The cytochemical localization of adenyl cyclase activity in rat sublingual gland.

Adenyl cyclase activity in mucous acinar cells and serous demilune cells of the rat sublingual gland was localized cytochemically. After incubation with adenylyl-imidodiphosphate (AMP-PNP) as substrate, deposits of reaction product are found along the cell membranes bordering the secretory surfaces of serous demilune cells. These are the membranes which participate directly in secretion by fusing with the granule membranes. The granule membranes of the demilune cells do not reveal reaction product, but the membranes of the granules which are fused with and become part of the cell membrane do show deposits. Thus, it appears that the cell membranes which fuse with granule membranes during secretion are associated with a high level of adenyl cyclase activity. In support of this, the luminal membranes of the mucous acinar cells which do not fuse with granule membranes during secretion are not associated with detectable amounts of adenyl cyclase activity.     

The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins

Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)-mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2 in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the complex exocytosis mechanisms.     

The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl-imidodi-phosphate (AMP-PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production. In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products. A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.     

Intracellular pathway and kinetics of protein secretion in the coagulating gland of the mouse

The coagulating gland of male rodents is part of the prostatic complex. Various mechanisms of secretion have been postulated, in part because organelles commonly involved in the secretory process possess unusual features, such as extreme distension of the rough endoplasmic reticulum. In the present study, the pathway, kinetics, and mode of secretion in the coagulating gland of the mouse were studied by electron microscope autoradiography at intervals between 5 min and 8 h after administration of 3H-threonine. The percentage of grains associated with the rough endoplasmic reticulum was initially high and generally decreased throughout the experiment, while a pronounced rise in the proportion of grains associated with the Golgi apparatus and secretory granules was observed 6 h after injection of precursor. In addition, there was a smaller elevation in the percentage of grains over the Golgi apparatus and secretory granules between 1 and 4 h, and radioactive material first reached the lumen of the gland 4 h after injection of the precursor. Although the general pathway of intracellular transport of secretory protein resembles that in other cells, the results indicate that there are several unusual aspects to the secretory process in the coagulating gland. First, the rate of transport was markedly slower than in most other exocrine gland cells, since the bulk of the labeled protein did not reach the Golgi apparatus and secretory granules until 6 h after administration of precursor. This reflected prolonged retention of secretory products in the endoplasmic reticulum. Second, in addition to the major bolus of labeled material that traversed the cells at about 6 h, a smaller wave of radioactivity appeared to pass through the Golgi apparatus and secretory granules and reach the lumen earlier, within the first few hours after the injection. Finally, the primary mode of secretion in the coagulating gland appears to be merocrine because the secretory granules contained much labeled protein.     

Fine structure of the silk gland in the mite Ornithocheyletia sp. (Prostigmata,Cheyletidae)

Silk spinning is widely-spread in trombidiform mites, yet scarse information is available on the morphology of their silk glands. Thus this study describes the fine structure of the prosomal silk glands in a small parasitic mite, Ornithocheyletia sp. (Cheyletidae). These are paired acinous glands incorporated into the podocephalic system, as typical of the order. Combined secretion of the coxal and silk glands is released at the tip of the gnathosoma. Data obtained show Ornithocheyletia silk gland belonging to the class 3 arthropod exocrine gland. Each gland is composed of seven pyramidal secretory cells and one ring-folded intercalary cell, rich in microtubules. The fine structure of the secretory cells points to intensive protein synthesis resulted in the presence of abundant uniform secretory granules. Fibrous content of the granules is always subdivided into several zones of two electron densities. The granules periodically discharge into the acinar cavity by means of exocytosis. The intercalary cell extends from the base of the excretory duct and contributes the wall of the acinar cavity encircling the apical margins of the secretory cells. The distal apical surface of the intercalary cell is covered with a thin cuticle resembling that of the corresponding cells in some acarine and myriapod glands. Axon endings form regular synaptic structures on the body of the intercalary cell implying nerve regulation of the gland activity.     

