Neuromodulators of the lingual von Ebner gland: an immunocytochemical study

Summary The serous lingual glands of von Ebner secrete lingual lipase, an enzyme that begins fat digestion in the stomach. The objective of this study was to characterize the neuromodulators in the rat tongue and von Ebner glands using immunocytochemical techniques. Rat lingual tissues were fixed in formalin, embedded in paraffin and sectioned at 4 m for light microscopic studies. Immunocytochemical localization of neuromodulators was performed with monospecific anti-rat neuromodulator IgG or control (preimmune) IgG as the primary antibody, using the peroxidase-antiperoxidase (PAP) technique. No staining was seen with control antirat IgG. Immunospecific staining for vasoactive intestinal peptide (VIP), tyrosine hydroxylase and choline acetyltransferase (CHAT) was observed in nerves in the tongue, and cells containing immunospecific staining for serotonin (5-hydroxytryptamine) were seen in the stroma between the lingual glands. Selected cells in the serous glands stained positively for the presence of substance P and somatostatin. Adrenergic, VIP-containing and cholinergic nerves appear to innervate the tongue and serous glands. Substance P and somatostatin were identified in cells of the lingual serous glands and may be additional local modulators regulating lingual lipase release.     

Development of secretory activity in serous cells of the rat tongue. Cytological differentiation and accumulation of lingual lipase

The development of the serous cells of the rat tongue was studied by light and electron microscopy and compared with the accumulation of lingual lipase, measured by triglyceride hydrolysis at pH 5.4. The lingual serous (von Ebner's) glands were initiated in 19- to 20-day fetuses as epithelial ingrowths from the vallate and foliate papillae. The cells contained mostly free polyribosomes, few RER cisternae, and a small Golgi apparatus. Branching of the cell cords began shortly after initiation, but formation of acini and production of secretory granules did not begin until 3–4 days postnatally. The acinar cells had abundant basal RER, a supranuclear Golgi apparatus, and apical secretory granules and attained adult appearance by 17–25 days. The serous demilune cells of the lingual mucous glands differentiated more rapidly than the lingual serous acinar cells, attaining functional secretory structure in Day 20 fetuses. Lipase activity was first detected in Day 20 fetuses and increased 14-fold by birth. The activity decreased 50% during the first suckling period, returned to birth levels 1 day later, and increased rapidly thereafter. By 17 days postnatally, lipase activity was 23% of the adult level, although activity per gram body weight was equal to the adult. The results suggest that lingual lipase is produced prenatally by the demilune cells of the lingual mucous glands and postnatally predominately by the lingual serous glands. Lingual lipase may play an important role in lipid digestion in neonates, when levels of pancreatic lipase are low.     

NADPH-diaphorase and NOS-1 Positive Ganglion Cells are Found in the Rat Vallate Papilla/von Ebner Gland Complex

The nervous system of the vallata papilla and von Ebner glands was investigated in the rat tongue. Cells involved in the production of nitric oxide were identified by immunohistochemical detection of neuronal nitric oxide synthase type-1 and by cytochemical detection of NADPH-diaphorase. The analysis of serial sections showed that a ganglion composed of about 180–190 neuronal cells was present between the vallata papilla and von Ebner glands. These cells were positive for nitric oxide synthase type-1 and NADPH-diaphorase. From the ganglion, we observed nitrergic fibres running: (a) in the lamina propria of the receptor-free mucosa; (b) just below the gustatory epithelium; (c) in the von Ebner glands; and (d) around the vascular system of the vallata papilla. Our study suggests that the nitrergic ganglion cells may mediate interactions between chemoreceptorial systems in the vallata papilla and secretory cells in the von Ebner glands and that nitric oxide could be involved in the regulation of the blood supply to the vallata papilla and in the regulation of the von Ebner glands.     

Secretion of carbonic anhydrase isoenzyme VI (CA VI) from human and rat lingual serous von Ebner's glands.

Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis. (J Histochem Cytochem 49:657-662, 2001)     

Developmental delay of lingual lipase expression after guanethidine-induced sympathectomy.

Rat lingual lipase increases during postnatal development. To evaluate the role of the sympathetic nervous system in the control of lingual lipase during development, suckling rats were chemically sympathectomized by chronic treatment with guanethidine. This treatment was found to be effective in suppressing the developmental increase of lingual lipase. The effect was age dependent and also related to the dose of guanethidine given (i.e., the higher the dose, the more effective the suppression is, up to 40 micrograms/g body wt). The effect of guanethidine on lingual lipase suppression was not a result of induced stress, since simultaneous treatment with RU-38486, a known glucocorticoid receptor antagonist, did not prevent the decrease in lingual lipase activity. Ephedrine, a known sympathomimetic agent, restored the lingual lipase to a near normal level in guanethidine-treated animals, confirming that guanethidine acts through the sympathetic nerves. Furthermore, histochemical studies showed that guanethidine-treatment resulted in the reduction or elimination of catecholaminergic fibers in the von Ebner's glands. The effect of guanethidine was found to be transient, in that the lingual lipase activity showed complete recovery upon withdrawal of the treatment for 1 week. Together, the results indicated that sympathetic nerves have an important regulatory role in lingual lipase in rat pups during development.     

Histology and mucosubstance histochemistry of ferret lingual glands.

The histology and mucosubstance histochemistry of the ferret lingual glands were studied. Both serous and mucous minor salivary glands were present in the posterior part of the tongue. In serous glands, acinar cells and a very few cells of the excretory ducts contained granules which gave reactions for neutral mucopolysaccharides only. The mucous glands, including the duct system, contained mainly weakly sulphated acidic mucin, some neutral mucin but no carboxylated mucin. Occasional goblet cells were present in the excretory ducts of both serous and mucous glands. They contained weakly sulphated mucin.     

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.     

Molecular cloning and nucleotide sequence of rat lingual lipase cDNA.

Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme.     

The tongue morphology and lingual gland histochemistry of Ligurian Sea odontocetes

The morphology and histomorphology of the tongue and the histochemistry of the lingual glands of eight specimens from four species of Ligurian Sea odontocetes (Stenella coeruleoalba, Tursiops truncatus, Grampus griseus, and Ziphius cavirostris) were studied. The shape of tongues and the appearance of their dorsal surfaces differed between species. The lingual glands differed in size, distribution, and histochemistry by species. In S. coeruleoalba and G. griseus, a strong alcianophilic mucous material was detected in the lingual glands, while neutral and acidic mucous substances were observed in the most proximal secretory acini. In G. griseus, small simple alveolar apocrine glands were also found, and the duct of the serous lingual glands in Z. cavirostris is of apocrine type. Numerous mechanoreceptors were observed. Only the tongue of the young specimens showed marginal papillae: their histomorphological composition is consistent with the hypothesis that they create a tight seal between the tongue and the roof of the cavity in order to create suction. This comparative study suggests that differences in tongue morphology and in the morphology and histochemistry of lingual glands might be related to feeding habits.     

Lingual and gastric lipases: species differences in the origin of prepancreatic digestive lipases and in the localization of gastric lipase

