and the CRF antagonist -helCRF9–41 were synthesized by solid-phase methodology (38) and kindly provided by Dr. J. Rivier (Salk Institute). The -helCRF9–41 blocks CRF-dependent biological responses in the brain but does not interfere with pituitary receptors involved in ACTH release (39, 40), which is an essential requirement for our studies. CRF was dissolved in 0.04 M PBS containing 0.01% ascorbic acid and 0.1% purified BSA. The -helCRF9–41 was first dissolved in apyrogenic water (neutralized to pH 7.0). NaOH (0.1 N) was then used to adjust the final pH to 7.0–7.2. Both CRF and its antagonist were injected at doses previously shown to be adequate for the desired effects (16, 41).
In vivo protocols
In preliminary studies, we compared the effect of the vehicle, the NF-B antagonist (SN50 ANT), or the scrambled peptide in different protocols which were based on studies previously published by other investigators (21). In the first protocol, we injected 50 μg of the SN50 ANT icv (1 μl over 10 sec, 5 μl total) once between 1500 and 1600 h in the afternoon before the assay. In the second protocol, the treatments (50 μg each time, 1 μl over 10 sec, 5 μl total) were infused icv between 1500 and 1600 h in the afternoon before the experiment, as well as between 0600 and 0700 h in the morning of the experiment. In the third protocol, the treatments (50 μg each time, 1 μl over 10 sec, 5 μl total) were administered icv between 1500 and 1600 h, 1 and 2 d before the experiment. Although all protocols produced comparable blunting of the ACTH response to alcohol (see partial results in Fig. 1), we decided to conduct most of the experiments described here with the third protocol (indicated in the legend as 2 x 50 μg) because it was technically easier to use. The only exception is the data illustrated in Fig. 1, in which we also used the first protocol.
FIG. 1. Effect of an NF-B antagonist (SN50 ANT, one or two 50-μg injections in a 5-μl vol, see Materials and Methods) on the ACTH response to alcohol (EtOH, 3.0 g/kg, ip, injected at t = 0). Because they were comparable, data from rats injected with the vehicle or SN50 ANT only were combined. Plasma ACTH levels were measured 0, 15, 30, 45, and 60 min after ip vehicle or alcohol injection. Each point represents the mean ± SEM of six to eight animals. **, P < 0.01 vs. alcohol alone.
ACTH assay
Plasma ACTH levels were determined by a commercially available two-site immunoradiometric assay (Allegro kit; Nichols Institute, San Juan Capistrano, CA), which has been validated for the measurement of rat ACTH (42). Assay sensitivity was 5 pg/ml, and the intra- and interassay coefficients of variation were 3.2 and 6.8%, respectively.
Measurement of cytokines
Brain transcripts.
Hypothalamic mRNA levels of TNF-, IL-1?, and IL-6 were measured by RNase protection assay. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA). Rat TNF-, IL-6, and IL-1? mRNAs were measured simultaneously by RNase protection assay, using rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32 as an internal loading control. A custom probe was synthesized from custom template (BD Biosciences PharMingen, San Diego, CA) with -32P-UTP using T7 RNA polymerase. The riboprobe was synthesized and the RNase protection assays were carried out in our laboratory. The fragment sizes protected by the custom probe for TNF-, IL-6, IL-1?, GAPDH, and L32 are 361, 202, 160, 112, and 97, respectively. RNA samples (15 μg of hypothalamus tissues) were hybridized in 30 μl hybridization buffer containing 24 μl deionized Formamide (Shelton Scientific, Shelton, CT), 3 μl 10xHyb buffer [4 M NaCl, 400 mM piperazine diethanesulfonic acid (pH 6.4), 10 mM EDTA, in diethylpyrocarbonate (DepC) water], 5 x 105 cpm custom probe, then brought to a final volume in DepC water. After heating at 85 C for 5 min, the samples were hybridized at 43 C for 16–18 h. After hybridization, 350 μl RNase solution [5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl (pH 7.5), 1.5 μg RNase A, 5 μl RNase T1 (Ambion, Austin, TX)] was added to the hybridization solution and incubated for 1 h at RT. To stop RNases, 10 μl 20% sodium dodecyl sulfate (SDS) and 2 μl proteinase K (20 mg/ml) were added, then samples were vortexed and incubated for 15 min at 37 C. Samples were cleaned and lyophilized. Six microliters of loading dye were added, and samples were resolved on 6% polyacrylamide urea gels. Image analysis was performed using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the IMAGEQUANT 5.2 software package.
Plasma levels.
TNF- and IL-6 plasma levels were measured by ELISA (TNF-, R & D Systems, Minneapolis, MN; IL-6, Endogen, Rockford, IL) that were validated in our laboratory by comparing standard curves with serial plasma dilutions (43).
EZ-Detect transcription factor assays
We measured activated NF-B p65 transcription factor in the nuclear fractions of control and alcohol-treated rats using the EZ-Detect chemiluninescent transcription factor assays (Pierce, 89859, Rockford, IL). This method has been published in detail by Rosenau et al. (44). The nuclear fraction of the hypothalamus was extracted by NE-PER Nuclear extraction reagents (Pierce, 78833). Fifteen micrograms of nuclear extract were then incubated in wells coated with consensus NF-B sequence for 1 h. Only the active form of NF-B transcription factor binds to the consensus sequence in the wells. After the first incubation, the wells were rinsed and incubated with specific primary antibody (NF-B p65) for 1 h. After incubation with the primary antibody, the wells were rinsed and incubated with a secondary HRP-conjugated antibody for another hour, then rinsed. A chemiluminescent substrate was added to the wells, and the resulting signal was detected using a luminometer (PerkinElmer, HTS 7000 Plus Bio Assay Reader). TNF--activated HeLa cell nuclear extract was used as a positive control.
Hypothalamic inhibitory factor B (IB) transcripts
The IB controls the activity of NF-B DNA binding. In the cytoplasm, IB forms an inactive trimer with the NF-B subunits p65 and p50, and activation of the complex results in IB degradation, nuclear NF-B translocation, and IB resynthesis. The expression of IB therefore reflects the activity of the NF-B and represents a well-accepted and powerful tool to investigate the regulation of this transcription factor (see Refs. 1 , 2 , and 45). Levels of IB mRNA were measured by Northern blot analysis. Brains from each group were obtained by decapitation under minimal stress, and hypothalami were dissected by the method of Glowinski and Iverson (69). The hypothalamus tissues were stored at –70 C until RNA extraction. Total RNA was isolated by Qiagen RNeasy Mini Kit. Ten to 15 μg RNA were denatured in 2.2 M formaldehyde and subjected to electrophoresis on a 1.2% agarose formaldehyde gel in 20 mM morpholinopropanesulfonic acid (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA buffer. After electrophoresis, RNA was transferred to a zeta-probe blotting membrane and UV cross-linked. The RNA blot was incubated at 65 C for 4 h in prehybridization buffer containing 5x saline sodium phosphate EDTA buffer (SSPE) (1x SSPE: 0.01 M phosphate buffer, pH 7.0/0.15 M NaCl), 10x Denhart’s solution (0.02%, wt/vol, each of BSA, Ficoll 400, and polyvinylpyrrolide), 50% formamide, 0.5% SDS, and 500 mg/ml denatured salmon sperm DNA. Hybridization was carried out with a cRNA probe and labeled with [-32P] UTP at 65 C in the same buffer overnight. Filters were washed successively in 1x SSPE/0.1% SDS, 0.1x SSPE/0.1% SDS, and 0.1x SSPE at 65 C. After posthybridization washes, membranes were air-dried and exposed. Quantitative analysis was performed using PhosphorImager system (Molecular Dynamics) and the ImageQuant 4.0 software packages. To obtain accurate measurements, hybridization with GAPDH probe was performed as an internal standard. The ratios of specific signal to GAPDH were calculated to estimate the levels of these signals.
Nerve growth factor (NGFI)-B mRNA transcripts
The use of this particular immediate early gene is based on the fact that the rat CRF gene contains one or more NGFI-B response element-like sequences (46, 47), thereby making NGFI-B an excellent marker to detect transcriptional activation in the PVN (48). Rats were deeply anesthetized with chloral hydrate, a drug that does not increase immediate early genes/peptides mRNA levels (our unpublished data). They were then perfused transcardially with saline followed by 4% paraformaldehyde/0.1 M borate buffer (pH 9.5). The brains were removed and postfixed in 4% paraformaldehyde for 4–5 d, then placed overnight in 10% sucrose/4% paraformaldehyde/0.1 M borate buffer. They were cut into 30-μm coronal slices obtained at 120-μm intervals throughout the hypothalamus and were stored at –20 C in a cryoprotectant solution (50% 0.1-M PBS, 30% ethylene glycol, and 20% glycerol) until histochemical analysis. Brains from control and experimental animals belonging to the same experiment were always analyzed in the same hybridization experiment. Hybridization histochemical localization of each transcript was carried out using 35S-labeled cRNA probes prepared as previously described (49, 50). A sense probe was used as a control for nonspecific signal in some adjacent sections for in situ hybridization.
