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Endocrinology Vol. 138, No. 12 5385-5397
Copyright © 1997 by The Endocrine Society

ARTICLES

The Role of {alpha}1 (Connexin-43) Gap Junction Expression in Adrenal Cortical Cell Function1

Ugochi A. Oyoyo, Uzma S. Shah and Sandra A. Murray

Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261

Address all correspondence and requests for reprints to: Sandra A. Murray, Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania l5261.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the relationship between adrenal cell function and gap junction expression, a bovine adrenal cell line (SBAC) was studied. Western blot and immunocytochemical techniques were used to demonstrate gap junction expression in SBAC cell populations. Cells expressed {alpha}1 (connexin 43) gap junction protein at points of cell-to-cell contact. Gap junction number and size increased in populations treated with ACTH (40 mU/ml) or dibutyryl cAMP (DbcAMP, 1.0 mM). Treatment with either ACTH or DbcAMP increased steroid production and cAMP levels. SBAC cell number, however, decreased in ACTH- or DbcAMP-treated populations. The number of cells increased in cultures transfected with {alpha}1-antisense complementary DNA. Neither ACTH nor DbcAMP treatment decreased cell number or increased steroidogenesis in {alpha}1-antisense complementary DNA-transfected cell populations. However, cell populations in which gap junctions were inhibited retained the capacity to increase cAMP production in response to ACTH (40 mU/ml) treatment. Hormone-stimulated gap junction expression and cell communication may represent an important factor in adrenal gland function and control of proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS been suggested that gap junctions play a role in the morphological and functional differentiation of developing adrenal cells, possibly by allowing the intercellular diffusion of ACTH mediators, such as cAMP (see Refs. 1–4). Cells in a communicating population have been demonstrated to share a pool of small ions and metabolites, such as nucleotides, amino acids, oligosaccharides, and second messengers (see Ref.6). Thus, gap junctions may increase the efficiency of cellular hormone responses by increasing the capacity for intercellular communication of material and facilitating amplification of hormone signals (1, 2, 3, 4, 5).

The method by which cells in a tissue regulate signaling to one another, to coordinate growth or function, is currently unknown. Gap junctions are thought to be ideally suited, however, for the direct passage of growth-controlling information through cell populations (6, 7, 8). Large numbers of gap junction plaques have been demonstrated in the adrenal gland with freeze fracture techniques (1, 9), thin section electron microscopy (1, 9, 10), and recently, with immunocytochemical localization (11, 12, 13, 14, 15). Furthermore, an inverse relationship between gap junction distribution and the proliferation rate in adrenal cortical zones has been reported (11, 12, 13, 14). In studies which used immunocytochemical techniques, the outer cortical zone (zona glomerulosa) had few gap junctions and greater proliferation rates than the inner two cortical zones (zona fasiculata and zona reticularis), which had an abundance of gap junctions. A decrease in ACTH-induced steroidogenesis was demonstrated when gap junction communication was inhibited in bovine adrenal cell populations (4, 5). This supported the suggestion that gap junctions play a role in adrenal function. Furthermore, the variation in gap junction size and distribution in adrenal tissue suggests that these structures may play a pivotal role in adrenal gland function (1, 9, 11, 12, 13, 14, 15). Nevertheless, relationships between adrenal cell-cell communication, morphology, and function are poorly understood. Hormonally stimulated interactions between adrenal cells may regulate glandular differentiation and development, perhaps by controlling proliferation rates. In this study, the role of gap junctions in changes in adrenocortical cell number and steroid production was studied in a bovine cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adrenal cells
Bovine adrenal cortical cells (SBAC) were purchased from ATCC and grown in Ham’s F-12 media supplemented with 10% FCS, 50 ng/ml basic fibroblast growth factor (Becton-Dickinson, Franklin, NJ), 200 U/ml penicillin, 200 µg/ml streptomycin, and 5 µg/ml fungizone (amphotericin B). This media subsequently will be referred to as complete F-12 media. Cells were fed every 2 or 3 days and incubated at 37 C with 5% CO2. Treatments included 40 mU/ml ACTH (Sigma, St. Louis, MO), 1.0 mM N6,2'-O-dibutyryl cAMP (DbcAMP, Sigma), or diluent.

