13 Gap junction proteins and cell–cell communication in the three functional zones of the

K T Davis, N Prentice,VLGayand S A Murray Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA (Requests for offprints should be addressed to S A Murray; Email: [email protected])

Abstract Mouse and monkey adrenal glands were used to study the yellow dye communication was most pronounced in the relationships between gap junction protein expression, inner /androgen-producing regions (zona intercellular communication and adrenal zonation. Dye fasciculata/), but was virtually absent in the communication patterns were determined by incubat- outer mainly -producing region (zona ing freshly excised and hemisected adrenal glands in glomerulosa). This pattern of dye communication was Lucifer yellow, a gap junction permeable fluorescent dye. coincident with immunohistochemical localization of the Immunohistochemical techniques were used to localize gap junction protein, 1Cx43. The variations in com- adrenal gap junction proteins. The combination of these munication and 1Cx43 expression within the adrenal two techniques permitted the correlation of gap junc- cortex are thought to be relevant to normal physiological tion proteins with dye transfer and hormone responses regulation of the adrenal gland. in specialized regions of the . Lucifer Journal of (2002) 173, 13–21

Introduction steroidogenesis in adrenal cell cultures (Decker 1976, Decker et al. 1978, Oyoyo et al. 1997, Shah & Murray It has been amply demonstrated that gap junctions provide 2001). pathways for the direct intercellular exchange of small As outlined above, direct evidence of cell–cell com- molecules (Goodenough 1978, Trautmann 1988) and munication in the adrenal gland, as in most organs, has it has been postulated that these small molecules include been limited to cells in culture and has not dealt with the ‘signals’ that modulate growth, differentiation and func- fact that, in the intact gland, adrenal cortical cells are tion in recipient cells (Larsen et al. 1977, Loewenstein segregated into separate zones with distinct functions 1979, Hellmann et al. 1999, Trosko et al. 2000). In (McNicol 1992). This unique zonation of the adrenal the case of the adrenal gland, it has been proposed gland has allowed us to demonstrate a differential distri- that such communication also facilitates and amplifies bution of gap junctions corresponding to differing func- hormonally stimulated cell responses (Decker et al. 1978, tions. Cells in the outermost and more rapidly growing Murray & Fletcher 1984, Usadel et al. 1993, Murray adrenal cortical zone, the zona glomerulosa, produce et al. 1995a, Munari-Silem & Rousset 1996, Oyoyo et al. (Malendowicz et al. 2001) but have few or no 1997). gap junctions (Murray et al. 1995a,b). Cells in the inner As demonstrated in vitro, procedures which compromise two zones, the zonae fasciculata and reticularis produce gap junction function consistently decrease adrenocortico- cortisol and sex hormones respectively (McNicol 1992), tropin (ACTH)-induced steroidogenesis (Oyoyo et al. and both the zones exhibit numerous gap junctions 1997). This is true for bovine adrenal cells either trans- (Murray et al. 1995a). Thus, although all three adrenal fected with 1Cx43 antisense cDNA or treated with the cortical zones may have ACTH receptors (Xia & Wikberg gap junction communication inhibitor, 18- glycerrhe- 1996, Chorvatova et al. 1999, Kau et al. 1999), cells with tinic acid (Munari-Silem et al. 1995, Shah & Murray abundant gap junctions (zonae fasciculata and reticularis) 2001) and also applies to a mouse adrenal tumor cell line are those which are most responsive to ACTH, while cells maintained in culture in which loss of gap junction which lack gap junctions (zona glomerulosa) are charac- functionality parallels loss of ACTH-stimulated steroid terized by their vigorous response to II and to production (Decker et al. 1978). Conversely, ACTH a lesser degree Na+,K+ and ACTH (Biglieri et al. 1987, treatment increases both gap junction protein number and McNicol 1992, Rocco et al. 1994).

