differential expression of gap junction connexins in endocrine and exocrine glands

8
0013.7227/93/1335-2371$03.00/0 Endocrinology Copyright 0 1993 by The Endocrine Society Vol. 133, No. 5 Printed in U.S.A. Differential Expression of Gap Junction Connexins in Endocrine and Exocrine Glands* PAOLO MEDA, MICHAEL S. PEPPER, OTTO TRAUB, KLAUS WILLECKE, DANIEL GROS, ERIC BEYER, BRUCE NICHOLSON, DAVID PAUL, AND LELIO ORCI Department of Morphology, University of Geneva, Geneva, Switzerland; Institute of Genetics, University of Bonn (O.T., K. W.), Bonn, Germany; Laboratoire de Biologie de la Differentiation Cellulaire, University of A&Marseille II (D.G.), Marseille, France; the Division of Hematology-Oncology, Washington University (E.B.), St. Louis, Missouri 63110; the Department of Biological Sciences, State University of New York (B.N.), Buffalo, New York 14260; and the Department of Neurobiology, Harvard Medical School (D.P.), Boston. Massachusetts 02115 ABSTRACT We have investigated the expression of three gap junction proteins and their corresponding mRNAs by secretory cells of a variety of endocrine and exocrine rat glands. By immunostaining cryostat sec- tions (indirect immunofluorescence) with antibodies against connexins (Cx) 26, 32, and 43 and by hybridizing total glandular RNA (Northern blot) with cRNAs for these proteins, we have found that several endocrine glands (pituitary, parathyroid, pancreatic islets, and adrenal) express Cx43, variable levels of Cx26, and no Cx32, whereas several exocrine glands (lacrimal gland, salivary glands, pancreas, prostate, and seminal vesicle) express high levels of Cx32 and variable levels of Cx26, but no Cx43. Thus, different sets of proteins comprise the gap junctions of endocrine and exocrine glands. Together with the findings that an endocrine gland (thyroid) that discharges secretory products extracellularly before releasing them in the vascular compartment expresses both Cx43 and Cx32 and that an exocrine gland (preputial gland) that has a pheromonal role expresses Cx43, these observations suggest that the differential expression of gap junction connexins may be required to specify the endocrine or exocrine differentiation of a secretory cell. (Endocrinology 133: 2371-2378, 1993) L IKE most other cell types, the cells comprising glandular epithelia are connected by gap junctions, the membrane domains where highly permeable channels required for direct intercellular exchanges of cytoplasmic ions and molecules are clustered (1, 2). Gap junctions are often unusually abun- dant in several endocrine and exocrine glands, even long after the morphogenetic and functional development of the secretory cells is completed (3-5). This extensive develop- ment together with the short half-life of gap junction proteins (6-8) suggest that the maintenance of gap junctions is im- portant for the proper functioning of adult glands. This hypothesis is further strengthened by the observations of parallel changes in gap junctions, cell to cell coupling, and secretion of someglands (4, 5, 9-11). We have recently found that insulin-producing B-cells of the endocrine pancreas express Cx43, a gap junction protein which is not detectable in the nearby cells of exocrine pan- creas (12). Conversely, the acinar cells of the latter tissue express Cx32 and Cx26, two gap junction proteins that are not found in endocrine pancreatic islets(12). This differential protein distribution may account for the markedly different characteristics of gap junctions and junctional coupling be- tween endocrine and exocrine pancreatic cells (4, 9-11) as Received January 21, 1993. Address requests for reprints to: Paolo Meda, M.D., Department of Morphology, University of Geneva, CMU, 1, rue Michel-Servet, CH- 1211 Geneva 4, Switzerland. * This work was supported by grants from the Swiss National Science Foundation (32-34090.92), the Sir Jules Thorn Charitable Overseas Trusts the Juvenile Diabetes Foundation International (192467), and the Sandoz Stiftung (to P.M.). well as for the opposite secretory changes in these systems after gap junction blockade (9-11). Here we have investi- gated whether the different pattern of connexin distribution observed in the insulin- and amylase-producing cells of the pancreasalso appliesto other types of endocrine and exocrine glands. To this end, we have screened a variety of multicel- lular glands with antibodies and cRNA probes for Cx26, Cx32, and Cx43, three of the proteins known to form mam- malian gap junctions (1, 2). Materials and Methods Tissue Normal male Sprague-Dawley rats, weighing 250-350 g, were anes- thetized by an ip injection of 37 mg/kg BW pentobarbital sodium (Vetanarcol, Veterinaria, Zurich, Switzerland). All glands were rapidly dissected after deeply anesthetized animals had been killed by sectioning the abdominal aorta, a procedure approved by our institutional commit- tee on animal care. Islets of Langerhans were isolated from the splenic portion of the pancreas by collagenase digestion and purification on Ficoll gradients (10). Localization of connexins For immunofluorescence labeling, small gland fragments were rapidly frozen by immersion in 2-methylbutane cooled with liquid nitrogen. The fragments were then stored in liquid nitrogen until cryostat sectioning (5 pm thickness), which was performed using a Cryocut 3000 (Leica Instruments, Nussloch, Germany). Frozen sections were collected on gelatin-coated slides and fixed for 3 min in -80 C acetone. Slides were rinsed in cold (4 C) PBS containing 0.1% BSA and processed for indirect immunofluorescence staining, as described previously (12). Briefly, sec- tions were incubated 2 h at room temperature with one of the following antibodies: 1) affinity-purified rabbit serum against liver Cx32 (13), 2371 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 October 2014. at 10:53 For personal use only. No other uses without permission. . All rights reserved.