The source of the lipase(s) acting in the stomach was investigated in five animal species: rat, mouse (rodents), rabbit (lagomorphs), guinea pig (caviidae), baboon and human (primates). The activity of lingual and gastric lipases was quantitated in homogenates of lingual serous glands and of gastric mucosa, respectively, by the hydrolysis of tri[3H]oleylglycerol and is expressed in units/g (1 U = 1 mumol [3H]oleic acid released/min) per g tissue wet weight, mean +/- S.E. There were marked differences in the activity level of lingual and gastric lipases among species: mouse and rat had high levels of lingual lipase activity (250 +/- 20 and 824 +/- 224 U/g) and only traces of gastric lipase activity (4.5 +/- 0.9 and 0.04 U/g, respectively), whereas rabbit and guinea pig had no lingual lipase activity and only gastric lipase activity (78 +/- 48 and 27 +/- 7.4 U/g, respectively). In the baboon and human, gastric lipase was the predominant enzyme (109 +/- 20 U/g and 118 +/- 8.8 U/g, respectively), whereas lingual lipase activity was present in trace amounts only (0.04 U/g and 0.3 U/g, respectively). In addition to species differences in the origin of the preduodenal lipases, there were also species differences in the distribution of gastric lipase in the stomach. Thus, while in the rabbit, gastric lipase was localized exclusively in the cardia and body of the stomach, it was diffusely distributed in the entire stomach of the guinea pig and baboon. A comparison between the level of activity of lipase and pepsin (the two chief digestive enzymes secreted by the stomach), showed differences in their localization in the species studied. The difference in source (tongue vs. stomach) and site (cardia-body vs. entire stomach) of lipase secretion must be taken into account in future studies of these digestive enzymes. Although the exact contribution of lingual and gastric lipases individually to fat digestion in species which contain both enzymes cannot yet be evaluated, the markedly higher levels of gastric lipase activity in the baboon and human suggests that, in primates, gastric lipase is probably the major non-pancreatic digestive lipase.     

Inhibition of lingual lipases by lidocaine in taste and non-taste epidermis from steer tongues

When epidermal tissue from several regions of the steer tonguewas incubated in buffer solutions, high percentages of freefatty acids were found. The presence of the verified phospholipaseinhibitor, lidocaine, prevented the accumulation of these freefatty acids. Using [¹⁴C]tripalmitin and [¹⁴C]phosphatidyl cholineas substrates for presumed lipases present in the tongue, weobserved that lidocaine inhibited this activity. Experimentscomparing homogenized and intact pieces of epidermal tissuesuggested that the lipase activity is of both serous gland andlysosomal origin. Highest levels of lipase activity in non-homogenizedtissues appeared in epidermis (taste containing) and dermisof the circumvallate papilla, reflecting the fact that the lingualserous glands which secrete a known lipase are located in andaround this structure. When integrity of the lipid species presentin mammalian lingual tissues needs to be preserved during biochemicalexperiments, it is suggested that lipase inhibitors such aslidocaine be present.     

A high resolution sem study of human minor salivary glands

By SEM we have investigated the human minor salivary glands using the NaOH method for the visualization of endpieces and myoepithelial cells, and the osmium maceration technique that reveals membranous intracellular structures. With the former method all minor glands, including the posterior deep (Ebner's) lingual glands, consist of tubules sometimes dilated into alveoli, while true acini of the kind observed in human major salivary glands, are absent. Tubules of the posterior deep lingual gland exhibit stellate myoepitelial cells that leave a substantial part of the secretory cells uncovered. The latter cells, at variance with serous cells of major glands, do not show basal folds. In contrast, tubules of the other minor glands, like the mucous ones of major glands, are covered almost completely by band-like myoepithelial cells. The osmium maceration method clearly demonstrates that posterior deep lingual glands are serous in character and that all the other minor glands, together with the predominant mucous cells, possess a variable number of seromucous cells that, despite variations among individuals, increase in order from palatine and posterior superficial lingual (Weber's), to minor sublingual, labial, anterior lingual (Blandin and Nuhn's), and buccal glands.     

The epithelia of the protrusible tongue of Eurycea longicauda guttolineata (Hoolbrook 1838) (Urodela: Plethodontidae)

