Granular Duct Cells Research Articles

IFT140+/K14+ cells function as stem/progenitor cells in salivary glands

Stem/progenitor cells are important for salivary gland development, homeostasis maintenance, and regeneration following injury. Keratin-14+ (K14+) cells have been recognized as bona fide salivary gland stem/progenitor cells. However, K14 is also expressed in terminally differentiated myoepithelial cells; therefore, more accurate molecular markers for identifying salivary stem/progenitor cells are required. The intraflagellar transport (IFT) protein IFT140 is a core component of the IFT system that functions in signaling transduction through the primary cilia. It is reportedly expressed in mesenchymal stem cells and plays a role in bone formation. In this study, we demonstrated that IFT140 was intensively expressed in K14+ stem/progenitor cells during the developmental period and early regeneration stage following ligation-induced injuries in murine submandibular glands. In addition, we demonstrated that IFT140+/ K14+ could self-renew and differentiate into granular duct cells at the developmental stage in vivo. The conditional deletion of Ift140 from K14+ cells caused abnormal epithelial structure and function during salivary gland development and inhibited regeneration. IFT140 partly coordinated the function of K14+ stem/progenitor cells by modulating ciliary membrane trafficking. Our investigation identified a combined marker, IFT140+/K14+, for salivary gland stem/progenitor cells and elucidated the essential role of IFT140 and cilia in regulating salivary stem/progenitor cell differentiation and gland regeneration.

Follistatin expressed in mechanically-damaged salivary glands of male mice induces proliferation of CD49f+ cells

Salivary glands (SGs) are very important for maintaining the physiological functions of the mouth. When SGs regenerate and repair from various damages, including mechanical, radiological, and immune diseases, acinar and granular duct cells originate from intercalated duct cells. However, the recovery is often insufficient because of SGs' limited self-repair function. Furthermore, the precise repair mechanism has been unclear. Here, we focused on CD49f, one of the putative stem cell markers, and characterized CD49f positive cells (CD49f+ cells) isolated from male murine SGs. CD49f+ cells possess self-renewal ability and express epithelial and pluripotent markers. Compared to CD49f negative cells, freshly isolated CD49f+ cells highly expressed inhibin beta A and beta B, which are components of activin that has anti-proliferative effects. Notably, an inhibitor of activin, follistatin was expressed in mechanically-damaged SGs, meanwhile no follistatin was expressed in normal SGs in vivo. Moreover, sub-cultured CD49f+ cells highly expressed both Follistatin and a series of proliferative genes, expressions of which were decreased by Follistatin siRNA. These findings indicated that the molecular interaction between activin and follistatin may induce CD49f+ cells proliferation in the regeneration and repair of mouse SGs.

First evidence of histological and histochemical intraspecific differences in salivary glands in Brown Norway and albino Wistar rats*

The size of salivary glands, acinar structure, and secretion particularities differ from one species to another and are influenced by diet and environment. In order to investigate whether there are histological or histochemical differences in the salivary glands of animals from the same species, bred in identical conditions of environment and diet, we assessed two laboratory rat strains. The salivary glands from two rat strains were processed for histological and histochemical investigations. Histologically, we noticed differences in the parotid gland: seromucous acini were present in Brown Norway rat and typical serous ones in Wistar rat. Histochemically, we observed that cells in the parotid gland present a moderate PAS-positive reaction only in Wistar rat. In the mandibular gland, the size and shape of the acini were similar, whereas the cytoplasm of acinar and granular duct cells was PAS-positive in both rat strains, but with different intensities. In the sublingual gland, both PAS and Alcian blue reactions were positive in the two rat strains, with a higher intensity in the Brown Norway strain. Our study highlighted intraspecific histological and histochemical differences in salivary glands from two rat strains bred and fed in identical conditions.

Changes in immunoglobulin A secretion induced by sympathetic and taste stimulation in the rat submandibular gland

Secretion of saliva is dependent on stimuli from autonomic nerves that are the effector arms of reflexes activated predominantly by taste and chewing. Nerve mediated stimulus evokes saliva secretion of water and proteins by different mechanisms. Secretion of submandibular saliva is controlled by the autonomic nervous system. The parasympathetic nervous system is the main controller of this secretion via impulses in the chorda tympani nerve that innervates it and releases acetylcholine and substance P. Both can evoke copious salivary secretion by activating muscarinic and tachykinin-1 receptors, respectively. In contrast, the sympathetic nervous system also controls salivary secretion by acting on αand βadrenergic receptors. Sympathetic nerve stimulation induced a relatively low flow of saliva that was rich in protein and was accompanied by extensive degranulation from both acinar and granular duct cells. The predominant secretory immunoglobulin on mucosal surfaces is immunoglobulin A (IgA). In the mouth, it is a component of the saliva secreted by the major and minor salivary glands. Secretory immunoglobulin (SIgA) is present in salivary secretions and, along with other glycoproteins such as mucin, lactoferrin and peroxidase, is responsible for helping maintain the integrity of mucosal surfaces against infectious agents. SIgA is the main component of the adaptive immune system. Polymeric, J-chain-conChanges in immunoglobulin A secretion induced by sympathetic and taste stimulation in the rat submandibular gland

