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Endocrinology Vol. 141, No. 10 3879-3886
Copyright © 2000 by The Endocrine Society

ARTICLES

Growth Hormone Increases Connexin-43 Expression in the Cerebral Cortex and Hypothalamus1

N. David Åberg2, Björn Carlsson, Lars Rosengren, Jan Oscarsson, Olle G. P. Isaksson, Lars Rönnbäck and Peter S. Eriksson

Institute of Clinical Neuroscience (N.D.Å., L.Ros., L.Rön., P.S.E.) and Research Center for Endocrinology and Metabolism, Department of Internal Medicine (B.C., O.G.P.I.), Sahlgrenska University Hospital, Göteborg University, and Department of Physiology and Pharmacology (J.O.), Göteborg University, SE-413 45 Göteborg, Sweden

Address all correspondence and requests for reprints to: Dr. Peter S. Eriksson, Institute of Clinical Neuroscience, Sahlgrenska University Hospital, Göteborg University, SE-413 45 Göteborg, Sweden. E-mail: per{at}neuro.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several studies indicate that systemic GH influences various brain functions. Connexin-43 forms gap junctions that mediate intercellular communication and establish the astroglial syncytium. We investigated the effects of peripheral administration of bovine GH (bGH) and recombinant human insulin-like growth factor I (rhIGF-I) on the expression of connexin-43 in the rat brain. Hypophysectomized female Sprague Dawley rats were substituted with cortisol (400 µg/kg·day) and L-T4 (10 µg/kg·day) and treated with either bGH (1 mg/kg·day) or rhIGF-I (0.85 mg/kg·day) for 19 days. The abundance of connexin-43 messenger RNA (mRNA) and protein in the brainstem, cerebral cortex, hippocampus, and hypothalamus was quantified by means of ribonuclease protection assays and Western blots. Treatment with bGH increased the amounts of connexin-43 mRNA and protein in the cerebral cortex and hypothalamus. No changes were found in the brainstem or hippocampus. Infusion of rhIGF-I did not affect connexin-43 mRNA or protein levels in any of the brain regions studied. These results show that administration of bGH increases the abundance of cx43 in specific brain regions, suggesting that GH may influence gap junction formation and thereby intercellular communication in the brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EFFECTS OF GH on postnatal growth and metabolism are well established (1, 2). In addition, both animal and human studies indicate that GH may exert important effects on the central nervous system (CNS). GH deficiency in adults is characterized by an impaired quality of life along with inability to concentrate, fatigue, lack of energy, poor memory, and irritability (3, 4). GH replacement therapy in GH-deficient adult humans reverses these conditions (3, 4). In analogy, GH administration improves certain memory parameters in rats (5).

GH affects the brain by various means. For example, Little mice (monodeficient in GH) exhibit both reduced brain weight and markers of myelination (6). After GH administration, in rats the expression of c-fos messenger RNA (mRNA) increases in the hypothalamus (7), and serotonin and norepinephrine levels are affected in specific brain regions (8). Furthermore, ß-endorphin levels rise in cerebrospinal fluid in GH-deficient humans after systemic administration of GH (9). These effects provide biochemical evidence for an effect of peripheral administration of GH on the brain. The mechanisms by which GH exerts these effects are unknown. However, the GH receptor is expressed in both glial cells and neurons, suggesting that GH can stimulate both cell types directly (10).

Insulin-like growth factor I (IGF-I) is believed to mediate some of the effects of GH. Indeed, locally produced IGF-I might mediate some of the effects of GH in the brain, as IGF-I levels are increased in major brain regions after systemic GH administration (11, 12). In addition, it was recently shown that peripherally infused radioactive IGF-I crosses the blood-brain barrier via a carrier-mediated uptake (13). The importance of IGF-I for normal brain development is further substantiated by the findings of hypomyelination, fewer oligodendrocytes, and reduction of total brain size in IGF-I knockout mice (14).