Probes
Protocols for riboprobe synthesis, hybridization, and autoradiography were adopted from Simmons et al. (70) and are routinely performed in our laboratory. All solutions were treated with DepC and autoclaved to prevent degradation of RNA. The pBluescript SK-1 vector (Stratagene, La Jolla, CA) containing a 1114-bp mouse IB cDNA (provided by Dr. A. Israel, Institut Pasteur, Paris, France) was linearized with BamHI. The mouse IB probe (51) is 92% homologous to the rat probe (52). Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in 6 mM MgCl2, 36 mM Tris (pH 7.5), 2 mM spermidine, 8 mM dithiothreitol, 25 mM ATP/GTP/CTP, [-32P]-UTP, 1 U RNasin (Promega, Madison, WI), and 10 U T7 for 60 min at 37 C. Unincorporated nucleotides were removed using Quick-Spin columns (Roche Molecular Biochemicals, Indianapolis, IN). PVN neuronal activity was assessed by measuring changes in mRNA levels of the immediate early gene NGFI-B (for example, see Refs. 25 and 48).
Quantitative analysis of in situ hybridization results
Semiquantitative densitometric analysis of hybridization signals for RNAs of interest was carried out in nuclear emulsion-dipped slides. Brain paste standards containing serial dilutions of 35S-UTP, used for quantification of mRNA signal, were prepared concurrently to ensure that OD was found within the linear range of the standard curve (53). In addition, analyses with emulsion-coated slides were carried out with 2–3 different exposure times to confirm that signals were not saturated. Densitometric analyses of autoradiographic signals were done over the confines of cells within the PVN using a Leitz optical system coupled to a Macintosh II computer and Image software [version 1.61; W. Rasband, National Institutes of Health (NIH)] (49). OD of hybridization signal was measured under dark-field illumination at a magnification of x100, as defined by redirected sampling from the corresponding Nissl-stained sections of the PVN under bright-field images (49). Data were expressed in OD values of 1–256. All OD values were corrected for background, which corresponded to the areas immediately adjacent to the PVN. Signals were measured in both sides of the PVN, and mean values for all animals (six to eight/group) were determined in three to four sections for each rat.
Statistical analysis
Statistical analyses were performed by one- or two-way ANOVA for the factors of group and time. The least-square-means post hoc tests were used to make comparisons between groups at a particular time point and between time points within a particular group. Differences were considered statistically significant from P < 0.05.
Results
Effect of pretreatment with an NF-B inhibitor on the ACTH response to alcohol
Preliminary experiments were conducted on the basis of in vivo doses of an NF-B inhibitor (SN50 ANT) published for another paradigm (21) (see Materials and Methods). This reagent was injected icv in a 5-μl vol as previously described (33). Although the fact that we injected SN50 ANT into the lateral brain ventricle does not allow us to identify its precise site of action, studies of the distribution of icv-injected compounds indicate that they readily access the PVN (54), which is the primary site of the CRF neurons that regulate ACTH release (15, 20, 55). First, we showed that this antagonist produced a decrease in alcohol-induced hypothalamic IB levels (vehicle/EtOH, 0.25 ± 0.03 arbitrary units, vs. NF-B antagonist/EtOH, 0.18 ± 0.04 arbitrary units, P = 0.086). The relatively large SEMs did not allow this difference to reach statistical significance, but these results nevertheless provide support for the concept that SN50 ANT interfered with NF-B protein activation. Although 25 μg already produced a significant (P < 0.01) decrease in the ACTH response to alcohol, the endocrine response to 50 μg was more reliable and reproducible (Fig. 1). Though we do not know whether the 50-μg dose of SN50 ANT provided maximum inhibition of alcohol-induced ACTH release, we decided not to test the effect of higher doses because they produced a temporary (<30 min) increase in locomotor activity and signs of agitation in some rats. In view of this behavioral response, we also tested the effect of a scrambled peptide that contained the same amino acids but a different sequence. This control peptide did not cause any behavioral changes, nor did it alter the ACTH response to alcohol (not shown). This suggests that the ability of SN50 ANT to blunt the aforementioned endocrine response was specifically due to inhibition of NF-B formation.
Effect of pretreatment with an NF-B inhibitor on the PVN neuronal response to alcohol
As we previously reported (32), alcohol significantly (P < 0.01) up-regulated PVN neuronal activity measured by the immediate early gene NGFI-B mRNA levels (Figs. 2 and 3). This response was significantly (P < 0.01) blunted by pretreatment with an NF-B inhibitor (SN50 ANT).
FIG. 2. Effect of an NF-B antagonist (two 50-μg injections, see Materials and Methods) on the PVN neuronal response to alcohol (EtOH, 3.0 g/kg, ip). These dark-field micrographs illustrate transcripts of the immediate early gene NGFI-B in the PVN of rats, 1 h after being injected with the vehicle (A), alcohol (B), the NF-B antagonist (C), or alcohol and the antagonist (D). III, Third ventricle.
FIG. 3. Statistical analysis of the data presented in Fig. 2. The icv injection of a cell-permeable peptide that inhibits NF-B translocation significantly decreases the PVN neuronal response to alcohol. Each point represents the mean ± SEM of six to eight rats. **, P < 0.01 vs. alcohol alone.
Effect of alcohol on hypothalamic IB mRNA levels
Adrenal-intact rats.
The observation that the ACTH response to alcohol was decreased by an NF-B inhibitor suggested that alcohol should increase this transcription factor. When we investigated endotoxin-induced changes in IB transcripts in the brain, we observed an increase in IB mRNA levels in many glial cells and in cells associated with the brain vasculature in the hypothalamus (data not shown), which corresponds to data reported by others (see Refs. 7 , 45 , 56 , and 57). However, by comparison, the increases in alcohol-induced hypothalamic IB mRNA levels, measured by in situ hybridization, were quite low. These transcripts were therefore measured by Northern blot analysis in whole hypothalami, an approach that allows a much better quantification of the data and comparison between various treatment groups. Preliminary studies were also conducted in adrenal-intact rats to delineate the time-course of hypothalamic NF-B responses to different doses of alcohol. Inconsistent increases were observed at 30 min, but reliable changes occurred at the 1- and 2-h time points after ip injection of 3.0 g/kg alcohol. One of these latter time points was used in subsequent experiments. This is illustrated in Fig. 4, which shows the ability of alcohol to significantly (P < 0.01) up-regulate hypothalamic IB transcripts.
FIG. 4. Effect of the vehicle or alcohol (3.0 g/kg, ip) on hypothalamic IB mRNA levels of adrenal-intact rats, measured 1 h later. A, Northern blot showing hybridization results for IB mRNA and GAPDH mRNA used for normalization. Each lane contains RNA from one individual hypothalamus. B, Quantitative data obtained by densitometry from the Northern blots shown in A. Each bar represents the mean ± SEM of five to six rats. **, P < 0.01.
ADX/corticosterone clamped rats.
Because of the possibility that circulating glucocorticoids mediated the influence of alcohol, we carried out an additional experiment in which hypothalami were collected 1 h after alcohol to adrenal-intact or ADX rats with corticosterone pellets (Fig. 5). When the adrenals were present, alcohol induced the expected increase in IB mRNA levels (vehicle/intact, 0.36 ± 0.03 arbitrary units; alcohol/intact, 0.65 ± 0.03 arbitrary units; P < 0.01). Basal IB transcripts were elevated in the hypothalami of ADX/corticosterone clamped rats, compared with adrenal-intact animals (vehicle/ADX, 0.50 ± 0.03 arbitrary units, P < 0.01 vs. vehicle/intact shown above). Alcohol significantly increased this response (alcohol/ADX, 0.62 ± 0.04 arbitrary units, P < 0.01), but the magnitude of this change was less than that measured in adrenal-intact animals (P < 0.05).
