Connexins and secretion

Connexin channels clustered at gap junctions are obligatory attributes of all macroscopic endocrine and exocrine glands investigated so far and also connect most types of cells which produce secretory products in other tissues. Increasing evidence indicates that connexins, and the cell-to-cell communications that these proteins permit, contribute to control the growth of secretory cells, their expression of specific genes and their differentiated function, including their characteristic ability to biosynthetize and release secretory products in a regulated manner. Since the previous reviews which have been published on this topic, several lines of evidence have been added in support of multiple regulatory roles of gland connexins. Here, we review this novel evidence, point to the many questions which are still open and discuss some interesting perspectives of the field.     

CELL CONTACTS IN THE MOUSE MAMMARY GLAND : I. Normal Gland in Postnatal Development and the Secretory Cycle

The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both.     

Role of gap junctions in fluid secretion of lacrimal glands

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.     

Intercellular junctions in the hypodermis,salivary gland and gené's organ of the cattle tick,Boophilus microplus

Summary The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.The authors would like to thank Mr. R. Lamb for preparing the plates. M.W.J. Megaw was supported by an S.R.C. Studentship     

Junctional types in the tissues of an onychophoran: the apparent lack of gap and tight junctions in Peripatus

The Onychophora are a rare group of primitive invertebrates, relatively little investigated. Tissues from a range of their digestive, secretory and excretory organs have been examined to establish the features of their intercellular junctions. Glutaraldehyde-fixed cells from the midgut and rectum, as well as the renal organ, mucous gland, salivary gland, epidermis, CNS and testis from specimens of Peripatus acacioi, have been studied by thin section and freeze-fracture electron microscopy. Adjacent cells in the epithelia of all these tissues are joined by apical zonulae adhaerentes, associated with a thick band of cytoskeletal fibrils. These are followed by regular intercellular junctional clefts, which, in thin sections, have the dense, relatively unstriated, appearance of smooth septate junctions (SSJ). However, freeze-fracture reveals that only the midgut has what appear to be characteristic SSJs with parallel alignments of closely-packed rows of intramembranous particles (IMPs); these IMPs are much lower in profile than is common in such junctions elsewhere. The mucous gland, testis, rectal and renal tissues exhibit, after freeze-fracture, the characteristic features of pleated septate junctions (PSJ) with undulating rows of aligned but separated junctional particles. Suggestions of tricellular septate junctions are found in replicas at the interfaces between 3 cells. In addition, renal tissues exhibit scalariform junctions in the basal regions of their cells. Between these basal scalariform and apical septate junctions, other junctions with reduced intercellular clefts are observed in these renal tissues as well as the rectum, but these appear not to be gap junctions. Such have not been unequivocally observed in any of the tissues studied from this primitive organism; the same is true of tight junctions.     

Fluorescence histochemical observations on catecholamine-containing cell bodies in Auerbach's plexus

Summary The nature and distribution of cell contacts have been examined in the human enamel organ in bell stage. The lateral cell surfaces of secretory ameloblasts are linked at their distal (apical) and proximal (basal) parts by junctional complexes consisting of tight junctions, large intermediate junctions (zonulae adherentes), occasional gap junctions and one or more series of desmosomes. Scattered desmosomes and large gap junctions link epithelial cells of the external enamel epithelium, stellate reticulum, stratum intermedium and internal enamel epithelium including secretory ameloblasts. Furthermore the above-mentioned layers are also linked together by desmosomes and gap junctions.With increasing maturation of the enamel organ an increase in size and number of gap junctions is observed. Some possible implications of the role of the different junctions are considered. The gap junctions probably participate in cell differentiation in the normal morphogenesis of the teeth as well as in metabolic and ionic coupling of the cells of the enamel organ. By means of tight junctions, adjacent secretory ameloblasts cooperate to form a physical barrier which might prevent the diffusion of some types of molecules or substances (e.g. secretory material distally and acid mucopolysaccharides proximally) through the interspaces between the cells. Adhering junctions might assist in regulation of the mechanical properties of the enamel organ as a whole.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen, and the Association for the Aid of the Crippled Children, New York.     

Novel structures in secreting the androgenic gland hormone

The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine structure of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda.     

Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells

Cells dissociated from normal prelactating mouse mammary glands or from spontaneous mammary adenocarcinomas, when grown at high density on an impermeable substrate, form nonproliferating, confluent, epithelial pavements in which turgid, blister-like domes appear as a result of fluid accumulation beneath the cell layer. To compare the structure of the fluid-segregating cell associations in normal and tumor cell cultures with that of lactating gland in vivo, we have examined such cultures alive and in thick and thin sections and freeze-fracture replicas. Pavement cells in all cases are polarized toward the bulk medium as a lumen equivalent, with microvilli and continuous, well-developed occluding junctions at this surface. Between the pavement and the substrate are other cells, of parenchymal or stromal origin, scattered or in loose piles; these sequestered cells are relatively unpolarized and never possess occluding junctions. Small gap junctions have been found in the pavement layer, and desmosomes may link epithelial cells in any location. Under the culture conditions used, development of the epithelial secretory apparatus is not demonstrable; normal and neoplastic cells do not differ consistently in any property examined. A dome's roof is merely a raised part of the epithelial pavement and does not differ from the latter in either cell or junction structure. We suggest that dome formation demonstrates the persistence of some transport functions and of the capacity to form effective occluding junctions. These basic epithelial properties can survive both neoplastic transformation and transition to culture.     