Immunohistochemical detection of arginase-I expression in formalin-fixed lung and other tissues

Arginases are a family of enzymes that convert l-arginine to l-ornithine and urea. Alterations in expression of the isoform arginase-I are increasingly recognized in lung diseases such as asthma and cystic fibrosis. To define expression of murine arginase-I in formalin-fixed tissues, including lung, an immunohistochemical protocol was validated in murine liver, a tissue that has distinct zonal arginase-I expression, making it a useful control. In the lung, arginase-I immunostaining was observed in airway surface epithelium and this decreased from large to small airways, with a preferential staining of ciliated epithelium versus Clara cells and alveolar epithelia. In submucosal glands, the ducts and serous acini had moderate immunostaining, which was absent in mucous cells. Focal immunostaining was observed in alveolar macrophages, endothelial cells, pulmonary vein cardiomyocytes, pulmonary artery smooth muscle, airway smooth muscle, and neurons of ganglia of the lung. Arginase-I immunostaining was also detected in other tissues including salivary glands, pancreas, liver, skin, and intestine. Differential immunostaining was observed between sexes in submandibular salivary glands; arginase-I was diffusely expressed in the convoluted granular duct cells of females, but was rarely noted in males. Strain specific differences were not detected. In a mouse with an incidental case of lymphoma, neoplastic lymphocytes lacked arginase-I immunostaining, in contrast to immunostaining detected in non-neoplastic lymphocytes of lymphoid tissues. The use of liver tissue to validate arginase-I immunohistochemistry produced consistent expression patterns in mice, and this approach can be useful to enhance consistency of arginase-I immunohistochemical studies.

The Structure of Tight Junctions in Mouse Submandibular Gland

Salivary gland cells are joined by junctional complexes consisting of a tight junction (TJ), zonula adherens and one or more desmosomes. TJs regulate paracellular permeability, maintain separate apical and basolateral membrane domains, and serve as signaling centers. We examined TJs of mouse submandibular glands (SMG) in thin sections and freeze-fracture replicas. TJs between acinar cells and between intercalated duct cells had 2-6 parallel strands on the protoplasmic fracture face, with occasional branches, interconnections and free ends, and corresponding grooves on the extracellular face. Granular duct cell TJs had 2-30 strands, a depth of <or=0.5 microm, and occasional loops extending further basally. Where 3 or 4 cells met, the TJs extended basally <or=1 microm and consisted of 2 parallel boundary strands into which the apical strands inserted. Quantitative analyses showed significant differences in TJ complexity, measured by fractal geometry, and strand number of acinar compared to granular duct cells, and a greater number of strands in male compared to female granular ducts. Pilocarpine stimulation increased TJ strand number in female acinar cells, and increased complexity of male granular duct cell TJs. As the salivary gland water channel aquaporin 5 (AQP5) has been proposed to functionally interact with TJs to regulate salivary fluid composition, we also studied glands from AQP5 knock-out mice. In males lacking AQP5, granular duct TJs were more complex than those of wild-type mice, and exhibited more strands following pilocarpine stimulation. The results demonstrate specific gender, cell type and genetic differences in TJ structure and response to stimulation.

Molecular and Functional Expression of the Best2 Ca2±activated Cl- Channel in Mouse Submandibular Salivary Gland

