Liver Diseases Unit, Departments of 1 Medicine and 2 Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3P5
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ABSTRACT |
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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 (3 and
) were expressed in human liver and one
subunit type (
3) in rat liver. PCR amplification of the human
GABAA receptor
3-subunit produced a single product
(molecular mass 53-59 kDa). In the case of the
-subunit, two
PCR products were identified. After partial hepatectomy,
GABAA receptor
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
3- and
-subunit types, whereas in the rat liver only the
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
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INTRODUCTION |
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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, ,
,
,
,
, and
with ~30-40% amino acid
homology between subunits. Within four of the subunit types, various
isoforms sharing 70% sequence identity have been described:
1-
6,
1-
3,
1-
3, and
1-
2
(31). Although the most ubiquitously expressed
GABAA receptor subunits are
1,
2,
3,
2,
3,
and
2 configured in a pentameric
(
)2(
)1(
)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.
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MATERIALS AND METHODS |
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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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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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[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 [-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.
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
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
GABAA
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.
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RESULTS |
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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 (3 and
) were found to
be expressed in both human brain and liver.
PCR amplification of the GABAA receptor 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
3-subunit transcript was further
confirmed by using an internal gene-specific oligonucleotide probe
(Fig. 1, B and C). GABAA
receptor
3-subunit expression was also found in the rat liver (Fig.
2).
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Figure 3 provides the results of Western
blot analyses for the human and rat GABAA receptor
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
2- and
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
-subunits (28) or
alternative splicing of the
3-subunit transcript in a region encoding a signal peptide (20).
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In addition to the 3-subunit, the
-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
-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
-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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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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DISCUSSION |
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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 3 and
GABAA receptor subunit types.
The
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
1-subunit
(3, 21). It possesses the highest affinity for GABA
compared with
1- or
2-containing receptors (36).
-Subunits have also been found to control the subcellular distribution of GABAA receptors. Interestingly,
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
-subunit cannot form a functional GABA-gated ion channel unless it
is coexpressed with other subunits such as
1 and
1 or
2
(35). The
-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
-subunit may also regulate GABA binding to the
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
-subunit cDNA and the expression of a
uniquely spliced
-subunit transcript in brain tissue, that a
functional
-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 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
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
-subunit and
-subunits carry the benzodiazepine binding sites,
the
-subunit carries only the GABA binding site (30, 33,
34). Thus our finding that rat liver contains only the
-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 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 3- and
-subunits and the results of this and previous studies involving GABA and hepatic regeneration, it can be
presumed that the coexpression of
3- and
-subunits leads to the
formation of a functional GABA-gated channel that serves to regulate
hepatic regenerative activity.
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ACKNOWLEDGEMENTS |
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The authors thank S. Zdanuk for assistance in preparation of the manuscript.
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FOOTNOTES |
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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.
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