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