Secretion of saliva by the rabbit mandibular gland in vitro: the role of anions

Salivary glands form their secretions by first elaborating an isotonic plasma-like primary fluid in the endpieces and then modifying the composition of this secretion during its passage along the gland duct system. We have studied the role of extracellular anions in both primary secretion and ductal modification with a recently developed technique for isolation and perfusion of the rabbit mandibular gland. Neither of the major extracellular anions (Cl- or HCO-3) is essential for primary fluid secretion. HCO-3 can be removed altogether and replaced with Cl- without diminution in secretory rate, provided that extracellular pH is maintained at 7.4, and its replacement with acetate actually enhances secretion. Complete replacement of Cl- with Br- also enhances secretion and replacement with I-, NO-3, CH3SO-4 or isethionate supports secretion but at progressively diminishing rates. Our data do not yet allow us to distinguish between an electroneutral Na+-Cl- cotransport model or a double countertransport (Na+-H+ plus Cl--HCO-3) model as the basis of primary salivary secretion, or to propose any more suitable alternative model. With respect to ductal modification of the primary saliva, HCO-3 omission inhibits ductal Na+ absorption (i.e. salivary Na+ concentration rises). This inhibition is probably related to an effect of pH on the postulated Na+-H+ exchanges mechanism in the luminal duct membrane since it can also be induced by lowering perfusate pH, and reversed by substitution of perfusate HCO-3 with acetate (which enters saliva) but not HEPES (which does not enter the saliva). Substitution of perfusate Cl- with other anions seems not to inhibit ductal Na+ and K+ transport markedly.     

Effects of substance P on Na and K transport in perfused main submandibular duct of rat

Substance P (2 and 4 micrograms/kg . min, iv) caused an inhibition of net efflux of Na and net influx of K in perfused main excretory duct of rat submandibular gland. These effects could not be blocked by atropine sulfate. The data suggest that substance P receptors are present in the duct cells and play a role in the regulation of transductal electrolyte transport.     

Effects of carbamylcholine and isoproterenol on electrolyte fluxes in perfused main duct of submandibular gland of reserpinized rat

Administration of reserpine (RES) at a dosage of 0.5 mg/kg body wt, ip daily for 7 days was found to lower the dose of carbamylcholine and isoproterenol that alters sodium and potassium transport by cells of the main duct of rat submandibular gland. In the perfused main excretory duct of the submandibular gland of the RES rat, administration of carbamylcholine at a dosage of 1 microgram/kg body wt, inhibited net efflux of sodium (17%) and administration of isoproterenol at a dosage of 2 micrograms/kg body wt increased net efflux of sodium (20%); these drugs, at the same dosages, did not induce significant change in electrolyte flux of normal rat. At a dosage of 5 micrograms/kg body wt, carbamylcholine decreased net influx of potassium (15%) in the RES rat but was without effect on normal rat. Isoproterenol at the dosage of 5 micrograms/kg body wt significantly inhibited net influx of potassium in both the RES rat and normal rat. The data suggested that the duct cells developed supersensitivity to sympathomimetic and parasympathomimetic stimulation after chronic RES treatment.     

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.     

Viscoelasticity of human whole saliva collected after acid and mechanical stimulation

The rheology of saliva is highly important due to its influence on oral health and physiochemical processes within the oral environment. While the rheology of human whole saliva (HWS) is considered important for its functionality, its measurement is often performed erroneously and/or limited to the viscosity at a single shear rate. To ensure accurate rheological measurements, it is necessary to test HWS immediately after expectoration and to apply a thin layer of surfactant solution around the rim of the rheometer plates so that protein adsorption is minimized at the air-liquid interface. It is shown for the first time that the viscosity and viscoelasticity of HWS depends greatly upon the method of stimulation. Mechanical action stimulates slightly shear-thinning and relatively inelastic saliva, while acidic solutions (e.g. 0.25% citric acid) stimulate secretion of saliva that is highly elastic and shear-thinning. However, both acidic solutions and mechanical action stimulate similar volumes of saliva. For acid-stimulated saliva, the ratio of the primary normal stress difference to the shear stress is of order 100 and the viscosity at high shear rates is only marginally above that of water. This extremely high stress ratio for such a low viscosity fluid indicates that saliva's elastic properties dominate its flow behavior and may assist in facilitating lubrication within the oral cavity. It is anticipated that the variation in saliva rheology arises because the individual glands secrete saliva of different rheology, with the proportion of saliva secreted from each gland depending on the method of stimulation. The steady-shear rheology and linear viscoelasticity of HWS are described reasonably well using a FENE-P constitutive model and a 3-mode Maxwell model respectively. These models indicate that there are several long relaxation modes within saliva, possibly arising from the presence of large flexible macromolecules such as mucin glycoproteins.     

