Connexins and secretion - Wiley Online Library

Biology of the Cell 94 (2002) 477–492 www.elsevier.com/locate/bicell

Review

Connexins and secretion Véronique Serre-Beinier a, Christophe Mas a, Alessandra Calabrese a, David Caton a, Juliette Bauquis a, Dorothée Caille a, Anne Charollais a, Vincenzo Cirulli b, Paolo Meda a,* a

Department of Morphology, University of Geneva, Medical School, C.M.U., 1 rue Michel, Servet, 1211 Genève 4, Switzerland b Department of Pediatrics, University of California, San Diego, 92093 La Jolla, CA, USA Received 20 June 2002; accepted 14 October 2002

Abstract 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. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Gap junctions; Exocrine glands; Endocrine glands; Pancreas

1. Introduction Most endocrine and exocrine secretions are multicellular events which depend on the coordinated activity of numerous cells. To achieve this coordination, secretory cells cross-talk in a variety of ways (Meda, 2001) which, with evolution, have been progressively integrated into a complex regulatory network that allows individual cells to sense the state of activity of their neighbors and to regulate, accordingly, their own level of functioning. Thus, gland cells may communicate with each other by interacting with hormones, neuromediators and other signals (NO, Ca2+,{), which diffuse in the extracellular spaces and are simultaneously sensed by all the cells bearing the appropriate receptors, transporters and channels or in which specific metabolic and effector steps are simultaneously activated by a signal diffusing through the lipid bilayer of the cell membrane (Fig. 1 ). These mechanisms ensure an efficient signaling network between both distant and nearby cells. Gland cells also communicate by mechanisms which depend on the physical contact between either the interacting cells or these cells and the extracellular * Corresponding author. E-mail address: [email protected] (P. Meda) © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S0248-4900(02)00024-2

matrix. In these cases, cell communication is mediated by the expression of several families of multigenic membrane proteins that have become specialized for cell-to-cell adhesion (Cell Adhesion Molecules and junctional proteins), sealing of the membrane and of the extracellular space (occludins, claudins), direct inter-cellular exchanges of cytoplasmic ions and molecules (connexins) and cell-to-matrix adhesion (integrins) (Fig. 1). These molecules also allow for the reciprocal transmission of signals between the cell membrane and the nucleus, via their interaction with the cytoskeleton network of the cross-talking cells (Fig. 1), thus linking extracellular signals to gene expression, and vice versa. Together, these mechanisms provide for the functional integration of individual cells into coordinated multicellular units and their accurate modulation as a function of the ever changing physiological demand. Still, the reasons as to why secretory cells require so many diverse control and signaling mechanisms, and what may be the hierarchical organization and interdependence of these systems remain to be fully elucidated (Meda and Bosco, 2001). In this perspective, the recognition that most secretions retain a close to normal regulation under in vitro conditions which perturb the native blood supply, innervation and flux of extracellular fluid which functionally interconnect gland cells in vivo, are

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Fig. 1. The mechanisms for communication of gland cells. The secretory cells of endocrine, exocrine, mixed endocrine–exocrine and pheromonal glands communicate with each other by a variety of indirect and direct mechanisms. Indirect cell-to-cell communication is mediated by extra-cellular signals (hormones, neuro-transmitters, Ca2+, NO,{) which simultaneously interact with either membrane or cytoplasmic components of both adjacent and distant cells. Direct cell-to-cell communication is mediated by integral membrane proteins that either link the surface of adjacent cells (Cell Adhesion Molecules, connexins, other junctional proteins,{) or cells to components of extra-cellular matrix (integrins). Most of these surface proteins associate to cytoskeleton elements (—) within the cytoplasms of the interacting cells.

markedly perturbed after the dispersion of secretory cells and may be rapidly improved upon cell reaggregation (Salomon and Meda, 1986; Bosco et al., 1989), indicates that those communication mechanisms which are dependent on cell contact play a major role in the control of secretory functioning in a variety of adult, fully differentiated glands. The finding that connexins, gap junctions and cell-to-cell coupling are obligatory features of all the endocrine, exocrine and pheromone-producing secretory cells so far investigated, has led to further hypothesis that cell-to-cell coupling mediated by the connexin channels which concentrate at gap junction domains of the cell membrane may be a central event in this control. Here, we review the main lines of evidence in favor of this hypothesis, with particular focus on

those studies which have been published since the previous reviews on this topic (Petersen, 1980; Meda et al., 1984; Meda, 1996; Munari-Silem and Rousset, 1996; Meda and Spray, 2000).

2. Connexins and coupling of exocrine gland cells The secretory cells of all exocrine glands so far investigated have been shown to co-express Cx26 and Cx32, two connexins of the b-group, and no connexin of either the a (Meda et al., 1993) or c groups (Fig. 2 ). Thus, Cx26 and Cx32 have been found to link the acinar cells of the exocrine pancreas, of the major salivary glands and of lacrymal


Fig. 2. The connexin patterns of gland cells. Vertebrate cells express at least 20 different connexin isoforms. At least six of these connexins are expressed in rodent and human glands. Epithelial cells producing exocrine secretions usually co-express Cx26 and Cx32, two connexins of the b-group. In contrast, most epithelial cells secreting hormones express only one connexin isoform of either the a (Cx43, Cx40) or the of c group (Cx36). However, some endocrine cells associated to central nervous system (pinealocytes and some secretory neurons) may be coupled by the b connexin Cx26. The most widespread “endocrine” connexin (Cx43) also associates the human (sebocytes) and rodent cells (of preputial glands) that produce pheromones. Epithelial cells with a dual exocrine and endocrine function (ovarian granulosa cells and hepatocytes) express either an a (Cx43, Cx37) or b type connexin (Cx26, Cx32). The follicular cells which produce thyroid hormones co-express both a (Cx43) and b connexins (Cx26, Cx32), in keeping with their endocrine function but exocrine origin and arrangement within the gland. Supportive gland cells (Sertoli cells of testis, fibroblasts and myoepithelial cells of exocrine glands) are linked by Cx43. Endothelial cells are coupled by Cx40 and Cx43 in both endocrine and exocrine glands. Duct cells of exocrine and pheromone glands usually express Cx26, even though other connexins are probably expressed in some cases, e.g. pancreas.

