Identification of gamma -aminobutyric acid receptor subunit types in human and rat liver

Ronit Erlitzki1, Yuewen Gong1, Manna Zhang1, and Gerald Minuk 1,2

Liver Diseases Unit, Departments of 1 Medicine and 2 Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3P5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GABA is a potent inhibitory neurotransmitter that binds to heterooligomeric receptors in the mammalian brain. In a previous study, we documented specific GABA binding to isolated rat hepatocytes that resulted in inhibition of hepatocyte proliferation. The purpose of the present study was to define the nature of hepatic GABAA receptors and to document their expression during rapid liver growth (after partial hepatectomy). PCRs with gene-specific primers derived from published sequences were performed with Marathon-ready human and rat liver cDNA. Two GABAA receptor subunit types (beta 3 and epsilon ) were expressed in human liver and one subunit type (beta 3) in rat liver. PCR amplification of the human GABAA receptorbeta 3-subunit produced a single product (molecular mass 53-59 kDa). In the case of the epsilon -subunit, two PCR products were identified. After partial hepatectomy, GABAA receptorbeta 3-subunit expression inversely correlated with regenerative activity (r = -0.527, P = 0.006). In conclusion, these results indicate that in the human liver GABAA receptors consist of the beta 3- and epsilon -subunit types, whereas in the rat liver only the beta 3-subunit type is expressed. The results also support the hypothesis that GABAergic activity serves to maintain hepatocytes in a quiescent state.

gamma-aminobutyric acid A receptor; hepatocytes; receptors; neurotransmitters


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GABA IS THE PRINCIPAL INHIBITORY neurotransmitter in the mammalian brain (18). GABA inhibits neuronal activity by activating specific GABAA receptor sites, resulting in increased chloride influx and, thereby, hyperpolarization of postsynaptic neuronal membranes (16). To date, at least 15 mammalian GABAA receptor genes have been identified (29). These can be subdivided into six major subunit types, alpha , beta , gamma , delta , rho , and epsilon  with ~30-40% amino acid homology between subunits. Within four of the subunit types, various isoforms sharing 70% sequence identity have been described: alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, and rho 1-rho 2 (31). Although the most ubiquitously expressed GABAA receptor subunits are alpha 1, alpha 2, alpha 3, beta 2, beta 3, and gamma 2 configured in a pentameric (alpha )2(beta )1(gamma )2 subunit stoichiometry, several GABAA receptor polypeptides have the capacity to form a GABA-gated homooligomeric chloride channel (31).

Recently, GABAergic activity has been described in various tissues beyond the central nervous system (reviewed in Ref. 9). Using standard receptor-ligand binding assays, we identified a sodium-independent GABAA receptor in isolated rat hepatocytes (23). Activation of this receptor with GABA or muscimol, a specific GABAA receptor agonist, resulted in prompt and marked hyperpolarization of the hepatocyte membrane, a result that could be prevented by preincubation of hepatocytes with bicuculline, a specific GABAA receptor antagonist. We also reported that increased GABAergic activity is associated with attenuation of hepatic regeneration after partial hepatectomy, whereas decreased activity is associated with enhanced hepatic regeneration after ethanol exposure or toxin-induced forms of acute and chronic liver disease (19, 24, 38, 39). Thus our data suggest that the liver possesses specific GABAA receptors and that these receptors are involved in regulating hepatic regenerative activity. However, the precise nature of the GABAA receptor subunit types expressed in either the human or rat liver and the expression of these subunit types during hepatic regeneration have yet to be reported.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Gene-specific PCR primers for each human and rat GABAA receptor subunit (Tables 1 and 2, respectively) were designed with the Oligo 5.1 program from cDNA sequences available at the NCBI GenBank nucleotide sequence database (http://www.ncbi.nlm.nih.gov/). Primers were then synthesized by Life Technologies (Burlington, ON, Canada). Marathon-ready human liver, human brain, or rat liver cDNA and Marathon PCR amplification kits were purchased from Clontech Laboratories (Palo Alto, CA).