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The clear morphological, architectural and functional adrenal preparations were assayed. Following the dye differences between adrenal cortical zones have permitted communication assay, the adrenal tissues were fixed in us to undertake studies designed to demonstrate the 4% paraformaldehyde for 1–2 h at room temperature, distribution of gap junctions as they relate to functional embedded in optimum cutting temperature tissue freezing communication within this organ. We demonstrate a medium (Triangle Biomedical Sciences, Durham, NC, close correlation between patterns of dye communication USA), flash frozen in liquid nitrogen, and stored at –80 C. and 1Cx43 gap junction distribution within the mouse Adrenal gland sections (8–10 µm thick) were cut and adrenal gland and thus confirm our hypothesis that dye collected on glass slides in preparation for analysis of communication would be limited in the zona glomerulosa, Lucifer yellow dye communication (Tsien & Weingart where very few 1Cx43 gap junctions were found. We 1976), gap junction immunohistochemistry (Oyoyo et al. also conclude that other members of the gap junction 1997) or lipid content determination by Oil Red O family of proteins either are not present in the zona staining (Troyer 1980). glomerulosa or are not functional under these conditions. As predicted from the results of our previous studies, we now report that in the freshly excised mouse adrenal gland, Gap junction antibodies and probes morphological structures (gap junction number) can be Affinity-purified polyclonal rabbit antibodies (IgG) closely correlated with function (extensive dye communi- were gifts from Drs Norton B Gilula and Nalin Kumar. cation and ACTH-stimulated response). In addition, we Preparation and characterization of these antibodies have present data on cell–cell communication of Lucifer yellow been described previously (Kumar & Gilula 1992). These dye between cells in the three zones of the adrenal gland antibodies were prepared against synthetic peptides corre- of the rhesus monkey (Macaca mulatta) which further sponding to the cytoplasmic domains of 1Cx43 peptide validates that our findings occur in species other than the extending from residue 370 to 381 and corresponding to mouse. the carboxyl terminus of the 1Cx43.

Immunohistochemistry of gap junction protein Materials and Methods The tissues in this study were prepared and stained Animals for connexin immunohistochemical analysis as previously described (Murray et al. 1995a, Oyoyo et al. 1997). Frozen Adult female C57Bl6 mice were obtained from the sections were collected on gelatinized slides and incubated Charles Rivers Laboratory (Wilmington, MA, USA). for 5 min in PBS before the addition of a blocking solution Adult ovariectomized female rhesus monkeys (Macaca (3% bovine serum albumin (BSA), 3% normal goat serum mulatta) were obtained from the Center for Research (Sigma-Aldrich, St Louis, MO, USA) in PBS). The in Reproductive Physiology, University of Pittsburgh 1Cx43 antibody or pre-immune rabbit IgG (5–10 µg/ml (Pittsburgh, PA, USA). The mice were fed Purina labora- in 3% normal goat serum with 3% BSA in PBS) was added tory chow and the monkeys were fed Purina monkey for 1 h. After rinsing with PBS, the sections were incu- chow (Ralston Purina, St Louis, MO, USA). Water was bated in secondary antibody (Cy3-conjugated goat anti- provided ad libitum. Animals were weighed, killed, and the rabbit IgG; Jackson Immunoresearch Laboratories, West adrenal glands were harvested. Grove, PA, USA) for 45 min at 37 C. Sections were washed thoroughly in PBS, stained with Hoechst nuclear dye and mounted in Fluoromount-G anti-quench Adrenal gland dye coupling assay mounting media (Southern Biotechnical Laboratory, Lucifer yellow dye loading for demonstration of cell– Birmingham, AL, USA). Gap junction distribution in cell coupling was performed with a modified procedure adrenal glands was visualized with a Zeiss-LSM confocal (Tsien & Weingart 1976). The adrenal glands were microscope or Nikon Microphot FXA fluorescence phase quickly removed from the animals, cut into segments and microscopes. Images were collected from the micro- incubated in 0·5% Lucifer yellow CH (Molecular Probes, scope interfaced to an Optimus Image Analysis (Media Eugene, OR, USA, or Sigma, St Louis, MO, USA) in Cybernetics, Silver Springs, MD, USA) program run on phosphate-buffered saline (PBS) (pH 7·4) and/or in 0·5% an IBM computer. rhodamine dextran at 37 C for 10 min. In some studies, the excised adrenal gland segments were preincubated in Histochemistry oleamide (100–200 µM), a gap junction communication inhibitor molecule (Boger et al. 1998) for 5 min. The For light microscopy, the tissue was sectioned on a cryostat tissues were then incubated for 10 min in 0·5% Lucifer (Minotome; International Equipment Co., Boston, MA, yellow assay mixture containing oleamide. Fourteen half USA). Sections were collected on gelatinized slides and gland, nine oleamide-treated and three whole gland stained with either hematoxylin–eosin or Oil Red O

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Figure 1 Demonstration of dye communication in an adrenal cell culture monolayer. Cells in culture communicate Lucifer yellow dye four to five cells from the cut edge.