Complementary DNA (cDNA) vectors
Purified rat antisense {alpha}1 cDNA HE9 fragments (residues 180-1864, 1.68-kb) were obtained as gifts from Drs. Norton B. Gilula and Nalin Kumar, as part of an ongoing collaboration. A pNUT vector, containing the rat {alpha}1-antisense cDNA HE9 fragment and encoding neomycin resistance, was then constructed (16).

Stable transfection
A Lipofectamine (GIBCO BRL, Grand Island, NY) transfection procedure was used to transfect the cell populations (17). To suppress gap junction expression, SBAC cells were cotransfected with the pNUT {alpha}1-antisense vector and with the pNUT Neo vector containing G418 (Geneticin, GIBCO BRL) resistance genes. Control populations were transfected with pNUT Neo vector only. Cells were selected for 1 week in F-12 complete media containing 400 µg/ml G418. The transfected cells were then maintained in Ham’s F-12 complete media with 300 µg/ml G418. Cells were fed every 2 to 3 days and incubated at 37 C with 5% CO2. Changes in cell function and gap junction expression, measured in {alpha}1-antisense-transfected cells, were compared with control cells transfected with pNUT Neo vector only, thus allowing for a control of variables related to the transfection procedure alone.

Antibody description
Affinity-purified polyclonal rabbit antibodies (IgG) were gifts from Dr. Norton B. Gilula and Dr. Nalin Kumar. Preparation and characterization of these antibodies have been previously described (18). These antibodies were prepared against synthetic peptides corresponding to cytoplasmic or extracellular domains of six different gap junction proteins. The following antibodies were used: {alpha}1 gap junction cDNA coding for 43,000 Mr protein (19), ß1 gap junction cDNA coding for 32,000 Mr protein (20), ß2 gap junction cDNA coding for 26,000 Mr protein (21), ß3 gap junction cDNA coding for 31,000 protein (22), {alpha}4 coding for 37,000 Mr protein (23), and {alpha}3 coding for 46,000 Mr protein (24).

Immunocytochemistry
SBAC cells were seeded at 1 x 105 cells/cm2 onto sterile coverslips and treated with ACTH (40 mU/ml), DbcAMP (1.0 mM), or diluent. The cells were fixed in 3% formaldehyde for 20 min at room temperature and permeabilized in anhydrous acetone for 7 min at -20 C. Cells were then incubated at 37 C for 60 min in primary antibody (rabbit IgG diluted 1:100 in PBS) or preimmune serum. After washing off the primary antibody with PBS, cells were incubated in secondary antibody (Cy3 conjugated goat antirabbit IgG; Jackson Immunoresearch Laboratories, West Grove, PA) for 45 min at 37 C.

Coverslips were washed thoroughly in PBS and mounted onto glass slides with a drop of Fluoromount-G anti-quench mounting media (Southern Biotechnical Lab, Birmingham, AL). Immunolabeling was viewed and photographed with Olympus IMT2 or Nikon Microphot FXA fluorescence phase microscopes (Olympus Optical Co., Ltd., Tokyo, Japan; and Fryer Co., Inc., Pittsburgh, PA). All photographs were taken with Kodak T Max 400 black and white film (Eastman Kodak, Rochester, NY).

Image analysis of gap junctions
Gap junction number, size, and distribution in adrenal cell populations were characterized with Olympus IMT2 or Nikon Microphot FXA fluorescence phase microscopes interfaced to an Optimas Image Analysis program run on an IBM PS2 computer. Statistical analysis between means was calculated by ANOVA followed by a Student’s t test, or by the Student’s t test alone. The data are expressed as: mean ± the SE of the mean (SEM). A value of P <= 0.05 was considered significant.

PCR analysis
cDNA probes (rat {alpha}1, human ß1, mouse ß2; Table 1Go) used in PCR analysis of adrenal samples were previously characterized (25, 26, 27) and obtained from Dr. Norton B. Gilula and Dr. Nalin Kumar as part of a collaboration project. The PCR protocol for detection of {alpha}1, ß1, and ß2 has been previously described (28). Adrenal tissues were pulverized in liquid nitrogen, homogenized in buffer, and sedimented by ultracentrifugation. One microgram of total RNA was transcribed into cDNA with oligo deoxythymidines, random primers, and MuMLV RT (GIBCO-BRL). The same amount of RNA was incubated in an enzyme-free control mixture. After incubation for 1 h at 37 C, MuMLV RT was inactivated by heating to 94 C for 3 min.