In most cells, including neurons and glial cells, there is cell to cell communication, which occurs by means of gap junctions. These are formed by two hexameric protein complexes, one from each cell membrane, that form aqueous pores, allowing ions and small molecules with mol wt less than 1000 to move from one cell to another. The proteins, connexins (cx), are encoded by distinct genes that are named by suffixing the mol wt in the thousands of the encoded protein. In the brain several types of cx genes form gap junctions, including cx26 (15), cx30 (16), cx32 (15), cx36 (17), cx40 (in vitro) (18), cx43 (15, 16, 19), and cx45 (20). cx43 is the most ubiquitously expressed connexin in the brain and is mainly found in astrocytes (15, 16, 19). The biological significance of connexins is illustrated by several pathological conditions associated with alterations in the expression of connexins or in the permeability of gap junctions (reviewed in Ref. 21). For example, blocking gap junctions in the brain restricts the zone of cell death after induced ischemia (22).

Based on reports that GH influences many aspects of brain function by, in many cases, unknown mechanisms, we investigated the possible effect of peripheral bovine GH (bGH) on cx43 as a ubiquitous biochemical marker for gap junction formation and intercellular communication in the rat brain. Furthermore, the effect of peripheral infusion of recombinant human IGF-I (rhIGF-I) on cx43 was investigated, as peripheral IGF-I also has direct effects on the brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental protocols
Female Sprague Dawley rats (Møllegaard Breeding Center Ltd., Ejby, Denmark) were maintained under standard conditions of temperature (24–26 C) and humidity (50% to 60%) and with lights on between 0500–1900 h each day. Both normal rats and hypophysectomized (hx) rats were kept to monitor effects of hypophysectomy per se. The effects of hormone administration were evaluated in rats that were hx at 50 days of age (n = 5–8 in each group). The rats had free access to standard laboratory chow and water (rat and mouse standard diet, B&K Universal Ltd., Sollentuna, Sweden). Hormone administration, which was maintained for 19 days, was initiated approximately 10 days after hypophysectomy.

All hx rats received substitution therapy with cortisol phosphate (C; 400 µg/kg·day; Solucortef, Upjohn, Puurs, Belgium) and L-T4 (10 µg/kg·day; Nycomed, Oslo, Norway), which were diluted in saline and administered sc once daily at 0800 h (23, 24). These rats were divided into a control group (designated hx in all figures) and a treatment group, which received an additional dose of bovine GH (bGH) or rhIGF-I. Bovine GH (recombinant), a generous gift from American Cyanamide Co. (Princeton, NJ), was diluted in a 0.05 M phosphate buffer of pH 8.6 with 1.6% glycerol and 0.02% sodium azide and given as a sc injection (1 mg/kg·day) once daily for 19 days (25). Additional hx rats living under identical conditions were used to evaluate the effect of rhIGF-I treatment. Miniosmotic pumps (Alzet 2004 model, Alza Corp.) were filled with rhIGF-I and implanted sc in the neck. Recombinant human IGF-I was provided by Genentech, Inc. (South San Francisco, CA), and administered at 0.85 mg/kg·day, a concentration shown to exert distinct biological effects in the periphery as well as the brain (26). All animals were weighed every 2–3 days to monitor the biological response in weight gain. As IGF-I has less of a growth-promoting effect than GH, we further analyzed serum IGF-I levels at decapitation. The concentration of IGF-I was determined using a RIA kit (Nichols Institute Diagnostics, San Juan, Capistrano, CA) (27). Finally, serum IGF-I levels were analyzed in a separate group of bGH-treated hx rats (bGH infusion, 1 mg/kg·day, and cortisol and T4 injections as described above).

After decapitation, the brain tissue was dissected, immediately frozen in liquid nitrogen, and stored at -80 C. All treatment procedures were approved by the board of animal ethics of Göteborg University.

RNA preparation
Total RNA was prepared essentially according to Chomczynski and Sacchi, with minor modifications (28, 29).

RNA probes
An EcoRI-HincII fragment corresponding to bp 398–985 of the cx43 complementary DNA (30) was subcloned using pBSK generating pBSK-c43. 35S- and 32P-labeled RNA antisense RNA was generated by in vitro transcription from EcoRI-digested pBSK-c43 using T7 polymerase. Similarly, sense RNA was generated from HincII-digested pBSK-c43 using T3 polymerase.

Northern blot and ribonuclease (RNase) protection assays (RPA)
We first determined the specificity of the probe used for analysis of cx43 gene expression using the 32P-labeled cx43 RNA probe in a Northern blot. RNA (15 µg) extracted from the brainstem was used in the assay as previously described (31). This analysis revealed one transcript of approximately 3 kb (Fig. 1aGo), which corresponds to the rat cx43 mRNA (30).