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0013.7227/93/1335-2371$03.00/0 Endocrinology Copyright 0 1993 by The Endocrine Society

Vol. 133, No. 5 Printed in U.S.A.

Differential Expression of Gap Junction Connexins in Endocrine and Exocrine Glands*

PAOLO MEDA, MICHAEL S. PEPPER, OTTO TRAUB, KLAUS WILLECKE, DANIEL GROS, ERIC BEYER, BRUCE NICHOLSON, DAVID PAUL, AND LELIO ORCI

Department of Morphology, University of Geneva, Geneva, Switzerland; Institute of Genetics, University of Bonn (O.T., K. W.), Bonn, Germany; Laboratoire de Biologie de la Differentiation Cellulaire, University of A&Marseille II (D.G.), Marseille, France; the Division of Hematology-Oncology, Washington University (E.B.), St. Louis, Missouri 63110; the Department of Biological Sciences, State University of New York (B.N.), Buffalo, New York 14260; and the Department of Neurobiology, Harvard Medical School (D.P.), Boston. Massachusetts 02115

ABSTRACT We have investigated the expression of three gap junction proteins

and their corresponding mRNAs by secretory cells of a variety of endocrine and exocrine rat glands. By immunostaining cryostat sec- tions (indirect immunofluorescence) with antibodies against connexins (Cx) 26, 32, and 43 and by hybridizing total glandular RNA (Northern blot) with cRNAs for these proteins, we have found that several endocrine glands (pituitary, parathyroid, pancreatic islets, and adrenal) express Cx43, variable levels of Cx26, and no Cx32, whereas several exocrine glands (lacrimal gland, salivary glands, pancreas, prostate,

and seminal vesicle) express high levels of Cx32 and variable levels of Cx26, but no Cx43. Thus, different sets of proteins comprise the gap junctions of endocrine and exocrine glands. Together with the findings that an endocrine gland (thyroid) that discharges secretory products extracellularly before releasing them in the vascular compartment expresses both Cx43 and Cx32 and that an exocrine gland (preputial gland) that has a pheromonal role expresses Cx43, these observations suggest that the differential expression of gap junction connexins may be required to specify the endocrine or exocrine differentiation of a secretory cell. (Endocrinology 133: 2371-2378, 1993)

L IKE most other cell types, the cells comprising glandular epithelia are connected by gap junctions, the membrane

domains where highly permeable channels required for direct intercellular exchanges of cytoplasmic ions and molecules are clustered (1, 2). Gap junctions are often unusually abun- dant in several endocrine and exocrine glands, even long after the morphogenetic and functional development of the secretory cells is completed (3-5). This extensive develop- ment together with the short half-life of gap junction proteins (6-8) suggest that the maintenance of gap junctions is im- portant for the proper functioning of adult glands. This hypothesis is further strengthened by the observations of parallel changes in gap junctions, cell to cell coupling, and secretion of some glands (4, 5, 9-11).