Junctional structures in digestive epithelia of a cephalopod

Intercellular junctions have been studied in the epithelia of digestive organs of Sepia officinalis (digestive gland, digestive duct appendages and caecum) by conventional staining, lanthanum tracer and freeze-fracturing techniques. In the three organs studied the same junctional complex occurs, consisting of a belt desmosome, a septate junction and gap junctions. The septate junction is of pleated-sheet type and the gap junction has its particles on the P face of the fracture. Circular structures have been found in the digestive gland septate junctions. Neither continuous nor tight junctions have been found. These results show that Cephalopods have junctional structures very close to those of other Molluscs and of Annelids. Some small differences between the septate junctions of the three organs could be related to their different physiology.     

Androgen-regulated formation and degradation of gap junctions in androgen-responsive human prostate cancer cells

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.     

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.     

Increase of gap junctions between pancreatic B-cells during stimulation of insulin secretion

The development of gap junctions between pancreatic B-cells was quantitatively assessed in freeze-fracture replicas of isolated rat islets under different conditions of insulin secretion. The results show that in resting B-cells, gap junctions are small and scarce but that these junctions increase when insulin secretion is stimulated. Both a short (90 min) stimulation by glucose in vitro and a prolonged (2.5 d) stimulation by glibenclamide in vivo raise the number of gap junctions; in addition, the glibenclamide stimulation causes an increase in the size of individual gap junctions. As a consequence, the total area occupied by gap junctions on the B-cell membrane and the ratio of this area to the cell volume were found significantly increased in the latter condition. The slight increase of these values observed after the glucose stimulation did not reach significance. These data indicate a change of gap junctions during the secretory activity of the pancreatic B-cells. The possibility that the coupling of the cells is affected by the treatment is discussed.     

A contribution to the functional morphology of the midgut gland in phalangiid harvestmen Gyas annulatus and Gyas titanus during their life cycle

The structure of the midgut gland and its changes in different seasons have been examined in the harvestmen Gyas annulatus and Gyas titanus (Arachnida: Opiliones: Phalangiidae). In both species, in the epithelium of the midgut gland two different types of cells are present: secretory and digestive ones. The secretory cells are characterized by plentiful rER and secretory granula. The digestive cells are characterized by an apical system of tubules. Both cells are connected by prominent specialized junctions. If a secretory cell is in contact with a digestive cell, rER cisterna are in close vicinity and parallel to these junctions. As found light- and electron microscopically and also histochemically, glycogen and lipids are stored in both cells. In both species, glycogen was seen to be used as energy compound during overwintering. At the end of their life, the digestive cells develop into excretory ones, containing metabolic wastes.     

Increasing Gap Junctional Coupling: A Tool for Dissecting the Role of Gap Junctions

Much of our current knowledge about the physiological and pathophysiological role of gap junctions is based on experiments where coupling has been reduced by either chemical agents or genetic modification. This has brought evidence that gap junctions are important in many physiological processes. In a number of cases, gap junctions have been implicated in the initiation and progress of disease, and experimental uncoupling has been used to investigate the exact role of coupling. The inverse approach, i.e., to increase coupling, has become possible in recent years and represents a new way of testing the role of gap junctions. The aim of this review is to summarize the current knowledge obtained with agents that selectively increase gap junctional intercellular coupling. Two approaches will be reviewed: increasing coupling by the use of antiarrhythmic peptide and its synthetic analogs and by interfering with the gating of gap junctional channels.     

Gap junctions between pinealocytes

Summary The intercellular junctions between the pinealocytes of male rats were investigated by freeze-fracture and conventional electron microscopy.Our findings reveal that the intercellular contacts between pineal cells, formerly described as zonulae adhaerentes or zonulae occludentes, are in fact gap junctions which are difficult to characterize in thin sections due to their peculiar geometrical arrangement, which is in the form of fenestrated communicating zonules.The arrangement of these communicating zonules around rudimentary lumina of pineal clusters and rare transitions between tight and gap junctions may point to phylogenetic transformations of occluding into communicating zonules, corresponding with the change of the pineal gland from a sensory to a secretory organ. Alternatively, these tight-to-gap junctional transitions may reflect the periodic (circadian or seasonal) activity of the pineal gland.These Studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

The role of cytoskeletal components in the maintenance of intercellular junctions in an insect