Distribution of S-100 protein and its subunits in bovine exocrine glands

 The distribution of S-100 protein and its α- and β-subunits in bovine exocrine glands was studied by indirect immunohistochemistry. The entire spectrum of salivary glands, glands of the respiratory tract, intestinal glands, male and female genital glands, and skin glands was examined. S-100 and its β-subunit were identified in most serous secretory cells of mixed salivary glands, although secretory acini in some serous glands remained unreactive for these antigens. Mucous cells were constantly negative; mucoid cells were positive in the lacrimal and Harderian gland. The α-subunit of S-100 protein was identified in serous cells but the staining reaction was faint. Subunits of S-100 showed a characteristic distribution along the excretory duct systems of compound glands: S-100 and the β-subunit were present in intercalated duct epithelium, while striated duct epithelium stained for S100-α. Therefore, it is suggested that S100-α is related to resorption and secretion in striated ducts, while S100-β may govern acinar exocytosis and probably regulates proliferation and differentiation of glandular cells. Differing staining intensities for S-100 and its subunits in secretory cells of exocrine glands most probably indicate functional differences with regard to secretory activity and the cell cycle. Accepted: 11 February 1997     

Visualizing form and function in organotypic slices of the adult mouse parotid gland

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.     

The harderian gland of the frog, Rana esculenta, during the annual cycle: histology, histochemistry and ultrastructure

The Harderian gland in Rana esculenta has been studied during the annual cycle at the histological, histochemical and ultrastructural levels. The Harderian gland has an acinar structure and is the only orbital gland in anuran amphibia. It develops at the medial corner of the orbit from the conjunctival epithelium at the premetamorphic stage. In the adult the glandular secretion reaches a maximum during the months of July and August, drops in September and resumes slowly from October onwards. The secretion is seromucoid and the secretory granules are released into the acinar lumen, mainly by exocytosis. Porphyrins were not detected. No sexual dimorphism was observed in the glandular cells. The resumption of secretory activity in October and the enhancement of secretion in May are marked by the appearance of "blue nuclei" (Mallory stain) in a relatively high percentage of glandular cells. This unusual blue colour, using the Mallory stain (by which nuclei stain red), disappears after digestion of paraffin sections with RNAase, but not with DNAase and trypsin. The blue staining may, therefore, indicate an increased amount of nuclear RNA. The Harderian gland in the frog most probably serves to lubricate and moisten the eye in the absence of the lacrimal gland. However, the gland may also represent an immunoactive organ owing to the presence of numerous mast cells and plasma cells in the interacinar spaces.     

Radioautographic study of glycoprotein synthesis and migration in the major sublingual gland of the Mongolian gerbil (Meriones unguiculatus)

Intracellular glycoprotein synthesis and migration in the major sublingual gland of the Mongolian gerbil were studied with light-microscopic radioautography, using L-4, 5-[3H]-leucine, D-1-[3H]-glucose and N-[3H]-acetyl-D-mannosamine as precursors. The time elapsed from the beginning of the synthesis of the secretion products to their release was longer in the mucous acinar cells than in any other exocrine cell investigated until now. A slow addition of the terminal sugars (especially sialic acid) to the glycoprotein molecule and a prolonged storage of the glycoproteins in the apical granules were assumed to be the reasons for this delay in schedule. The secretion in the serous demilune cells was much faster than in the mucous acinar cells. The lack of uptake of labelled mannosamine showed that the demilunes are likely to produce substances different from those of the mucous acinar cells, presumably a protein-rich material with a high turnover.     

Signal transduction of mucous secretion by bronchial gland cells

Bronchial glands, which consist of mucous and serous cells, are abundant in human airways, playing a major role in the airway secretion. Cl(-) secretion is accompanied by water transport to the lumen in the acinar cells of bronchial glands. Agonists that increase [Ca(2+)]i induce the Cl(-) secretion in bronchial glands. Ca(2+) release from a IP(3)-sensitive Ca(2+) pool at the apical portion stimulates and opens Ca(2+)-sensitive Cl(-) channels at the apical membrane, producing Cl(-) secretion in bronchial glands. K(+) channels at the basolateral membranes are Ca(2+)-sensitive and activated by Ca(2+) release from a cADPribose-sensitive Ca(2+) pool, maintaining the Cl(-) secretion in bronchial glands. Further, cADP ribose in concert with IP(3) induce [Ca(2+)]i oscillation, inducing Cl(-) secretion in bronchial glands. Some tyrosine kinases are involved in the Cl(-) secretion in bronchial glands. Mucous and serous cells in bronchial glands take part in mucin secretion and the secretion of defensive substances (glycoconjugates), respectively. [Ca(2+)]i oscillations are shown to play a central role in the exocytosis of secretory granules in serous cells of bronchial glands. Other signal transductions of mucin and glycoconjugates in airway gland cells remain to be studied, although agonists which increase [cAMP]i are also well known to induce mucin and glycoconjugate secretion from airway glands.     

