Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Dye-coupling between cells of the salivary glands in the cockroach Periplaneta americana

Cockroaches have acinar salivary glands. The acini consist of peripheral cells specialized for electrolyte and water transport and central cells contributing proteinaceous components to the saliva. Salivary duct cells probably modify the primary saliva. The acinar cells in Nauphoeta cinerea had been shown to be electrically coupled and dye-coupled. Since intercellular communication via gap junctions between acinar cells is difficult to reconcile with previous findings that dopamine and serotonin selectively stimulate the secretion of either protein-free or protein-rich saliva in Periplaneta americana, we investigated whether dye-coupling occurs between both acinar cell types and between duct cells. We iontophoretically loaded Lucifer yellow into impaled cells and tested for dye-coupling by confocal laser scanning microscopy. We found that: (1) peripheral and central cells within an acinar lobulus of the gland in P. americana are dye-coupled; and (2) salivary duct cells are dye-coupled.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands

Background information. TSPO (translocator protein), previously known as PBR (peripheral‐type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High‐affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. Results. Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam‐binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, ³H‐labelled PK 11195, as shown by B_max and K_d values of 10.0±0.5 pmol/mg and 4.0±1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and α‐adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K⁺, Na⁺, Cl⁻ and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. Conclusions. High‐affinity ligand binding to mitochondrial TSPO modulates neurotransmitter‐induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.     

Dissection of Calcium Signaling Events in Exocrine Secretion

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca²⁺] ([Ca²⁺]_i) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca²⁺]_i are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca²⁺ release, and Ca²⁺ entry, as well as those that maintain cellular Ca²⁺ homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca²⁺]_i signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca²⁺]_i in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca²⁺ signaling mechanisms that determine salivary gland fluid secretion.     

Calcium signaling defects underlying salivary gland dysfunction

Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Saliva secretion is regulated by stimulation of specific signaling mechanisms within the acinar cells of the gland. Neurotransmitter-stimulated increase in cytosolic [Ca²⁺] ([Ca²⁺]_i) in acinar cells is the primary trigger for salivary fluid secretion from salivary glands, the loss of which is a critical factor underlying dry mouth conditions in patients. The increase in [Ca²⁺]_i regulates multiple ion channel and transport activities that together generate the osmotic gradient which drives fluid secretion across the apical membrane. Ca²⁺ entry mediated by the Store-Operated Ca²⁺ Entry (SOCE) mechanism provides the essential [Ca²⁺]_i signals to trigger salivary gland fluid secretion. Under physiological conditions depletion of ER-Ca²⁺ stores is caused by activation of IP3R by IP3 and this provides the stimulus for SOCE. Core components of SOCE in salivary gland acinar cells are the plasma membrane Ca²⁺ channels, Orai1 and TRPC1, and STIM1, a Ca²⁺-sensor protein in the ER, which regulates both channels. In addition, STIM2 likely enhances the sensitivity of cells to ER-Ca²⁺ depletion thereby tuning the cellular response to agonist stimulation. Two major, clinically relevant, conditions which cause irreversible salivary gland dysfunction are radiation treatment for head-and-neck cancers and the autoimmune exocrinopathy, Sjögren's syndrome (pSS). However, the exact mechanism(s) that causes the loss of fluid secretion, in either condition, is not clearly understood. A number of recent studies have identified that defects in critical Ca²⁺ signaling mechanisms underlie salivary gland dysfunction caused by radiation treatment or Sjögren's syndrome (pSS). This chapter will discuss these very interesting and important studies.     

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.     

Ca signaling and fluid secretion by secretory cells of the airway epithelium

Cytoplasmic Ca²⁺ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca²⁺ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca²⁺. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca²⁺ as a second messenger. Changes in intracellular Ca²⁺ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca²⁺-activated K⁺ channels and Cl⁻ channels. We also review evidence of interactions of Ca²⁺ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca²⁺ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.     

The apical Na+–HCO3− cotransporter Slc4a7 (NBCn1) does not contribute to bicarbonate transport by mouse salivary gland ducts

The HCO₃ ⁻ secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. Slc4a7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, Slc4a7 was not detected in acinar cells, and correspondingly, Slc4a7 disruption did not affect fluid secretion in response to cholinergic or β-adrenergic stimulation in the submandibular gland (SMG). Much of the Na ⁺-dependent intracellular pH (pH _i) regulation in SMG duct cells was insensitive to 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO ₃ ⁻. Consistent with these latter observations, the Slc4a7 null mutation had no impact on HCO ₃ ⁻ secretion nor on pH _i regulation in duct cells. Taken together, our results revealed that Slc4a7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH _i regulation or stimulated HCO ₃ ⁻ secretion.     