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  		Figure 1. Specificity of the radioactive cx43 RNA probe. a, Northern blot using RNA (15 µg) isolated from the brainstem and 32P-labeled cx43 RNA probe. The Northern blot was performed as described in Materials and Methods. The blot shows one RNA transcript, approximately 3 kb, corresponding to the size previously reported for cx43 mRNA. b, RPA run on PAGE using a 35S-labeled cx43 RNA probe. The samples were treated as described in Materials and Methods. The lanes from the left: the hybridization fragment of the probe and 15 µg RNA from the brainstem (Brst RNA), the hybridization fragment of the probe and synthetic sense cx43 RNA (cx43 RNA, positive control), the hybridization fragment of the probe and transfer RNA (tRNA; negative control), and, last lane, the probe itself directly loaded onto the gel (probe). This assay shows that the probe hybridizes to a fragment of the expected size (610 bp) with little nonspecific hybridization.

 
The RNase solution hybridization was performed using a 35S-labeled cx43 probe. Protected radioactive hybridization products were handled in two procedures after they were precipitated with 7% trichloroacetic acid. In the first procedure (gel-RPA) samples were treated in the Ambion, Inc., RPA II kit (Austin, TX), where hybridization conditions are essentially identical to the protocol below (29, 32). The protected hybridization fragments were then electrophoresed on PAGE (6%) and detected on a PhosphorImager screen (Molecular Dynamics, Inc., Sunnyvale, CA). The hybridization product of the antisense 35S-labeled cx43 probe was of the expected size in a sample from the brainstem (~610 bp; Fig. 1bGo). The hybridization signal showed the same appearance and size regardless of sample origin (RNA from heart, liver, brainstem, cerebellum, and astroglial primary cultures from cerebral cortex were used; data not shown). In the second procedure (filter-RPA), double samples of 10–25 µg total RNA from each brain region were incubated with the 35S-labeled cx43 probe, and the hybridization products of the RPA were collected on glass-fiber filters (GF/C, Whatman International Ltd., Maidstone, UK) (29, 32). Each sample was subsequently analyzed for radioactivity in a scintillation counter. The amount of cx43 mRNA was calculated from a standard curve based on hybridization to known amounts of synthetic sense cx43 RNA. Relative changes between treatment groups were only compared with samples within the same assay. The intraassay coefficient of variation was 14.1%.

Western blot
After the RNA isolation procedure, the remaining phenol phase (containing protein) was used to isolate protein as modified from Chomczynski and Sacchi (33). DNA was precipitated using 96% (1:3, vol/vol) ethanol and gently centrifuged for 5 min at approximately 200 x g. The supernatant was transferred to a second tube and precipitated with isopropanol (1:1, vol/vol) and centrifuged at 3150 x g for 10 min. The supernatant was discarded, and the protein pellet was washed three times for 20 min each time and centrifuged as before in 0.3 M guanidinium hydrochloride in 96% ethanol and finally in 96% ethanol. The protein pellets were then dissolved in a urea-saturated buffer (9.0 M urea, 0.23 M sucrose, 97 mM dithiothreitol, 33 mM SDS, and 10 mM Tris, pH 8.0), and the total protein concentration was determined as described previously (29).

The Western blot was performed using PAGE (with 10% separation gel, pH 8.8, and 4% stacking gel, pH 6.8, in 0.1% SDS) run for 2–3 h at 20 mA using a Protean Cell apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). Protein was transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore Corp., Bedford, MA) at 80 mA overnight. The membranes were washed and probed with a monoclonal mouse cx43 antibody at 1:2000 in 10 ml TBS blocking solution (1% polyvinylpyrrolidone 40 in 0.05% Tween-20 in 0.5 M Tris-buffered saline, pH 7.5). Three washes in TBS were performed before the secondary antibody, POD-conjugated sheep antimouse (Roche Molecular Biochemicals, Mannheim, Germany), was applied at a 1:5000 dilution in blocking solution. After five washes with 0.1% Tween-20 in TBS, the membranes were applied to a chemiluminescence kit (BM chemiluminescence blotting substrate, Roche Molecular Biochemicals).