FIG. 5. Effect of the vehicle or alcohol (3.0 g/kg, ip) on hypothalamic IB mRNA levels of intact and adrenalectomized/corticosterone-replaced rats, measured 1 h later. A, Northern blot showing hybridization results for IB mRNA and GAPDH mRNA used for normalization. Each lane contains RNA from one individual hypothalamus. B, Quantitative data obtained by densitometry from the Northern blots shown in A. Each bar represents the mean ± SEM of five rats. a, P < 0.01 from corresponding vehicle; b, P < 0.01 from vehicle/intact.
Effect of alcohol on active NF-B p65 DNA binding in the hypothalamus
Glucocorticoids have been reported to be able to stimulate IB synthesis in the absence of NF-B translocation, i.e. through induction of the glucocorticoid response element in the IB promoter (7). To verify that nuclear NF-B DNA binding had taken place, we used the new Pierce EZ-Detect NF-B p65 transcription factor kit, which is a chemiluminescent ELISA-based assay, to measure active NF-B transcription factors (44). We found that in adrenal-intact rats, alcohol activated NF-B p65 transcription factor in the nuclear fractions of the hypothalamus (Fig. 6). This correlates well with Northern blot data showing increases in hypothalamic IB mRNA levels when treated with alcohol. Therefore, we can assume that alcohol-induced activation in IB mRNA levels led to an activation of nuclear NF-B DNA binding activity.
FIG. 6. Effect of the vehicle or alcohol (3.0 g/kg, ip) on hypothalamic NF-B p65 DNA binding activity expressed as relative light units (RLU). The hypothalami were obtained 30 and 60 min after ip treatment. Each bar represents the mean ± SEM of four rats. **, P < 0.01 vs. vehicle.
Role of CRF and corticosterone in mediating the NF-B response to alcohol CRF
Although the results illustrated above in ADX animals indicated a role of corticosterone in mediating the influence of alcohol on IB transcripts, we also wanted to probe the importance of CRF, a hypothalamic factor whose transcripts are increased by alcohol (23). First, we studied the effect of this peptide by injecting it icv (1 μg). As illustrated in Fig. 7, A and B, in adrenal-intact rats, CRF significantly up-regulated hypothalamic IB mRNA levels 1 h later. We then investigated the role played by endogenous CRF, by injecting the CRF antagonist -helCRF9–41 icv before alcohol. This approach has the advantage of blocking brain CRF receptors without significantly altering corticotroph activity (39). It therefore allows us to dissociate CRF-mediated responses in the hypothalamus from those taking place in the anterior pituitary, which causes ACTH and corticosterone release. Control rats received the appropriate vehicles. As noted in a number of different systems (for example, see Ref. 39), the CRF antagonist alone displayed some agonistic activity, but this effect did not reach statistical significance (P > 0.05). Alcohol produced the expected significant (P < 0.01) increase in hypothalamic IB transcripts, and this response was blocked by the CRF antagonist (P < 0.01) (Fig. 8).
FIG. 7. Effect of the icv injection of r/hCRF (1 μg) on hypothalamic IB mRNA, measured 1 h later. A, Northern blot showing hybridization results for IB mRNA and GAPDH mRNA used for normalization (veh, vehicle). Each lane contains RNA from one individual hypothalamus. B, Quantitative data obtained by densitometry from the Northern blots shown in A. Each bar represents the mean ± SEM of six rats. **, P < 0.01.
FIG. 8. Effect of the icv injection of the CRF antagonist -helCRF9–41 (5 μg) on hypothalamic IB mRNA levels of vehicle and alcohol-injected rats (3.0 g/kg, ip). The antagonist was administered 15 min before alcohol, and hypothalami were collected 1 h after alcohol. Each point represents the mean ± SEM of five rats. a, P < 0.01 vs. vehicle alone; b, P < 0.01 vs. alcohol alone.
Corticosterone.
Although these results might suggest a role of endogenous CRF in the IB response to alcohol, we further tested this hypothesis by determining whether a CRF antagonist would block alcohol-induced IB synthesis by attenuating the corticosterone response that is normally observed in response to this drug. Although, as indicated above, this would be highly unlikely in view of the inability of icv injected to significantly alter ACTH release, we nevertheless measured plasma ACTH and corticosterone in groups of rats injected with alcohol in the presence or absence of the CRF antagonist. As we had previously reported (58), blockade of hypothalamic CRF receptors with doses of an antagonist that blocks brain CRF-dependent behaviors (40) while not altering the pituitary response to this peptide (39, 40) did not decrease alcohol-induced ACTH and corticosterone release [cumulative hormone levels measured 15, 30, and 45 min after alcohol injection: 1) ACTH (pg/ml): vehicle, 1059 ± 172; -helCRF9–41, 1147 ± 157; P > 0.05; and 2) corticosterone (ng/ml): 1456 ± 96; -helCRF9–41, 1625 ± 98; P > 0.05]. Collectively, these results indicate that blockade of brain CRF receptors attenuated the NF-B response to acute alcohol injection in the absence of significant changes in ACTH/corticosterone levels.
Effect of alcohol on circulating and hypothalamic cytokine levels
Because NF-B activation is often associated with increased transcription of genes linked to immune responses, such as those transcribing proinflammatory cytokines (7), we considered the possibility that the influence of alcohol on NF-B might be, at least in part, due to a proinflammatory influence of the drug comparable with that of pathogens and tissue injuries. This hypothesis was tested by measuring circulating and hypothalamic levels of TNF- and IL-6. Plasma cytokine levels were not significantly different in vehicle- or alcohol-injected rats (TNF-: vehicle, 205 ± 42 pg/ml; alcohol, 169 ± 38 pg/ml; P > 0.05; and IL-6: vehicle, 2017 ± 385 pg/ml; alcohol, 1823 ± 395 pg/ml; P > 0.05); data represent the sum of levels measured 15, 30, 60, and 90 min after vehicle or alcohol injection. TNF- and IL-6 transcripts were also measured in hypothalamic extracts, but mRNA levels were not detectable under basal conditions or 1, 3, or 6 h after alcohol injection (not shown).
Discussion
The present work illustrates several aspects of the interactions between alcohol and NF-B, which collectively help us understand how this transcription factor may modulate the influence of this drug on the HP axis. First, we show that alcohol significantly up-regulated NF-B DNA binding activity in the hypothalamus of adrenal-intact rats. However, we were unable to observe a clear pattern of activation in brain areas specifically linked to the HP axis, such as the PVN of the hypothalamus. Indeed, in contrast to the reported ability of neuronal cell cultures to contain constitutively active forms of NF-B in the nucleus of these cells (59), the cells that expressed this transcription factor in the tissues that we examined by in situ hybridization all pertained to glia and the vasculature [on the other hand, Bhakar et al. (60) also found NF-B activity in neurons of the murine brain]. Although our findings agree with a recent report by Laflamme et al. (6), the absence of NF-B in PVN CRF neurons should not be taken to suggest a lack of influence of this signal on these perikarya. Indeed, there are several examples where factors not necessarily present close to cell bodies nevertheless represent important modulating secretagogues of these perikarya [the most recent example pertains to nitric oxide (25)]. To test the hypothesis that despite its absence from CRF and VP neurons, NF-B was able to mediate the ability of alcohol to stimulate the HPA axis, we injected a peptide that inhibits its translocation. We show here that this antagonist blunted the ACTH and PVN neuronal responses to this drug. Although these results suggest a physiological role of NF-B in these responses, several caveats need to be taken into account. First, the temporal relationship between alcohol-induced activation of the HPA axis and NF-B-induced activation of CRF neurons has not been established. The apparent absence of NF-B expression within CRF cell bodies, the time required for the effects of NF-B on the target gene transcription and protein processing, and the rapid (within 30 min) influence of alcohol on PVN neurons argue against an influence of acute changes in IB/NF-B during the HP axis response to one alcohol injection. Furthermore, because we administered an NF-B inhibitor (SN50 ANT) over at least an 18-h time frame, our experiments do not pertain to acute activation of NF-B. On the other hand, SN5O ANT clearly interfered with the PVN neuronal and ACTH response to alcohol. Thus, whereas it is unlikely that NF-B facilitates the acute response of CRF neurons and/or their afferents to alcohol, our data nevertheless provide strong evidence for a tonic influence of this transcription factor. Finally, although we cannot exclude the possibility that this antagonist might also act on other signaling cascades, such as the transduction events that lead to c-fos activation, or interact with other proteins that share a similar nuclear localization domain, the demonstrated ability of this reagent to inhibit NF-B translocation by competing with the NF-B complexes for the cellular machinery responsible for nuclear translocation of NF-B (33) suggests that a significant part of its effect was due to a specific influence on NF-B signaling.
