Acinar cell response to liquid diet during rats' growth period differs in submandibular and sublingual glands from that in parotid glands.

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.

Effects of glucocorticoid on postnatal development of rat sublingual glands.

Hormones have been reported to be involved in salivary gland's growth and development, but few studies have investigated the effects of glucocorticoids on the morphology of the sublingual glands around the weaning period. The objective of this study was to ascertain the effects of glucocorticoid administration on rat sublingual glands around the weaning period. Male Wistar rats were administered triamcinolone, a glucocorticoid, once every other day from 8 days after birth (experimental group). A control group was given vehicle only. The sublingual glands were then extracted at 15, 20, 25, and 30 days after birth. Samples thus obtained were subjected to Alcian blue and periodic acid-Schiff staining, lectin staining, and immunohistochemical staining to assess cellular proliferative potential. And acinar cell circumferences were measured. We found that glucocorticoid had no effect on the production of acid or neutral mucopolysaccharides by acinar cells around the weaning period. Glucocorticoid administration resulted in hypertrophy of acinar cells between 15 and 30 days after birth. Early appearance of changes in α-mannose, α-glucosamine, and N-acetylglucosamine in secretory granules suggested that glucocorticoid may have acted to promote cell differentiation. The glucocorticoid had no effect on the proliferative potential of sublingual gland acinar cells around the weaning period.

The sld genetic defect: two intronic CA repeats promote insertion of the subsequent intron and mRNA decay.

The autosomal recessive mutation, sld, attenuates mucous cell expression in murine sublingual glands with corresponding effects on mucin 19 (Muc19). We conducted a systematic study including genetic mapping, sequencing, and functional analyses to elucidate a mutation to explain the sld phenotype in neonatal mice. Genetic mapping and gene expression analyses localized the sld mutation within the gene Muc19/Smgc, specifically attenuating Muc19 transcripts, and Muc19 knock-out mice mimic the sld phenotype in neonates. Muc19 transcription is unaffected in sld mice, whereas mRNA stability is markedly decreased. Decreased mRNA stability is not due to a defect in 3'-end processing nor to sequence differences in Muc19 transcripts. Comparative sequencing of the Muc19/Smgc gene identified four candidate intronic mutations within the Muc19 coding region. Minigene splicing assays revealed a novel splicing event in which insertion of two additional repeats within a CA repeat region of intron 53 of the sld genome enhances retention of intron 54, decreasing the levels of correctly spliced transcripts. Moreover, pateamine A, an inhibitor of nonsense-mediated mRNA decay, inhibits degradation of aberrant Muc19 transcripts. The mutation in intron 53 thus enhances aberrant splicing leading to degradation of aberrant transcripts and decreased Muc19 message stability, consistent with the sld phenotype. We propose a working model of the unique splicing event enhanced by the mutation, as well as putative explanations for the gradual but limited increase in Muc19 glycoprotein expression and its restricted localization to subpopulations of mucous cells in sld mice during postnatal gland development.

Expression of Muc19/Smgc gene products during murine sublingual gland development: cytodifferentiation and maturation of salivary mucous cells.

Muc19/Smgc expresses two splice variants, Smgc (submandibular gland protein C) and Muc19 (mucin 19), the latter a major exocrine product of differentiated murine sublingual mucous cells. Transcripts for Smgc were detected recently in neonatal sublingual glands, suggesting that SMGC proteins are expressed during initial salivary mucous cell cytodifferentiation. We therefore compared developmental expression of transcripts and translation products of Smgc and Muc19 in sublingual glands. We find abundant expression of SMGC within the initial terminal bulbs, with a subsequent decrease as Muc19 expression increases. During postnatal gland expansion, SMGC is found in presumptive newly formed acinar cells and then persists in putative acinar stem cells. Mucin levels increase 7-fold during the first 3 weeks of life, with little change in transcript levels, whereas between postnatal days 21 and 28, there is a 3-fold increase in Muc19 mRNA and heteronuclear RNA. Our collective results demonstrate the direct transition from SMGC to Muc19 expression during early mucous cell cytodifferentiation and further indicate developmentally regulated changes in Muc19/Smgc transcription, alternative splicing, and translation. These changes in Muc19/Smgc gene expression delineate multiple stages of salivary mucous cell cytodifferentiation and subsequent maturation during embryonic gland development through the first 4 weeks of postnatal life.

Localization of FGF-6 and FGFR-4 during prenatal and early postnatal development of the mouse sublingual gland.