Total protein samples were also dialyzed into 0.1% SDS overnight, as immunoprecipitation was not compatible with the urea-saturated buffer above. The protein samples (~1000 µg) were then subsequently immunoprecipitated with magnetic beads (Dynabead kit, DynAl, Oslo, Norway) with antirabbit antibodies. The beads were then coated with a rabbit polyclonal anti-cx43 antibody 71–0700(71–0700, Zymed Laboratories, Inc., South San Francisco, CA) to which the protein samples were added for 1 h. After washing away the unbound surplus protein, the immunoprecipitate was eluted, electrophoresed, and blotted as described above (Western blot). Double samples were run on Western blots, and the PVDF membrane was cut into two pieces. One of the membrane pieces were preincubated with 160 µg control peptide (amino acids 252–270 of rat cx43; Chemicon International, Temecula, CA) with the mouse monoclonal anti-cx43 antibody in 10 ml blocking solution (1:2000 for 2 h at room temperature). This solution was then added to the strip of PVDF membrane, and the other strip was incubated under identical conditions without the control peptide (Fig. 2aGo). Both strips were then processed with POD-conjugated secondary antiserum and developed with chemiluminescence as described above. Addition of the control peptide with or without immunoprecipitation abolished the signal on the Western blot. These control experiments confirm that bands with mol wt of 42,000–44,000 represent cx43 protein (Fig. 2aGo), in line with previous studies using antibodies raised against the 252–270 amino acid epitope of cx43 (31, 34, 35). The detected immunoreactive band was scanned and analyzed with integrated OD of the bands using Image Alpha 9 (Scion Corp., CA). The densitometry of a protein sample was always compared with that of samples within the same gel. The intraassay coefficient of variation in the densitometry of the Western blot was 20%.



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  		Figure 2. Specificity of the mouse monoclonal anti-cx43 antibody shown with Western blots using immunoprecipitation and control peptide block, and immunohistochemistry. a, Western blot with immunoprecipitate and control peptide block. The immunoprecipitation and control peptide block were performed as described in Materials and Methods. The different lanes are treated as indicated by the signs. Ip, Immunoprecipitation using a rabbit anti-cx43 antibody; Protein, total protein from the hippocampus; C. P., control peptide (amino acids 252–270 of cx43); Art, immunoprecipitation artifact bands, possibly proteolytic fragments of the Igs. In lanes 2 and 4, 15 µg total protein were loaded, and in lanes 3 and 5 all of the immunoprecipitate (originating from ~1000 µg total protein) was loaded. Lanes 1 and 6 contain immunoprecipitation yield run without any brain protein added (showing the immunoprecipitation artifact bands). The blots were all incubated with the monoclonal cx43 antibody with or without control peptide block as indicated and developed using chemiluminescence (see Materials and Methods). The detected band has a mol wt of approximately 42,000–44,000, as previously reported. The mol wt are given in thousands. b, Confocal image of the hilus region of the hippocampus in a normal animal using mouse monoclonal antiserum against cx43 (amino acids 252–270). Immunoreactivity was detected with FITC-conjugated secondary antimouse antiserum, as described in Materials and Methods. The staining shows a typical punctuate distribution, with abundant immunoreactivity in the hilus, very little staining in the granule cell layer, and somewhat more immunoreactivity outside the granule cell layer. c, A control section from the same animal treated in the same procedure, with the primary antibody replaced by 1% swine serum. The section shows only faint background staining compared with b. GCL, Granule cell layer. Scale bar, 50 µm.

 
Immunohistochemistry
One normal rat was anesthetized with ip pentothal (40 mg/kg) and perfusion-fixed in 4% paraformaldehyde in PBS (pH 7.5). The brain was dissected and further fixed in 4% paraformaldehyde-PBS for 24 h at 4 C, after which 20-µm coronal sections of the hippocampus were prepared and treated with 1% polyvinylpyrrolidone-40 and 0.5% Tween-20 in PBS for 19 h. After two washes in PBS, the sections were blocked with 1% normal swine serum in PBS for 45 min. The sections were then incubated with primary mouse monoclonal antirat cx43 (Chemicon) antibody in a 1:200 blocking solution (1% polyvinylpyrrolidone-40 and 0.01% Tween-20) for 21 h at 4 C. As a control, to exclude the possibility of nonspecific background staining, we substituted 1% swine serum for the primary anti-cx43 antibody using identical conditions. After four washes in blocking solution, the secondary fluorescein isothiocyanate (FITC)-conjugated antimouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added 1:100 in blocking solution for 21 h at 4 C in all of the sections. Immunofluorescence was detected and processed using a Nikon Diaphot microscope (Melville, NY) and confocal laser-scanning microscopy system (model 1024, Bio-Rad Laboratories, Inc.; Fig. 2Go, b and c). The immunofluorescence staining of the hippocampus sections showed that the monoclonal antibody used (Chemicon) not only recognizes the correct band on Western blots, but also reveals the typical punctuate appearance of cx43-like immunoreactivity (15).