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  		Table 1. cDNA probes used in PCR analysis of adrenal samples

 
Taq polymerase (Promega, Madison WI), sense and antisense primers (final concentrations of 0.5–1 µM), 0.2 M 2'-deoxynucleoside 5'-triphosphates (dNTP), 2 mM MgCl2, and PCR buffer (50 mM KCl, 0.1% Triton X-100, 10 mM Tris, pH 8.4) were then added to the samples. Amplification reactions were performed with a Perkin-Elmer-Cetus Thermocycler [for human {alpha}1, ß1, and ß2 primers: 35 cycles, 20 sec at -95 C, followed by 1 min at -60 C; for mouse/rat {alpha}1 primers: 35 cycles, 1 min at -92 C, followed by 1 min at -55 C and 2 min at -72 C; for mouse/rat ß1 and ß2 primers: 35 cycles, 1 min at -92 C, followed by 1 min at -50 C and 2 min at -72 C].

Reaction products were separated by electrophoresis on an agarose gel, transferred to a nylon membrane (0.2-µm pore size), and hybridized overnight at 37 C with respective cDNA probes in 50% formamide, 5x SSPE, 5x Denhardt’s, 100 µg/ml yeast RNA. Blots were subjected to autoradiography at -70 C using Kodak XAR-5 film with an intensifying screen. After incubation membranes were washed in 2x SSC and 0.1% SDS once (20 min) at 37 C and twice (20 min) at 55 C.

Cell number analysis
The number of viable cells was determined by Trypan Blue dye exclusion cell counting techniques after trypsinization (29). The data were analyzed with the Student’s t test and expressed as: average number of cells x 104 ± SEM. A level of P <= 0.05 was considered significant.

Steroid production analysis
Culture media from SBAC populations seeded at 1 x 105 cells/cm2 and treated with ACTH (40 mU/ml), DbcAMP (1.0 mM), or diluent was collected and analyzed for {Delta}4, 3 ketosteroid production with a modified procedure of Vernikos-Danellis and colleagues, 1966 (30). Data from the steroid assay was analyzed with the Student’s t test and expressed as: µg steroids/104 cells ± the SE of the mean (SEM). A level of P <= 0.05 was considered significant.

cAMP production analysis
SBAC cells were seeded at 1 x 105 cells/cm2 and treated with ACTH (40 mU/ml), DbcAMP (1.0 mM), or diluent. The cells were harvested and analyzed for cAMP production. The GIBCO-BRL Non-Isotopic Immunoassay cAMP determination system was used for measuring cAMP production (31). Data from the cAMP immunoassay was analyzed with the Student’s t test and expressed as: pmol cAMP/103 cells ± the SE of the mean (SEM). A level of P <= 0.05 was considered significant.

Western blot analysis of gap junction expression
Cells were lysed in 200 µl of 2x Laemmli Sample buffer and boiled for 5, min. The cell lysates were then shaken in a vortex mixer (Eppendorf, Madison, WI) for 15 min at 4 C to shear the DNA. The lysates were cleared of aggregates by the addition of 25 µl CL2B (50% slurry) and centrifuged in a microfuge for 5 min at room temperature. Equivalent protein amounts of each lysate were resolved by SDS-PAGE (10% gels). The gels were then equilibrated in 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid - NaOH buffer, pH 11.0 (CAPS buffer), for 10 min at room temperature before proteins were transferred to Immobilon-P membrane (Millipore; Bedford, MA) for 75 min at 375 mA constant current in CAPS buffer. The Immobilon-P membrane was blocked overnight in 5% BSA dissolved in Dulbecco’s PBS and then incubated with a 1:1000 dilution of polyclonal anti-{alpha}1 connexin-43 (gift from Drs. Gilula and Kumar) or monoclonal mouse anti-{alpha}1 connexin-43 IgG (Transduction Laboratories, Lexington, KY) antibody in 1% dry nonfat milk/PBS for 120 min at room temperature. Unbound primary antibody was removed by three 15-min washes in Tris-buffered saline with Tween (25 mM Tris, pH 8.0, 500 mM NaCl, 25 mM KCl, 0.05% [wt/vol) Tween-20) and three washes with PBS. The Immobilon-P membrane was then incubated 60 min at room temperature with goat-antirabbit-horseradish peroxidase (Jackson Immunoresearch Laboratories) diluted 1:25,000 in 1% dry nonfat milk/PBS. After three 15-min washes in Tris-buffered saline with Tween, connexin was detected using the SuperSignal chemiluminescent detection system (Pierce, Rockford, IL), following the protocol described by the manufacturer. A "CH" chemiluminescent detection screen (BioRad, Hercules, CA) was exposed to the Immobilon membrane and connexin protein quantified using a GS525 phosphorimager (BioRad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gap junction expression and cell function
To characterize gap junction family type in SBAC populations, PCR (Fig. 1Go, A–C), Western blot (Fig. 1Go, D–F), and immunocytochemical analyses were performed (Fig. 2Go). PCR analysis revealed the presence of an abundant amount of {alpha}1, but only trace amounts of ß2, in SBAC populations (Fig. 1Go, A and C). The transcript for ß1 was not detected in the SBAC cell cultures (Fig. 1BGo). A protein consistent with {alpha}1 was detected with Western blot and immunocytochemical analysis (Figs. 1Go, D and E). Other connexin species were not detected by either immunohistochemistry or Western blot analysis (data not shown). The amount of {alpha}1 gap junction protein expression, as measured by phosphorimaging of populations prepared with Western blotting, increased 38% and 42% after 24 h ACTH (40 mU/ml) or DbcAMP (1 mM) treatment (Fig. 1FGo), respectively.