Figure 3 Lucifer yellow dye coupling in adrenal cortex. The dye is excluded from the cells of the zona glomerulosa but is evident in the connective tissue capsule (arrowheads) and trabeculae (arrow). Note that numerous individual cells and packages of cells are not coupled to neighboring cells (as indicated by asterisks in B). (A) bar=10 µm; (B) bar=20 µm.

Stereological methods The number of gap junctions was determined by computer-assisted image analysis of the immunolabeled adrenal glands. Gap junction number in immunohisto- chemically prepared samples was expressed either as number of plaques per measured area or as the levels of 1Cx43 fluorescence per measured area. To quantitate the dye within the adrenal cortical zones, computer-assisted image analysis of the Lucifer yellow dye fluorescence was measured with the Optimas program area morphometric function. Statistical comparisons of mean values were carried out with a one-way analysis of variance and Duncan’s multiple comparison procedure. To determine the significance of differences, Student’s two-tailed t-test

Figure 2 Immunohistochemical localization of 1Cx43 gap was also employed. junction antigen (A and C) and Lucifer yellow dye (green in B and D) in two representative mouse adrenal gland sections. Extensive expression of 1Cx43 is observed primarily in the zonae fasciculata/reticularis (ZF/ZR). ZG, zona glomerulosa; arrowheads, Results connective tissue capsule; arrows, trabeculae; blue stain, nucleus. (A and B) bar=50 µm; (C and D) bar=30 µm. Proceeding from the observations that cells in monolayer culture communicate Lucifer yellow dye from the cut edge (Fig. 1), we have modified the technique of Tsien hematoxylin (Troyer 1980). Images were captured with an & Weingart (1976) in order to study the relationship Optimas Image Analysis program interfaced to an IBM between gap junction protein expression and cell–cell computer. communication patterns in intact adrenal tissue from two www.endocrinology.org Journal of Endocrinology (2002) 173, 13–21

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Figure 4 Graphs of Lucifer yellow dye communication (A) and Figure 5 Graph of the average number of 1Cx43 gap junction plaques detected by immunohistochemistry and expressed per 1Cx43 gap junction distribution (B). Fluorescent intensity analyses revealed Lucifer yellow (LY) dye levels that were minimal in the measured area. Note the abundance of gap junctions in the zonae zona glomerulosa (ZG) and maximal in the zonae fasciculata/ fasciculata and reticularis while the zona glomerulosa had virtually reticularis (ZF/ZR). Note the proportionate fluctuations in Lucifer undetectable levels of 1-Cx43. yellow dye transmission and 1-Cx43 fluorescence. INT, interface between the ZT and ZR interface. As anticipated, incubation of excised adrenal glands in species. As assessed in this ‘gland dye coupling’ assay, oleamide, a potent gap junction communication inhibitor Lucifer yellow dye was found to be communicated exten- (Boger et al. 1998), eliminated most appreciable dye sively in the inner two cortical zones (zonae fasciculata and movement except in small foci of tissue injury (Fig. 8). reticularis) (Figs 2–4). A similar distribution of 1Cx43 gap Occasional dye communication could be seen within the junctions was revealed with immunohistochemical analysis inner two adrenal cortical zones of oleamide-incubated (Figs 2, 4 and 5). Remarkably, both dye coupling and adrenal glands (Fig. 8C). Tissue next to injury sites in the 1Cx43 gap junction plaques were virtually absent among presence of oleamide did not contain dye or appeared to cells of the outermost cortex (zona glomerulosa) (Figs 2 contain less dye then that observed in non-oleamide- and 4). At the interface of the and zona treated controls. glomerulosa, we observed cells and/or packets of cells In the adrenal capsule, an abundance of 1Cx43 gap which lacked dye and therefore were not thought to junction plaques was evident, as was the transfer of Lucifer be functionally coupled to other cells in the region (Fig. 3). yellow dye (Figs 2 and 3). However, neither the con- The number of 1Cx43 gap junction plaques was counted nective tissue cells of the capsule nor the penetrating by computer-assisted image analysis and, in addition, the trabeculae appear to communicate dye to cells of the zona extent of the 1Cx43 staining was also measured as a glomerulosa (Fig. 3). Dye in the adrenal connective tissue function of fluorescence intensity per area in order to capsule or penetrating trabeculae was rarely transferred to determine gap junctional distribution. When compared, cells within the cortex, and the zona glomerulosa cells the fluorescence indices of Lucifer yellow dye communi- were consistently excluded from the extensive coupling of cation and that of the 1Cx43 plaque presence were cells in the zona fasciculata. This was most apparent in the almost directly proportional throughout the three cortical monkey adrenal cortex (Fig. 9). Although not shown here, zones (Figs 2 and 4). it should be noted that medullary staining with Lucifer The presence of Lucifer yellow within the ovoid clusters yellow was highly inconsistent and, when present, seemed of zona glomerulosa cells was rare and, when evident, it to be within blood vessels or non-specific staining. was typically confined to a single cell within a given The observed close correlation between the zonal packet of cells (Fig. 3). Although dye was clearly evident distribution of gap junctions and dye communication, at the cut edge of injured zona glomerulosa cells, dye did observed in the mouse, was also evident in the freshly not enter neighboring cells (Figs 6 and 7). Under identical excised monkey adrenal gland (Fig. 9). In the monkey, experimental conditions, extensive dye communication as in the mouse, dye communication was observed occurred between cells of the inner two cortical zones. between cells located in the zonae fasiculata and reticularis.