Summary Accessory glands of the cockroach are composed of secretory and supportive cells, the latter providing a skeleton-like framework of attentuated cytoplasmic processes into which the former are positioned. These two cell types are associated with one another laterally by adhaering, pleated septate, and gap junctions. Hemi-adhaerens junctions are also found on both luminal and basal surfaces of the gland; the former are associated with the cuticular lining of the lumen and the latter with extracellular matrix. The adhering and septate junctions are flanked by both filaments and microtubules; the former insert into the junctional membranes and are actin-like, binding both rhodamine-conjugated phalloidin and the S₁ subfragment of rabbit heavy meromyosin. The role of this cytoskeletal protein with the cellular junctions has been explored by treatment with a disruptive agent, cytochalasin D. Dissociation of actin leads to changes in septate junctions and in microtubular distribution. This suggests that the latter act as anchors for the actin filaments which, in turn, appear bound to certain of the intramembranous junctional components.Supported by a Conicet/Royal Society Visiting Fellowship     

VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

Genes, gene knockouts, and mutations in the analysis of gap junctions

Gop junctions are cell junctions found between most cells and tissues. They contain membrane channels that mediate the cell-to-cell diffusion of ions, metabolites, and small cell signaling molecules. Cell-cell communication mediated by gap junctions has been proposed to have a variety of functions, including roles in regulating events in development, cell differentiation, and cell growth and proliferation. The analysis of these possibilities has been confounded by the fact that there are over a dozen connexin genes encoding polypeptides that make up vertebrate gap junctions. This complexity, coupled with the fact that most cells express multiple connexin isotypes, likely explains why recent studies using reverse genetic and genetic approaches to disrupt connexin gene function have yielded only limited insights into the physiological roles of gap junctions. Nevertheless, studies in vivo and in vitro together have provided evidence for gap junctions being involved in the regulation of cell metabolism, growth, and differentiation in restricted cell and tissue types. Surprisingly, studies in invertebrates suggest that their gap junctions are encoded not by connexins, but by a family of proteins referred to as innexins. Analysis of various Drosophila and C. elegans mutants suggest that innexins may be functional homologs to the connexins. However, whether innexins are the elusive invertebrate gap junction proteins or, rather, accessory proteins that facilitate gap junction formation remains an open question. Given the rapid progress being made in the cloning and functional analysis of gap junctions in many diverse species, confusion and difficulties with nomenclature are coming to a head in this rapidly expanding field. It may be timely to form a Nomenclature Committee to establish a uniform classification scheme for naming gap junction proteins.     

Physiological and morphological evidence for coupling in mouse salivary gland acinar cells

Three experimental techniques were employed to examine coupling between acinar cells of the mouse salivary gland. Passage of DC current pulses via intracellular microelectrodes between neighboring cells showed that small ions could be directly passed from one cell to another. Intracellular iontophoresis of the dye Lucifer Yellow CH into a single cell indicated that small molecules could spread by means of intercellular cytoplasmic bridges througout an acinus and, occasionally, into cells of adjacent acini. Freeze-fracture replicas of acinar cell membranes indicated the presence of gap junctions which were correlated with both electrical and dye coupling experiments. Suggestions are made for the function of direct intercellular exchange in salivary secretory cells. The role of electrical coupling in coordination of the activity of different secretory cell types is discussed as one possible function.     

Cell Junctions in Arthropod Ion-transport Systems

The epithelial cells involved in the movement of ions and waterform a major subset of all epithelial cell types. Both the formand the functions of cell junctions present in these cells areessentially the same as those found elsewhere. Gap junctionsare believed to regulate intercellular communication; desmosomesand hemidesmosomes provide mechanical anchorage to other cellsand the extracellular matrix; septate junctions play roles inproviding cell to cell anchorage, and perhaps in sealing thelateral surfaces of adjacent cells together to prevent paracellularfluid and solute movement; tight junctions (of limited distributionin insects) are seals between adjacent cells. They form a barrierto the paracellular movement of solutes and water. Examination of the junctions in salivary glands and midgut provideinsight into the roles of these junctions in the developmentand function of ion transport systems. In Manduca sexta (Johannsen)the cells of the salivary gland are joined by pleated septateand gap junctions. Individual salivary cells have numerous foldsand canaliculi. The walls of the canaliculi consist of extensivelyfolded plasma membrane in intimate association with mitochondria.Gap junctions connect adjacent parts of the same cell acrossmembrane folds, effectively shortening diffusion distances inthe cells. Hemidesmosomes are present in the walls of developingcanaliculi. They are attached to pore filaments that occupythe lumen of the developing canaliculi. The hemidesmosomes andpore filaments may have a morphogenetic role as they disappearafter the canaliculi are formed. In Manduca sexta the midgut cells are joined by gap and septatejunctions. These junctions differ in morphology from their counterpartsin the salivary gland; physiological studies show the gobletcells are not coupled to neighboring tall columnar cells. Wehave shown the gap junctions joining them are typical of non-couplingjunctions. Preliminary studies suggest that the gap junctionschange form when the cells are coupled.