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  		Figure 1. A–C, PCR analysis of SBAC populations for the presence of {alpha}1 (Cx43) (A), ß1 (Cx32) (B), and ß2 (Cx26) (C) mRNA. PCR analysis was performed as described in Materials and Methods. mRNA from sterile water (lane 1), mouse genomic DNA (lane 2), mouse heart in the presence (lane 3) or absence (lane 4) of RT enzyme, mouse liver in the presence (lane 5) or absence (lane 6) of RT enzyme, SBAC in the presence (lane 7) or absence (lane 8) of RT enzyme. {alpha}1 and ß2 mRNA were detected, but not ß1, in SBAC populations. D, Western blot analysis of rat heart (lane 1), rat liver (lane 2), rat adrenal gland (lane 3), and SBAC populations (lanes 4–6). The membrane was probed with monoclonal mouse anti-{alpha}1 connexin-43 IgG (Transduction Laboratories). Note that {alpha}1 (Cx43) protein products were detected in SBAC populations. E, Western blot analysis of the Immobilon-P membrane probed with mouse anti-{alpha}1 connexin-43 IgG polyclonal anti-{alpha}1 connexin-43 (gift from Drs. Gilula and Kumar). Control SBAC (nontransfected) (lane 1), SBAC cells transfected with the control vector(pNUT) (lane 2), pNUT {alpha}1-antisense transfected SBAC (pNUT {alpha}1-antisense) (lanes 3 and 4), ACTH-treated (24 h) nontransfected SBAC population (lanes 5 and 6), DbcAMP treated (24 h) nontransfected SBAC population (lanes 7 and 8). Note the presence of {alpha}1 (Cx43) protein product in nontransfected and control-transfected SBAC Cells. There is a notable absence of {alpha}1 (Cx43) protein product in {alpha}1-antisense-transfected SBAC populations. F, Phosphorimage analysis of Western blot of {alpha}1 (Cx43) expression. This table presents the quantitation of {alpha}1 (Cx43) expression in transfected and nontransfected SBAC populations. Cells were stably transfected with pNUT (controls) or {alpha}1-antisense or nontransfected cells were treated 24 h, with or without ACTH (40 mU/ml) or DbcAMP (1 mM). The populations were processed using the procedures described in Materials and Methods. Data from Western blot analysis was quantified in a phosphorimager. Values are expressed as mean % ± SEM {alpha}1 protein expression change from respective control populations. {alpha}1-antisense transfected ({alpha}1 (Cx43)AS) compared with pNUT transfected control, nontransfected ACTH treated compared with ACTH nontreated, nontransfected DbcAMP treated compared with non-DbcAMP treated).