Activation of Cl− channels in salivary acinar and duct cells is essential for saliva production. Anion efflux through an apical Ca2+-dependent Cl− channel (CaCC) is the rate limiting step for fluid secretion by acinar cells. The ionic composition of the primary saliva is then modified by salivary ducts. CaCC may support electrolyte reabsorption by duct cells of several types that constitute the duct system. The molecular identity of salivary CaCC is currently under vigorous examination. Here we explored the function of Best2, a member of the Bestrophin family of CaCCs, in the mouse submandibular salivary gland. Heterologous expression of the Best2 transcript in HEK293 cells produced Ca2+-activated Cl− current with the biophysical and pharmacologic properties that closely resembled the current found in native salivary cells. A recently developed Best2−/− mouse where the gene was disrupted by insertion of Lac Z was used to further characterize the role of this channel in the exocrine salivary gland. Even though Best2 expression was abolished, the amplitude and properties of the Ca2+-activated Cl− current in the acinar cells obtained from Best2-deficient mice were the same as the Ca2+-activated Cl− current in wildtype cells. Consistent with the observation the fluid secretion rate was not significantly different in Best2 null mice. Best2 gene was highly expressed in the duct cells of submandibular glands as revealed by X-gal staining. While, the ionic composition and osmolality of the saliva was not significantly altered in mice lacking Best2, the possibility of the functional compensation has been investigated in duct cells. Granular duct cells failed to present Ca2+-sensitive component of anion conductance. The properties of Cl− channels in intercalated, striated and excretory duct cells are currently under investigation.

Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5alpha-dehydrotestosterone (DHT) and triiodothyronine (T(3)), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T(3), and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.

Spontaneous Ca2+ oscillations via purinergic receptors elicit transient cell swelling in rat parotid ducts

Rat parotid ductal cells were found to exhibit spontaneous Ca(2+) oscillations. These oscillatory Ca(2+) responses were observed during continuous perfusion with physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. These Ca(2+) oscillations were completely blocked by the purinergic receptor inhibitors, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) (PPADS) and suramin, but were not blocked by the muscarinic antagonist, atropine, nor the alpha-adrenergic antagonist, phentolamine. Simultaneous observation with fura-2 fluorescence and differential interference contrast (DIC) images showed that the spontaneous elevations of [Ca(2+)]i were well correlated with the shape changes of the ductal cells. Using a plasma membrane fluorescence probe, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Electron microscopic analysis after Ca(2+) imaging indicated that the spontaneously oscillating duct cells contained numerous granules at the luminal side, which is characteristic of the granular duct cells. These results indicate that the spontaneous [Ca(2+)]i increase occurs through purinergic receptors, and activates Ca(2+)-dependent ion transporters and/or channels. Our findings present the possibility that spontaneous Ca(2+) oscillations via purinergic receptors are involved in the regulation of the electrolyte composition of saliva in resting states.

Hormone-induced granular convoluted tubule-like cells in mouse parotid gland

Most striated duct (SD) cells in the adult mouse parotid gland (PAG) have a few small secretory granules. These granules, however, are usually too small and sparse to be detected using light microscopy. Our serial studies have suggested that these PAG SD cells belong to a group of hormone-responsive granular duct cells, similar to the granular convoluted tubule (GCT) cells found in the submandibular gland. These studies also indicate that and some PAG SD cells may be capable of developing a granular cell phenotype under supraphysiological conditions of androgenic and thyroid hormones, leading to more abundant, and more kinds of GCT-specific secretory polypeptides. Here, the cytology of hormone-modulated SD cells, the immunocytochemistry of their secretory products, and their secretory responses to some autonomic agents are reviewed. Finally, the close similarity of the duct systems of the three major salivary glands in mice is critically emphasized.

Clcn2encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.

Apical maxi-K (KCa1.1) channels mediate K+ secretion by the mouse submandibular exocrine gland

The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3(-) efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3(-), demonstrating that neither K+/HCO3(-) cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3(-) did not appear to participate in this process, we tested whether a K channel is required. Indeed, K+ secretion was inhibited >75% in mice with a null mutation in the maxi-K, Ca2+-activated K channel (KCa1.1) but was unchanged in mice lacking the intermediate-conductance IKCa1 channel (KCa3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the K+ concentration in submandibular saliva. The K+ concentration of saliva is well known to be flow rate dependent, the K+ concentration increasing as the flow decreases. The flow rate dependence of K+ secretion was nearly eliminated in KCa1.1 null mice, suggesting an important role for KCa1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of K+ secretion, but we found that KCa1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of K+ requires and is likely mediated by KCa1.1 potassium channels localized to the apical membranes of striated and excretory duct cells in the mouse submandibular exocrine gland.

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70-100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the composition of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell-cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.

Immunohistochemical localization of senescence marker protein-30 (SMP30) in the submandibular gland and ultrastructural changes of the granular duct cells in SMP30 knockout mice.

