Immunohistochemical localization of rat submandibular gland esterase B (homologous to the RSKG-7 kallikrein gene) in relation to other serine proteases of the kallikrein family.

The rat submandibular gland contains several members of the kallikrein family. In the present study we purified and raised an antiserum against one of these enzymes, i.e., esterase B, which was first described by Khullar et al. in 1986. N-terminal amino acid analysis revealed complete homology between esterase B and the kallikrein family gene RSKG-7. For characterization of the antiserum, flat-bed isoelectrofocusing with immunoblotting was superior to immunoelectrophoresis and double immunodiffusion in detecting and identifying crossreacting proteins. This was due to the fact that kallikrein-like enzymes were readily separated by isoelectrofocusing, and immunoreactivity was easily detected by the sensitive peroxidase-anti-peroxidase staining after blotting onto nitrocellulose membrane. Immunohistochemical controls were carried out accordingly, including homologous as well as crossreacting antigens. In the submandibular gland, esterase B was detected exclusively in all granular convoluted tubular cells, co-localized with tissue kallikrein and tonin. Some staining was also observed in striated duct cells; however, this staining reaction was induced by cross-reactivity with kallikrein, since staining was abolished by addition of kallikrein as well as esterase B to the primary antiserum. It was therefore concluded that like tonin and antigen gamma, but unlike kallikrein, esterase B was not detected in the striated ducts of the submandibular, parotid, or sublingual glands. This separation in anatomic distribution between esterase B and kallikrein may indicate that prokallikrein activation is not the only biological function of esterase B.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Tonin has been localized in salivary glands and kidney by the indirect immunofluorescence technique of Coons and by the unlabeled antibody technique of Sternberger. Both techniques gave identical results. Immunoreactive tonin was localized in the cytoplasm of granular convoluted tubular cells and on the apical surface of striated duct cells and collecting duct cells of the submandibular gland. In the parotid and sublingual glands, which lack granular cells, tonin was only found on the apical surface of striated duct and collecting duct cells. In the kidney, immunoreactive tonin was found only associated with cells of the distal convoluted tubules. After fixation with Bouin fluid or with ethanol, tonin was found not only on the apical surface of the cells but also in the apical and perinuclear cytoplasm. This cytoplasmic staining has been attributed to artefactual diffusion since, after fixation with formol-picric acid, the enzyme could only be localized on the apical surface of the tubular cells.     

Immunohistochemistry of tonin in rat submandibular gland during development, lactation and secretion

Tonin is a serine protease found in high concentrations in the submandibular gland (SMG) of the adult rat where it has been localized by immunohistochemistry in the granular ducts. The present study examined the development of tonin in the SMG, the effect of lactation and of stimulation of tonin release, using the peroxidase-antiperoxidase technique and antitonin. Tonin-like immunoreactivity first appeared in the primitive striated duct of the SMG on day 19 foetal and increased in intensity as the ducts developed into granular ducts. Reaction product in granules was seen on day 17 postpartum. Its localization within granules was established by immunochemistry of Sepharose beads to which had been coupled the contents of granules isolated from adult rats. The granular ducts of female rats, which are less developed than in the male, showed a marked increase in tonin-immunoreactivity during lactation. Stimulation of tonin secretion by isoprenaline caused massive discharge of tonin-like immunoreactivity into the lumen of the granular ducts during in vitro incubation. However, within one hour complete regranulation was apparent. The secretion was prevented by propranolol. The results indicate that tonin or a tonin-like substance appears in the rat submandibular gland late in gestation in ducts that presumably develop into granular ducts where it is found in abundance in granules in the adult, that the amount in females is increased during lactation, and that most of the granules are discharged during stimulation, only to be rapidly replaced.     

Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.     

Localization of kallikrein in the guinea-pig submandibular gland

The localization of kallikrein in the acinar and the ductal components of the guinea-pig submandibular gland was investigated by a microdissection technique and an esterase assay. The results indicated that a major portion of the kallikrein is located in the striated duct cells. These results disagree with previous studies in the guinea-pig but are consistent with findings in the rat and cat submandibular gland.     

Occurrence and Nuclear Localization of cAMP Response Element- Binding Protein in the Post-natal Development of the Rat Submandibular Gland

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5 promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the post-natal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1–2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the r352;-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar ce lls that are regulated by r352;-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.     

Identification and expression of kallikrein gene family in rat submandibular and prostate glands using monoclonal antibodies as specific probes

Panels of monoclonal antibodies to three vasoactive peptide-producing enzymes: tissue kallikrein, tonin and arginine esterase A were developed, characterized and used as probes for identification of tissue-specific expression. In addition, immunoblot analyses were performed, using monospecific monoclonal antibodies which did not show cross-reactivity to related-purified enzymes in enzyme-linked immunosorbant assay (ELISA), and radioimmunoassay. We obtained the following results. In rat submandibular gland extract, the expression of 38 kDa kallikrein, 32 kDa tonin, and 18 kDa heavy chain of esterase A was identified by monoclonal antibodies to kallikrein (V₄D₁₁), tonin (1F₁₁), and esterase A (5A₁₀, 6C₁₁, and 4B₁₂), respectively. In the prostate gland, a 32 kDa kallikrein-like protein was identified by monoclonal antibodies to esterase A (5A₁₀, 6C₁₁ and 4B₁₂) and by antibodies recognizing both tonin and esterase A (5A₅), but not by antibody to kallikrein (V₄D₁₁) or to tonin (1F₁₁, 1G₆) in Western blot analysis. The esterase A-like enzyme in the prostate gland was found within the cytoplasm of ductal epithelial cells by using monoclonal anti-esterase A antibody (5A₁₀) but not by employing anti-tonin antibody (1F₁₁). These results indicate that tissue kallikrein, tonin, and esterase A are all expressed in the submandibular gland, while only esterase A or an esterase A-like enzyme is expressed in the prostate gland. The specific monoclonal antibodies can be used as probes for the identification and expression of the kallikrein gene-family enzymes.     