We have recently found that insulin-producing B-cells of the endocrine pancreas express Cx43, a gap junction protein which is not detectable in the nearby cells of exocrine pan- creas (12). Conversely, the acinar cells of the latter tissue express Cx32 and Cx26, two gap junction proteins that are not found in endocrine pancreatic islets (12). This differential protein distribution may account for the markedly different characteristics of gap junctions and junctional coupling be- tween endocrine and exocrine pancreatic cells (4, 9-11) as

Received January 21, 1993. Address requests for reprints to: Paolo Meda, M.D., Department of

Morphology, University of Geneva, CMU, 1, rue Michel-Servet, CH- 1211 Geneva 4, Switzerland.

* This work was supported by grants from the Swiss National Science Foundation (32-34090.92), the Sir Jules Thorn Charitable Overseas Trusts the Juvenile Diabetes Foundation International (192467), and the Sandoz Stiftung (to P.M.).

well as for the opposite secretory changes in these systems after gap junction blockade (9-11). Here we have investi- gated whether the different pattern of connexin distribution observed in the insulin- and amylase-producing cells of the pancreas also applies to other types of endocrine and exocrine glands. To this end, we have screened a variety of multicel- lular glands with antibodies and cRNA probes for Cx26, Cx32, and Cx43, three of the proteins known to form mam- malian gap junctions (1, 2).

Materials and Methods

Tissue

Normal male Sprague-Dawley rats, weighing 250-350 g, were anes- thetized by an ip injection of 37 mg/kg BW pentobarbital sodium (Vetanarcol, Veterinaria, Zurich, Switzerland). All glands were rapidly dissected after deeply anesthetized animals had been killed by sectioning the abdominal aorta, a procedure approved by our institutional commit- tee on animal care. Islets of Langerhans were isolated from the splenic portion of the pancreas by collagenase digestion and purification on Ficoll gradients (10).

Localization of connexins

For immunofluorescence labeling, small gland fragments were rapidly frozen by immersion in 2-methylbutane cooled with liquid nitrogen. The fragments were then stored in liquid nitrogen until cryostat sectioning (5 pm thickness), which was performed using a Cryocut 3000 (Leica Instruments, Nussloch, Germany). Frozen sections were collected on gelatin-coated slides and fixed for 3 min in -80 C acetone. Slides were rinsed in cold (4 C) PBS containing 0.1% BSA and processed for indirect immunofluorescence staining, as described previously (12). Briefly, sec- tions were incubated 2 h at room temperature with one of the following antibodies: 1) affinity-purified rabbit serum against liver Cx32 (13),

2371

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2372 GLANDULAR GAP JUNCTIONS Endo. 1993 Voll33. No 5

diluted 1:lOO; 2) rat monoclonal R5.21C against liver Cx32 (14), undi- luted supernatant; 3) rat monoclonal 12-lC5 against liver Cx32 (15), undiluted supernatant; 4) affinity-purified rabbit serum against liver Cx26 (S), diluted 1:50; 5) rabbit serum against residues 101-119 of liver Cx26 (a gift from Drs. A. Kuraoka and Y. Shibata, Kyushu University, Fukuoka, Japan), diluted 1:lOO; 6) rabbit serum against residues 252- 271 of heart Cx43 (16), diluted 1:lOO; 7) rabbit serum against residues 314-322 of heart Cx43, diluted 1:lOO (17); or 8) rabbit serum against residues 360-382 of heart Cx43, diluted 1:lOO (18). In all cases, the second incubation was carried out for 1 h at room temperature using fluoresceinated antirabbit or antirat antibodies, all diluted 1:200. After rinsing, sections were mounted with 0.02% paraphenylenediamine in PBS-glycerol (1:2, vol/vol) and photographed on a Zeiss Axiophot microscope fitted with filters for fluorescein detection (Zeiss, Ober- kochen, Germany).