 


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  		Figure 2. Immunocytochemical localization of {alpha}1 (connexin 43) gap junction antigen in SBAC populations. Cell populations were cultured for 1 day (A, D, and G), 3 days (B, E, and H), or 6 days (C, F, and I) in the presence or absence of ACTH (40 mU/ml; D, E, and F), DbcAMP (1 mM; G, H, and I) or diluent (A, B, and C). Note the areas of punctate fluorescence, indicative of {alpha}1 gap junction antigen at sites of cell-cell contact. Gap junction plaques increased with culture time and after stimulation with either ACTH (E and F) or DbcAMP (H and I). Bar = 5 µm.

 
To further characterize the changes in {alpha}1 gap junction protein expression seen with Western blotting after hormonal stimulation, immunocytochemical analysis was performed. SBAC cells prepared for immunocytochemistry had {alpha}1 gap junction plaques at points of cell-cell contact and within the cytoplasm of some cells (Fig. 2Go). Gap junction number increased with time, after seeding, whereas both gap junction size and number increased after ACTH or DbcAMP treatment (Figs. 2Go and 3Go).



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  		Figure 3. Average number (A) and size (B) of gap junctions in SBAC populations. Both ACTH (40 mU/ml) and DbcAMP (1.0 mM) treatment for 24 h increased the average gap junction number per cell and size, compared with control populations. a, P < 0.01, compared with respective treatment group on day 1; b, P < 0.01, compared with non-ACTH or DbcAMP-stimulated (control) cultures on day 6; c, P < 0.01, compared with non-ACTH or DbcAMP-stimulated cultures on day 3.

 
Image analysis was used to quantitate the changes in gap junction expression observed with immunocytochemical analysis on days 1, 3, and 6, after seeding. In control populations, the number of gap junction plaques per cell doubled after 3 days in culture (Fig. 3AGo). The average number of gap junctions did not, however, change significantly from day 3 to day 6 (Fig. 3AGo). In contrast to these changes in number, the average gap junction plaque size in control populations did not significantly change from days 1–6 (Fig. 3BGo).

Treatment with ACTH (40 mU/ml) or DbcAMP (1 mM), however, approximately doubled the average number of gap junction plaques per cell, compared with control populations on day 6. In control, ACTH- and DbcAMP-treated populations, gap junction number increased significantly between days 1 and 3 of treatment. As can be seen in Fig. 3BGo, the average gap junction plaque size was increased significantly above control levels on days 3 and 6 after DbcAMP treatment. Gap junction size increased with ACTH treatment by day 6. There was no significant difference between ACTH and DbcAMP treatment at any of the time points measured.

To analyze the effects of gap junction inhibition on SBAC cell functions, cell populations were cotransfected with pNUT {alpha}1-antisense and pNUT Neo vectors and compared with cells containing pNUT Neo vectors only (vector transfection control populations). The morphological appearance of the transfected cells was indistinguishable from that of untransfected SBAC cells, with phase microscopy (Fig. 4Go). Immunocytochemical techniques were used to analyze gap junction protein expression in the transfected SBAC populations. SBAC cells transfected with {alpha}1-antisense had very few {alpha}1 gap junction plaques, compared with those transfected with control vector only (Fig. 5Go). Few gap junction plaques were observed at sights of cell contact, and very little (if any) punctate staining was observed in the cytoplasm of these cells. Similarly, {alpha}1-antisense-transfected SBAC populations showed no band at MW 43 kDa with Western blot analysis (1E, lanes 3 and 4), whereas the control pNUT Neo-transfected populations did (1E, lane 2). Quantitative Western blot analysis confirmed that {alpha}1 expression was decreased by 94% in cells transfected with antisense, compared with control (pNUT Neo-transfected) populations (Fig. 1FGo).



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  		Figure 4. Phase micrographs of nontransfected (A), pNUT Neo-transfected (B), and {alpha}1-antisense-transfected (C) SBAC populations.

 


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  		Figure 5. Gap junction expression in transfected SBAC populations. SBAC cells transfected with pNUT {alpha}1-antisense (B) demonstrated significantly fewer {alpha}1 gap junction plaques at points of cell-cell contact than SBAC populations transfected with pNUT Neo only (A). Bar = 3 µm.