Senescence Marker Protein-30 (SMP30) is a calcium-regulating protein that decreases in an androgen-independent manner as aging occurs. An enzyme-labeled antibody technique has demonstrated that SMP30 localized to the ducts (granular, intercalated, and striated ducts) of mouse submandibular glands. Immunoelectronmicroscopy demonstrated that the granular duct cells were strongly positive for SMP30, but that pillar cells in the granular duct were negative for the protein. In SMP30-knockout (KO) mice, the granular ducts were smaller in diameter. Swelling of mitochondria in the granular duct cells was observed; however, this phenomenon was not observed in the pillar cells. After administration of alpha-isoproterenol, a beta-adrenergic stimulant, a large numbers of small secretory granules were present in the granular duct cells and an expansion of the rough endoplasmic reticulum in SMP30-wild type (WT) mice; in contrast, little change was observed in SMP30-KO mice. These results suggest that SMP30 may be closely related to a signal transduction pathway in the granular duct cells of submandibular glands.

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice.

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.

Secretion of IgA by rat parotid and submandibular cells in response to autonomimetic stimulation in vitro

The major antibody in saliva is IgA, which is actively transported by pIgR expressed by parenchymal cells within the salivary glands. The rate of IgA secretion into saliva is regulated by the autonomic nerves supplying the glands in vivo. This study examined the mechanism of increased IgA secretion into saliva with autonomimetic stimulation. In vitro stimulation of IgA secretion from cells prepared by digestion of rat salivary glands found submandibular cell preparations responded to α- and β-adrenergic stimuli whereas the parotid cells responded only to β-adrenergic stimulation, although cells from both glands responded similarly to cholinergic stimulation. The additional responsiveness of submandibular cells to α-adrenergic stimulation probably reflects the presence of granular duct cells (absent in parotid glands) which are known to secrete protein in response to high frequency sympathetic stimulation. The increased secretion of IgA was not dependant upon increased plasma cell activation since isolated salivary gland plasma cells did not respond to agonists. Further evidence for the regulating role of parenchymal cells in IgA secretion into saliva was revealed by analysis of polymeric immunoglobulin receptor (pIgR) levels expressed on cells. Following in vivo nerve stimulation, there was an increased amount of pIgR expressed on the membrane surface. This was functionally demonstrated in vitro by increased uptake of human IgA by acutely prepared rat salivary cells following stimulation by adrenaline, indicating increased mobilisation of pIgR with stimulation. This study confirms that salivary cells increase the delivery of IgA into saliva by a pIgR-mediated mechanism in response to autonomic stimulation.

Thyroid hormone regulation of granular intercalated duct cells in the submandibular glands of female mice.

Intercalated ducts (IDs) in the submandibular glands (SMGs) of mice exhibit a sexual dimorphism, in which a few cells in the IDs of females, but not of males, possess secretory granules. The effects of a hypophysectomy (Hypox) followed by the administration of triiodo- l-thyronine (T3) on such granular intercalated duct (GID) cells in the female gland were histologically examined. Semithin sections stained with Heidenhain's iron hematoxylin revealed that Hypox resulted in the complete disappearance of the GID cells. In Hypox females, electron-microscopy examination of the ID cells whose localization corresponded to that of the GID cells in normal females showed that these cells had a pale, centric nucleus, poorly developed rough-surfaced endoplasmic reticulum (RER) and Golgi apparatus, and no secretory granules. When T3 (1 mg/kg body weight) was given to Hypox female mice every other day for 2 weeks, the GID cells reappeared in most of the ID segments. Electron microscopy revealed that these cells had abundant secretory granules in their apical cytoplasm, a nucleus located near the base of the cell, and layered cisterna of RER and segments of Golgi apparatus in the perinuclear cytoplasm. The localization and distribution of the GID cells in the T3-treated Hypox females were almost the same as those in normal females. Taken together, these results suggest that thyroid hormones upregulate the GID phenotype, and that thyroid hormones are essential for the exocrine activities of GID cells.

Acidic β-galactosidase in the developing mouse submandibular gland

Acidic β-galactosidase (β-gal) is expressed at high concentrations in the salivary glands, where its function is unknown but probably related to lysosomal catabolism of glycolipids or glycoproteins such as mucin. Here, the developmental pattern of the enzyme activity in the submandibular gland of B10A/SGSNJ mice was investigated in relation to the expression of mucin. Biochemical studies indicated that the activity of acidic β-gal increases significantly from birth to 3 weeks of age ( P<0.05). Histochemical staining of the submandibular glands of embryonic day 15 (E15), E18, 3-week-old and adult mice showed a trend of increasing staining intensity accompanied by a change in the cellular distribution of enzyme activity from E15 to adulthood. Acidic β-gal activity was first evident at E18, around the time mucin production starts, primarily in terminal-bud epithelial cells. The spatial and temporal coordination between the appearance of β-gal and embryonic mucin in the terminal buds of acinar cells might be related to a role of this enzyme in lysosomal degradation of embryonic mucin. In contrast, in adult mice, β-gal activity was primarily found in granular-duct cells. The function of β-gal in duct cells is unclear, but the change in the cellular distribution of β-gal in the mouse submandibular gland could reflect a change in the substrate specificity of this enzyme between E18 and adulthood.