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glands, as well as the alveolar cells of mammary glands, the parietal cells of gastric pits and the hepatocytes of liver (Meda et al., 1993; Fujikura et al., 1993; Monaghan et al., 1994; Pozzi et al., 1995; Shimono et al., 1996; Muramatsu et al., 1996; Radebold et al., 2001). It appears therefore that a same set of connexins has been selected during the evolution by all major exocrine glands, in spite of major differences in their embryological origin, developmental chronology, adult architecture, composition of their secretory products (Table 1 ), rate and control of secretion. The reason for this consistent choice remains to be understood. Conceivably, it could be related to the specific conductance, permeability and regulatory characteristics that Cx26 and Cx32 impart to homomeric connexons and complete inter-cellular channels (Veenstra et al., 1998; Harris et al., 1998; Goldberg et al., 1999), and which, in turn, may be required to control the cell-to-cell exchange of specific signal molecules. Recent experiments on transgenic mice have shown that the functional deletion of either the gene coding for Cx32 (Nelles et al., 1996); or the coding for Cx26 (Willecke and Ott, personal communication) did not markedly alter the embryological development, growth and differentiation of the glands, inasmuch as mice lacking one of these genes featured apparently normal exocrine glands. However, at least in the case of Cx32, this deletion induced marked changes in the secretion of hepatocytes (Nelles et al., 1996; Stumpel et al., 1998), as well as of acinar cells of both pancreas (Chanson et al., 1998) and lacrymal glands (Walcott et al., 2002), in spite of the retained expression of Cx26 and of a reduced, but persistent coupling (Nelles et al., 1996; Chanson et al., 1998).

2.1. Pancreas In the case of pancreatic secretion, the effect was sufficiently important to increase the basal circulating levels of amylase, providing direct evidence that at least Cx32 plays a significant role in the proper in vivo functioning of adult glands (Chanson et al., 1998). These observations attribute to specific connexin species (Cx32) effects that were previously observed both in vivo and in vitro, using various preparations of normal rodent pancreas. Thus, pancreatic cells were found to be electrically and dye coupled (Fig. 3 ) throughout each pancreatic acinus under resting conditions (Petersen, 1980; Meda et al., 1987; Chanson et al., 1991), but to rapidly uncouple during maximal stimulation by a variety of secretagogues which promote amylase release (Petersen, 1980; Meda et al., 1987; Chanson et al., 1991), including after the in vivo stimulation of the vagal nerve, a procedure that releases the major secretagogue acetylcholine in a manner similar to that occurring physiologically (Chanson et al., 1991). Since alkanols blocking acinar cell coupling also increased amylase release in the absence of other secreta-

gogues (Meda et al., 1987), the data clearly indicate that acute uncoupling of acinar cells is somehow required to initiate, maintain or enhance the increase in the secretion of pancreatic enzymes which is elicited by both endogenous hormones and neurotransmitters and pharmacological agonists. Intriguingly, the same secretagogues that uncouple acinar cells also increase in their cytosol the free Ca2+ concentration, leading to coordinated waves of the cation that propagate from one cell to another (Nathanson et al., 1992; Kasai et al., 1993). Since the frequency of the Ca2+ oscillations within an acinus is thought to be at least in part dependent on the presence of opened gap junction channels (Stauffer et al., 1993), it is possible that uncoupling may take place only after the stimulation by the agonists has started. In this case, the persistence of junctional coupling at the beginning of the agonist action may allow for the transmission of a threshold signal produced in few acinar cells to the entire acinus. This inter-cellular amplification could lead to the functional recruitment of additional secreting cells, an effect consistent with the finding that dispersed acinar cells secrete poorly, compared to cell clusters and isolated acini, when exposed to the same secretagogues (Bosco et al., 1988;Yule et al., 1996). In this perspective, the subsequent uncoupling may be needed to terminate the agonist action. It has indeed been reported that the gap junctional conductance of acinar cell pairs decreases after the agonist-induced Ca2+ peak and is the lowest during the intracellular Ca2+ plateau (Chanson et al., 1999). However, it is equally possible that among the endogenous signal molecules that permeate connexin channels, some may negatively control the secretion of enzymes produced by acinar cells. In this view, uncoupling, by hindering the inter-cellular exchange of these signals, would allow a number of acinar cells to escape inhibition and enter activated secretion. Eventually, uncoupling may be needed to functionally isolate those acinar cells that are highly sensitive to secretagogues, in order to prevent the dilution of the agonist-induced second messengers into less sensitive cells of the acinus (Chanson et al., 1998). It has indeed been shown that, when exposed simultaneously to a secretagogue, individual acinar cells show a rather variable secretory function and that this functional heterogeneity decreases as acinar cells aggregate into clumps expressing connexins (Bosco et al., 1988, 1994). In summary, the evidence gathered using various preparations of exocrine pancreas has provided compelling evidence for a physiological role of connexindependent signaling in the control of both basal and stimulated release of digestive enzymes. Much less evidence has been gathered in other cell systems, that would allow to understand whether the implication of gap junctional coupling is a general requirement for the proper functioning of all exocrine glands. The following models provide, however, some valuable support to this possibility.