Histomorphometrical study of the submandibular gland ductal system in the rat

The duct system of murine submandibular gland is composed, in contrast with other mammals, by four types of ducts, among which the granular duct is unique for rodents. The granular duct shows a typical secretory structure with a clear intersex morphological diversity on which we carried out a morphometrical study in order to determine the relative area of each duct in rats in comparison with the rest of ducts and the whole gland. Our results, in both sexes, show that the duct with the broadest surface is the granular duct, followed by the excretory, striated and the intercalated ducts. In addition, we found a significant intersex difference between the relative surface of the granular and the excretory ducts, being bigger in males than in females. Finally, in both sexes, there is a greater variation in the data related to the excretory ducts than to the other ducts.     

Salivary myeloperoxidase of young adult humans

Leukocytes, principally polymorphonuclear leukocytes (PMNs), enter the oral cavity where they release a portion of their constituents, including myeloperoxidase, into oral fluids. A greater number of PMNs in the oral cavity are associated with oral inflammation. However, the quantitative contribution of the PMN to oral fluids, including saliva, during various conditions is poorly understood. An assay method based on the adsorbance loss at 278 nm from the reaction of the myeloperoxidase product hypochlorous acid with monochlorodimedon to yield dichlorodimedon was developed for the quantitation of salivary myeloperoxidase. Myeloperoxidase was determined in supernatants of whole saliva obtained at low and moderate flow rates and in parotid saliva collected during moderate and pronounced stimulation from young adults with minimal oral inflammation. The greatest myeloperoxidase activity was in whole saliva supernatants collected at low flow rates where PMN products have an opportunity to accumulate. Lesser quantities of myeloperoxidase were found in both the whole saliva supernatants and parotid saliva obtained at the faster flow rates. Low flow rate whole saliva supernatants contained about 25% of the myeloperoxidase in the PMNs which enter the oral cavity. Myeloperoxidase is responsible for a significant portion (15-20%) of the total peroxidase activity in supernatants of whole saliva obtained at low flow rates. Preliminary results indicate that young adults with phenytoin-associated gingival overgrowth or who smoke have more myeloperoxidase activity in low flow rate whole saliva.     

Fine structure of the Caenorhabditis elegans secretory-excretory system

The secretory-excretory system of C. elegans, reconstructed from serial-section electron micrographs of larvae, is composed of four cells, the nuclei of which are located on the ventral side of the pharynx and adjacent intestine. (1) The pore cell encloses the terminal one-third of the excretory duct which leads to an excretory pore at the ventral midline. (2) The duct cell surrounds the excretory duct with a lamellar membrane from the origin of the duct at the excretory sinus to the pore cell boundary. (3) A large H-shaped excretory cell extends bilateral canals anteriorly and posteriorly nearly the entire length of the worm. The excretory sinus within the cell body joins the lumena of the canals with the origin of the duct. (4) A binucleate, A-shaped gland cell extends bilateral processes anteriorly from cell bodies located just behind the pharynx. These processes are fused at the anterior tip of the cell, where the cell enters the circumpharyngeal nerve ring. The processes are also joined at the anterior edge of the excretory cell body, where the excretory cell and gland are joined to the duct cell at the origin of the duct. Secretory granules may be concentrated in the gland near this secretory-excretory junction. Although the gland cells of all growing developmental stages stain positively with paraldehyde-fuchsin, the gland of the dauer larva stage (a developmentally arrested third-stage larva) does not stain, nor do glands of starved worms of other stages. Dauer larvae uniquely lack secretory granules, and the gland cytoplasm is displaced by a labyrinth of large, transparent spaces. Exit from the dauer stage results in the return of active secretory morphology in fourth-stage larvae.     