Descnsitization of Noradrenaline-Induced Secretory Response Coincides with Redistribution of Subcellular IP3 Receptor and Compound Exocytosis in Granular Duct Cell of Rat Suhmandibular Gland

Double immunohistochemical staining detected inositol 1, 4, 5-trisphosphate receptor type 2 immunoreactivity (IP 3 R2 IR) and chromogranin A-like immunoreactivity (CgA-like IR) on the same sections prepared from the isolated and perfused submandibular gland of rat. In the control sections prepared from resting state, an intense IP 3 R2 IR was localized at the apical pole of granulated duct cell containing CgA-like IR. Electron-microscopically, the granular cells in the resting state stored numerous membrane-bound granules in the apical cytoplasm. The combined stimulation with 1 μM noradrenaline and 0.1 μM acetylcholine caused immediate maximum increase in secretory response (CgA-like IR secretion, flow, and protein secretion). When the sections were prepared from the gland at the peak of secretory response, the apically converged IP 3 R2 IR became diffuse and indistinguishable. Ultrastructure of the maximally stimulated cells exhibited extensive compound exocytosis in the apical half of the granular duct cells. The secretory responses to the combined stimulation were significantly inhibited by 100 μM 2-aminoethyl diphenylborinate, an inhibitory modulator of IP 3 -mediated Ca 2 + release from intracellular stores, and the intense IP 3 R2 IR was well preserved in the apical pole of granular duct cells containing CgA-like IR. These results may be compatible with the view that the divergence of apically convergent IP 3 R2 with the progress of compound exocytosis may result in decay of [Ca 2 + ], due to divergence of IP 3 R2-sensitive Ca 2 + pool, and this may provide a novel paradigm for desensitization of G protein-coupled signaling system.

Contributions of intercalated duct cells to the normal parenchyma of submandibular glands of adult rats.

The parenchyma of the submandibular gland in the adult male rat is self-renewing, with most newly formed acinar and granular duct cells believed to differentiate from the rapidly proliferating intercalated duct (ID) compartment. Since the ID cells are phenotypically diverse, based on their different expression of perinatal secretory proteins, we systemically injected tritiated thymidine for 24 hours, and followed the pattern of thymidine distribution in cells by autoradiography and immunocytochemistry of defined cellular phenotypes over a 1-month chase period. Proliferating cells were found within all parenchymal cell compartments; they were most numerous in ID, and primarily in those cells lacking immunoreactivity for the perinatal proteins SMG-B1, -C, and -D. The labeling index (LI) of the ID cells reached a peak at 7 days postinjection, and then decreased over the next 3 weeks. Concurrently, the LI increased significantly in those cells at the junctions of ID with both acini and granular ducts, and also within these larger parenchymal elements. We conclude that the ID cells not reactive for perinatal proteins proliferate to expand the ID compartment, and that ID cells at the ends of the ducts differentiate into both acinar and granular duct cells. Our data provide no evidence for the differentiation of ID cells into cells of striated ducts (SD); however, the small number of excretory duct (ED) profiles seen in our preparations showed extremely high LI (>25%), suggesting that more extensive data might reveal a precursor role for the ED in replacement of SD cells. In addition to the stepwise passage of cells from ID to other parenchymal elements at their junctions, the reported occurrence of occasional clusters of B1-positive acini (BAC) among the typical B1-negative acini had suggested an alternate pathway, in which entire segments of newly expanded ID might develop directly into a recapitulated perinatal stage of B1-reactive cell, pursuant to becoming mature acinar cells. Consistent with this suggestion, the BAC had a fourfold greater LI than typical adult acini; moreover, when analyzed by electron microscopic immunocytochemistry, they appeared similar to the novel perinatal Type III cells both ultrastructurally and in their pattern of B1-immunogold labeling. In contrast, the less common acini showing a sublingual gland phenotype had no significant difference in LI from typical acinar cells. Overall, our results emphasize the importance of the nonimmunoreactive ID cells in normal cellular replacement, and the possibility that ID can undergo en bloc differentiation into replacement acini as well as incremental addition of single cells at the boundaries of ID with acini and with granular ducts.