In this study the lingual and sublingual glands, the lingual stem and the epithelial surface of the protrusible secondary tongue were investigated by light, scanning and transmission electron microscopy. The quality of the secretions of the epithelia was characterized histochemically. The lingual epithelium is formed by superficial (pavement) and goblet cells and at the margin of the tongue pad are also regions covered by ciliated cells. On the dorsal part of the tongue there are goblet cells of type A with mainly acidic secretions and of type B containing neutral secretions. Most of the goblet cells on the ventral side of the tongue (hypoglottis) show a strong alcian blue/PAS positive reaction (type I) and some produce neutral secretions (type II). The glandular cells of the lingual gland react positively to alcian blue and PAS in the apical region of the gland. In contrast there is only alcian blue-positive staining in the basal part of the gland. The size and complexity of the inclusion bodies of the secretory granules increase in a basal direction. In addition, there are ciliated cells in the glandular epithelium. Although the epithelium of the lingual stem is thin, it is double-layered. The cell types observed in this region are identical to those of the ventral part of the protrusible tongue. At the margin of the sublingual gland are trough-like structures. In the center, tubular parts are observed. The cells of this gland are stain strongly with alcian blue (pH 1.0) mainly in the basal part of the gland. The results of this are compared to the tongue pad and the lingual gland of Salamandra salamandra and Ambystoma mexicanum.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Importance of lipolysis in oral cavity for orosensory detection of fat

Lingual lipase is usually secreted from von Ebner's glands, although there is great variation between species. Lingual lipase is thought to be an auxiliary enzyme for fat digestion and absorption in mammals; however, the reason for lipolysis in the oral cavity is not known. We focused on the gustatory sense and investigated the significance of lingual lipase in the perception of a fat taste by using orlistat, a potent lipase inhibitor. Five-minute two-bottle preference tests demonstrated that the addition of orlistat diminished the preference for triacylglycerides but not for free fatty acids. Radioactive triolein applied on rats' circumvallate papilla revealed that lingual lipase was released continuously to generate significant amounts of fatty acids and other lipolytic products within 1-5 s, which was enough time to taste fat. These findings suggest that lingual lipase is released to perceive the taste of triacylglycerides and to find nutritive lipids in food.     

A neonatal secretory protein associated with secretion granule membranes in developing rat salivary glands

In the perinatal submandibular gland, the secretion granules of Type I cells contain protein C (89 KD) and those of Type III cells have Bl-immunoreactive proteins (Bl-IP, 23.5-27.5 KD). In this report we used immunocytochemistry at the light and electron microscopic levels to describe the developmental distribution and localization of protein D (175 KD), which is secreted by both Type I and Type III cells. At its first appearance in Type I cells at 18 days and in Type III cells at 19 days post conception, protein D immunoreactivity (D-IR) is associated with secretion granule membranes; this is more pronounced in Type I than in Type III cells. In early postnatal life the label remains membrane associated, but as Type III cells differentiate into seromucous acinar cells, the lower level of label present in these cells is found in the granule content. Label is found associated with the membrane in secretion granules of Type I cells as long as these cells are identifiable in acini, and subsequent to this similarly labeled cells are seen in intercalated ducts. In the sublingual gland (SLG), D-IR is membrane associated in secretion granules of serous demilune cells, and is present in the secretion granule content in mucous acinar cells. D-IR is also found in the lingual serous (von Ebner's) glands, lacrimal gland, and tracheal glands, primarily in the ducts, where it is localized in the content of secretion granules.     

Light and scanning electron microscopic study of the structure of the ostrich (Strutio camelus) tongue

The ostrich's tongue is situated in the posterior part of the oropharyngeal cavity and its length is only about a quarter of the beak cavity. The triangular shortened tongue has retained the usual division into the apex, the body and the root. There are no conical papillae between the body and the root of the tongue, and the presence of the flat fold with lateral processes sliding over the tongue root in the posterior part of the lingual body is a unique morphological feature. All lingual mucosa covers non-keratinised stratified epithelium, and the lamina propria of the mucosa is filled with mucous glands whose round or semilunar openings are found on both the dorsal and ventral surface of the tongue. The complex glands found in the lingual body are composed of alveoli and/or tubules. Moreover, simple tubular glands seen in the posterior part of the tongue root are an exception. Numerous observations have shown that the ostrich's tongue is a modified structure, though not a rudimentary one, whose main function is to produce the secretion moisturising the beak cavity surface and the ingested semidry plant food in this savannah species.