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.     

Localization of kallikrein in the coagulating and submandibular glands of the guinea pig.

Antibody to pure kallikrein from the coagulating gland of the guinea pig was used to localize kallikrein in the gland by immunofluorescence techniques. This antibody also reacted with the guinea pig's submandibular gland kallikrein. The specific fluorescence in the coagulating gland was present diffusely in all secretory cells lining the crypts. In contrast to its diffuse location in the coagulating gland, kallikrein in the submandibular gland was specifically located in the luminal border of striated and some larger duct cells, whereas the acinar cells and interstitial tissue showed no significant fluorescence.     

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.     

Purification and characterization of a kallikrein-like T-kininogenase

A T-kininogenase has been purified to homogeneity from rat submandibular gland extracts by DEAE-Sepharose chromatography and preparative gel electrophoresis. The purified protein has an apparent Mr of 28,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and splits into heavy and light chains with Mr of 22,000 and 6,000 in the presence of dithiothreitol. It is an acidic glycoprotein with pI of 4.65-4.75. The carbohydrate moiety is located on the light chain and binds concanavalin A and wheat germ agglutinin. The active site serine residue of the heavy chain is labeled with [14C]diisopropylfluorophosphate and visualized by fluorography. NH2-terminal amino acid sequences of the light and heavy chains reveal 74-84% identity to rat tissue kallikrein, tonin, and other kallikrein-related enzymes. The enzyme cleaves T-kininogen to release T-kinin which was separated by high performance liquid chromatography on a reverse phase C18 column and identified by a kinin radioimmunoassay. Its T-kininogenase but not N-tosyl-L-arginine methyl ester esterase activity can be enhanced 10-fold in the presence of dithiothreitol. The esterolytic activity of the enzyme is inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, and antipain; whereas lima bean and ovomucoid trypsin inhibitors stimulate its activity. The enzyme is localized at the granular convoluted tubule and striated duct cells in rat submandibular glands by immunohistochemistry. The results indicate that T-kininogenase belongs to the group of structurally similar yet distinct kallikrein-like serine proteases.     

Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Immunocytochemical localization of nerve growth factor in mouse salivary glands

Summary The submandibular glands of female mice and the sublingual and parotid glands of adult male and female mice have been examined by light microscopical immunocytochemistry for nerve growth factor (NGF). In female submandibular glands, staining for NGF was observed in granular convoluted tubule and striated duct cells. Sublingual glands of the mouse contained relatively few granular cells staining for NGF compared with submandibular glands. However, such granular cells appeared to be more numerous in male sublingual glands than in female glands. The remainder of the intralobular duct cells in both male and female sublingual glands exhibited apical subluminal staining for NGF as well as light basal plasmalemmal staining. Parotid glands in both male and female mice exhibited a similar pattern of staining for NGF in striated duct cells. However, the glands did not contain granular cells nor did they exhibit any pattern of staining which reflected a sexual dimorphism. Immunodot staining of salivary gland extracts confirmed the presence of immunoreactivity for NGF in all three of the major salivary glands.     

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.     

Immunohistochemical localization of kallikrein in human pancreas and salivary glands

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Androgen receptor in rat Harderian and submandibular glands

Summary Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.     

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.     

Immunoelectron microscopy of carbonic anhydrase isozyme VI in rat submandibular gland: comparison with isozymes I and II.

Carbonic anhydrase (CA) was purified from the saliva of pilocarpine-treated rats by inhibitor-affinity chromatography, and its localization in the rat submandibular gland was studied by the indirect immunoperoxidase technique using a monoclonal antibody (MAb) raised against the enzyme. SDS-polyacrylamide gel electrophoresis of the CA VI gave three bands of 33, 39, and 42 KD. Enzyme digestion experiment showed that the 42 KD molecule was degraded into the 39 KD molecule and the 39 KD molecule into the 33 KD molecule. The cleavage of the 42 KD molecule was independent and that of the 39 KD molecule was dependent on endo-beta-N-acetylglucosaminidase F. The 42 KD molecule was detected in the CA purified from the pilocarpine-treated but not the untreated salivary gland. The MAb recognized all the three components of the enzyme. Immunostaining for CA VI was seen in the cytosol and secretory granules of serous acinar cells and in the duct luminal contents. Staining specific for erythrocyte CA (CA I and CA II) was observed in the cytosol of the epithelial cells of granular, striated, and excretory ducts. Among these duct cells, the agranular varieties in the granular and excretory ducts were essentially devoid of the immunoreactivity.