Controls included exposure of sections during the first incubation to one of the following reagents: 1) purified rabbit or rat immunoglobulin G, 2) nonimmune rabbit or rat serum, 3) preimmune rabbit serum (when available), or 4) the fluoresceinated antirabbit or antirat antibodies normally used during the second incubation. None of these control incubations resulted in a specific labeling of glandular cells (not shown).

The expression of each connexin was considered trustworthy when- ever it was reliably observed with antibodies to at least two different epitopes of a connexin molecule. The extent of immunolabeling was evaluated by two independent observers using an arbitrary scale rating the numerical density of immunofluorescent spots per microscope field from - (no specific spot) to + (few spots), ++ (numerous spots), and +++ (very numerous spots).

Identification of connexin mRNAs

Total cellular RNA was extracted according to a modification of the method reported previously (19). Gland fragments were homogenized in 2.5 ml 0.1 M Tris-HCl, pH 7.4, containing 2 M P-mercaptoethanol and 4 M guanidinium thiocyanate. After addition of solid CsCl (0.4 g/ml), the homogenate was layered onto 2 ml of a 5.7 M CsCl-0.1 M EDTA (pH 7.4) cushion and centrifuged in a Beckman SW55 rotor (Fullerton, CA) at 35,000 rpm for 20 h at 20 C. Pelleted RNA was resuspended in 300 ~1 10 rnM Tris-HCl, pH 8.1, supplemented with 5 rnM EDTA and 0.1% sodium dodecyl sulfate, extracted twice with phenol-chloroform, precip- itated in ethanol, and resuspended in water.

A probe for Cx26 (pSP65Cx26AS), was constructed by subcloning a 600.basepair (bp) PstI-EcoRI fragment of a 1.1.kilobasepair (kbp) rat liver gap junction cDNA (20) into plasmid pSP65 (21). A probe for Cx32 (pSP64Cx32AS) was constructed by subcloning a 504.bp EcoRI-SmaI fragment of a 1.5-kbp rat liver gap junction cDNA (22) into plasmid pSP64 (21). Two probes for Cx43 were constructed by subcloning either a 1.4.kbp (clone G2) rat heart gap junction cDNA (23) into the EcoRI site of Bluescript Ml3 (probe pBSG2A) or a 509-bp SacI-EcoRI fragment of G2 in plasmid pSP65 (21) (probe pSP65Cx43-3’AS). Plasmids pSP65Cx26AS, pSP64Cx32AS, pBSG2A, and pSP65Cx43-3’AS were linearized using PstI, EcoRI, BamHI, and SacI, respectively. Linearized plasmids were used as templates for bacteriophage SP6 (in the case of uSP65Cx26AS, uSP64Cx32AS, and uSP65Cx43-3’AS) or T3 RNA DO- jymerase (in the-case of pBSG2A). Tianscription was performed exaitly as described previously (24).

For Northern blots, total cell RNA was denatured with glyoxal, electrophoresed in a 1.2% agarose gel (5 pg RNA/lane), and transferred overnight onto nylon membranes (Hybond, Amersham, Arlington Heights, IL), as described previously (25). Filters were baked under vacuum at 80 C for 2 h, exposed to UV light (302 nm) for 30 set, and stained with methylene blue to reveal 18s and 28s ribosomal RNA. Filters were prehybridized for 6 h at 65 C and then hybridized for 18 h at the same temperature with 2 X lo6 cpm/ml ?-labeled probe (24). The filters were washed twice at 65 C with 3 x SSC solution (0.45 M NaCl and 0.045 M Na citrate, pH 7.0) supplemented with 2 X Denhardt’s solution (26) and three times at 75 C with a 0.2 x SSC solution (0.03 M NaCl and 0.005 M Na citrate, pH 7.0) supplemented with 0.1% sodium dodecyl sulfate and 0.1% sodium pyrophosphate. Filters were exposed to Kodak XAR-5 films (Eastman Kodak, Rochester, NY), between inten- sifying screens, at -80 C.