                              
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Table 1.   Gene-specific PCR primers for human GABAA receptor subunits


                              
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Table 2.   Gene-specific PCR primers for rat GABAA receptor subunits

PCR analysis of GABAA receptor subunit mRNA expression. PCR reactions were performed with Marathon PCR amplification kits in a final volume of 50 µl with 5 µl of Marathon-ready cDNA, 1 µM each sense and antisense primers, 0.2 mM dNTPs, and 1.5 mM MgCl2. The reaction was run through 94°C for 45 s, the annealing temperature recommended by the Oligo-5.1 program for each pair of primers, and 72°C for 1 min for 30 times with a final extension step at 72°C for 5 min. Twenty-microliter aliquots of each PCR product were analyzed by electrophoresis through a 2.0% agarose gel in 1× Tris-acetate-EDTA buffer.

Southern blot analysis. After PCR products were electrophoresed through 2.0% agarose gels, which were then denatured and neutralized, DNA was transferred to GT-zeta-probe nylon membranes by capillary diffusion in 10× sodium chloride-sodium citrate (SSC) (Bio-Rad, Mississauga, ON, Canada). The membranes were cross-linked by exposure to ultraviolet light (150 s) by GS Genelinker (Bio-Rad) and hybridized for 18-24 h at 45°C in a hybridization buffer of 6× SSC, 50.0 mM NaPO4 (pH 6.5), 5× Denhardt's solution, 0.1 mg/ml salmon sperm DNA, and 1.25 pmol/ml of [32P]5'-labeled internal gene-specific oligonucleotides (Table 3). After hybridization, membranes were washed once with 6× SSC-0.1% SDS at 45°C for 10 min and then once in 2× SSC-0.1% SDS at 45°C for 10 min. Audioradiography was performed by exposure of the membrane to Kodak X-AR film for 0.5-5.0 h at -70°C using an intensifying screen.

                              
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Table 3.   Internal gene-specific probe for selected human and rat GABAA receptor subunits

[32P]5'-labeling of internal gene-specific oligonucleotides. Ten picomoles of gene-specific probes (Table 3) were labeled at their 5' terminus by ten units of T4 polynucleotide kinase (Life Technologies) and 1.2 µCi/µl [gamma -32P]dATP (3,000 Ci/mmol) in a final volume of 25.0 µl for 30 min at 22°C. Unincorporated nucleotides were removed by gel filtration through a G-50 nick column (Pharmacia, Baie d'Urfe, QC, Canada).

Western blot analysis. Livers and brains were removed from Sprague-Dawley male rats (200-250 g), washed with Tris-EDTA-sucrose (TES) buffer (0.05 M Tris · HCl pH 8.0, 0.001 M EDTA, 0.3 M sucrose) and immediately homogenized or frozen at -70°C. Homogenizations were performed at 4°C in 5.0 volumes of TES buffer containing proteinase inhibitors [1.0 mM phenylmethylsulfonyl fluoride (PMSF), 5.0 µg/ml benzamidine, 1.0 µg/ml pepstatin, 2.0 µg/ml aprotinin, and 2.0 µg/ml leupeptin] in a glass tube using a motor-driven Teflon pestle. Homogenates were centrifuged for 15 min at 1,000 g, and the pellets were discarded. Protein concentrations were determined by a Bradford reagent (Bio-Rad), and aliquots were stored at -70°C.

Aliquots of liver or brain crude extracts (50.0 µg protein) were electrophoresed through 10.0% polyacrylamide-SDS gels. Resolved proteins were transferred to Nitro-plus 2000 membranes (Micron Separations, Westborough, MA). Membranes were blocked with 5.0% skim milk in Tris-buffered saline (TBS; 0.02 M Tris-base, 0.5 M NaCl, pH 7.6) for 1 h at room temperature and incubated overnight at 4°C in the presence of 20.0 mg/ml of mouse anti-GABAA receptor beta 2/3 subunit monoclonal antibody (Cedarlane Laboratories, Mississauga, ON, Canada; Refs. 5, 10). Subsequently, membranes were washed in TBS with 0.05% Tween 20 and 0.5% skim milk, and bands were visualized by horseradish peroxidase-conjugated goat anti-mouse IgG and an enhanced chemiluminescence (ECL) kit (Amersham, Burlington, ON).