Enzyme-linked immunosorbent assay (ELISA)
Aliquots of the protein samples from cerebral cortex were used to measure glial fibrillary acidic protein (GFAp) by ELISA as previously described (36). Microtest plates were coated with hen anti-GFAp IgG. Samples and a dilution series taken from one sample were added. After washing them in sequential order, the wells were incubated with rabbit anti-GFAp, peroxidase-conjugated donkey antirabbit IgG and enzyme substrate (o-phenylenediamine and H2O2). Absorbance was measured at 490 nm using a computerized ELISA reader (Molecular Devices, Menlo Park, CA), and the relative concentrations of antigen in the samples were interpolated from the standard curve.

Computerized analysis of potential binding sites of the cx43 promoter
Putative binding sites for transcription factors were searched for in the 5'-flanking sequence of the cx43 gene (L36949, GenBank, National Center for Biotechnology Information) using the 1300 bp of the cx43 promoter region and TFSEARCH: Searching Transcription Factor Binding Sites, http://www.rwcp.or.jp/papia/, written by Yutaka Akiyama) and the TRANSFAC database (37).

Statistics
Statistical analysis of the effects of the treatments was performed with log10-transformed data where appropriate, followed by one-way ANOVA. The values are presented as the mean and as SEM. The mean value of the T4/C-treated hx group was designated 100%, and the relative change in the treatment (bGH or rhIGF-I) groups was expressed relative to this value. The mRNA and protein data for the normal and hx animals were pooled individually to statistically examine the tendency toward a decrease in cx43. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH and cx43
Comparing normal and hx rats, we found a modest decrease in both cx43 mRNA and protein, with an overall decrease in cx43 protein (23 ± 14%; P < 0.05), whereas the decrease in cx43 mRNA (12 ± 21%) failed to reach significance. These data show that hypophysectomy per se has a general effect on cx43 abundance when comparing brain tissues from normal animals with brain tissues from hx animals.

It is possible that locally produced GH in the brains of hx rats increases the baseline expression of cx43 in different regions of the CNS, because GH is expressed, although at low levels, at extrapituitary sites in the brain (38). As hypophysectomy had a general effect on cx43, and preliminary results of administration of bGH indicated that bGH increased cx43, we examined the effect of peripheral administration of bGH on cx43 expression in the brainstem, cerebral cortex, hippocampus, and hypothalamus. Female hypophysectomized rats substituted with T4 and C (hx) were given daily injections of bGH for 19 days (1 mg/kg), and the effect of bGH on cx43 expression was analyzed. Comparing the first and last days of the treatment, the hx rats showed no weight gain (139 ± 2.0 vs. 136 ± 1.9 g), whereas the bGH-treated hx rats showed the expected response in body growth (134 ± 1.3 vs. 191 ± 0.9 g; P < 0.001). These data show that bGH had appropriate systemic activity.

Administration of bGH resulted in increased cx43 mRNA in the cerebral cortex (Fig. 3aGo) and hypothalamus (Fig. 3bGo), whereas no statistically significant effects of bGH administration were detected in the brainstem or hippocampus (data not shown). We also performed identical experimental protocols with 6 days of bGH treatment and found similar responses in cx43 mRNA (data not shown). Moreover, the administration of bGH increased the amount of cx43 protein in the cerebral cortex (Fig. 3cGo) and in the hypothalamus (Fig. 3dGo), as analyzed by Western blot. In line with the effects on cx43 mRNA, no statistically significant changes were detected in cx43 protein abundance in the brainstem or hippocampus (data not shown). Thus, administration of bGH to adult hx female rats increased the amounts of cx43, both mRNA and protein, in the cerebral cortex and hypothalamus, whereas the brainstem and hippocampus were unaffected.