How is the role exerted? In view of the disseminated nature of the NF-B response throughout the brain, it is unlikely that this factor acts directly on PVN neurons. Rather, NF-B probably initiates a cascade of events that triggers the release of intermediate modulators, which in turn stimulate PVN neuronal activity. At present, we have no basis for speculating on the nature of these neurotransmitters or their sites of origin, though injecting specific antagonists or lesioning predetermined brain areas will undoubtedly provide information in this regard. We therefore focused on another aspect of the functional relationship between alcohol, NF-B, and the HPA axis, namely the possibility that alcohol might up-regulate this transcription factor through corticosterone or through CRF, both of which are stimulated by alcohol (16, 23, 24, 61). Glucocorticoids represent potent endogenous inhibitors of NF-B signaling, via the transcriptional activation of IB, to form the complex made of the two subunits p50/RelA, thereby preventing nuclear translocation of the dimer (1). Glucocorticoids also inhibit NF-B directly by protein-protein interactions (62). Thus, one could argue that because alcohol is known to elevate circulating corticosterone levels (61), it might stimulate IB synthesis without altering NF-B nuclear translocation, for example through GC-induced glucocorticoid response element in the IB promoter. We report here that whereas removal of the adrenals induced an increase in basal IB transcripts, it remained compatible with a stimulatory (though diminished) effect of alcohol. These results suggest that the hypothesis mentioned above is not valid. With regard to the role of CRF, we first showed that the icv injection of this peptide (a model chosen to correspond to increased CRF levels induced by alcohol) increased hypothalamic IB levels, thereby mimicking the influence of alcohol. Interestingly, CRF was recently shown to stimulate NF-B in another system, epidermal keratinocytes (63). We also probed the functional role of CRF by determining whether blocking CRF receptors would prevent the effect of alcohol. We report here that the icv injection of a potent CRF antagonist abolished the hypothalamic response to the drug, which supports the concept that alcohol stimulates NF-B synthesis through a CRF-dependent mechanism. Because this antagonist did not alter the corticosterone response to alcohol, these results point to a separate role of brain CRF and circulating glucocorticoids in mediating the influence of alcohol on NF-B signaling pathways (in this regard, it is important to note that we purposefully chose a CRF antagonist that only exhibits very weak potency on the corticotrophs so that we could dissociate pituitary and brain sites of action of CRF in our model).
Another point that warrants discussion pertains to the fact that NF-B activation is usually associated with increased transcription of genes linked to the innate immune response (7, 11). Our finding that alcohol up-regulates NF-B DNA binding activity might therefore indicate a proinflammatory role of this drug, either by itself and/or through induction of tissue injury. Acute systemic alcohol injection has been reported to increase clearance of proinflammatory cytokines, which results in elevated IL-6 content in the liver, kidney, and blood (64). However, though chronic alcohol consumption elevates circulating cytokine levels through hepatic injury (65), most investigators concur that this drug acutely inhibits the release of cytokines induced by endotoxemia (for example, see Refs. 66, 67, 68). As we show here, there were no significant changes in circulating or hypothalamic levels of TNF-, IL-1?, or IL-6 after ip alcohol injection. This rules out the possibility that alcohol might have acted as a proinflammatory agent in stimulating NF-B, and indeed the results discussed above point to the important, but distinct, modulating role of circulating corticosteroids and brain CRF in mediating the influence of the drug.
In conclusion, we have provided evidence that in rats, acute alcohol injection up-regulates hypothalamic IB levels (and hence, it is surmised, NF-B) and that the ability of alcohol to release ACTH is mediated, at least in part, by activation of this latter transcription factor. Because the icv injection of an NF-B peptide inhibitor is likely to reach not only the hypothalamus, but other brain areas as well (54), this functional interaction could, in theory, take place within the PVN and/or at the level of its afferent. However, our inability to detect IB signals in the PVN suggests that the latter hypothesis is more likely. We also show that whereas adrenal glucocorticoids play some role in mediating the influence of alcohol on NF-B signaling pathways, the influence of brain CRF appears more important and that, furthermore, this peptide acts independently from these steroids. Indeed, our observation that CRF increased hypothalamic IB transcripts, and that blockade of CRF receptors abolished the ability of alcohol to up-regulate these transcripts without altering circulating corticosterone levels, provides novel information regarding the role of NF-B in modulating the HPA axis response to alcohol.
Acknowledgments
The authors gratefully acknowledge the excellent technical assistance of Keith Hansen, Michael Rothwell, Anh-Khoi Nguyen, James Mathews, Elaine Law, Melissa Herman, and Yaira Haas and are indebted to Dr. Jean Rivier (Salk Institute, La Jolla, CA) for his generous gifts of r/hCRF and -helCRF9–41.
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Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier@salk.edu.
Abstract
The DNA binding protein nuclear factor-B (NF-B) is a transcription factor translocated from the cytosol to the nucleus in response to stressors. Here we determined whether the known ability of alcohol to activate the hypothalamic-pituitary axis was mediated by NF-B, tested the hypothesis that this phenomenon was accompanied by increased hypothalamic NF-B transcripts, and investigated some of the mechanisms involved in this response. We found that alcohol-induced increase in plasma ACTH was blunted by the intracerebroventricular (icv) injection of a cell-permeable peptide that inhibits NF-B translocation. Alcohol also increased hypothalamic inhibitory factor B (IB) mRNA levels, a factor that regulates NF-B protein activation and the activity of NF-B DNA binding and whose expression is thought to reflect NF-B activity. This response, which was not accompanied by detectable changes in brain levels of proinflammatory cytokines, was partially retained in adrenalectomized/corticosterone-replaced rats. The icv injection of corticotropin-releasing factor (CRF), a hypothalamic peptide that is released by alcohol and mediates its influence on ACTH secretion, also stimulated hypothalamic IB transcription. We therefore determined whether brain CRF played a role in the influence of alcohol on NF-B signaling pathways. Indeed, the icv injection of the CRF antagonist -helCRF9–41 decreased alcohol-induced hypothalamic IB transcripts. Because this antagonist did not alter corticosterone levels, our data suggest that the role played by CRF was not modulated by this steroid. Collectively, our results provide evidence for a functional interaction between alcohol and NF-B-dependent pathway in stimulating the rat hypothalamic-pituitary axis activity that involves independent roles of corticosterone and CRF.
Introduction
THE DNA BINDING protein nuclear factor-B (NF-B) is a transcription factor that is present in the cytoplasm of most resting cells. NF-B forms a heterodimer that includes one 50-kDa (p50 or NF-B1) and one 65-kDa (p65 or RelA) protein (1, 2, 3). In the cytosol, NF-B dimers form a complex with one of the isoforms of the inhibitory protein IB, which becomes phosphorylated and degraded upon extracellular stimulation (4). This causes the release of free NF-B, which is translocated to the nucleus and binds to specific B sequences of the DNA to initiate the transcription of various genes, including IB (5). Commensurate with the role of NF-B as a central regulator for the expression of various genes involved in inflammation and infection, its brain levels are increased by proinflammatory cytokines, as well as bacterial and viral products (see Refs. 6 and 7). In addition, a vast array of categorically different stressors (for example, see Ref. 8) also up-regulate brain NF-B transcripts (see Refs. 9, 10, 11, 12, 13). Because stressors, in general (14, 15), and alcohol, in particular (16), are powerful activators of the hypothalamic-pituitary (HP) axis, we became interested in testing the hypothesis that there were functional connections between the expression of NF-B and the HP axis response to alcohol. An earlier report has indicated that acute, but not chronic, alcohol activated NF-B in the nuclear fraction of homogenized rat brains (17). In the present work, we first sought to determine whether our acute alcohol paradigm (the ip injection of a moderate dose of the drug) would alter NF-B levels in the endocrine hypothalamus, where the peptides that regulate the HP axis are synthesized (18, 19, 20), and whether manipulation of NF-B production by the intracerebroventricular (icv) injection of an inhibitor peptide (21) would alter the ACTH response to an acute alcohol injection. In view of the ability of glucocorticoids to interfere with NF-B signaling events (22), some of these experiments were done in both adrenal-intact and adrenalectomized (ADX) rats with replacement corticosterone pellets to determine the role played by this adrenal steroid in the responses induced by alcohol. The rationale for the second part of our work was that because corticotropin-releasing factor (CRF) stimulates ACTH synthesis and release (23, 24), we determined whether this peptide would increase tissue NF-B levels; and having observed that it did so, we investigated the role of endogenous CRF in the NF-B response to alcohol. Collectively, our results provide novel information regarding the role of NF-B, and its possible interaction with CRF, in mediating the hypothalamic-pituitary-adrenal (HPA) axis response to alcohol.