GABAA mRNA expression in regenerating livers. Adult male Sprague-Dawley rats (200-250 g) underwent 70% partial hepatectomy (PHx) as described by Higgins and Anderson (17) or sham surgery while under ether anesthesia. Rats were then killed by exsanguination in groups of six on days 1, 3, 5, and 7 after PHx. Resected livers were snap-frozen in liquid nitrogen and stored at -70°C. [3H]thymidine incorporation into hepatic DNA was determined at each time interval as described previously (24). GABAA mRNA expression was documented by Northern blot analysis using a rat beta 3 cDNA probe kindly provided by Dr. P. H. Seeburg, University of Heidelberg, Germany. Briefly, total RNA was extracted from liver tissues by the lithium chloride-urea method (2). RNA was resolved in 1% formaldehyde-agarose gels and transferred onto GT nylon membranes (Bio-Rad, Hercules, CA). Membranes were hybridized with a radiolabeled human GABAAbeta 3 cDNA probe and 28S rRNA cDNA probe in a hybridization solution consisting of 50% formamide, 0.2 M NaCl, 0.12 M Na2HPO4, and 7% SDS. Membranes were washed twice with 2× SSC-0.1% SDS at room temperature for 15 min and 1.2× SSC-0.1% SDS at 42°C for 15 min. X-ray films exposed to membranes were scanned, and the bands were quantitated by an NIH Image program (National Institutes of Health, Bethesda, MD). All bands were standardized against concurrently run 28S rRNA. GABAA receptor protein expression was documented by Western blot analyses as described in Western blot analysis.

This study was approved by the University of Manitoba Conjoint Ethics Committee for animal experimentation.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the expression of GABAA receptors in the human liver, 13 pairs of primers derived from different subunits of human brain GABAA receptors were used (Table 1). After comparative PCR amplification of the human brain and liver cDNAs with these primers, all GABAA receptor subunits were confirmed to be present in the brain, but only two (beta 3 and epsilon ) were found to be expressed in both human brain and liver.

PCR amplification of the GABAA receptor beta 3-subunit produced a single 633-bp PCR product. The expression of this subunit appeared to be higher in the brain than in the liver (Fig. 1A). The presence of the GABAA receptor beta 3-subunit transcript was further confirmed by using an internal gene-specific oligonucleotide probe (Fig. 1, B and C). GABAA receptorbeta 3-subunit expression was also found in the rat liver (Fig. 2).


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Fig. 1.   Expression of human GABAA receptor beta 3-subunit in the liver. A: gene-specific PCR primers for the human GABAA receptor beta 3-subunit were used for PCR amplification of human brain cDNA (B) and human liver cDNA (L). Standard molecular mass markers (M) and PCR products were resolved by electrophoresis in 1.5% agarose gel and stained with ethidium bromide. Control assays were performed without cDNA (C). The arrow points toward a 633-bp PCR product. B: Southern blot analysis of the agarose gel seen in A probed with an internal gene-specific probe for the human GABAA receptor beta 3-subunit. The autoradiogram was exposed for 30 min at -70°C. C: the same blot as in B, exposed for 3 h at -70°C.



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Fig. 2.   Expression of rat GABAA receptorbeta 3-subunit in the liver. A: gene-specific primers for the rat GABAA receptorbeta 3-subunit were used for PCR amplification of rat brain cDNA (B) and rat liver cDNA (L). See legend to Fig. 1 for details. B: Southern blot analysis of the agarose gel seen in A probed with an internal gene-specific probe for the human GABAA receptor beta 3-subunit. The autoradiogram was exposed for 30 min at -70°C.

Figure 3 provides the results of Western blot analyses for the human and rat GABAA receptor beta 3-subunit protein. When crude tissue extracts from human liver, rat brain, and rat liver were analyzed using a commercially available antibody (monoclonal antibody bd 17) with activity against both GABAA receptor beta 2- and beta 3-subunits, bands with the appropriate molecular masses of 53-59 kDa were identified. Presumably, the different molecular masses represent different extents of glycosylation of the two beta -subunits (28) or alternative splicing of the beta 3-subunit transcript in a region encoding a signal peptide (20).