 
Image analysis was used to quantitate the number and size of gap junctions in transfected cell populations observed with immunocytochemical techniques on days 1 and 6 (Fig. 6Go). After {alpha}1-antisense treatment, the number of gap junctions was reduced to 10% of that in cells transfected with the control vector. The average size of the few gap junctions present in {alpha}1-antisense-containing cells tended to be smaller than those in vector control populations. These size differences, however, failed to reach levels of significance. Neither treatment with ACTH nor DbcAMP resulted in significant changes in gap junction number or size in {alpha}1-antisense-transfected cells (Fig. 6Go). In contrast, control-transfected cells, like nontransfected cells, significantly increased gap junction number and size after ACTH or DbcAMP treatment (Fig. 6Go). Overall, the average size and number of junctions in SBAC populations, following transfection procedures, were decreased (compared with nontransfected cultures).



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  		Figure 6. Average gap junction number (A) and size (B) in hormonally treated transfected cell populations. Although ACTH (40 mU/ml) and DbcAMP (1 mM) treatment for 6 days significantly increased the average number (A) and size (B) of gap junctions in cells transfected with the control vector (pNUT), they did not significantly change gap junction expression in {alpha}1-antisense containing SBAC populations (AS). x, P < 0.01, compared with control.

 
Changes in cell number
The number of SBAC cells in control population increased steadily from day 1 to day 8 (Fig. 7AGo). SBAC populations in which gap junction expression was suppressed by transfection with {alpha}1-antisense grew faster than control vector-transfected populations (Fig. 7Go, B and C). Whereas treatment with ACTH (40 mU/ml) or DbcAMP (1 mM) significantly decreased the number of cells in control vector-transfected and nontransfected cell populations, {alpha}1-antisense-transfected cells did not decrease cell number in response to hormone treatment (Fig. 7CGo). In fact, there is a slight increase in the number of cells in ACTH- and DbcAMP-treated antisense-containing cultures.



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  		Figure 7. Cell numbers. ACTH (40 mU/ml) and DbcAMP (1.0 mM) treatment resulted in fewer numbers of cells in the nontransfected SBAC populations (A). {alpha}1-antisense-transfected SBAC populations (AS) contained significantly more cells than SBAC populations transfected with the control vector only (B). ACTH (40 mU/ml) and DbcAMP (1.0 mM) treatment did not result in a smaller population number in {alpha}1-antisense-transfected cells (C).

 
As can be seen in Fig. 7AGo, the greatest effect of ACTH or DbcAMP on cell number was observed between days 2 and 4 of treatment in cells containing gap junctions. At day 8, the ACTH and DbcAMP cultures remained relatively sparse, whereas antisense-transfected cell populations were confluent.

Steroidogenesis
Not only were the accumulation of cells reduced in response to ACTH treatment in populations in which {alpha}1 gap junction expression had been eliminated, but also the steroidogenic capacity of cells was decreased. Both ACTH (40 mU/ml) and DbcAMP (l mM) treatment significantly increased steroid production per cell, compared with unstimulated populations (Fig. 8AGo). ACTH- and DbcAMP-treated cells produced two to three times as much steroid as control populations (Fig. 8AGo). On the other hand, {alpha}1-antisense-transfected SBAC did not significantly increase steroid production after ACTH or DbcAMP treatment. The basal levels of steroids per cell in nonhormone-stimulated control and antisense-transfected SBAC populations were not significantly different (Fig. 8Go).



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  		Figure 8. Steroid production. SBAC populations treated with either ACTH (40 mU/ml) or DbcAMP (1.0 mM) for 24 h produced more steroids per cell than control populations (A) but did not significantly increase steroid production in {alpha}1-antisense containing SBAC cells (B). x, P< 0.01, compared with control.

 
cAMP production
ACTH treatment brings about its effect by activation of cAMP and the subsequent dissociation of cAMP-dependent protein kinase holoenzyme into regulatory and catalytic subunits (32). Because the {alpha}1-antisense-transfected cells no longer responded to ACTH with either increased steroid synthesis or inhibition of population growth, as in control populations, it was necessary to characterize the capacity of transfected cells to generate cAMP in response to hormone.

ACTH treatment resulted in an increase in cAMP levels in nontransfected cell populations (Fig. 9AGo). ACTH treatment of {alpha}1-antisense-transfected cells slightly increased cAMP levels, compared with non-ACTH treated {alpha}1-antisense-transfected cells on day 1. On day 6, hormonal treatment did not significantly alter cAMP production (Fig. 9BGo) in cells in which gap junctions were inhibited. The level of cAMP decreased in all transfected cell populations, with culture time, after seeding. Furthermore, the basal level of cAMP in transfected cell populations was decreased, compared with nontransfected populations. Cells containing the antisense had the least amount of cAMP.