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 compostion 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.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

Pulmonary epithelial sieving of small solutes in rat lungs

Transport and consumption of glucose from the air spaces of isolated, fluid-filled lungs can result in significantly lower glucose concentrations in the air spaces than in the perfusate compartment (11). This concentration difference could promote the osmotic movement of water from the air spaces to the perfusate, but the rate of fluid extraction from the air spaces would then be limited by the rates of electrolyte transport through the epithelium. In the present study, measurements were made of solute and water losses from the air spaces of fluid-filled rat lungs and the transport of these solutes and water into the vasculature after addition of hypertonic glucose or sucrose to the perfusate. Increases in the concentrations of Na+, Cl-, K+, and labeled mannitol in the air space were initially comparable to those of albumin labeled with Evans blue. Similarly, decreases in electrolyte concentrations in the perfusate were comparable to those of labeled albumin, indicating that very little solute accompanied the movement of water out of the lungs. Nor was evidence found that exposure of the vasculature to hypertonic glucose resulted in an increase in the rate at which fluid was reabsorbed from the air spaces over a 1-h interval, aside from an initial, abrupt loss of solute-free water from the lungs. These observations suggest that perfusion of fluid-filled lungs with hypertonic solutions of small solutes results in the extraction of water from the air spaces and pulmonary parenchyma across membranes that resist the movement of electrolytes and other lipophobic solutes.     

An ultrastructural analysis of the salivary system of the terrestrial mollusc, Limax maximus.

The bilateral salivary glands, ducts, and nerves of the giant garden slug Limax maximus control the secretion of saliva and its transport to the buccal mass. Each salivary nerve, which originates at the buccal ganglion, contains over 3000 axon profiles. The axons innervate the musculature of the duct and branch within the gland. The salivary duct is composed of several muscular layers surrounding an epithelial layer which lines the duct lumen. The morphology of the duct epithelium indicates that it may function in ion or water balance. The salivary gland contains four major types of secretory cells. The secretory products are released from vacuoles in the gland cells, and are presumably transported by cilia in the collecting ducts of the gland into the larger muscular ducts.     

Functional study of the Caenorhabditis elegans secretory-excretory system using laser microsurgery

Individual cells of the Caenorhabditis elegans secretory-excretory system were ablated by laser microbeam in various larval stages. Effects on growth, molting, osmoregulation, fertility, longevity, and dauer larva formation were tested. Single-cell ablations did not prevent subsequent molting, but ablation of the pore cell or the duct cell resulted in the absence of the normal cuticular lining of the excretory duct following a molt. When the pore cell, duct cell, or excretory cell was ablated, the animals filled with fluid within 12-24 hr and died within a few days, producing very few progeny. Ablation of the excretory gland cell, on the other hand, had no obvious developmental or behavioral effects. Excretory activity was monitored in dauer larvae by observing pulsation of the excretory duct in conditions of differing osmolarity. The rate of pulsation was quite variable over time in conditions of low osmotic strength, but average five- to six-fold higher than that observed in buffered saline. These observations, combined with the effects of laser ablation, lead to the conclusion that one function of the excretory system is osmoregulation.     

Autosomal recessively inherited electrolyte excretory defect in the parotid of the "cribriform degeneration" mouse mutant--possible analogy to cystic fibrosis

The mutation in the DBA/2J mice characterized by cribriform degeneration of the central nervous system also fxhibits a genetically determined defect in the transport on Na⁺ by the parotid gland duct. This defect results in the increased Na⁺ concentration of final parotid saliva. The sweat gland of the dorsal foot pad of the mutant mouse does not exhibit this defect. The apparent homology of the electrolyte disturbance in this animal model to the one seen in the autosomal recessively inherited disorder cystic fibrosis of the pancreas is elaborated upon.     