Histology,histochemistry, and ultrastructure of the harderian gland of the snake Coluber viridiflavus

Analyses of the histology, histochemistry, and ultrastructre of the Harderian gland of Coluber viridiflavus prove the gland to be compound acinar and to produce a seromucous secretion. Acinar cells (type I) contain secretory granules that are composite, consisting ultrastructurally of three distinct parts that are sharply separated. They are similar to the “special secretory granules” described in the cells of the Harderian gland of the lizard Podarcis s. sicula. Some acini of the most anterior and posterior parts of the gland are mucous. Acinar cells (type II) of this type contain secretory granules that are Alcian blue/PAS positve. At the ultrastructural level, they appear homogeneous and of low density, characteristic of mucous secretions. These mucus-secreting anterior and posterior parts of the Harderian gland may by considered as regions of intial differentiation of the anterior and posterior lacrimal galnds.     

Development of secretory units of mouse submandibular gland

Summary The fine structure of the secretory units of the mouse submandibular gland was studied according to the developmental sequence. The embryonic submandibular gland consists of terminal tubules and ducts. Myoepithelium is associated only with the terminal tubules, and the cells of the primary intercalated ducts show characteristics of the young striated duct cells. The major changes shortly after birth consist of: 1) opening of the secretory lumina, 2) increasing rough ER and its altered configuration, 3) dilatation of Golgi cisternae and 4) changes in the granular structure. These findings suggest that the salivary secretion first occurs after birth, and acinar differentiation or transformation of the secretory cells of the terminal tubules is induced and profoundly affected by the commencement of the secretory activity. In the intercalated ducts this process is somehow inhibited, and the granular cells found in the adult can be considered as the remnants of the secretory cells of the terminal tubules.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

cDNA and genomic cloning of lacritin, a novel secretion enhancing factor from the human lacrimal gland.

Multiple extracellular factors are hypothesized to promote the differentiation of unstimulated and/or stimulated secretory pathways in exocrine secretory cells, but the identity of differentiation factors, particularly those organ-specific, remain largely unknown. Here, we report on the identification of a novel secreted glycoprotein, lacritin, that enhances exocrine secretion in overnight cultures of lacrimal acinar cells which otherwise display loss of secretory function. Lacritin mRNA and protein are highly expressed in human lacrimal gland, moderately in major and minor salivary glands and slightly in thyroid. No lacritin message or protein is detected elsewhere among more than 50 human tissues examined. Lacritin displays partial similarity to the glycosaminoglycan-binding region of brain-specific neuroglycan C (32 % identity over 102 amino acid residues) and to the possibly mucin-like amino globular region of fibulin-2 (30 % identity over 81 amino acid residues), and localizes primarily to secretory granules and secretory fluid. The lacritin gene consists of five exons, displays no alternative splicing and maps to 12q13. Recombinant lacritin augments unstimulated but not stimulated acinar cell secretion, promotes ductal cell proliferation, and stimulates signaling through tyrosine phosphorylation and release of calcium. It binds collagen IV, laminin-1, entactin/nidogen-1, fibronectin and vitronectin, but not collagen I, heparin or EGF. As an autocrine/paracrine enhancer of the lacrimal constitutive secretory pathway, ductal cell mitogen and stimulator of corneal epithelial cells, lacritin may play a key role in the function of the lacrimal gland-corneal axis.     

Cytodifferentiation of the acinar cells of the rat submandibular gland

The present study examines the cytodifferentiation of the acinar exocrine cells of the rat submandibular gland (SMG) at the ultrastructural level. Submandibular glands from rats at 14 days of gestation through 12 weeks postpartum were examined. The acinar cells of the SMG begin to develop at 15–16 days of gestation and are not fully differentiated until 3–4 weeks after birth. The earliest cells show multiple Golgi zones and a few strands of widely dilated rough endoplasmic reticulum (rer). Subsequently, the cells show an orderly sequence of organelle changes and rearrangement which leads to fully differentiated exocrine cells. A series of five morphologically distinct secretory granules is observed during differentiation and these granules serve as markers of the functional maturity of the cells. Attention is given in this study to the development of the apical-basal polarity of functional organelles typically seen in exocrine cells and its relationship to secretory granule production. The findings of the current study are compared with similar reports in the literature on the developing pancreas and parotid gland of the rat. It is concluded that different developmental pathways are followed to attain a similar functional capacity in the three organs.