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  		Figure 9. cAMP production. SBAC populations treated with ACTH (40 mU/ml) produced significantly more cAMP than control populations (A). ACTH (40 mU/ml) stimulation significantly increased cAMP production in {alpha}1-antisense containing SBAC (AS), compared with non-ACTH treated {alpha}1-antisense-transfected cells, on day 1 but not in cultures treated for 6 days (B). x, P< 0.01, compared with non-ACTH treated cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results presented from gap junctional analyses indicate that {alpha}1 (Cx43) gap junction expression in SBAC cells is dynamic. Of the connexins studied, {alpha}1 was the most abundant in SBAC populations and observed on the surface of cells at points of cell contact. Bands corresponding to {alpha}1 (Cx43), and a weaker band corresponding to ß2 (Cx26) messenger RNA (mRNA), were found with PCR analysis. However, immunocytochemistry revealed only the presence of {alpha}1 (Cx43). Similarly, {alpha}1 (Cx43) protein product was detected with Western blot analysis.

Although {alpha}1 (Cx43) was detected in intact bovine adrenal glands (14), this is the first report demonstrating connexin type and behavior of adrenal cortical cells in culture. This is also the first demonstration of changes in adrenal cell function after incorporation of antisense cDNA directed against gap junction transcript. It was demonstrated with immunocytochemical analysis that the number of {alpha}1 gap junction plaques at points of cell-cell contact increased with time, after seeding. Moreover, gap junction number and size increased after stimulation with either ACTH or DbcAMP. The increased gap junction expression, after ACTH treatment, also was noted by Western blot analysis.

These results of dynamic connexin expression are consistent with past freeze fracture findings of an increased gap junction plaque number between adjacent adrenal cells in primary culture (33, 34) and in Y-1 adrenal tumor cell populations treated for 1 day with ACTH (3).

The effects of DbcAMP treatment on the size and number of gap junctions are similar and parallel to the changes induced by ACTH treatment. The DbcAMP-stimulated changes in gap junction number and size, however, were slightly higher than changes seen after ACTH treatment. The greater effects of DbcAMP treatment on gap junction expression, compared with ACTH, may reflect the absence of ACTH receptors in some cells of the population. In this case, more cells in the DbcAMP-treated population could respond than in ACTH-treated cell populations, because some cells may have been ACTH nonresponsive. Future studies directed at determining receptor number and hormone binding would disprove or confirm this theory. Based on the observed ability of DbcAMP to mimic the ACTH effect on gap junction number and size, we propose that ACTH actions in the SBAC are mediated by cAMP-dependent mechanisms, presumably via a protein kinase A (pKA) activation and catalytic subunit protein phosphorylation.

In several other cell types in culture, an increase in gap junction expression has been correlated to peptide hormone treatment and elevation of cAMP levels (35, 36). For example, In’t Veld and colleagues (37) demonstrated an increase in the total number of gap junctional plaques, measured with freeze fracture techniques, when pancreatic islet tissue was cultured with DbcAMP. Also, FSH treatment increased gap junctional membrane in intact and cultured granulosa cells (38). A decreased proliferation rate and a corresponding increase in the average size and fractional area of gap junctions and forming gap junctions (formation plaque) have been quantitated after DbcAMP treatment in human adrenal tumor cells (39). From these data, it is suggested that elevations in cellular cAMP levels correlate with observed increases in gap junction number and size and decreases in cell proliferation rate in adrenal tumor cells (SW-13).

Not only have increases in gap junction number and size been reported, but also cAMP-mediated changes in gap junctional communication have been described (40, 41). Electrical conductivity (40) and dye transfer (41) increased in cardiac myocytes after cAMP injection. In hepatocytes, increased junctional conductance occurred within minutes of glucagon or cAMP analog treatment (42). Treatment with 8-bromo-cAMP resulted in increased {alpha}1 (Cx43) gap junctional distribution and permeability in rat hepatocyte, granulosa, coronary venular endothelial cells, and primary rat myometrial cells (43).

In further support of cAMP-mediated effects on gap junction expression, overexpression of exogenous gap junctions, by introducing {alpha}1 (Cx43) gap junction cDNAs into the Morris Hepatoma cell by DNA transfection or by retroviral mediated gene transfer, did not induce communication unless cAMP levels were increased (43). Injection of the catalytic subunits of pKA into Mauthner cells increased electrical coupling (44), further support that pKA is the effector of the cAMP effect on gap junctions.