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Table 1 Connexin pattern and secretory products of exocrine and endocrine gland cells Cx isoform

Gland

Secretory cell

Secretory product

Cx26

Stomach Salivary glands Pancreatic acini

Parietal cell Acinar cell Acinar cell

Mammary gland Lacrymal glands Seminal vesicle Prostate Pineal gland Hypothalamus

Alveolar cell Acinar cell Alveolar cell Alveolar cell Pinealocyte Neuron

Thyroid Liver

Thyrocyte Hepatocyte

Stomach

Parietal cell

Salivary glands Pancreatic acini

Acinar cell Acinar cell

Mammary gland Lacrymal glands Seminal vesicle Prostate Pineal gland Hypothalamus

Alveolar cell Acinar cell Alveolar cell Alveolar cell Pinealocyte Neuron

Thyroid Liver

Thyrocyte Hepatocyte

Pancreatic islets Adrenal medulla Hypothalamus

b-cell Cathecolamineproducing cell Neuron

Exocrine gastic juice (HCl, ions, glycoproteic intrinsic factor,...) blood - transported HCO3– Exocrine saliva (ions, proteins, mucins, enzymes,...) Exocrine pancreatic juice (ions, amylase, lipase and > 20 other proteic and glycoproteic enzymes,...) Exocrine milk (ions, peptides, gylcoproteins, lipids,...) Exocrine lacrymal fluid (ions, peptides, glycoproteins,...) Exocrine semen (fructose, peptides, prostaglandins,...) Exocrine fluid (peptides, enzymes, fibrinolysin,...) Indolamine hormones (melatonin, serotonin) Peptide homones (GnRH, CRH, TRH, GHRH, somatostatin) Amine hormone (PIH) ? hormone (PRF) Glycoproteic hormones (thyroxin, triiodothyronin) Blood-transported molecules (glucose, albumin,...) Exocrine bile (ions, cholesterol, phospholipids, bilirubin, cholic and chenodeoxycholic acids) Exocrine gastic juice (HCl, ions, glycoproteic intrinsic factor,...) Blood - transported HCO3– Exocrine saliva (ions, proteins, mucins, enzymes,...) Exocrine pancreatic juice (ions, amylase, lipase and > 20 other proteic and glycoproteic enzymes,...) Exocrine milk (ions, peptides, gylcoproeteins, lipids,...) Exocrine lacrymal fluid (ions, peptides, glycoproteins,...) Exocrine semen (fructose, peptides, prostaglandins,...) Exocrine fluid (peptides, enzymes, fibrinolysin,...) Indolamine hormones (melatonin, serotonin) Peptide homones (GnRH, CRH, TRH, GHRH, somatostatin) Amine hormone (PIH) ? hormone (PRF) Glycoproteic hormones (thyroxin, triiodothyronin) Blood-transported molecules (glucose, albumin,...) Exocrine bile (ions, cholesterol, phospholipids, bilirubin, cholic and chenodeoxycholic acids) Peptide hormone (insulin) Amine hormones (epinephrine, norepinephrine)

Cx37

Ovary

Granulosa cell

Cx40

Kidney

Cx43

Testis Adrenal cortex Parathyroid Thyroid Pituitary (pars distalis) Hypothalamus

Myoepithelial cells of afferent arteriole Sertoli cell Leydig cells Spongiocyte Chief cell C cells Basophil cell Acidophil cell Neuron

Ovary

Granulosa cell

Pancreatic islets (?) Skin Preputial glands

Luteal cells b-cell (?) Sebocyte Alveolar cell

Cx32

Cx36

Peptide homones (GnRH, CRH, TRH, GHRH, somatostatin) Amine hormone (PIH) ? hormone (PRF) Steroid hormone (estradiol) Exocrine antral fluid (ions, glycoproteins) Glycoproteic hormone (renin) Peptidic exocrine fluid (inhibins, activins,...) Steroid hormone (testosterone) Steroid hormones (aldosterone, angiotensin II, cortisol, steroid sex hormones) Peptide hormone (parathormone) Peptide hormone (calcitonin) Glycoproteic hormones (FSH, LH, TSH, POMC, ACTH, b- lipotropin) Peptide hormones (growth hormone, prolactin) Peptide homones (GnRH, CRH, TRH, GHRH, somatostatin) Amine hormone (PIH) ? hormone (PRF) Steroid hormone (estradiol) Exocrine antral fluid (ions, glycoproteins) Steroid hormones (progesterone, estradiol) Peptide hormone (insulin) Lipidic pheromenones (sebum) Lipidic pheromones (farnesenes)

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Fig. 3. Connexins and coupling differ in the endocrine and exocrine pancreas. Cx32 is localized by immunofluorescence between the secretory cells of the exocrine acini, but not between those of an endocrine islet of a rat pancreas (B, A is the phase-contrast view of the field shown in B). In contrast, Cx36 is detected between the islet cells, but not between the cells of the surrounding pancreatic acini of a mouse pancreas (E, D is the phase-contrast view of the field shown in E). Microinjection of the fluorescent, gap junction-permeant tracer LuciferYellow, revealed that all secretory cells are dye coupled within an exocrine pancreatic acinus (C). In contrast, when the experiment is repeated with an isolated rat islet, the tracer delineated restricted territories of coupled cells, each resulting from the microinjection of a distinct islet cell (three of such territories, due to three successive injections, are seen in F. The bar represents 20 µm in A, B, D and E and 60 µm in C and F).

2.2. Salivary glands Connexin involvement in salivary secretion appears quite similar to that of pancreas. Thus, the acinar cells of the two types of glands express the same connexins (Meda et al., 1993; Shimono et al., 1996, 2000), individually show a heterogeneous function (Segawa et al., 2002), become uncoupled during the stimulation of salivary secretion by cholinergic agonists (Petersen, 1980; Sasaki et al., 1995) and

show inhibited secretion in the presence of octanol (Kim et al., 1999). To what extent this effect is only due to cell uncoupling remains to be determined, inasmuch as the uncoupling drug also inhibited the capacitive entry of Ca2+ into salivary acinar cells (Kim et al., 1999). The use of the knock out transgenic mice lacking one or another of the connexins connecting the acinar cells of salivary glands (Nelles et al., 1996; Ott and Willecke, personal communication) should provide some insights into this question.