Materials and Methods
Animals
Adult male Sprague Dawley rats (200–220 g) were kept under standard light (lights on, 0630–1830 h) and feeding (rat chow and water ad libitum) regimens. Surgeries included the stereotaxic placement, 8–10 d before the assay, of an icv cannula that allowed delivery of treatment to the right lateral brain ventricle (25); aseptic insertion of an ip or intragastric (ig) cannula, 7–10 d before the assay; and a right jugular iv catheter, 2–3 d before testing (26). The iv, ip, and ig surgeries were performed under isoflurane anesthesia, whereas icv cannulae were implanted under sc administration of ketamine (100 mg/kg)/acepromazine (4 mg/kg)·xylazine (10 mg/kg). Adrenalectomy was performed under isoflurane anesthesia via the lumbar approach, and the rats were implanted at that time with corticosterone pellets (35 mg/3 wk; Innovative Research of America, Sarasota, FL) that maintained steroid levels at 25.2 ± 2.9 ng/ml. All animals were singly housed to prevent chewing of the cannulae. All protocols were approved by the Salk Institute Institutional Animal Care and Use Committee.
Experimental protocols
On the day of the experiment, the animals were removed to a soundproof room, singly housed in opaque buckets, and left undisturbed for 3 h. All experiments were conducted in nonhandled, freely moving rats that were not exposed to nonspecific stressors. When appropriate, blood samples (0.3 ml) were taken through the iv cannula and immediately replaced with an equivalent volume of apyrogenic isotonic saline. Alcohol was administered through indwelling cannulae (see below) to otherwise undisturbed, unhandled rats. It is important to note that although alcohol activates the HPA axis (16) and can thus be classified as a stressor, the procedure of injecting this drug is not itself stressful because delivery is carried out through the indwelling cannula. Blood was drawn into tubes that contained EDTA (10 μl of a 60 mg/ml solution) and placed on ice. They were centrifuged at 4 C, and plasma was stored at –20 C until assayed. When tissues were collected at the end of an experiment, the animals were either rapidly decapitated and tissues were removed for Northern blot analysis and ribonuclease (RNase) protection assay or they were anesthetized for perfusion and tissue collection for in situ hybridization (see below).
Reagents
Alcohol was diluted with saline so that its final injected concentration was 18% (vol/vol). Alcohol was injected ip at a dose (3.0 g/kg) that is commonly used by us (16) and others (for example, see Refs. 27, 28, 29, 30). It induces a moderate degree of intoxication (31, 32) and significantly stimulates ACTH release (16) as well as paraventricular nucleus (PVN) CRF neuronal activity (23). Several experiments were repeated with injection of the drug through an indwelling permanent ig cannula to verify that results were not specific to one mode of administration. In all cases, results were comparable between the ip and ig routes. However, because not all experiments were performed in both models, here we only illustrate data obtained in ip-injected rats. For studies that investigated the role played by NF-B, a survey of the published literature indicated the availability of several well-defined cell-permeable peptides that inhibit NF-B translocation by competing with the NF-B complexes for the cellular machinery responsible for nuclear translocation of NF-B (for example, see Refs. 21 and 33, 34, 35). We chose the well-known SN50 NF-B antagonist (SN50 ANT) with the amino acid sequence AAVALLPAVLLALLAPVQRKQKLMP because it has already been tested and validated in rats (21), as opposed to only being used in isolated cells. This antagonist was originally shown to inhibit nuclear induction of the NF-B proteins (36), although at high doses, it may also compete for proteins generally involved in nuclear import (37). The route and regimen of administration described by these authors (21) were used as a basis for our preliminary studies and were later modified as needed (see Results). The active peptide antagonist (molecular weight, 2781.5) and its corresponding inactive control scrambled peptide were purchased from Calbiochem (La Jolla, CA) and dissolved in sterile apyrogenic water. Rat/human (r/h)CRF and the CRF antagonist -helCRF9–41 were synthesized by solid-phase methodology (38) and kindly provided by Dr. J. Rivier (Salk Institute). The -helCRF9–41 blocks CRF-dependent biological responses in the brain but does not interfere with pituitary receptors involved in ACTH release (39, 40), which is an essential requirement for our studies. CRF was dissolved in 0.04 M PBS containing 0.01% ascorbic acid and 0.1% purified BSA. The -helCRF9–41 was first dissolved in apyrogenic water (neutralized to pH 7.0). NaOH (0.1 N) was then used to adjust the final pH to 7.0–7.2. Both CRF and its antagonist were injected at doses previously shown to be adequate for the desired effects (16, 41).
In vivo protocols
In preliminary studies, we compared the effect of the vehicle, the NF-B antagonist (SN50 ANT), or the scrambled peptide in different protocols which were based on studies previously published by other investigators (21). In the first protocol, we injected 50 μg of the SN50 ANT icv (1 μl over 10 sec, 5 μl total) once between 1500 and 1600 h in the afternoon before the assay. In the second protocol, the treatments (50 μg each time, 1 μl over 10 sec, 5 μl total) were infused icv between 1500 and 1600 h in the afternoon before the experiment, as well as between 0600 and 0700 h in the morning of the experiment. In the third protocol, the treatments (50 μg each time, 1 μl over 10 sec, 5 μl total) were administered icv between 1500 and 1600 h, 1 and 2 d before the experiment. Although all protocols produced comparable blunting of the ACTH response to alcohol (see partial results in Fig. 1), we decided to conduct most of the experiments described here with the third protocol (indicated in the legend as 2 x 50 μg) because it was technically easier to use. The only exception is the data illustrated in Fig. 1, in which we also used the first protocol.
FIG. 1. Effect of an NF-B antagonist (SN50 ANT, one or two 50-μg injections in a 5-μl vol, see Materials and Methods) on the ACTH response to alcohol (EtOH, 3.0 g/kg, ip, injected at t = 0). Because they were comparable, data from rats injected with the vehicle or SN50 ANT only were combined. Plasma ACTH levels were measured 0, 15, 30, 45, and 60 min after ip vehicle or alcohol injection. Each point represents the mean ± SEM of six to eight animals. **, P < 0.01 vs. alcohol alone.
ACTH assay
Plasma ACTH levels were determined by a commercially available two-site immunoradiometric assay (Allegro kit; Nichols Institute, San Juan Capistrano, CA), which has been validated for the measurement of rat ACTH (42). Assay sensitivity was 5 pg/ml, and the intra- and interassay coefficients of variation were 3.2 and 6.8%, respectively.
Measurement of cytokines
Brain transcripts.
Hypothalamic mRNA levels of TNF-, IL-1?, and IL-6 were measured by RNase protection assay. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA). Rat TNF-, IL-6, and IL-1? mRNAs were measured simultaneously by RNase protection assay, using rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32 as an internal loading control. A custom probe was synthesized from custom template (BD Biosciences PharMingen, San Diego, CA) with -32P-UTP using T7 RNA polymerase. The riboprobe was synthesized and the RNase protection assays were carried out in our laboratory. The fragment sizes protected by the custom probe for TNF-, IL-6, IL-1?, GAPDH, and L32 are 361, 202, 160, 112, and 97, respectively. RNA samples (15 μg of hypothalamus tissues) were hybridized in 30 μl hybridization buffer containing 24 μl deionized Formamide (Shelton Scientific, Shelton, CT), 3 μl 10xHyb buffer [4 M NaCl, 400 mM piperazine diethanesulfonic acid (pH 6.4), 10 mM EDTA, in diethylpyrocarbonate (DepC) water], 5 x 105 cpm custom probe, then brought to a final volume in DepC water. After heating at 85 C for 5 min, the samples were hybridized at 43 C for 16–18 h. After hybridization, 350 μl RNase solution [5 M NaCl, 0.5 M EDTA, 1 M Tris-HCl (pH 7.5), 1.5 μg RNase A, 5 μl RNase T1 (Ambion, Austin, TX)] was added to the hybridization solution and incubated for 1 h at RT. To stop RNases, 10 μl 20% sodium dodecyl sulfate (SDS) and 2 μl proteinase K (20 mg/ml) were added, then samples were vortexed and incubated for 15 min at 37 C. Samples were cleaned and lyophilized. Six microliters of loading dye were added, and samples were resolved on 6% polyacrylamide urea gels. Image analysis was performed using the PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) and the IMAGEQUANT 5.2 software package.
Plasma levels.
TNF- and IL-6 plasma levels were measured by ELISA (TNF-, R & D Systems, Minneapolis, MN; IL-6, Endogen, Rockford, IL) that were validated in our laboratory by comparing standard curves with serial plasma dilutions (43).