Channels and transporters in salivary glands

According to the two-stage hypothesis, primary saliva, a NaCl-rich plasma-like isotonic fluid is secreted by salivary acinar cells and its ionic composition becomes modified in the duct sytem. The ducts secrete K⁺ and HCO₃^- and reabsorb Na⁺ and Cl^- without any water movement, thus establishing a hypotonic final saliva. Salivary secretion depends on the coordinated action of several channels and transporters localized in the apical and basolateral membrane of acinar and duct cells. Early functional studies in perfused glands, followed by the molecular cloning of several transport proteins and the subsequent analysis of mutant mice, have greatly contributed to our understanding of salivary fluid and the electrolyte secretion process. With a few exceptions, most of the key channels and transporters involved in salivary secretion have now been identified and characterized. However, the picture that has emerged from all these studies is one of a complex molecular network characterized by redundancy for several transport proteins, compensatory mechanisms, and adaptive changes in health and disease. Current research is directed to the molecular interactions between the determinants and the ways in which they are regulated by extracellular signals and intracellular mediators. This review focuses on the functionally and molecularly best-characterized channels and transporters that are considered to be involved in transepithelial fluid and electrolyte transport in salivary glands.     

Expression of transforming growth factor beta 1 in the submandibular gland of the rat

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.     

Obestatin is present in saliva: alterations in obestatin and ghrelin levels of saliva and serum in ischemic heart disease

Ghrelin and obestatin are a single gene products and are a multiple functional peptides that regulates energy homeostasis, and food intake. In the present work, we studied the secretion of ghrelin and its co-secreted peptide obestatin in 44 patients with ischemic heart disease with that of 27 healthy matched controls. Here we first conducted using an immunohistochemistry assay to screen whether human salivary glands have any obestatin immunoreactivity. Then, serum and saliva obestatin and acylated ghrelin levels were determined by using Radioimmunoassay. Our immunohistochemical analysis demonstrated that obestatin was localized in the striated and excretory duct of human salivary gland. We also report for the first time that obestatin, like ghrelin, is present in human salivary gland and saliva. No evidence of the role of obestatin or ghrelin saliva levels in the context of ischemic heart disease was found. Salivary ghrelin and obestatin levels are correlated in controls with the blood levels. Determination of salivary values could represent a non-invasive alternative to serum ones that can be useful in clinical practice.     

Excretion and degradation of exogenous adenosine 3',5'-monophosphate by isolated perfused rat kidney

To determine the role played by the kidney in the metabolism and excretion of plasma adenosine 3′,5′-monophosphate (cAMP) we have studied the fate of this nucleotide (0.01–1.0mM) when it is perfused in a recirculating medium through the isolated rat kidney. cAMP was rapidly taken up and degraded by the kidney, the rate of its disappearance from the perfusate being at least twice its rate of excretion in the urine. Nevertheless, the cAMP excretory rate exceeded the filtration rate by 1.5 to 2 fold, and thus net secretion (transtubular transport) was demonstrated. The rates of filtration, perfusate clearance, and degradation of cAMP were proportional to its perfusate concentration. Methyl xanthines (caffeine and aminophylline) at 10mM, and probenecid at 0.9mM abolished transtubular transport of cAMP and greatly retarded disappearance of the nucleotide from the perfusate. It is concluded that there is a ready penetration of cAMP into renal cells from peritubular capillaries. Depending on the perfusate concentration of cAMP, transtubular transport may or may not exceed the simultaneous intra-renal breakdown of the compound. A low rate of cAMP excretion in the urine may accompany a considerably higher rate of cAMP clearance from the perfusate by the kidney.     

A calcium ionophore-induced secretion in rabbit lacrimal gland in vivo

Local administration of the calcium ionophore, A-23187 increased basal fluid secretion (non-stimulated) from the cannulated main excretory duct of rabbit lacrimal gland in vivo. A-23187 also facilitated fluid secretion induced by submaximal dose of methacholine (0.1 μg/kg, intraarterially). The stimulatory effect of A-23187 was dependent on the extracellular calcium concentration. Lowering the extracellular calcium by addition of EGTA markedly depressed or abolished the responses to the ionophore while increasing the extracellular calcium with CaCl₂ enhanced it. The results suggest that A-23187 causes increase in cell membrane permeability to extracellular calcium and the rise in intracellular calcium activates the secretory process(es) by an unknown mechanism to produce fluid secretion in the rabbit lacrimal gland.