Results

Endocrine glands

Incubation of cryosections with antibodies against Cx43 consistently resulted in a punctate labeling of the membrane of secretory cells in parathyroid (Fig. lA), anterior pituitary (Fig. lD), pancreatic islets (Fig. lF), adrenal cortex (Fig. lG), adrenal medulla (Fig. lH), and thyroid (Fig. 2, A and D). Labeling for Cx43 varied from sparse to very abundant between different glands (e.g. thyroid IIS. parathyroid), as well as between different regions/cells (e.g. zona fasciculata IIS. zona glomerulosa of the adrenal gland) of the same gland (Table 1). No specific pattern of Cx43 distribution was no- ticeable in most glands, except the thyroid, in which Cx43 predominated in the juxtapical locale of the lateral membrane domain of follicular cells (Fig. 2, A, D, and E). Discrete fluorescence labeling was also observed with antibodies against Cx26 in parathyroid (Fig. lC), anterior pituitary (Fig. lE), and thyroid (Fig. 2B). However, Cx26 labeling was not detectable in other types of endocrine glands (Table 1). No specific immunostaining with antibodies against Cx32 was observed in parathyroid (Fig. lB), pancreatic islets (Fig. 4H), or any other endocrine gland tested (Table l), except the thyroid. In this gland, follicular cells showed high levels of Cx32, which appeared distributed throughout the basolateral domain of the plasma membrane (Fig. 2, C, F, and G).

To assess the steady state levels of connexin transcripts, total cellular RNA extracted from endocrine glands was analyzed by Northern blot hybridization using cRNA probes for Cx43, Cx32, and Cx26. We found that endocrine glands expressed substantial levels of the mRNAs for Cx43 and Cx26, and that the level of expression of the two transcripts varied from one gland to another (Fig. 3). Northern blots also revealed the presence of abundant Cx32 mRNA in the thyroid, but failed to detect this message in pituitary and adrenal glands (Fig. 3). Traces of Cx32 transcript were also detected in enzymatically isolated islets of Langerhans and mechanically isolated parathyroids (Fig. 3). As Cx32 could not be detected by immunofluorescence in either tissue (Figs. 1B and 4H and Table l), these traces are presumably ac- counted for by the unavoidable contamination of the isolated islet and parathyroid preparations by minimal amounts of pancreatic acini and thyroid follicles, respectively.

Exocrine glands

Antibodies against Cx32 resulted in a substantial punctate labeling of secretory cells in parotid (Fig. 4B), submandibular gland (Fig. 4D), lacrymal gland (Fig. 4E), prostate (Fig. 4G), pancreatic acini (Fig. 4H), and all other exocrine glands tested (Table 1). The level of immunolabeling was variable from gland to gland (Table 1). Whenever it was high, comparison of the immunofluorescence and phase contrast photographs of the same region (e.g. Fig. 4, E and F) revealed that Cx32 was distributed throughout the basolateral domain of the plasma membrane of secretory cells. Antibodies to Cx26 also labeled the acinar cells of parotid (Fig. 4C) and those of most other exocrine gland tested (Table 1) in a pattern comparable to that observed for Cx32. In submandibular glands and

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FIG. 1. Immunostaining of rat endocrine glands for gap junction connexins. In parathyroids, peptide-producing princi- pal cells showed a punctate immunola- beling with antibodies to Cx43 (A) and Cx26 (C), but not with antibodies to Cx32 (B). Cx43 (D) and Cx26 (E) im- munoreactivity were also detected in the in the peptide-producing cells of anterior pituitary. By contrast, only Cx43 was detected in the peptide-producing cells of pancreatic islets (F), the steroid-pro- ducing cells of adrenal cortex (G), and the amine-producing cells of adrenal me- dulla (H). The bar represents 40 pm in all figures.

GLANDULAR GAP JUNCTIONS 2313

pancreas, the abundance of both Cx32 and Cx26 in exocrine cells markedly contrasted with the consistent lack of detec- tion of these connexins in the endocrine cells that were intermixed with the exocrine tissue. This was the case for both the epidermal growth factor-producing cells comprising the convoluted granular tubules of submandibular glands (Fig. 4D) and the insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells of the islets of Lan- gerhans (Fig. 4H). Antibodies to Cx43 did not elicit specific immunostaining of secretory cells in parotid (Fig. 4A) or any of the other exocrine glands tested (Table l), with the excep- tion of the preputial gland, which is discussed below. This lack of labeling could not be accounted for by technical

reasons, as these antibodies conspicuously labeled some con- nective tissue cells in the very same gland sections (not shown). In all glands tested, intralobular (Fig. 4, A-C) and interlobular excretory ducts (Fig. 4H) were not detectably decorated by the anticonnexin antibodies used, including two antibodies that were directed to Cx40 (not shown).