EZ-Detect transcription factor assays
We measured activated NF-B p65 transcription factor in the nuclear fractions of control and alcohol-treated rats using the EZ-Detect chemiluninescent transcription factor assays (Pierce, 89859, Rockford, IL). This method has been published in detail by Rosenau et al. (44). The nuclear fraction of the hypothalamus was extracted by NE-PER Nuclear extraction reagents (Pierce, 78833). Fifteen micrograms of nuclear extract were then incubated in wells coated with consensus NF-B sequence for 1 h. Only the active form of NF-B transcription factor binds to the consensus sequence in the wells. After the first incubation, the wells were rinsed and incubated with specific primary antibody (NF-B p65) for 1 h. After incubation with the primary antibody, the wells were rinsed and incubated with a secondary HRP-conjugated antibody for another hour, then rinsed. A chemiluminescent substrate was added to the wells, and the resulting signal was detected using a luminometer (PerkinElmer, HTS 7000 Plus Bio Assay Reader). TNF--activated HeLa cell nuclear extract was used as a positive control.
Hypothalamic inhibitory factor B (IB) transcripts
The IB controls the activity of NF-B DNA binding. In the cytoplasm, IB forms an inactive trimer with the NF-B subunits p65 and p50, and activation of the complex results in IB degradation, nuclear NF-B translocation, and IB resynthesis. The expression of IB therefore reflects the activity of the NF-B and represents a well-accepted and powerful tool to investigate the regulation of this transcription factor (see Refs. 1 , 2 , and 45). Levels of IB mRNA were measured by Northern blot analysis. Brains from each group were obtained by decapitation under minimal stress, and hypothalami were dissected by the method of Glowinski and Iverson (69). The hypothalamus tissues were stored at –70 C until RNA extraction. Total RNA was isolated by Qiagen RNeasy Mini Kit. Ten to 15 μg RNA were denatured in 2.2 M formaldehyde and subjected to electrophoresis on a 1.2% agarose formaldehyde gel in 20 mM morpholinopropanesulfonic acid (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA buffer. After electrophoresis, RNA was transferred to a zeta-probe blotting membrane and UV cross-linked. The RNA blot was incubated at 65 C for 4 h in prehybridization buffer containing 5x saline sodium phosphate EDTA buffer (SSPE) (1x SSPE: 0.01 M phosphate buffer, pH 7.0/0.15 M NaCl), 10x Denhart’s solution (0.02%, wt/vol, each of BSA, Ficoll 400, and polyvinylpyrrolide), 50% formamide, 0.5% SDS, and 500 mg/ml denatured salmon sperm DNA. Hybridization was carried out with a cRNA probe and labeled with [-32P] UTP at 65 C in the same buffer overnight. Filters were washed successively in 1x SSPE/0.1% SDS, 0.1x SSPE/0.1% SDS, and 0.1x SSPE at 65 C. After posthybridization washes, membranes were air-dried and exposed. Quantitative analysis was performed using PhosphorImager system (Molecular Dynamics) and the ImageQuant 4.0 software packages. To obtain accurate measurements, hybridization with GAPDH probe was performed as an internal standard. The ratios of specific signal to GAPDH were calculated to estimate the levels of these signals.
Nerve growth factor (NGFI)-B mRNA transcripts
The use of this particular immediate early gene is based on the fact that the rat CRF gene contains one or more NGFI-B response element-like sequences (46, 47), thereby making NGFI-B an excellent marker to detect transcriptional activation in the PVN (48). Rats were deeply anesthetized with chloral hydrate, a drug that does not increase immediate early genes/peptides mRNA levels (our unpublished data). They were then perfused transcardially with saline followed by 4% paraformaldehyde/0.1 M borate buffer (pH 9.5). The brains were removed and postfixed in 4% paraformaldehyde for 4–5 d, then placed overnight in 10% sucrose/4% paraformaldehyde/0.1 M borate buffer. They were cut into 30-μm coronal slices obtained at 120-μm intervals throughout the hypothalamus and were stored at –20 C in a cryoprotectant solution (50% 0.1-M PBS, 30% ethylene glycol, and 20% glycerol) until histochemical analysis. Brains from control and experimental animals belonging to the same experiment were always analyzed in the same hybridization experiment. Hybridization histochemical localization of each transcript was carried out using 35S-labeled cRNA probes prepared as previously described (49, 50). A sense probe was used as a control for nonspecific signal in some adjacent sections for in situ hybridization.
Probes
Protocols for riboprobe synthesis, hybridization, and autoradiography were adopted from Simmons et al. (70) and are routinely performed in our laboratory. All solutions were treated with DepC and autoclaved to prevent degradation of RNA. The pBluescript SK-1 vector (Stratagene, La Jolla, CA) containing a 1114-bp mouse IB cDNA (provided by Dr. A. Israel, Institut Pasteur, Paris, France) was linearized with BamHI. The mouse IB probe (51) is 92% homologous to the rat probe (52). Radioactive cRNA copies were synthesized by incubation of 250 ng linearized plasmid in 6 mM MgCl2, 36 mM Tris (pH 7.5), 2 mM spermidine, 8 mM dithiothreitol, 25 mM ATP/GTP/CTP, [-32P]-UTP, 1 U RNasin (Promega, Madison, WI), and 10 U T7 for 60 min at 37 C. Unincorporated nucleotides were removed using Quick-Spin columns (Roche Molecular Biochemicals, Indianapolis, IN). PVN neuronal activity was assessed by measuring changes in mRNA levels of the immediate early gene NGFI-B (for example, see Refs. 25 and 48).
Quantitative analysis of in situ hybridization results
Semiquantitative densitometric analysis of hybridization signals for RNAs of interest was carried out in nuclear emulsion-dipped slides. Brain paste standards containing serial dilutions of 35S-UTP, used for quantification of mRNA signal, were prepared concurrently to ensure that OD was found within the linear range of the standard curve (53). In addition, analyses with emulsion-coated slides were carried out with 2–3 different exposure times to confirm that signals were not saturated. Densitometric analyses of autoradiographic signals were done over the confines of cells within the PVN using a Leitz optical system coupled to a Macintosh II computer and Image software [version 1.61; W. Rasband, National Institutes of Health (NIH)] (49). OD of hybridization signal was measured under dark-field illumination at a magnification of x100, as defined by redirected sampling from the corresponding Nissl-stained sections of the PVN under bright-field images (49). Data were expressed in OD values of 1–256. All OD values were corrected for background, which corresponded to the areas immediately adjacent to the PVN. Signals were measured in both sides of the PVN, and mean values for all animals (six to eight/group) were determined in three to four sections for each rat.
Statistical analysis
Statistical analyses were performed by one- or two-way ANOVA for the factors of group and time. The least-square-means post hoc tests were used to make comparisons between groups at a particular time point and between time points within a particular group. Differences were considered statistically significant from P < 0.05.
Results
Effect of pretreatment with an NF-B inhibitor on the ACTH response to alcohol
Preliminary experiments were conducted on the basis of in vivo doses of an NF-B inhibitor (SN50 ANT) published for another paradigm (21) (see Materials and Methods). This reagent was injected icv in a 5-μl vol as previously described (33). Although the fact that we injected SN50 ANT into the lateral brain ventricle does not allow us to identify its precise site of action, studies of the distribution of icv-injected compounds indicate that they readily access the PVN (54), which is the primary site of the CRF neurons that regulate ACTH release (15, 20, 55). First, we showed that this antagonist produced a decrease in alcohol-induced hypothalamic IB levels (vehicle/EtOH, 0.25 ± 0.03 arbitrary units, vs. NF-B antagonist/EtOH, 0.18 ± 0.04 arbitrary units, P = 0.086). The relatively large SEMs did not allow this difference to reach statistical significance, but these results nevertheless provide support for the concept that SN50 ANT interfered with NF-B protein activation. Although 25 μg already produced a significant (P < 0.01) decrease in the ACTH response to alcohol, the endocrine response to 50 μg was more reliable and reproducible (Fig. 1). Though we do not know whether the 50-μg dose of SN50 ANT provided maximum inhibition of alcohol-induced ACTH release, we decided not to test the effect of higher doses because they produced a temporary (<30 min) increase in locomotor activity and signs of agitation in some rats. In view of this behavioral response, we also tested the effect of a scrambled peptide that contained the same amino acids but a different sequence. This control peptide did not cause any behavioral changes, nor did it alter the ACTH response to alcohol (not shown). This suggests that the ability of SN50 ANT to blunt the aforementioned endocrine response was specifically due to inhibition of NF-B formation.