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It has been recently reported that the parietal cells of gastric glands, which secrete H+ in the stomach lumen and HCO3– in the vessels of the gastric mucosa, are joined by Cx26 and Cx32 (Radebold et al. 2001). Intriguingly, the channels made by these proteins appear not to be permeable to Lucifer Yellow under resting conditions, when acid secretion is at the basal level. Stimulation of isolated glands with gastrin elicited the inter-cellular propagation of a Ca2+ wave and of microinjected Lucifer Yellow, two events that were blocked in the presence of the uncoupling drug 18-a -glycyrrhetinic acid (Radebold et al. 2001).

2002). Accordingly, cumulus–oocyte complexes exposed to drugs blocking gap junction channels were reported not to normally progress to the metaphase II stage (Vozzi et al., 2001). Moreover, after in vitro transduction with an antisense cDNA, which reduced Cx43 expression and oocyte–cumulus cell coupling, these complexes showed retarded oocyte maturation (Vozzi et al., 2001). In vivo grafting into control females of ovaries of transgenic mice lacking Cx43, also resulted in the aborted development of follicles and retarded oocyte growth, in spite of the persistence of some coupling between granulosa cells (Ackert et al., 2001). Together, the data support a specific role of Cx43 in sustaining the expansion of granulosa during the formation of antral follicles, which is a prerequisite for proper oocyte maturation that, in turn, is required for fertilization (Ackert et al., 2001). Intriguingly, the levels of expression of Cx43 and Cx37 are differentially regulated during folliculogenesis (Nuttinck et al., 2000) and oocytes from mice lacking Cx37 are unable to initiate meiotic maturation (Simon et al., 1997), unless treated with a phosphatase inhibitor, suggesting that Cx37 may be specifically involved in the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence (Carabatsos et al., 2000).

2.5. Lacrymal glands

2.7. Testis

In mice, the acinar cells of exorbital lacrimal glands are extensively coupled (Petersen, 1980; Neyton and Trautmann, 1985) by Cx32 and Cx26 channels (Meda et al., 1993; Takayama et al., 2002). Loss of Cx32 after homologous recombination of the cognate gene locus (Nelles et al., 1996), caused the electrical uncoupling of lacrimal acinar cells and decreased their fluid secretion in response to low, but not to high doses of carbachol, a difference which was accounted for by the variable diffusion of the drug, which was applied at the surface of the gland, in the center of the organ (Walcott et al., 2002). Intriguingly, the secretory defect was only observed in female mice and did not appear to impact on the integrity of the cornea of the knock out animals.

Sertoli and basal germ cells of testis are connected by Cx43, and the expression of this protein, as well as coupling, change during differentiation of the seminiferous epithelium (Batias et al., 1999). This finding and the observation that the levels of Cx43 were reduced in the testes of mutant mice with impaired spermatogenesis (Batias et al., 2000; BravoMoreno et al., 2001), suggests an in vivo role of connexin signalling in the control of spermatogenesis. Accordingly, both the inactivation of the Cx43 gene by homologous recombination (Roscoe et al., 2001; Juneja et al., 1999) and its replacement by either Cx32 or Cx40, under control of the native Cx43 promoter (Plum et al., 2000), have been reported to affect the subsequent growth and differentiation of testes. It remains to be shown whether this effect is specifically due to the effect of the connexin loss on the cells of the seminiferous epithelium or a secondary effect of alterations of the numerous types of accessory cells (endothelial and smooth muscle cells of vessels, mesenchymal cells of testis interstitium,{) that are required for proper testicular function and which also express Cx43 (Fig. 2).

2.3. Mammary glands Gap junction changes have been described by comparing mammary glands during pregnancy, lactation and after weaning of the pups (Pitelka et al., 1980). In agreement with these observations, Cx26 and Cx32 were found to be co-expressed in the mammary alveolar cells of lactating rodents but were not observed in nonpregnant animals (Pozzi et al., 1995; Yamanaka et al., 2001; Locke et al., 2000). 2.4. Gastric glands

2.6. Ovary In the developing follicles of ovary, gap junctions couple the growing oocyte and its surrounding cumulus cells by Cx37 channels and the mural granulosa cells of the entire follicle via Cx43 channels (Risek et al., 1990; Simon et al., 1997; Kidder and Mhawi, 2002). Thus, a- and b-type connexins are segregated, possibly to define distinct cellular compartments (Wright et al., 2001). The arrest of follicular development at early preantral stages and the production of incompetent oocytes after loss of either Cx43 (Juneja et al., 1999; Ackert et al., 2001) or Cx37 (Simon et al., 1997) indicate that connexin signaling has a physiopathological relevance for the preantral to antral transition of follicles which is needed to ensure a normal meiotic maturation of oocytes (Kidder and Mhawi, 2002; Klinger and De Felici,

3. Connexins and coupling of endocrine gland cells The secretory cells of most multicellular, endocrine glands express only one connexin isoform of either the a or of c group (Fig. 2). Cx43 is by far the most widespread and has been found to connect the Leydig cells of testis, the cells of adrenal cortex, the chief cells of parathyroid, the thyroid cells making calcitonin, the cells of the anterior, intermediate