Northern blot hybridizations showed that all of the exo- crine glands tested expressed mRNAs for Cx32 and/or Cx26 (Fig. 3). The levels of these transcripts varied from one exocrine gland to another (Fig. 3). Northern blots did not detect Cx43 transcripts in exocrine glands, with the noticea- ble exception of the preputial gland (Fig. 3).

In spite of a typical exocrine architecture, the preputial

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2374 GLANDULAR GAP JUNCTIONS Endo. 1993 Vol133. No 5

FIG. 2. Connexin immunostaining in rat thyroid. The follicular cells of thyroid were sparsely labeled by antibodies to Cx43 (A) and Cx26 (B), and much more abundantly by antibodies to Cx32 (C). Comparison of immunofluorescence photographs with the corresponding phase contrast views revealed that Cx43 (D and E) predominated in the upper juxtapical locale of the lateral membrane of follicular cells, whereas Cx32 (F and G) was distributed throughout the baso- lateral membrane domain. The bar rep- resents 40 pm in all figures.

gland, which secretes lipid products with a pheromonal action, stood apart all other exocrine glands in that its secre- tory cells were not immunostained by antibodies to either Cx32 (Fig. 5C) or Cx26 (Fig. 5D). The latter antibodies, however, markedly labeled the stratified epithelium lining the excretory ducts of the gland (Fig. 5, D and E). In contrast and consistent with the Northern blot findings (Fig. 3), the secretory cells of the preputial gland showed abundant im- munostaining for Cx43 (Fig. 5A). This immunostaining was prominent over the differentiating secretory cells comprising the outermost layers of the acini and was not detected over the more centrally located and fully differentiated cells, which were crowded with highly regringent lipid inclusions (Fig. 5, A and B).

Discussion

We have studied several endocrine and exocrine glands for the distribution of three proteins comprising mammalian

gap junctions. Using antibodies to different epitopes on each of these proteins, as well as probes that specifically hybridize to their corresponding mRNAs, we have found that connex- ins and their transcripts are differentially expressed in en- docrine and exocrine secretory cells. All of the endocrine cells we investigated expressed sizable amounts of Cx43 and, in general, no Cx32. Conversely, exocrine cells expressed Cx32, but usually no Cx43. Cx26 showed a more widespread distribution, being detected in several endocrine and exocrine glands.

The distribution of Cx26, Cx32, and Cx43 throughout the body is complex. Thus, different tissues with divergent origins and functions may express the same connexin in both developing and adult organs, whereas different connexins may be found within the same tissue or even the same cell (23, 27-30). This surprising diversity does not provide a simple clue to the reason(s) why different cell types differ- entially regulate the expression of different connexin genes. In this context, the present identification of consistently

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GLANDULAR GAP JUNCTIONS 2375

TABLE 1. Distribution of connexins in rat endocrine and exocrine glands as determined by immunofluorescence labeling

Gland Region (cell) Cx43 Cx32 Cx26

Pituitary

Parathyroid

Pancreatic is- lets

Adrenal

Thyroid

Preputial

Liver

Lacrimal

Parotid

Submandib- ular

Sublingual

Pancreas

Prostate

Se;? vesi-

Adenohypophysis Intermediate lobe Neurohypophysis (pituicytes)

Principal cells

Central core (B-cells) Peripheral mantle

Zona glomerulosa Zona fasciculata Zona reticularis Medulla

Follicles Interfollicular regions (C-

cells)

Acini Ducts

Centrolobular (hepatocytes) Perilobular (hepatocytes)

Serous acini Ducts

Serous acini Ducts

Seromucous acini Convoluted granular tubules Ducts

Mucous acini Ducts

Serous acini Ducts

Alveolae

Secretory tubules

++ +

++

++

+ +

++ +

++ +

+ +

+ -

-

-

-

-

-

-

-

-

t++

-

++ t++

++ -

++ -

++

+

+++ -

++

+

+

++

-

- -

+

++

++

++

++

++

+

++ -

-

-

+ and - indicate the presence and absence of immunofluorescence spots, respectively.