Effect of pretreatment with an NF-B inhibitor on the PVN neuronal response to alcohol
As we previously reported (32), alcohol significantly (P < 0.01) up-regulated PVN neuronal activity measured by the immediate early gene NGFI-B mRNA levels (Figs. 2 and 3). This response was significantly (P < 0.01) blunted by pretreatment with an NF-B inhibitor (SN50 ANT).
FIG. 2. Effect of an NF-B antagonist (two 50-μg injections, see Materials and Methods) on the PVN neuronal response to alcohol (EtOH, 3.0 g/kg, ip). These dark-field micrographs illustrate transcripts of the immediate early gene NGFI-B in the PVN of rats, 1 h after being injected with the vehicle (A), alcohol (B), the NF-B antagonist (C), or alcohol and the antagonist (D). III, Third ventricle.
FIG. 3. Statistical analysis of the data presented in Fig. 2. The icv injection of a cell-permeable peptide that inhibits NF-B translocation significantly decreases the PVN neuronal response to alcohol. Each point represents the mean ± SEM of six to eight rats. **, P < 0.01 vs. alcohol alone.
Effect of alcohol on hypothalamic IB mRNA levels
Adrenal-intact rats.
The observation that the ACTH response to alcohol was decreased by an NF-B inhibitor suggested that alcohol should increase this transcription factor. When we investigated endotoxin-induced changes in IB transcripts in the brain, we observed an increase in IB mRNA levels in many glial cells and in cells associated with the brain vasculature in the hypothalamus (data not shown), which corresponds to data reported by others (see Refs. 7 , 45 , 56 , and 57). However, by comparison, the increases in alcohol-induced hypothalamic IB mRNA levels, measured by in situ hybridization, were quite low. These transcripts were therefore measured by Northern blot analysis in whole hypothalami, an approach that allows a much better quantification of the data and comparison between various treatment groups. Preliminary studies were also conducted in adrenal-intact rats to delineate the time-course of hypothalamic NF-B responses to different doses of alcohol. Inconsistent increases were observed at 30 min, but reliable changes occurred at the 1- and 2-h time points after ip injection of 3.0 g/kg alcohol. One of these latter time points was used in subsequent experiments. This is illustrated in Fig. 4, which shows the ability of alcohol to significantly (P < 0.01) up-regulate hypothalamic IB transcripts.
FIG. 4. Effect of the vehicle or alcohol (3.0 g/kg, ip) on hypothalamic IB mRNA levels of adrenal-intact rats, measured 1 h later. A, Northern blot showing hybridization results for IB mRNA and GAPDH mRNA used for normalization. Each lane contains RNA from one individual hypothalamus. B, Quantitative data obtained by densitometry from the Northern blots shown in A. Each bar represents the mean ± SEM of five to six rats. **, P < 0.01.
ADX/corticosterone clamped rats.
Because of the possibility that circulating glucocorticoids mediated the influence of alcohol, we carried out an additional experiment in which hypothalami were collected 1 h after alcohol to adrenal-intact or ADX rats with corticosterone pellets (Fig. 5). When the adrenals were present, alcohol induced the expected increase in IB mRNA levels (vehicle/intact, 0.36 ± 0.03 arbitrary units; alcohol/intact, 0.65 ± 0.03 arbitrary units; P < 0.01). Basal IB transcripts were elevated in the hypothalami of ADX/corticosterone clamped rats, compared with adrenal-intact animals (vehicle/ADX, 0.50 ± 0.03 arbitrary units, P < 0.01 vs. vehicle/intact shown above). Alcohol significantly increased this response (alcohol/ADX, 0.62 ± 0.04 arbitrary units, P < 0.01), but the magnitude of this change was less than that measured in adrenal-intact animals (P < 0.05).
FIG. 5. Effect of the vehicle or alcohol (3.0 g/kg, ip) on hypothalamic IB mRNA levels of intact and adrenalectomized/corticosterone-replaced rats, measured 1 h later. A, Northern blot showing hybridization results for IB mRNA and GAPDH mRNA used for normalization. Each lane contains RNA from one individual hypothalamus. B, Quantitative data obtained by densitometry from the Northern blots shown in A. Each bar represents the mean ± SEM of five rats. a, P < 0.01 from corresponding vehicle; b, P < 0.01 from vehicle/intact.
Effect of alcohol on active NF-B p65 DNA binding in the hypothalamus
Glucocorticoids have been reported to be able to stimulate IB synthesis in the absence of NF-B translocation, i.e. through induction of the glucocorticoid response element in the IB promoter (7). To verify that nuclear NF-B DNA binding had taken place, we used the new Pierce EZ-Detect NF-B p65 transcription factor kit, which is a chemiluminescent ELISA-based assay, to measure active NF-B transcription factors (44). We found that in adrenal-intact rats, alcohol activated NF-B p65 transcription factor in the nuclear fractions of the hypothalamus (Fig. 6). This correlates well with Northern blot data showing increases in hypothalamic IB mRNA levels when treated with alcohol. Therefore, we can assume that alcohol-induced activation in IB mRNA levels led to an activation of nuclear NF-B DNA binding activity.
FIG. 6. Effect of the vehicle or alcohol (3.0 g/kg, ip) on hypothalamic NF-B p65 DNA binding activity expressed as relative light units (RLU). The hypothalami were obtained 30 and 60 min after ip treatment. Each bar represents the mean ± SEM of four rats. **, P < 0.01 vs. vehicle.
Role of CRF and corticosterone in mediating the NF-B response to alcohol CRF
Although the results illustrated above in ADX animals indicated a role of corticosterone in mediating the influence of alcohol on IB transcripts, we also wanted to probe the importance of CRF, a hypothalamic factor whose transcripts are increased by alcohol (23). First, we studied the effect of this peptide by injecting it icv (1 μg). As illustrated in Fig. 7, A and B, in adrenal-intact rats, CRF significantly up-regulated hypothalamic IB mRNA levels 1 h later. We then investigated the role played by endogenous CRF, by injecting the CRF antagonist -helCRF9–41 icv before alcohol. This approach has the advantage of blocking brain CRF receptors without significantly altering corticotroph activity (39). It therefore allows us to dissociate CRF-mediated responses in the hypothalamus from those taking place in the anterior pituitary, which causes ACTH and corticosterone release. Control rats received the appropriate vehicles. As noted in a number of different systems (for example, see Ref. 39), the CRF antagonist alone displayed some agonistic activity, but this effect did not reach statistical significance (P > 0.05). Alcohol produced the expected significant (P < 0.01) increase in hypothalamic IB transcripts, and this response was blocked by the CRF antagonist (P < 0.01) (Fig. 8).
FIG. 7. Effect of the icv injection of r/hCRF (1 μg) on hypothalamic IB mRNA, measured 1 h later. A, Northern blot showing hybridization results for IB mRNA and GAPDH mRNA used for normalization (veh, vehicle). Each lane contains RNA from one individual hypothalamus. B, Quantitative data obtained by densitometry from the Northern blots shown in A. Each bar represents the mean ± SEM of six rats. **, P < 0.01.
FIG. 8. Effect of the icv injection of the CRF antagonist -helCRF9–41 (5 μg) on hypothalamic IB mRNA levels of vehicle and alcohol-injected rats (3.0 g/kg, ip). The antagonist was administered 15 min before alcohol, and hypothalami were collected 1 h after alcohol. Each point represents the mean ± SEM of five rats. a, P < 0.01 vs. vehicle alone; b, P < 0.01 vs. alcohol alone.
Corticosterone.
Although these results might suggest a role of endogenous CRF in the IB response to alcohol, we further tested this hypothesis by determining whether a CRF antagonist would block alcohol-induced IB synthesis by attenuating the corticosterone response that is normally observed in response to this drug. Although, as indicated above, this would be highly unlikely in view of the inability of icv injected to significantly alter ACTH release, we nevertheless measured plasma ACTH and corticosterone in groups of rats injected with alcohol in the presence or absence of the CRF antagonist. As we had previously reported (58), blockade of hypothalamic CRF receptors with doses of an antagonist that blocks brain CRF-dependent behaviors (40) while not altering the pituitary response to this peptide (39, 40) did not decrease alcohol-induced ACTH and corticosterone release [cumulative hormone levels measured 15, 30, and 45 min after alcohol injection: 1) ACTH (pg/ml): vehicle, 1059 ± 172; -helCRF9–41, 1147 ± 157; P > 0.05; and 2) corticosterone (ng/ml): 1456 ± 96; -helCRF9–41, 1625 ± 98; P > 0.05]. Collectively, these results indicate that blockade of brain CRF receptors attenuated the NF-B response to acute alcohol injection in the absence of significant changes in ACTH/corticosterone levels.