The mechanism by which increased cAMP levels result in an increase in gap junction expression remains unclear. Possibly, pKA activation results in phosphorylation of proteins involved in gap junction insertion into the plasma membrane, or an increase in the production of gap junction proteins, and/or a preservation of existing gap junctions (42, 43, 45). In addition, cAMP may regulate gap junctions directly at the channel by controlling the pore size (43).

Our findings of increased gap junction expression, after peptide hormone treatment and subsequent cAMP elevation, are consistent with an increased need or capacity for communication in hormonally stimulated populations. Such increases in communication may facilitate proper hormonal response, as well as regulate proliferation.

In support of the role of gap junctions in regulation of adrenal cell proliferation, after transfection with {alpha}1-antisense cDNA, the SBAC populations grew faster than transfected controls but no longer had ACTH- or DbcAMP-induced suppression of cell population growth. In addition, the ACTH- and DbcAMP-induced increases in gap junction number and size observed in nontransfected populations were no longer detected in {alpha}1-antisense-transfected SBAC populations.

In this study, both ACTH and DbcAMP treatment of cells possessing gap junctions resulted in increased steroid production but not in gap junction-inhibited populations. These results are consistent with the idea that gap junction-mediated communication may play a pivotal role in adrenal steroidogenesis. The more numerous and large the gap junction plaques, the greater the ACTH- and DbcAMP-induced increases in steroidogenesis. This finding is reasonable because gap junctions are known to transfer second-messenger molecules (2, 46, 47), as well as other regulatory substances (3, 6, 48, 49) between cells. Thus, the larger and more numerous the junctions between SBAC cells, the more signaling molecules that could pass between them, amplifying the response within the population. This would be particularly useful in a population of cells with heterogeneity of ACTH-receptor number, binding capacity, or variations in factors needed for steroid production and growth regulation. Increased ACTH-mediated responses could occur in such populations by both the direct effect of hormone on the membrane receptors and/or by intercellular movement of information between cells. Likewise, DbcAMP treatment resulting in increased gap junction communication capacity may allow for the movement of regulatory molecules between contacting cells that would participate in growth regulation and control of steroid production within the population. In support of an amplification of hormone effect related to gap junction communication, Munari-Silem and colleagues (4, 5) found that the concentration of ACTH required to obtain the half-maximum cortisol production varied, depending on the level of cell-to-cell coupling within the cell population. They suggested that these results provide at least one explanation for the known discrepancy between the kDa value for the interaction of ACTH with its receptor and the ACTH concentrations active in steroidogenesis, both under physiological conditions (50, 51) and in Cushing’s disease (52).

Both transfected and nontransfected SBAC cells in our study significantly increased cAMP levels after stimulation with ACTH. In transfected populations, however, increases in cAMP levels after stimulation with ACTH were smaller in magnitude than those in nontransfected populations. This suggests that the inability of {alpha}1-antisense-transfected cells to evoke significant ACTH-mediated responses (increased gap junction number, size, and possible decreased proliferation rate and increased steroidogenesis) may be caused by insufficient quantities of cAMP being generated after ACTH treatment. It is also possible that the defect in the hormone-second messenger-cell response pathway may be post-cAMP activation.

In summary, SBAC populations in which gap junction expression was inhibited grew more rapidly and were less sensitive to hormone stimulation, compared with transfected control cells which expressed gap junctions. Hormone stimulated-intercellular communication may therefore represent an important factor in adrenal function. Communication of information molecules via gap junction also may increase the efficiency of hormone response by facilitating the amplification of pKA-mediated hormone signaling (2, 4, 42) and other important molecule communication. Moreover, intercellular communication via gap junctions could control adrenal gland function by regulating cellular concentrations of, or subcellular distributions of, regulatory molecules. Future studies are in progress to elucidate the relationship between hormone stimulation, hormone binding, signal transduction, gap junction expression, and endocrine tissue function.


   Acknowledgments
 
The expert assistance of Ambra Pozzi and Stacey Pharrams is gratefully acknowledged.


   Footnotes
 
1 This research was supported by NSF Grant MCB-9514285. Back

Received April 14, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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