different patterns of expression of two major connexins in endocrine and exocrine glands provides a new lead to address this question. The alternative distribution of Cx43 and Cx32 was observed in glands derived from the three germ layers, regardless of the spatial arrangement of secretory cells or whether these cells released peptides, glycoproteins, or lipids. These observations indicate that the expression of gap junc- tion proteins is not necessarily related to the embryological origin of a gland, its three-dimensional architecture, or the type of metabolic differentiation of its constitutive secretory cells. Rather, it appears that connexin expression may be differentially regulated depending on whether a secretory cell becomes part of an endocrine or an exocrine gland.

The mechanism of this differentiation is unknown. Phy- logenetically, endocrine and exocrine secretions are analo- gous processes (31). With evolution, most endocrine cells

032 *

CA26 c

- 18s

FIG. 3. Northern blot hybridization of 3ZP-labeled connexin probes to total cellular RNA from endocrine (right panels) and exocrine (left panels) glands. Upper panels, The Cx43 probe hybridized to a 3.0- kilobase (kb) fragment in heart, liver, and all endocrine glands tested. By contrast, this probe did not hybridize to total RNA from exocrine glands, except the preputial gland. Middle panels, The Cx32 probe hybridized to a 1.6-kb fragment in liver and all exocrine glands tested, except the preputial gland. By contrast, it did not hybridize to total RNA from endocrine glands, except the thyroid. A small signal was also detected in samples of isolated pancreatic islets and parathyroid glands, presumably as a result of a minimal contamination of these preparations by pancreatic acini and thyroid follicles, respectively. Lowerpanels, The Cx26 probe hybridized to a 2.5kb transcript in liver and most of the endocrine and exocrine glands tested, except prostate, seminal, and adrenal glands. The arrowheads on the left indicate the positions of Cx43, Cx32, and Cx26 mRNAs, respectively. The arrow- heads on tne right indicate the positions of the 28s and 1% ribosomal RNA subunits. All lanes were loaded with 5 pg total cellular RNA. Equal loading was verified by staining the filters with methylene blue (not shown).

have become segregated into distinct and discrete organs, leading, at the level of vertebrates, to the endocrine glands we have studied here (31). Strikingly, comparison of con- nexin sequences suggests that it is at that time or just before that a gene duplication permitted the divergence of Cx43 (and other connexins of group I or a) from Cx26 and Cx32 (and other connexins of group II or /3) (1, 28). This temporal coincidence suggests that the expression of a given connexin type may be required to specify the endocrine or exocrine nature of a secretory cell. In this perspective, it is intriguing that the acinar cells of the preputial gland, an organ that participates in pheromone production and release (32), ex- press Cx43 and no detectable levels of Cx32, in spite of a histological organization typical of exocrine glands. This finding may not be limited to the rat, as we have recently

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2376 NCTIONS Endo. 19% Vol 13x. No R

FIG. 4. Immunostaining of rat exocrine glands for gap junction connexins. In parotids, the enzyme-producing cells of serous acini showed abundant labeling with antibodies to Cx32 (B) and Cx26 (C), but were unstained by antibodies to Cx43 (A). In submandibular glands (D), Cx32 was observed in the glycoprotein- secreting cells of seromucous acini, but not in the epidermal growth factor-pro- ducing cells of convoluted granular tu- bules (cgt). Cx32 was also detected in the serous acinar cells of lacrimal glands (E), where, as shown by comparison with the corresponding phase contrast photo- graph (F), it appeared distributed throughout the basolateral domain of the plasma membrane. Cx32 was also abun- dant in the enzyme-producing cells of prostate (G) and pancreatic acini (H). In the same pancreas sections (H), this con- nexin was not detectable in either en- docrine pancreatic islets (i) or ducts (d). The bar represents 40 wrn in all figures.