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  		Figure 3. Effect of 19-day bGH treatment on cx43 mRNA and protein abundance in the cerebral cortex and hypothalamus of T4/C-treated hypophysectomized rats. a and b, Effect of bGH treatment (+) on cx43 mRNA levels in the cerebral cortex (Cortex; a) and hypothalamus (b) of T4/C-treated hx rats (-). The amount of cx43 transcript was determined by a solution hybridization assay as described in Materials and Methods. c and d, Effect of bGH treatment (+) on cx43 protein levels in the cerebral cortex (Cortex; c) and hypothalamus (d) of T4/C-treated hx rats (-). The amount of cx43 protein was determined by densitometry of Western blots, as described in Materials and Methods. Representative samples of cx43 in the Western blot are shown. Data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01. The mean of the bGH treatment group is expressed as a percentage of the mean of the T4/C-treated hx rats (=100).

 
IGF-I and cx43
As IGF-I has some effects similar to those of GH on the brain (see introduction), and peripheral IGF-I crosses the blood-brain barrier (13) and affects the brain (26, 39), IGF-I could also affect cx43 in the brain. Therefore, the effect of systemic administration of rhIGF-I on cx43 expression was investigated. Female hx rats were given rhIGF-I (0.85 mg rhIGF-I/kg) sc via miniosmotic pumps for 19 days. Comparing the first and last days of the treatment, hx rats did not gain any weight (140 ± 1.4 vs. 139 ± 2.0 g) whereas rhIGF-I administration in the hx rats resulted in a weight gain (141 ± 1.9 vs. 150 ± 2.3 g; P < 0.001), indicating a systemic effect of rhIGF-I. In addition, serum IGF-I levels were significantly increased in the rhIGF-I-treated animals compared with those in animals hx on the day of death (157 ± 17 vs. 63 ± 4 ng/ml; P < 0.001). However, administration of rhIGF-I did not cause any statistically significant changes in cx43 mRNA or protein in any of the brain regions studied (Fig. 4Go). Serum IGF-I in bGH-treated rats was analyzed in a separate cohort of bGH-treated rats. These rats gained 2.7 ± 0.2 g/day, and serum IGF-I was 712 ± 48 ng/ml on the day of decapitation.



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  		Figure 4. Effect of 19-day rhIGF-I treatment on the amounts cx43 mRNA and protein in the cerebral cortex and hypothalamus. a and b, Effect of rhIGF-I infusion (+) on cx43 mRNA levels in the cerebral cortex (Cortex; a) and hypothalamus (b) of T4/C-treated hx rats (-). Cx43 transcript abundance was determined by a solution hybridization assay, as described in Materials and Methods. c and d, Effect of rhIGF-I infusion (+) on cx43 protein levels in the cerebral cortex (Cortex; c) and hypothalamus (d) of T4/C-treated hx rats (-). The amount of cx43 protein was determined by densitometry of Western blots as described in Materials and Methods. Representative samples of cx43 in the Western blot are shown. Data are presented as the mean ± SEM. The mean of the rhIGF-I treatment group is expressed as a percentage of the mean for the T4/C-treated hx (=100) rats.

 
GH and IGF-I and GFAp
Finally, to investigate whether the effect of bGH on cx43 abundance in the cerebral cortex was due to a more general effect on astroglial cells, we analyzed the possible effect of bGH and rhIGF-I on GFAp using ELISA. However, none of the treatments had any effect on the abundance of GFAp (Fig. 5Go).



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  		Figure 5. Effect of 19 days of bGH and rhIGF-I treatment on the amount of GFAp in the cerebral cortex as analyzed by ELISA. a, Differences in GFAp expression in animals given daily injections of T4/C/bGH (+) vs. T4/C-treated (-) hx animals. b, Differences in GFAp expression in animals treated with T4/C/rhIGF-I (+) vs. T4/C-treated (-) hx animals. Data are presented as the mean ± SEM. The mean for each treatment group is expressed as a percentage of that for the T4/C-treated hx (=100) rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several studies have demonstrated that the amount of cx43 correlates with gap junction formation and cell to cell communication (29, 40). In this study the peripheral administration of bGH was followed by an increase in the amount of cx43 in specific regions of the rat brain. These results suggest that GH increases gap junction formation in the brain and thereby enhances cell to cell communication in the CNS.