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adrenal anatomical zonation so critical to secretory func- tion and have permitted all cell types within the adrenal cortex to be exposed to identical conditions of preservation, staining and analysis, thus providing direct comparisons of dye transfer and gap junction expression. A considerable body of evidence has been presented to demonstrate that gap junctions are indeed the conduits for intercellular transfer of small molecules (Goodenough 1975, 1978, Peracchia 1980, Hertzberg et al. 1981, Willecke et al. 1983, TenBroek et al. 1994, Nagy et al. 1996). Most of this evidence comes from in vitro studies of primary cell cultures or cell lines (Murray & Fletcher 1984, Bouille et al. 1991, Baldwin & Calabrese 1994, Munari-Silem et al. 1995, Veenstra 1996, Willecke & Haubrich 1996, Tomai et al. 1999). Because cell functions in cultures of immortalized cell lines are, by definition, significantly altered as are the three-dimensional relation- ships of primary cells in culture compared with the intact gland, assessment of in vivo conditions is often compromised. By utilizing the organ system, we have been able to compare directly the patterns of dye distribution and 1Cx43 gap junction location. We found that there is a regional specificity of dye transfer in the adrenal cortex such that there is little or no dye transfer in the zona glomerulosa and extensive transfer in the inner two zones. Furthermore, although the connective tissue cells have 1Cx43 gap junction plaques and appear to readily com- municate with one another, they lack a comparable communication with the cells of the zona glomerulosa. Figure 6 Lucifer yellow dye coupling in mouse adrenal cortex (A) In the inner zones, the relationship was less clear, given with corresponding Oil Red O-stained section (B). Dye diffused the fact that virtually all cells were clearly labeled and into all damaged cells at the cut edge (indicated by arrows) but communication of dye between connective tissue cells did not transfer to adjacent cells in the zona glomerulosa (ZG). In the inner two zones, however, dye transfer is extensive. or between connective tissue cells and the cortical Bar=50 µm. parenchyma could not be distinguished. The areas of the adrenal gland that we find to efficiently transmit dye and to exhibit numerous 1Cx43 gap junc- However, the zona glomerulosa cells neither communicate tions are characterized by their production of glucocorti- with one another nor with cells of the connective tissue coids and androgens in response to ACTH (Garren et al. capsule (Fig. 9). Occasionally, individual cells within an 1971, Le Roy et al. 2000). This is of particular interest ovoid cluster of zona glomerulosa cells contained Lucifer given the observations that ACTH treatment increased yellow dye (Fig. 9). Thus, the similarity of the dye 1Cx43 gap junction plaques in primary cell cultures distribution and gap junctions observed in the monkey (enriched for the two inner cortical adrenal zones), and in tissue validates our findings in the mouse adrenal glands. established adrenal cell lines, as well as in adrenal glands of hypophysectomized mice (Murray et al. 2000b). The ACTH-induced increase in 1Cx43 gap junction protein Discussion levels has been correlated with a decrease in proliferation in adrenal cell lines (Oyoyo et al. 1997, Shah & Murray The studies presented here were designed to monitor the 2001), and it is suggested that the increased communi- movement of a gap junction-permeable molecule between cation of regulatory molecules through gap junctions cells of the adrenal gland and to assess the presumptive role following ACTH stimulation plays a pivotal role in of gap junctions in making such movement possible. Using hormone responsiveness, differentiation and cytogenesis immunohistochemistry for gap junction visualization and (Decker 1976, Usadel et al. 1993, Murray et al. 2000a,b). Lucifer yellow dye coupling assay, we have assessed these Cytogenesis in the adrenal is generally thought to processes in the intact cells of the freshly excised adrenal involve the migration and differentiation of cells from glands. The techniques applied here have preserved the the outer to the inner cortex (Salmon & Zwemer 1941, www.endocrinology.org Journal of Endocrinology (2002) 173, 13–21