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and posterior lobes of pituitary, the cells of ovarian corporea lutea, the cells of the pineal gland (Meda et al., 1993; Berthoud and Saez, 1993; Murray et al., 1995; Munari-Silem et al., 1995; Mayerhofer and Garfield, 1995; Morand et al., 1996). Cx43 also associates the secretory cells that produce pheromones in humans (sebaceous glands of skin) and rodents (preputial glands) (Meda et al., 1993; Salomon et al., 1994). The distribution of Cx40 is so far restricted to the renin-producing cells of kidney arterioles (Haefliger et al., 2001), whereas that of Cx36 has been shown between the pancreatic b-cells (Serre-Beinier et al., 2000), most other secretory neurons (Belluardo et al., 2000) and may also take place in the adrenal medulla (Martin et al., 2001). Particularly, no macroscopic endocrine gland other than the thyroid (Fig. 2), which embryologically develops as an exocrine gland and maintains throughout life an architectural organization (extracellular lumen of follicle storing the prehormones) partially reminiscent of an exocrine function, expresses a connexin of the b group (Meda et al., 1993; Munari-Silem et al., 1994). However, cells of pineal gland express the b connexin Cx26 (Saez et al., 1991). It appears therefore that various connexin species have been selected during evolution by different endocrine cells (Fig. 2). Presently, the reason for this variable choice cannot be explained as a function of either the embryological origin, chronology of development or architecture of the adult glands. It cannot be either related to the biochemical nature of the hormones produced by each gland (Table 1), the rate of their secretion and the control of either the release or biosynthesis of these secretory products. The development of lines of transgenic mice lacking either Cx43 (Reaume et al., 1995; Theis et al., 2001a, b), Cx32 (Nelles et al., 1996) or both connexins (Houghton et al., 1999), and the more recent development of Cx36 knock out mice (Deans et al., 2001; Guldenagel et al., 2001) have failed to reveal obvious alterations of the normal pre-natal development, morphogenesis and differentiation of endocrine glands, suggesting that connexin-dependent signaling is not obligatory for these processes. However, increased tissue development was detected by morphometric analysis and measurements of hormone content in the endocrine pancreas of transgenic mice in which the insulin-producing b-cells were selectively forced to ectopically overexpress Cx32, suggesting a role of connexin-dependent cell communication in the in vivo, postnatal growth of the endocrine cells of pancreatic islets (Charollais et al., 2000). 3.1. Pancreatic islets This alteration was paralleled by a decrease in the insulin secretion stimulated by glucose, which was sufficiently important to cause an intolerance to the sugar in the living animals (Charollais et al., 2000), providing the first in vivo evidence that connexin-dependent signaling contributes to control insulin secretion. In view of the recent identification that Cx36 is the prominent, if not the sole connexin protein

expressed by adult pancreatic b-cells (Serre-Beinier et al., 2000), this contribution should now be tested in transgenic mice in which this connexin has been deleted by a homologous recombination approach (Guldenagel et al., 2001). At any rate, spontaneous changes in the in vivo function of insulin-secreting cells are associated with changes in Cx36 expression and b-cell coupling (Calabrese et al., 2001, Caton et al., in press). Conversely, in vitro inhibition of Cx36 expression alters glucose-induced insulin release (Calabrese et al., 2001, Caton et al., in press). Several other lines of evidence indicate a contribution of b-cell coupling to the control of insulin secretion. Thus, single b-cells show alterations in secretion and insulin gene expression, which are rapidly corrected after restoration of b-cell contacts (Salomon and Meda, 1986; Bosco and Meda, 1991; Philippe et al., 1992) and are mimicked by exposure of either intact islets or entire pancreas to a drug which blocks gap junction channels (Meda et al., 1990). Also, a number of cell lines which show defects in insulin secretion do not express connexins (Vozzi et al., 1995). However, when implanted in living rats, these cells gained the ability to express Cx43 and formed tumors that released levels of insulin increasing with those of connexin expression (Vozzi et al., 1997). Recently, a marked increase with expression of the Cx43 protein has also been correlated with increased insulin secretion from neonatal islets exposed to prolactin (CollaresBuzato et al., 2001). These findings are intriguing, inasmuch as the presence of Cx43, which has been consistently documented at the transcript level (Serre-Beinier et al., 2000; Meda et al. 1993; Vozzi et al., 1995; Charollais et al., 2000; Collares-Buzzato et al., 2001) has been recently questioned in transgenic mouse islets. Thus, the Cre/loxP-dependent deletion of the Cx43 coding region under conditions activating the expression of a reporter gene (Theis et al., 2001a, b), as well as the expression of either Cx32 or Cx40 under control of the endogenous Cx43 promoter (Plum et al., 2000), have failed to reveal a Cx43-specific signal in pancreatic islets. These negative findings contrast with the earlier observation of sparse immunolabeling for Cx43 in islets of normal rats (Meda et al., 1993 and Fig. 3) and with the finding of sizable levels of this protein between the still undifferentiated endocrine cells of embryonic human pancreas (unpublished data). The reason for this apparent discrepancy remains to be elucidated, since there is no obvious evidence for the lack of specificity of the previously published immunolabeling data. The findings made in the prolactin-treated rats (Collares-Buzzato et al., 2001) and the more recent observation that Cx36 replaces Cx43, as the insulin-producing cells of the human endocrine pancreas achieve full differentiation (unpublished data), now call for further experiments to carefully evaluate whether the levels of Cx43 expression may be selectively increased under specific physio-(patho-)logical conditions. At any rate, it appears that, in both laboratory rodents and humans, normal adult pancreatic b-cells express the Cx36 protein (Fig. 3) at


levels significantly higher than those of the Cx43 protein (Serre-Beinier et al., 2000). Recent work has indicated that stimulation by glucose induces secretory and metabolic responses from either intact pancreatic islets or clusters of islet cells, which are markedly more uniform than those of single b-cells (Bennett et al., 1996; Valdeolmillos et al., 1996, Fernandez and Valdeolmillos, 2000; Jonkers et al., 1999, Jonkers and Henquin, 2001; Andreu et al., 1997). Thus, coupling between pancreatic b-cells contributes both to increase the acute release of insulin after stimulation, and to synchronize the activity of individual cells which, taken individually, are metabolically and secretory heterogeneous (Meda, 1996; Meda and Bosco, 2001). Additional evidence for the participation of connexindependent signaling in the function of other endocrine organs has also been gathered in the following cell systems.