Effect of alcohol on circulating and hypothalamic cytokine levels
Because NF-B activation is often associated with increased transcription of genes linked to immune responses, such as those transcribing proinflammatory cytokines (7), we considered the possibility that the influence of alcohol on NF-B might be, at least in part, due to a proinflammatory influence of the drug comparable with that of pathogens and tissue injuries. This hypothesis was tested by measuring circulating and hypothalamic levels of TNF- and IL-6. Plasma cytokine levels were not significantly different in vehicle- or alcohol-injected rats (TNF-: vehicle, 205 ± 42 pg/ml; alcohol, 169 ± 38 pg/ml; P > 0.05; and IL-6: vehicle, 2017 ± 385 pg/ml; alcohol, 1823 ± 395 pg/ml; P > 0.05); data represent the sum of levels measured 15, 30, 60, and 90 min after vehicle or alcohol injection. TNF- and IL-6 transcripts were also measured in hypothalamic extracts, but mRNA levels were not detectable under basal conditions or 1, 3, or 6 h after alcohol injection (not shown).
Discussion
The present work illustrates several aspects of the interactions between alcohol and NF-B, which collectively help us understand how this transcription factor may modulate the influence of this drug on the HP axis. First, we show that alcohol significantly up-regulated NF-B DNA binding activity in the hypothalamus of adrenal-intact rats. However, we were unable to observe a clear pattern of activation in brain areas specifically linked to the HP axis, such as the PVN of the hypothalamus. Indeed, in contrast to the reported ability of neuronal cell cultures to contain constitutively active forms of NF-B in the nucleus of these cells (59), the cells that expressed this transcription factor in the tissues that we examined by in situ hybridization all pertained to glia and the vasculature [on the other hand, Bhakar et al. (60) also found NF-B activity in neurons of the murine brain]. Although our findings agree with a recent report by Laflamme et al. (6), the absence of NF-B in PVN CRF neurons should not be taken to suggest a lack of influence of this signal on these perikarya. Indeed, there are several examples where factors not necessarily present close to cell bodies nevertheless represent important modulating secretagogues of these perikarya [the most recent example pertains to nitric oxide (25)]. To test the hypothesis that despite its absence from CRF and VP neurons, NF-B was able to mediate the ability of alcohol to stimulate the HPA axis, we injected a peptide that inhibits its translocation. We show here that this antagonist blunted the ACTH and PVN neuronal responses to this drug. Although these results suggest a physiological role of NF-B in these responses, several caveats need to be taken into account. First, the temporal relationship between alcohol-induced activation of the HPA axis and NF-B-induced activation of CRF neurons has not been established. The apparent absence of NF-B expression within CRF cell bodies, the time required for the effects of NF-B on the target gene transcription and protein processing, and the rapid (within 30 min) influence of alcohol on PVN neurons argue against an influence of acute changes in IB/NF-B during the HP axis response to one alcohol injection. Furthermore, because we administered an NF-B inhibitor (SN50 ANT) over at least an 18-h time frame, our experiments do not pertain to acute activation of NF-B. On the other hand, SN5O ANT clearly interfered with the PVN neuronal and ACTH response to alcohol. Thus, whereas it is unlikely that NF-B facilitates the acute response of CRF neurons and/or their afferents to alcohol, our data nevertheless provide strong evidence for a tonic influence of this transcription factor. Finally, although we cannot exclude the possibility that this antagonist might also act on other signaling cascades, such as the transduction events that lead to c-fos activation, or interact with other proteins that share a similar nuclear localization domain, the demonstrated ability of this reagent to inhibit NF-B translocation by competing with the NF-B complexes for the cellular machinery responsible for nuclear translocation of NF-B (33) suggests that a significant part of its effect was due to a specific influence on NF-B signaling.
How is the role exerted? In view of the disseminated nature of the NF-B response throughout the brain, it is unlikely that this factor acts directly on PVN neurons. Rather, NF-B probably initiates a cascade of events that triggers the release of intermediate modulators, which in turn stimulate PVN neuronal activity. At present, we have no basis for speculating on the nature of these neurotransmitters or their sites of origin, though injecting specific antagonists or lesioning predetermined brain areas will undoubtedly provide information in this regard. We therefore focused on another aspect of the functional relationship between alcohol, NF-B, and the HPA axis, namely the possibility that alcohol might up-regulate this transcription factor through corticosterone or through CRF, both of which are stimulated by alcohol (16, 23, 24, 61). Glucocorticoids represent potent endogenous inhibitors of NF-B signaling, via the transcriptional activation of IB, to form the complex made of the two subunits p50/RelA, thereby preventing nuclear translocation of the dimer (1). Glucocorticoids also inhibit NF-B directly by protein-protein interactions (62). Thus, one could argue that because alcohol is known to elevate circulating corticosterone levels (61), it might stimulate IB synthesis without altering NF-B nuclear translocation, for example through GC-induced glucocorticoid response element in the IB promoter. We report here that whereas removal of the adrenals induced an increase in basal IB transcripts, it remained compatible with a stimulatory (though diminished) effect of alcohol. These results suggest that the hypothesis mentioned above is not valid. With regard to the role of CRF, we first showed that the icv injection of this peptide (a model chosen to correspond to increased CRF levels induced by alcohol) increased hypothalamic IB levels, thereby mimicking the influence of alcohol. Interestingly, CRF was recently shown to stimulate NF-B in another system, epidermal keratinocytes (63). We also probed the functional role of CRF by determining whether blocking CRF receptors would prevent the effect of alcohol. We report here that the icv injection of a potent CRF antagonist abolished the hypothalamic response to the drug, which supports the concept that alcohol stimulates NF-B synthesis through a CRF-dependent mechanism. Because this antagonist did not alter the corticosterone response to alcohol, these results point to a separate role of brain CRF and circulating glucocorticoids in mediating the influence of alcohol on NF-B signaling pathways (in this regard, it is important to note that we purposefully chose a CRF antagonist that only exhibits very weak potency on the corticotrophs so that we could dissociate pituitary and brain sites of action of CRF in our model).
Another point that warrants discussion pertains to the fact that NF-B activation is usually associated with increased transcription of genes linked to the innate immune response (7, 11). Our finding that alcohol up-regulates NF-B DNA binding activity might therefore indicate a proinflammatory role of this drug, either by itself and/or through induction of tissue injury. Acute systemic alcohol injection has been reported to increase clearance of proinflammatory cytokines, which results in elevated IL-6 content in the liver, kidney, and blood (64). However, though chronic alcohol consumption elevates circulating cytokine levels through hepatic injury (65), most investigators concur that this drug acutely inhibits the release of cytokines induced by endotoxemia (for example, see Refs. 66, 67, 68). As we show here, there were no significant changes in circulating or hypothalamic levels of TNF-, IL-1?, or IL-6 after ip alcohol injection. This rules out the possibility that alcohol might have acted as a proinflammatory agent in stimulating NF-B, and indeed the results discussed above point to the important, but distinct, modulating role of circulating corticosteroids and brain CRF in mediating the influence of the drug.
In conclusion, we have provided evidence that in rats, acute alcohol injection up-regulates hypothalamic IB levels (and hence, it is surmised, NF-B) and that the ability of alcohol to release ACTH is mediated, at least in part, by activation of this latter transcription factor. Because the icv injection of an NF-B peptide inhibitor is likely to reach not only the hypothalamus, but other brain areas as well (54), this functional interaction could, in theory, take place within the PVN and/or at the level of its afferent. However, our inability to detect IB signals in the PVN suggests that the latter hypothesis is more likely. We also show that whereas adrenal glucocorticoids play some role in mediating the influence of alcohol on NF-B signaling pathways, the influence of brain CRF appears more important and that, furthermore, this peptide acts independently from these steroids. Indeed, our observation that CRF increased hypothalamic IB transcripts, and that blockade of CRF receptors abolished the ability of alcohol to up-regulate these transcripts without altering circulating corticosterone levels, provides novel information regarding the role of NF-B in modulating the HPA axis response to alcohol.
Acknowledgments
The authors gratefully acknowledge the excellent technical assistance of Keith Hansen, Michael Rothwell, Anh-Khoi Nguyen, James Mathews, Elaine Law, Melissa Herman, and Yaira Haas and are indebted to Dr. Jean Rivier (Salk Institute, La Jolla, CA) for his generous gifts of r/hCRF and -helCRF9–41.
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