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Figure 7 Lucifer yellow dye coupling in the adrenal cortex. Lucifer yellow dye diffused into damaged cells at the cut edge but did not transfer to cells deep within the zona glomerulosa (ZG) (A and B). In contrast, Lucifer yellow dye was observed deep within the inner zones (zonae fasciculata/reticularis, ZF/ZR). (C) This illustrates that rhodamine (Rhod)-dextran, which exceeds gap junctional pore size, is limited to damaged cells at the cut edge. The nuclei of cells seen in (B) and (C) were stained with Hoescht dye (D). Bar=50 µm.

Bertholet 1980, Zajicek et al. 1986). A functional tran- significance of such a zone is not known but we now sition zone has been described in the rat adrenal gland in report that gap junction plaques in this transition area are which cells differ both morphologically and physiologically less abundant than those located in deeper adrenal cortical from either zona fasciculata or zona glomerulosa cell type zones and that intercellular communication within this (Mitani et al. 1994). Certainly, if such a migration does area was more variable. The relationships between endo- occur a transition zone should exist in which both, or crine function, cytogenetic fate and dye communication of neither, zona glomerulosa and zona fasciculata type cells these uncoupled cells remains to be elucidated. The would be found. In our study, such a transition zone was observation that some of the cells of the outer zona also evident to the extent that intercellular dye transfer fasciculata were uncoupled, however, would suggest in the outermost zona fasiculata was inconsistent. The that these cells do not exchange low molecular weight majority of cells in this area were coupled while other molecules with their neighbors. These cells may be zona single cells or packets of cells were uncoupled. The fasciculata cells that are devoid of gap junctions, zona

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Figure 8 Location of Lucifer yellow dye in adrenal cortex incubated in oleamide. Dye (A) was restricted to areas of tissue injury (arrow) as indicated by absence of nuclear staining in the same tissue section (B). The oleamide inhibition was not totally effective as indicated by minimal staining in the inner cortical zones (C). (A and B) bar=80 µm; (C) bar=30 µm. glomerulosa cells devoid of gap junctions, or cells that those metabolic pathways used in steroidogenesis and/or possess gap junction channels which are in a closed cellular differentiation. It appears that the cells of the inner conformation. The latter hypothesis seems more likely zones have a greater capacity for, and presumably a greater since it has been demonstrated in cell populations functional dependence on, intercellular communication that communication was established in a sub-population of than cells of the zona glomerulosa. The specific benefits thyroid cells following hormone treatment (Munari-Silem derived from the presence of such communication is not et al. 1991). totally clear, but might include the ability to control We have previously proposed that cell–cell communi- proliferation and the capacity to transmit hormonal cation may be of particular importance for hormonally signaling between cells. responsive glandular tissues (Murray & Fletcher 1984, From a more technical standpoint, the importance of Oyoyo et al. 1997). In such tissues, individual cells with a these studies may relate to the fact that we have demon- decreased number of cell surface receptors might be less strated the specificity of dye movement within a single compromised if neighboring cells were able to com- ‘block’ of tissue. The distribution of dye movement municate the presence of hormonal stimulation and initiate appears to be precisely correlated with the presence of gap the process of amplification. In addition, the selective junctions, a pattern which we interpret as a demonstration communication of molecules through gap junctions that cell damage at the cut edge of the tissue block alone may inhibit some cellular activities, i.e. proliferation is not adequate to promote cell–cell transfer of Lucifer (Loewenstein & Rose 1992, Tsuda et al. 1995, Shah & yellow dye. The fact that this relationship holds true in a Murray 2001), and thus direct cellular activities toward primate adrenal cortex, although limited to a small sample, www.endocrinology.org Journal of Endocrinology (2002) 173, 13–21

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