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of a direct link between the direct communication of luteal cells and their progesterone production (Grazul-Bilska et al., 2001). Consistent with this view, the experimental inhibition of Cx43 expression decreased the LH-induced steroid secretion of luteal cells (Khan-Dawood et al., 1998). Interestingly, mice lacking Cx37, the connexin that forms gap junctions between oocytes and granulosa cells, fail to ovulate and develop numerous inappropriate corpora lutea (Simon et al., 1997). 3.5. Thyroid gland

Drugs that block gap junctional conductance have been reported to impair adrenocorticotropic hormone (ACTH)induced secretion of cortisol from clusters of adrenal cells but not from single cells in culture (Murray et al., 1995; Munari-Silem et al., 1995). The study further revealed that the drugs did not affect the secretion of cells stimulated by 8-bromo-cAMP, suggesting that the coupling effect was somehow related to the chain of events triggered by the binding of ACTH to the adrenal cell receptors (Munari-Silem et al., 1995). Consistent with these data, another study has shown that the transfection of adrenal cells with an antisense Cx43 cDNA which decreased cell coupling, inhibited the secretion of steroids induced by ACTH (Oyoyo et al., 1997).

Cells of thyroid follicles are unusual among endocrine cells since they co-express Cx32 and Cx43 in different domains of the lateral cell membrane (Meda et al., 1993; Guerrier et al., 1995). Since these connnexins cannot form heterotypic channels with each other (White and Bruzzone, 1996) and provide homotypic channels with rather distinct conductance, permeability and regulatory characteristics (Veenstra et al., 1998; Harris et al., 1998, Harris, 2001; Goldberg et al., 1999), they presumably fulfill different physiological functions. In both primary thyrocytes and the derived line of FRTL-5 cells, expression of Cx32 (but not Cx43) and coupling are lost with culture time, in parallel with the ability to form follicular structures (Munari-Silem et al., 1994; Green et al., 2001), whereas exposure to TSH induces cell monolayers to form follicular structures in parallel with the novel expression of Cx32 (Munari-Silem et al., 1994). Moreover, transfection of communication-incompetent FRT cells with Cx32 leads to the formation of follicular type organoids, which did not form after transfection of Cx43 (Tonoli et al., 2000). These data indicate that Cx32 may be required for proper morphogenesis of thyroid follicles. However, it remains to be understood whether a difference in animal species or in the in vitro vs. in vivo control of follicular morphogenesis explains as to why transgenic mice lacking Cx32 do not show obvious defects in thyroid architecture (Nelles et al., 1996; Houghton et al., 1999). It has also been reported that stimulation by TSH increases the dye coupling of primary thyrocytes in a time- and concentration-dependent manner (Munari-Silem et al., 1991) and that a spontaneous mutation of Cx32, leading to loss of coupling, reduced the release of thyroxin (Green et al., 2001). Eventually, transfection of Cx32 in cell lines was shown to increase the expression of the thyroglobulin gene (Statuto et al., 1997).

3.4. Corpus luteum

3.6. Pituitary gland

The secretory cells producing steroids in the corporea lutea of ovary are connected by Cx43 up to their atretic involution (Mayerhofer and Garfield, 1995; Khan-Dawood et al., 1996). The findings that pharmacological treatments enhancing cell coupling also increase progesterone secretion and, conversely, that drugs inhibiting junctional channels also decrease steroid release, have been taken as a suggestion

Several types of anterior pituitary cells have been shown to be coupled (Morand et al., 1996; Fauquier et al., 2001) via channels made of Cx43, Cx26 and Cx36 (Meda et al., 1993; Yamamoto et al., 1993; Belluardo et al., 2000). This coupling increases the synchrony of fast peaking Ca2+ transients, that spontaneously initiate within individual cells, across groups of coupled cells (Guerineau et al., 1998). Ca2+ waves may

3.2. Adrenal medulla A study of rat adrenal slices, has revealed the simultaneous occurrence of both spontaneous and electrically evoked Ca2+ transients in neighboring chromaffin cells, which was attributable to gap junctional communication (Martin et al., 2001). This coupling was activated after a synaptic-like application of nicotine to a single cell, and triggered catecholamine release in coupled cells, suggesting that connexin channels act to amplify catecholamine secretion after synaptic stimulation of individual cells (Martin et al., 2001). 3.3. Adrenal cortex

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also propagate throughout the entire gland via a network of folliculo-stellate cells, coupled by Cx43 channels (Fauquier et al., 2001). Changes in the expression of Cx43 have been shown to parallel changes in prolactin secretion, suggesting a contribution of connexin-dependent signaling in the control of the seasonal changes of pituitary activity (Vitale et al., 2001). 3.7. Hypothalamus Coupling of hypothalamic neurons has been implicated in the pulsatile release of neuropeptides, including gonadotropin-releasing hormone (Taylor and Dudek, 1982; Perez et al., 1990; Matesic et al., 1993, 1997). Accordingly, drugs blocking gap junction channels have been shown to reversibly abolish the periodic burst of electrical activity of secretory neurons (Shinohara et al., 2000). There is some uncertainty about the specific connexin species that may mediate this effect since Cx26, Cx36 and Cx32 have been described between hypothalamic neurons (Wetsel et al., 1992; Matesic et al., 1993, Hu et al., 1999, Belluardo et al., 2000; Colwell, 2000; Hosny and Jennes, 1998). Recently, one case of moderate growth retardation associated with congenital hearing loss due to a Cx26 mutation, has been attributed to partial hypogonadotrophic hypogonadism of hypothalamic origin (Houang et al., 2002). This report provides support for the presence of at least Cx26, and the first suggestion that the control of the secretion of gonadotropinreleasing hormone by this connexin may significantly contribute to puberty initiation. However, it is unclear as to why the many other hearing-deficient patents that also carry the same mutation, appear not to have developed the same endocrine disorder. Neurons of the suprachiasmatic nucleus are extensively coupled during the day, when the cells exhibit synchronous activity, but become minimally coupled during the night, when the cells are electrically silent, suggesting that coupling may also be involved in the regulation of circadian rhythms (Colwell, 2000).