Analysis of loss of function after hypophysectomy combined with a subsequent restitution mediated by the administration of a pituitary hormone is the most common and is an important experimental paradigm to determine the physiological role of pituitary hormones. However, some issues require careful consideration when interpreting the biological effects of the hormones administered. As hypophysectomy had a general, but modest, effect on cx43 expression in the brain, it was unexpected that administration of bGH would raise the levels of cx43 to a large extent. Several explanations may account for these findings. The effects of bGH could be pharmacological. However, this is unlikely, because the normal 24-h secretion of rat GH is about 1.3 mg/day (calculated from GH secretion and GH clearance rates) (41, 42), which is above the 24-h dose we administered. Furthermore, hypophysectomy may disrupt the effects of other hormones that normally inhibit the expression of cx43. The effect of hypophysectomy may also be blurred by the production of local GH in the brain, which may be unaffected by hypophysectomy. Indeed, substantial evidence for the presence of extrapituitary production of GH has accumulated, as GH mRNA (43, 44, 45) as well as immunoreactive GH (38, 45, 46, 47) have been detected in several brain regions. The highest concentrations of GH in the rat brain, apart from the pituitary, have been found in the amygdala, hypothalamus, caudate nucleus, thalamus, hippocampus, and cerebral cortex (38).

bGH administration was accompanied by an increase in the concentration of cx43 in the hypothalamus and cerebral cortex, whereas no effect was seen in the brainstem or hippocampus. Regional differences in GH activity are not surprising, because the brain is functionally highly heterogeneous. Several potential mechanisms, e.g. differences in the blood-brain barrier permeability and GH receptor expression, may account for the differences in the effects of bGH on cx43 expression in specific brain regions. Indeed, systemic GH may have a greater accessibility to the hypothalamus to exert feedback on its own release (48, 49). Alternatively, the effect of bGH on cx43 in the hypothalamus and cerebral cortex could result from a stronger cellular response due to high concentrations of GH receptors in the hypothalamus and frontal lobe (10). However, a high GH receptor concentration has also been found in parts of the brainstem (10), where we were unable to detect any effect of bGH on cx43 expression. Therefore, the distribution of the GH receptors per se cannot explain the regional differences in the effects of bGH on cx43 expression. Additionally, the amount of GH-binding protein, a splice variant of the GH receptor, affects the response to GH by regulating the bound fraction of GH. However, although GH-binding protein is widespread throughout the brain (50), to our knowledge, detailed quantitative data are not available.

It could be argued that the higher IGF-I levels in bGH-treated rats than in rhIGF-I-infused rats are responsible for the effect on cx43. However, in the present study IGF-I of both treatments probably had a maximal or near-maximal effect on the brain, as it was recently shown that IGF-I passes the blood-brain barrier via a carrier-mediated uptake that is saturated at a serum IGF-I level of 150 ng/ml (13). Therefore, the difference in serum IGF-I levels between bGH and rhIGF-I treatments is unlikely to explain the difference in effect on cx43 in the brain. In addition, we know that the brain is affected by this particular administration of rhIGF-I because neurogenesis is increased in the hippocampal region of the brain after identical infusions of rhIGF-I with similar serum IGF-I levels and growth rate (26). Therefore, the present regimen of rhIGF-I administration is known to access the brain, although cx43 was not affected. These results suggest that GH has a direct effect on cx43 expression, which is not primarily mediated by circulating IGF-I. However, our results do not exclude the possibility of local production of brain IGF-I or other hormones that may mediate the effect of GH. This idea is supported by the facts that 1) the biological role of circulating hepatic IGF-I appears to be different from that of local IGF-I (51, 52); 2) IGF-I probably also has autocrine and paracrine effects, as it is expressed in the brain (53, 54); 3) GH treatment induces the expression of a luciferase transgene driven by the IGF-I promoter in the brain (12); and 4) rhIGF-I may not have the same activity as locally produced IGF-I variants in the brain (53, 54). This is due to a protease present in the brain that cleaves IGF-I and makes the product, des(1, 2, 3)IGF-I, unable to associate with IGF-binding proteins (54, 55), thereby possibly enhancing bioactivity. To date it is unclear which form of IGF-I dominates in the rat brain.