Ca<Superscript>2+</Superscript>-CaMKKβ pathway is required for adiponectin-induced secretion in rat submandibular gland

Adiponectin functions as a promoter of saliva secretion in rat submandibular gland via activation of adenosine monophosphate-activated protein kinase (AMPK) and increased paracellular permeability. Ca²⁺ mobilization is the primary signal for fluid secretion in salivary acinar cells. However, whether intracellular Ca²⁺ mobilization is involved in adiponectin-induced salivary secretion is unknown. Here, we found that full-length adiponectin (fAd) increased intracellular Ca²⁺ and saliva secretion in submandibular glands. Pre-perfusion with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) combined with thapsigargin (TG), an endoplasmic reticulum Ca²⁺-ATPase inhibitor, abolished fAd-induced salivary secretion, AMPK phosphorylation, and enlarged tight junction (TJ) width. Furthermore, in cultured SMG-C6 cells, co-pretreatment with EGTA and TG suppressed fAd-decreased transepithelial electrical resistance and increased 4-kDa FITC-dextran flux responses. Moreover, fAd increased phosphorylation of calcium/calmodulin-dependent protein kinase (CaMKKβ), a major kinase that is activated by elevated levels of intracellular Ca²⁺, but not liver kinase B1 phosphorylation. Pre-perfusion of the isolated gland with STO-609, an inhibitor of CaMKKβ, abolished fAd-induced salivary secretion, AMPK activation, and enlarged TJ width. CaMKKβ shRNA suppressed, whereas CaMKKβ re-expression rescued fAd-increased paracellular permeability. Taken together, these results indicate that adiponectin induced Ca²⁺ modulation in rat submandibular gland acinar cells. Ca²⁺-CaMKKβ pathway is required for adiponectin-induced secretion through mediating AMPK activation and increase in paracellular permeability in rat submandibular glands.     

Clcn2 encodes 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.     

Taste of a Pill: ORGANIC CATION TRANSPORTER-3 (OCT3) MEDIATES METFORMIN ACCUMULATION AND SECRETION IN SALIVARY GLANDS*

Drug-induced taste disturbance is a common adverse drug reaction often triggered by drug secretion into saliva. Very little is known regarding the molecular mechanisms underlying salivary gland transport of xenobiotics, and most drugs are assumed to enter saliva by passive diffusion. In this study, we demonstrate that salivary glands selectively and highly express OCT3 (organic cation transporter-3), a polyspecific drug transporter in the solute carrier 22 family. OCT3 protein is localized at both basolateral (blood-facing) and apical (saliva-facing) membranes of salivary gland acinar cells, suggesting a dual role of this transporter in mediating both epithelial uptake and efflux of organic cations in the secretory cells of salivary glands. Metformin, a widely used anti-diabetic drug known to induce taste disturbance, is transported by OCT3/Oct3 in vitro. In vivo, metformin was actively transported with a high level of accumulation in the salivary glands of wild-type mice. In contrast, active uptake and accumulation of metformin in salivary glands were abolished in Oct3^−/− mice. Oct3^−/− mice also showed altered metformin pharmacokinetics and reduced drug exposure in the heart. These results demonstrate that OCT3 is responsible for metformin accumulation and secretion in salivary glands. Our study uncovered a novel carrier-mediated pathway for drug entry into saliva and sheds new light on the molecular mechanisms underlying drug-induced taste disorders.     

Why Mouse Airway Submucosal Gland Serous Cells Do Not Secrete Fluid in Response to cAMP Stimulation

Airway submucosal glands are important sites of cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl⁻) channel expression and fluid secretion in the airway. Whereas both mouse and human submucosal glands and their serous acinar cells express CFTR, human glands and serous cells secrete much more robustly than mouse cells/glands in response to cAMP-generating agonists such as forskolin and vasoactive intestinal peptide. In this study, we examined mouse and human serous acinar cells to explain this difference and reveal further insights into the mechanisms of serous cell secretion. We found that mouse serous cells possess a robust cAMP-activated CFTR-dependent Cl⁻ permeability, but they lack cAMP-activated calcium (Ca²⁺) signaling observed in human cells. Similar to human cells, basal K⁺ conductance is extremely small in mouse acinar cells. Lack of cAMP-activated Ca²⁺ signaling in mouse cells results in the absence of K⁺ conductances required for secretion. However, cAMP activates CFTR-dependent fluid secretion during low-level cholinergic stimulation that fails to activate secretion on its own. Robust CFTR-dependent fluid secretion was also observed when cAMP stimulation was combined with direct pharmacological activation of epithelial K⁺ channels with 1-ethyl-2-benzimidazolinone (EBIO). Our data suggest that mouse serous cells lack cAMP-mediated Ca²⁺ signaling to activate basolateral membrane K⁺ conductance, resulting in weak cAMP-driven serous cell fluid secretion, providing the likely explanation for reduced cAMP-driven secretion observed in mouse compared with human glands.     

Effect of Neural Stimulation on Acinar Cell Membrane Potentials in Isolated Pancreas and Salivary Gland Segments

The effect of cholinergic neural excitation by field stimulation on the acinar cell membrane potential was investigated in superfused segments of mouse pancreas and salivary glands (sublingual, submaxillary, and parotid glands).

Responses of acinar cells in both exocrine pancreas and salivary glands to the neural excitation obtained by field stimulation were similar to responses previously described in each gland to the externally applied acetylcholine.