4. Connexins and coupling of mixed exocrine-endocrine gland cells Variable patterns of connexin expression are observed in glands producing a parallel, though independent exocrine and endocrine secretions (Fig. 2), irrespective of whether the two types of secretions are accounted for by segregated populations of distinct secretory cell types (pancreas, testis) or by a single type of secretory cell (liver, ovary). Thus, the exocrine acinar cells of pancreas express the b-type Cx26 and Cx32, whereas the endocrine islet cells of the gland express the c-type Cx36 and possibly the a-type Cx43, two proteins which do not form functional channels with b connexins (White and Bruzzone, 1996). In contrast, in the testis, both the exocrine (Sertoli and germ cells) and the endocrine

Leydig cells express Cx43. In the liver, hepatocytes are always connected by Cx32 and in the peri-portal regions, by Cx26. In the ovary, granulosa cells form Cx37 channels with oocytes and Cx43 channels with the companion cells forming most of the follicle walls (Fig. 2). As yet, however, few studies have investigated in parallel the involvement of connexin-signaling in both the endocrine (released in the vascular compartment) and exocrine secretion (released in the external milieu) of the same mixed gland. The following are the available data. 4.1. Pancreas Vascular perifusion of the entire pancreas in situ with drugs blocking gap junctional channels, resulted in a simultaneous increase in the amylase and fluid secretions from the exocrine acini and in a decrease in the insulin secretion from the endocrine islets of the very same organ (Bruzzone and Meda, 1988). 4.2. Liver The expression of hepatocyte-specific marker proteins was shown to correlate with the co-expression by liver cells of Cx26 and Cx32, and to be lost under culture conditions leading to the replacement of these two connexins by Cx43 (Stutenkemper et al. 1992). Since this protein is not observed in normal hepatocytes, full functional differentiation of these cells appears to require the native connexin pattern which is observed in vivo. Analysis of transgenic mice lacking Cx32 (Nelles et al., 1996) has further revealed that these animals have a reduced glucose output (one of the numerous secretory products the liver releases in the blood stream), resulting from hydrolysis of the glycogen stores of hepatocytes, in response to noradrenaline and glucagon (Stumpel et al., 1998). The finding of this difference when the two hormones were perifused at less than half-saturating concentration and not at saturating concentrations, suggests that connexin channels may propagate both a neural (sympathetic) and a hormonal signal (cAMP?) from peri-portal to centro-lobular hepatocytes (Stumpel et al., 1998). Interestingly, hepatocytes lacking Cx32 and featuring abnormal sensitivity to hormones, also exchanged IP3 and transmitted Ca2+ waves less efficiently than control cells (Niessen and Willecke, 2000). Pharmacological blockade of gap junctional channels has been reported not to affect basal bile secretion (the exocrine secretion of liver), but to alter the increase and decrease in bile flow induced by glucagon and vasopressin, respectively (Nathanson et al., 1999). Since the treatment did not alter bile flow when the levels of the second messengers used by the two hormones were experimentally increased throughout the hepatic lobule, the data suggest that gap junction communication may be important for the diffusion towards peri-portal hepatocytes of signals (cAMP?, Ca2+?) generated in centrolobular cells (Nathanson et al., 1999). Accordingly, electrical stimulation of sympathetic nerves in mice lacking Cx32


leads to an abnormally low decrease of bile flow, but not of total bile output, suggesting that the contraction of bile canaliculi is somehow impaired in the absence of the connexin (Temme et al., 2001). This absence should not impair the cells of the extrahepatic biliary tract, which have been recently shown to be coupled via Cx43 channels (Bode et al., 2002). Eventually, loss of Cx32 has also been shown to promote the rate of hepatocyte proliferation (Temme et al., 1997), an effect that presumably accounts for the higher susceptibility of the knock out mice to develop spontaneous, as well as chemically induced hepatocarcinomas (Temme et al., 1997; Moennikes et al., 2000a). Intriguingly, however, subpopulations of tumor cells were shown to similarly proliferate, irrespective of the levels of Cx32 expression (Moennikes et al., 2000b). 4.3. Ovary The granulosa cells, that form the wall of ovarian follicles are connected by at least Cx43 and Cx37 (Risek et al., 1990; Simon et al., 1997; Kidder and Mhawi, 2002), whose expression varies with the stage of follicle development. Previous experiments have provided evidence that these two connexins may be involved in both the exocrine (fluid of follicular antrum) and endocrine secretions (steroid hormones) of granulosa cells (Simon et al., 1997; Juneja et al., 1999; Ackert et al., 2001; Vozzi et al., 2001; Kidder and Mhawi, 2002; Klinger and De Felici, 2002).

5. Other types of secretory cells Connexins are also expressed by many cell types which secrete outside macroscopic multicellular glands, including fibroblasts, osteocytes and lymphoid cells. These cells, which release secretory products acting in a paracrine manner or contributing to the formation of extra-cellular matrix, almost consistently express Cx43 plus variable levels of Cx45 and Cx40 (Lecanda et al., 2000; Ehrlich et al., 2000; Meda and Spray, 2000).

6. Effects of secretory products on connexin expression and coupling Many effects of other endocrine secretory products, neurotransmitters, growth factors, enzymes and cytokines have been reported on the transcription, mRNA stability, translation and cytoplasmic trafficking of connexins, as well as on the gating and regulation of the cell-to-cell channels formed by these proteins (Petersen, 1980; Stagg and Fletcher, 1990; Meda et al., 1984; Meda, 1996; Munari-Silem and Rousset, 1996; Bruzzone et al., 1996; Saez et al., 1998, Meda and Spray, 2000; Harris, 2001). The effect of a given secretory product and the molecular mechanism underlying it may vary in different cell types, possibly depending on the cell-

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specific pattern of connexin expression, and the reader is referred to the previous reviews mentioned above for a comprehensive discussion of these differences.