cgt

I d

observed that the sebaceous glands of human skin, which are functionally and phylogenetically related to preputial glands (33), also express Cx43 and no Cx32 (34). Also intrigu- ing is the observation that the main secretory cells of thyroid, an endocrine gland that discharges its secretory products extracellularly (a characteristic feature of exocrine glands) before releasing them in the circulation, express both Cx43 and Cx32. However, whereas Cx32 was distributed through- out the basolateral domain of the membrane of follicular cells, Cx43 was prominent in the upper juxtapical locale of the lateral cell membrane. The expression of two different gap junction proteins by thyroid follicular cells is but another example of coexpression of multiple connexins by a normal cell (2), including several types of gland cells (Ref. 35 and

our present data). In at least one such cell type, we know that two different connexins can be colocalized at the same gap junction (8). In contrast, there is as yet no precedent evidence that different connexins are sorted to distinct mem- brane domains within a single cell, as seen in thyroid follicles where connexins show a polarized distribution, as other unrelated membrane proteins. It is, therefore, possible that the characteristics of gap junction channels and of the cell to cell communications they mediate are influenced not only by the type of connexin expressed (36), but also by the concentration of a given gap junction protein in a specific membrane domain (37). The dual location of connexins observed in thyroid follicles is consistent with the distribution of gap junctions, which, by electron microscopy, have been

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2317

FIG. 5. Connexin immunostaining in rat preputial gland. Antibodies to Cx43 labeled the pheromone-producing cells of preputial glands (A) at the periphery (where cells are differentiating) but not in the center of acini, where fully differentiated cells are crowded with refringent lipid vacuoles (B). Preputial acinar cells were not marked by antibodies to Cx32 (C) and Cx26 (D). However, the latter antibodies readily detected Cx26 in the stratified squamous epithelium (D), which lines the excretory ducts (d) of preputial glands (E). The bar represents 40 pm in all figures.

found in both the juxtabasal and juxtapical locales of the lateral cell membranes of follicular cells (38, 39). In the latter domain, gap junctions are closely associated with the belt of tight junction fibrils that surround the follicular lumen and appear somewhat smaller than those found elsewhere within the membrane. Whether these phenotypic characteristics are related to a unique connexin composition (as could be deter- mined by immunolabeling at the electron microscopy level) and change with the pattern of connexins when the func- tioning and/or arrangement of thyroid cells is modified re- mains to be assessed.

Differential expression of distinct connexin genes is likely to impart specific characteristics to gap junctions and cell to cell communications (1, 2, 28, 36). Therefore, the control, if not the function, of junctional coupling may be different in endocrine and exocrine glands. Certainly, gap junctions, coupling, and their regulation under conditions affecting secretion are very different in the only endocrine and exo- crine systems in which such a regulation has so far been studied to some extent, i.e. in the insulin- and amylase-

producing cells of the pancreas (4, 5, 9-11). Also, exposure of these cells to the same pharmacological blocker of junc- tional coupling induced distinct and opposite changes in secretory function (4, 9-l 1).

At present, about nine homologous connexin sequences have been identified in the mammal genome in addition to those coding for Cx26, Cx32, and Cx43 (1, 2). Whether these new connexins are actually expressed, at any significant level, by the secretory cells of glands is still undetermined and awaits the availability of specific antibodies for immunolo- cation at both the biochemical and histological levels (using two antibodies to Cx40, we have failed to detect this connexin in a number of exocrine and endocrine secretory cells). Future screening should, therefore, determine whether connexins other than Cx32 and Cx43 are also alternatively expressed, as secretory cells become part of an exocrine or endocrine gland.

Acknowledgments

We thank L. Burkhardt, A. Charollais, S. De Mitri, C. Di Sanza, J.-P. Gerber, and E. Sutter for excellent technical assistance.

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2378 GLANDULAR GAP JUNCTIONS Endo. 1993 Voll33. No 5

Zhang JT, Nicholson BJ 1989 Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J Cell Biol 109:3391-3401 Melton DA, Krieg P A, Rebagliati MR, Maniatis T, Zinn K, Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacterio- phage SF6 promoter. Nucleic Acids Res 12:7035-7056 Paul DL 1986 Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol 103:123-134 Beyer EC, Paul DL, Goodenough DA 1987 Connexin43: a protein from rat heart homologous to agap junction protein from-liver. J Cell Biol 105:2621-2629

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