In the pancreatic acinar cell, electrical field stimulation induced depolarization with a latency of 0.3 to 1.2 sec. This depolarization was accompanied by a marked decrease in membrane resistance. The equilibrium potential of the depolarization induced by stimulation was between -10 and -20 mV. In the sublingual gland, field stimulation induced depolarization of the acinar cell with a latency of 0.2 to 0.3 sec. The stimulus induced depolarization was blocked by the addition of atropine. In the submaxillary and parotid glands, field stimulation induced depolarization in some acinar cell and hyper-polarization in other cells.

The results support evidence previously presented by Petersen and his colleagues that acetylcholine acts to increase Na⁺ and K⁺ or Na⁺, K⁺, and Cl^- permeabilities in the pancreatic acinar cell and to increase K⁺ and Na⁺ permeabilities in the salivary gland [11,24].     

Analysis of parotid and mixed saliva in Roe deer (Capreolus capreolus L.)

In ruminants, different functions have been ascribed to the different salivary glands according to the feeding type. In this context, possible adaptations of salivary functions were investigated regarding the secretion of various proteins by different types of salivary glands. To yield uncontaminated parotid saliva in large quantities, a non-surgical method has been developed. Parotid gland secretions were collected via endoscopic placement of guide wires into each parotid duct, which were subsequently used for placement of collection catheters. Salivary flow was stimulated by intra-glandular administration of the parasympathomimetic compound pilocarpine-hydrochloride into the parotid gland. Mixed saliva (excluding parotid saliva) was collected into sterile tubes by normal outflow during the sampling of parotid saliva. The total flow volume, flow rate and the content of proteins as well as of several ions (Na⁺, K⁺, Ca²⁺, inorganic phosphate) of both types of saliva were measured in sheep, fallow deer and roe deer. Roe deer secreted the highest amount of total salivary proteins relative to body mass [mg/kg body mass] and the highest relative volume [ml/10 min/kg body mass], both in parotid and mixed saliva, of all ruminant species examined. Additionally, the protein profile and the tannin-binding properties of parotid and mixed saliva in roe deer were investigated. Parotid saliva bound almost twice as much tannin as mixed saliva, underlining the importance of yielding uncontaminated parotid saliva for tannin-binding studies. Accepted: 6 January 1998     

Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

Ano6 disruption impairs acinar cell regulatory volume decrease and protein secretion in murine submandibular salivary glands

The widely expressed Anoctamin 6 (Ano6) supports different Ca²⁺-dependent functions, but little is known about its role in salivary glands. Mouse submandibular gland (SMG) acinar cells exhibited a robust regulatory volume decrease (RVD) following cell swelling that was reduced approximately 70% in Ano6^–/– mice. Ca²⁺-free conditions nearly eliminated the RVD response suggesting that Ano6 is an obligatory component of the cell volume-activated, Ca²⁺-dependent RVD pathway in salivary gland acinar cells. Ex vivo agonist-stimulated secretion of water and ions was unaffected by Ano6 disruption under both isotonic and hypotonic conditions suggesting that Ano6 does not play a major role in fluid and electrolyte secretion. In contrast, the total amount of β-adrenergic-dependent protein secretion by the SMG was significantly reduced in Ano6^–/– mice. Closer inspection of these latter results revealed that protein secretion was affected only in the female SMG by Ano6 disruption. These results indicate that Ano6 modulates the RVD response and protein secretion by salivary gland acinar cells.     

Tubular Fluid Secretion in the Seminiferous Epithelium: Ion Transporters and Aquaporins in Sertoli Cells

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K⁺ than the blood and express various membrane and water transporters (Na⁺/K⁺-ATPase; Ca²⁺-ATPase; V-type ATPase; Cl⁻ channels; CFTR Cl⁻ channels; K⁺ channels; L-, T- and N-type Ca²⁺ channels; Na⁺/H⁺ exchangers; Na⁺-driven HCO₃ ⁻/Cl⁻ exchangers (NDCBEs); Na⁺/HCO₃ ⁻ cotransporters (NBCes); Na⁺–K⁺–2Cl⁻ cotransporter; Na⁺/Ca²⁺ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.     

The aminergic control of cockroach salivary glands

The acinar salivary glands of cockroaches receive a dual innervation from the subesophageal ganglion and the stomatogastric nervous system. Acinar cells are surrounded by a plexus of dopaminergic and serotonergic varicose fibers. In addition, serotonergic terminals lie deep in the extracellular spaces between acinar cells. Excitation-secretion coupling in cockroach salivary glands is stimulated by both dopamine and serotonin. These monoamines cause increases in the intracellular concentrations of cAMP and Ca(2+). Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, two elementary secretory processes, namely electrolyte/water secretion and protein secretion, are triggered by different aminergic transmitters. Because of its simplicity and experimental accessibility, cockroach salivary glands have been used extensively as a model system to study the cellular actions of biogenic amines and to examine the pharmacological properties of biogenic amine receptors. In this review, we summarize current knowledge concerning the aminergic control of cockroach salivary glands and discuss our efforts to characterize Periplaneta biogenic amine receptors molecularly.