7. Conclusions, questions and perspectives The studies summarized above have provided increasing evidence for a physiologically relevant role of cell-to-cell communication mediated by connexins in the secretion of a variety of exocrine, endocrine and mixed glands. They have also opened up the following series of questions, many of which do not yet have an adequate answer but which provide new leads for exciting investigations to come: 7.1. Why do secretory cells use multiple mechanisms to communicate? Most likely, the multiplicity of communication mechanisms (Fig. 1) permits gland cells to properly adapt their secretion to the needs of the organism, which continuously change throughout the day depending on the physiological condition. It also provides these cells with several independent signaling pathways that, if somewhat redundant and costly, may be instrumental in preserving an adequate secretory function under pathophysiological conditions that may perturb one or several systems for cell communication (Meda and Bosco, 2001). 7.2. Why do connexins significantly contribute to this signaling network? As compared to other forms of cell-to-cell communication, coupling achieves a rapid inter-cellular equilibration of electrochemical gradients of cytoplasmic ions and molecules which may be crucial for secretion. This equilibration, in turn, could ensure the recruitment and coordination of a population of cells that, taken individually, would otherwise function asynchronously. Although secretory cells of a given type are generally considered to form homogeneous populations, there is some evidence that they may also differ in several respects, including the ability to release secretory products (Salomon and Meda, 1986; Bosco et al., 1988, 1989, 1994; Bosco and Meda, 1991). 7.3. How is the connexin-dependent signaling achieved? Many of the endogenous molecules (cAMP, nucleotides, Ca2+, K+, IP3,{) that are known to permeate connexin channels are also important players for many types of secretions (Petersen, 1980; Stagg and Fletcher, 1990; Meda, 1996; Meda and Spray, 2000; Meda and Bosco, 2001), complicating the identification of the signal(s) that couple the connexin-dependent communication to changes in secretion. Other signals that are generated in the early steps of the

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glycolytic pathway have also been considered (Kohen et al., 1979; Goldberg et al., 1999). 7.4. Do connexins affect secretory cells by a mechanism independent of cell coupling? Recent studies have proposed that connexins may control cell functioning, and particularly gene expression and cell growth (Huang et al. 1998; Omori and Yamasaki, 1998; Moorby and Patel 2001), even in the absence of significant cell-to-cell communication. The underlying mechanism remains to be elucidated and may involve opened hemiconnexin channels that allow the passage of second messengers (Ca2+? others?) able to regulate gene transcription and/or the interaction of connexins, or fragments of these proteins, with transcriptions factors. While these possibilities are consistent with some observations recently also made in endocrine (Calabrese et al., 2001, in press) and exocrine gland cells (Qin et al., 2002), it is fair to stress that the formation of inter-cellular channels is, so far, the only proven function of connexins. It is certainly conceivable that connexins may not need to be assembled into functional gap junction plaques in order to exert a biological effect. However, much additional experimental data are required to eventually validate this putative, alternative role of connexins. In particular, the systems referred to above need to be revisited with highly sensitive electrophysiological approaches to exclude the fact that changes in low levels of cell coupling, falling below the detection threshold of the previously widely used dye injection procedures (Meda 2001) could account for the significant biological effects which have been reported. This situation has clearly been demonstrated at least for a line of insulin-producing cells (Calabrese et al., in press). 7.5. Why are exocrine and endocrine cells are linked by distinct connexin patterns? Increasing evidence indicates that different connexin isoforms, including Cx32 and Cx43 that are preferentially selected by exocrine and endocrine cells, respectively, form channels with distinct conductance, permeability and regulatory characteristics (Veenstra et al., 1998; Harris et al., 1998, Harris, 2001; Goldberg et al., 1999; Niessen and Willecke, 2000). Therefore, it is plausible that, during evolution, secretory cells have selected those sets of channels that, by favoring the inter-cellular exchange of specific signals while preventing the diffusion of others, better fit the requirements of their secretory machinery. 7.6. Are connexins implicated in clinical situations involving glands? Changes in connexin expression have been reported under a variety of pathological conditions which were caused by a gland dysfunction or that affected it, including in humans (e.g. Lee et al., 1992; Nagahara et al., 1996; Murray et al.,

2000; Carystinos et al, 2001; Houang et al., 2002; Habermann et al., 2002). As yet, however, this evidence remains circumstantial and does not provide a direct link between any alteration of a connexin and a secretory disease. 7.7. Could connexins be used to correct defective secretion in patients? If alterations in connexins and/or in the direct communications that these proteins permit were shown to cause secretory defects, one would be left with the problem of identifying means for restoring a normal situation in vivo. The task is formidable since connexins are widespread and are controlled by a large variety of factors. Thus, methods will have to be developed to specifically interact with individual proteins, on individual cell types. Recent evidence shows that at least some types of secretory cells are certainly experimentally amenable to such a selective expression of a membrane protein. The development of novel tools which should permit to extend these experiments to in vivo conditions opens the exciting perspective that this correction may also be envisaged in humans in a not too far future. Initial clinical tests with connexins have already provided encouraging information that these signaling proteins may be effectively used to improve the therapeutic treatment of some lethal human disorders (Meda and Spray, 2000).

Acknowledgements The authors thank Mrs. P. Severi-De Marco for excellent secretarial assistance. This work was supported by grants from the Swiss National Foundation (3100-06-67788.02), the Juvenile Diabetes Research Foundation International (12001-622), the National Institute of Health/NIDDK (DK 063443-01) the European Union (QLGT-CT-1999-00516) and the Swiss Academy of Medical Sciences (Theodor Ott Fund 07/01).

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