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Fig. 3.   Western blot of rat and human GABAA receptor with mouse anti-GABAA receptor beta 2/3 subunit monoclonal antibody (MAb) bd 17. Aliquots [50.0 µg protein of rat brain (rB), rat liver (rL), and human liver (hL) crude extracts] and standard molecular mass markers (M) were electrophoresed through a 10.0% polyacrylamide-SDS gel. Resolved proteins were electroblotted to nitrocellulose and incubated with the mouse anti-GABAA receptor beta 2/3-subunit MAb, bd 17, that reacts against both GABAA receptor beta 2- and beta 3-subunits. A typical broad band, 53-59 kDa, was detected in the rat brain.

In addition to the beta 3-subunit, the epsilon -subunit was also detected in the human liver by PCR amplification (Fig. 4). However, in this case, two PCR products were detected, a major product of 632 bp and an additional product of ~900 bp. These results were anticipated because blasting of the 632-bp sequence with the complete sequence of the human chromosome band Xq28 (locus U82696), where the GABA receptor epsilon -subunit has been mapped, revealed that this sequence extends over three exons. These exons are separated by two introns, of 138 bp and 284 bp, and alternative splicing of the 284-bp intron would result in a PCR product of 916 bp. These observations were confirmed by Southern blot analyses (Fig. 4B) and are in keeping with previous reports describing multiple patterns of alternative splicing of the epsilon -subunit transcript in adult human tissues including the electrical conduction system of the heart (11). Although the 632-bp product was more abundant in the liver, the expression of the 916-bp band was higher in the brain (Fig. 4A).


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Fig. 4.   Expression of human GABAA receptor epsilon -subunit in the liver. A: gene-specific PCR primers for the human GABAA receptor epsilon -subunit were used for PCR amplification of human brain cDNA (B) and human liver cDNA (L). Standard molecular mass markers (M) and PCR products were resolved by electrophoresis in 1.5% agarose gel and stained with ethidium bromide. Control assays were performed without cDNA (C). The arrows point toward 916-bp and 632-bp PCR products. B: Southern blot analysis of the agarose gel seen in A probed with an internal gene-specific probe for the human GABAA receptor epsilon -subunit. The autoradiogram was exposed for 30 min at -70°C.

Figure 5 outlines changes in GABAA mRNA expression after PHx and the correlation that exists between [3H]thymidine incorporation into hepatic DNA and GABAA mRNA expression. GABAA mRNA expression decreased after PHx and remained decreased from baseline until day 7 after PHx. A significant negative correlation (r = -0.527, P < 0.01) was observed between [3H]thymidine incorporation and GABAA mRNA expression. GABAA receptor protein expression paralleled these findings (Fig. 6).


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Fig. 5.   Correlation between [3H]thymidine incorporation and GABAA receptorbeta 3-subunit mRNA levels. Regression analysis of [3H]thymidine incorporation into rat liver DNA and GABAA receptorbeta 3-subunit mRNA expression by Northern blot analyses at various times after partial hepatectomy (PHx) was performed. r = -0.5275; P = 0.0061.



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Fig. 6.   Effect of PHx on the expression of the GABAA receptorbeta 3-subunit in the rat liver. Representative blot of GABAA receptorbeta 3-subunit protein expression by Western blot analysis with MAb bd17 at various times after PHx is shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

That the liver might contain specific GABAA receptors was first suggested by Bondy and Harrington (1), who documented [3H]GABA binding to a number of peripheral tissues including rat liver. Although Defeudis et al. (6) were unable to confirm these findings, in 1987 we (23) reported the presence of specific, sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes. We (23) also reported that activation of these sites resulted in prompt and marked hyperpolarization of the resting hepatocyte membrane potential. Nevertheless, the precise nature and subunit composition of these sites, the identification of which may provide insights into their physiological relevance, remained to be determined. Indeed, the nature of GABAA receptor subunit types in any tissues beyond the central nervous system and neuronal conduction systems in peripheral tissues has hitherto not been reported.