To further investigate the possibility of direct GH or IGF-I regulation of the cx43 gene, we searched the identified 1300 bp of the cx43 promoter region (L36949, GenBank) for motifs that may act as putative binding sites for transcription factors (37). Among these was activator protein-1, which is induced in neural systems (56), and specifically after GH treatment in a preadipocyte cell culture (57). However no STAT (signal transducer and activator of transcription) response element was found. These data support the idea that cx43 gene transcription may be under the control of GH and possibly IGF-I.

The effect of bGH on cx43 may originate from up-regulation of cx43 in several types of cells in the brain. Astrocytes, endothelial cells, leptomeningeal cells (which were discarded in the dissection procedure), and ependymocytes are reported to express cx43 (reviewed in Ref. 15). Accordingly, abundant cx43 mRNA has been reported in astrocytes of most nuclei in the hypothalamus and in astrocytes of all layers of the cerebral cortex; however, it is most abundant in layers 2 and 3 (19). In recent years it has been argued that cx43 is also expressed by neurons (58, 59), especially early in development (58, 59). However, in adult animals, neuronal gap junctions (possibly composed of minor amounts of cx43) are much less numerous and are separated from the gap junctions of astrocytes (60). Together these data show that bGH has a profound effect on cx43 expression in major brain regions, which suggests that several cell types could be involved in the up-regulation of cx43. However, based on previous reports, astrocytes are probably involved in the increase in cx43 expression, although further studies on the ultrastructural level are needed to prove this suggestion.

It could be argued that the changes in cx43 observed during the present study reflect only a general glial hypertrophy or gliosis, of which excess GFAp is representative. However, we observed no increase in GFAp levels in the cerebral cortex after 19 days of bGH administration to hx animals. Thus, astrocytes are not generally affected in their GFAp content by bGH or rhIGF-I treatment.

Does a change in cx43 concentration correlate with astrocyte function in terms of gap junction coupling? No study has yet been able to show this in vivo using current techniques. However, in vitro, we and others reported a positive correlation between the amount of cx43 protein and the spreading of tracer dyes between coupled cells (29, 40). These studies suggest that increased cx43 protein concentrations in astroglial cultures correlate positively with the number of gap junction channels that transfer substances less than 1000 mol wt from cell to cell. With this in mind, the regulation of the expression of gap junction proteins in vivo becomes interesting, as several important phenomena have been associated with gap junctions. For instance, when gap junctions are blocked in vivo, the spreading depression is attenuated, and the cerebral infarct volume that develops after induced cerebral ischemia is also attenuated (22). Moreover, calcium waves, which are partly mediated by astrocytic gap junctions, might be able to affect neurons. For example, both neuronal excitability and neuronal intracellular calcium are elevated in vitro after passage of astroglial calcium waves (61). The effects of altered cx43 expression and gap junctional coupling may therefore have a wide impact on the interplay between neurons and astrocytes in normal as well as pathological brain physiology.

In summary, this study shows that peripheral administration of bGH increases cx43 transcript abundance and that this is accompanied by a similar increase in the amount of cx43 protein. Taken together with previous studies demonstrating a correlation between the abundance of cx43 and gap junction coupling, these results suggest that GH may enhance intercellular communication in specific brain regions.


   Acknowledgments
 
The authors are grateful to Prof. David Paul for supplying the cx43 complementary DNA. Dr. Kliment Gatzinsky and laboratory technician, Ann-Marie Alborn, are thanked for supplying the protocol on immunohistochemistry and technical assistance, respectively. Gunnel Hellgren is thanked for advice on the immunoprecipitation kit. Finally, we thank John Gulliver, Authorized Translator, for careful correction of the English manuscript.


   Footnotes
 
1 This work was supported by the Swedish Medical Research Council (Projects 14X-06005, 04X-13015, and, in part, 8269) and by grants from the Novo Nordisk Foundation, the Åke Wiberg Foundation, the Tore Nilsson Foundation, and the Magnus Bergvall Foundation. Back

2 Supported by the Swedish Society of Medicine and the Göteborg Medical Society. Back

Received February 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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