The results of the present study indicate that the human liver possesses both beta 3 and epsilon  GABAA receptor subunit types. The beta 3-subunit, like other GABAA receptor subunits, can form a functional homomeric GABA-gated ion channel when expressed alone in Xenopus oocytes (31). This subunit is able to spontaneously gate in the absence of ligand, as can the beta 1-subunit (3, 21). It possesses the highest affinity for GABA compared with beta 1- or beta 2-containing receptors (36). beta -Subunits have also been found to control the subcellular distribution of GABAA receptors. Interestingly, beta 3-subunit-containing receptors were documented to have the specific capability to transcytose, suggesting that this subunit may be involved in rapid relocation of the GABAA receptor (3).

In contrast to other GABAA-receptor subunits, the epsilon -subunit cannot form a functional GABA-gated ion channel unless it is coexpressed with other subunits such as beta 1 and alpha 1 or alpha 2 (35). The epsilon -subunit has also been found to confer insensitivity to the potentiating effect of anesthetic agents, which is achieved through muscimol/GABA binding to different subunit combinations (4). Thus there is a possibility that the epsilon -subunit may also regulate GABA binding to the beta 3-subunit of the liver GABA receptor in humans. Of note, the identification of this subunit type in the human liver refutes the assumption, based on the structure of the 5' region of epsilon -subunit cDNA and the expression of a uniquely spliced epsilon -subunit transcript in brain tissue, that a functional epsilon -subunit polypeptide, e.g., a product of a completely spliced transcript, is only expressed in brain or neuronal tissues outside the central nervous system (11, 35, 37).

Using RNase protection analysis, others have described beta 3-subunit transcripts in several human, rat, and mouse immortalized cell lines but not in the liver (14). This may result from the relatively low sensitivity of the RNase protection assay compared with the PCR analysis used in the present study, although our positive findings on Northern blot analyses argue against that explanation. The detection of beta 3-subunit transcripts in immortalized cells may imply that this subunit is involved, via changes in its expression, in cell proliferation typical of malignancy, or in the regenerative process that is unique to the liver.

The GABAA receptor in the brain contains binding sites for both GABA and benzodiazepine ligands (15, 18). Photoaffinity labeling studies have revealed that although the alpha -subunit and gamma -subunits carry the benzodiazepine binding sites, the beta -subunit carries only the GABA binding site (30, 33, 34). Thus our finding that rat liver contains only the beta -subunit type is in keeping with previous data indicating that benzodiazepines do not significantly alter 3[H]GABA binding to isolated rat hepatocytes (26).

The precise role of GABAA receptors in the liver remains to be determined. In other peripheral tissues in which GABAergic activity has been described, changes in motility and hormonal function are thought to be relevant (7, 9). In the liver, however, portal and systemic infusions of GABA had no effect on either bile flow or hormonal activity, rendering these possibilities less likely (22, 25). Perhaps more relevant were findings of increased GABAergic activity attenuating hepatic regeneration whereas decreased GABAergic activity enhanced hepatic regeneration (19, 24, 38, 39). These findings, together with our present data documenting a negative correlation between [3H]thymidine incorporation and GABAA receptor beta 3-subunit mRNA expression in regenerating rat livers, are consistent with the hypothesis that GABA serves to regulate hepatic growth and development (8, 12, 13).

In summary, the results of this study indicate that GABAA receptors are not confined to the central or peripheral nervous systems but also exist in the liver, a tissue that is generally considered to be poorly innervated. The precise function of GABAA receptors in the liver is unclear. However, considering the unique nature of both beta 3- and epsilon -subunits and the results of this and previous studies involving GABA and hepatic regeneration, it can be presumed that the coexpression of beta 3- and epsilon -subunits leads to the formation of a functional GABA-gated channel that serves to regulate hepatic regenerative activity.


    ACKNOWLEDGEMENTS

The authors thank S. Zdanuk for assistance in preparation of the manuscript.


    FOOTNOTES

This work was supported by a grant from the Medical Research Council of Canada.

Present address of R. Erlitzki: Children's Hospital Oakland Research Institute, 5700 Martin Luther King, Jr. Way, Oakland, CA 94609-1673.

Address for reprint requests and other correspondence: G. Y. Minuk, Liver Diseases Unit, Rm. 803 F, John Buhler Research Centre, 715 McDermot Ave., Winnipeg, MB, Canada R3E 3P5 (E-mail: gminuk{at}cc.umanitoba.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4 January 2000; accepted in final form 28 June 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 279(4):G733-G739
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