A number of fibroblast growth factors (FGFs) are involved in regulatory mechanisms of the salivary gland development. However, the role of FGF-6 unique in myogenic cells has not been elucidated in the developing sublingual gland. In the present study, temporo-spatial expression of FGF-6 and its receptor (FGFR)-4, in conjunction with some related histo-chemical properties, were investigated in the sublingual gland of the prenatal and early postnatal mice. The earliest expression of both FGF-6 and FGFR-4 was detected in immature acinar cells at gestational day 17 (GD17). The staining intensity increased gradually and some acinar cells showed a distinct staining at postnatal day 0 (PD0). The immunopositive cells had a relatively round profile and were assumed to be acinar cells. The positive staining decreased thereafter and disappeared completely by PD11. To confirm the identity of cells positive for FGF-6, double immunolabeling with anti-alphasmooth muscle actin (alphaSMA) and anti-FGF-6 antibodies was performed. The positive staining of alphaSMA, a marker of myoepithelial cells, was detected in the flattened cells surrounding the acini but not in the cells positive for FGF-6. The staining properties of secretory granules in acinar cells were also examined with periodic acid-Shiff (PAS) and alcian blue (AB). PAS-positive granules abundant in the late gestational stages (GD17 to PD0) began to be replaced with AB-positive mucous granules at early neonatal days (PD0-3), when the FGF-6/FGFR-4 expression was the strongest. These findings suggest that FGF-6/FGFR-4 might be involved in the changes of secretory granule content of acinar cells in the sublingual gland during the late gestational and early neonatal stages.

Granule types and their morphological changes in terminal cluster and acinar cells in the late pre- and early postnatal rat sublingual gland.

The developmental characteristics of serous cells appearing in the rat sublingual gland from the late prenatal to the early postnatal period were investigated in this study. Particular attention was paid to the morphological changes observed in the secretory granules at the histochemical and ultrastructural level. On prenatal day 18, granules with homogeneous high electron density (Type I granules), and mottled granules (Type II granules) with heterogeneous electron density appeared in the narrow luminar cytoplasm of cells constituting the terminal clusters. On prenatal day 19, these granules decreased in number and were replaced by bipartite granules (Type III granules) composed of a highly electron-dense core and a more electron-lucent rim. Pronase treatment almost completely digested the Type I and II granules and the electron-dense core of the Type III granules, although some of the Type I and II granules in serous demilunes at a later stage were insufficiently digested. On prenatal day 19.5, homogeneous granules of low electron density (Type IV granules) appeared in the terminal clusters and acini, and increased in number daily, making up 92.8% of the total granules on postnatal day 28. The granule morphology on electron microscopy, Alcian blue, and periodic acid-Schiff staining strongly suggested that Type I and II granules were serous granules, Type IV granules were mucous granules, and Type III granules were transforming-type granules. None of the secretory cells showed chromatin condensation, which is a characteristic of apoptosis. These findings suggest that the developing rat sublingual gland from the late prenatal to early postnatal period has numerous serous granules in the terminal clusters and acini, and that the majority of granules are replaced by mucous granules through transforming-type granules. In addition, because apoptotic figures of secretory cells could not be detected, it appears that most of the serous cells in the developing rat sublingual gland might have changed to mucous cells.

Involvement of hepatocyte growth factor in branching morphogenesis of murine salivary gland.

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage.

Growth rate of the cell populations of the rat sublingual glan during the early postnatal period / Taxa de crescimento das populações celulares da glândula sublingual de rato durante o período postnatal inicial

O desenvolvimento da glândula sublingual do rato durante o primeiro mês de vida pós-natal foi analisado pela morfometria. O número absoluto de células nos vários compartimentos morfológicos glandulares - ácinos mistos com demiluas serosas, ductos intercalares, ductos estriados e estroma - foi determinado usando o método morfométrico II de Aherne de contagem de partículas. Os dados de volume glandular à fresco e de número de células foram analisados pela regressão linear, exponencial e parabólica, sendo que os melhores ajustesforam obtidos pela equação linear (Y=a + bx). De posse dessas equções calculamos a velocidade de crescimento de volume glandular e de cada população celular. O volume glandular cresceu ao redor 12 vezes no período de 2 a 30 dias de vida pós natal, a uma velocidade de 1,34 mm3/dia. Esse crescimento volumétrico da glândula se deveu em grande parte ao aumento significante ao redor de 16 vezes, 10 vezes, 4 vezes, 7 vezes e 8 vezes, respectivamente, no número absoluto de células acinosas mucosas, das demiluas serosas, dos ductos intercalares, dos ductos estriados e estromais, a uma velocidade, respectivamente, de 301 x 10 ao cubo células/dia e 228 x 10 ao cubo células/dia, 19 x 10 ao cubo células/dia, 54 x 10 ao cubo células/dia e 247 x 10 ao cubo células/dia. Baseados nos resultados apresentados aqui, concluímos que durante o desenvolvimento pós-natal das glândulas sublinguais do rato, as populações de células mucosas e serosas dos ácinos mistos crescem com velocidades próximas a das células estromais, mas sensivelmente maiores do que as das populações dos ductos intercalares e dos ductos estriados, sendo que a população que exibe a menor velocidade de crescimento é a dos ductos intercalares

Homeobox protein, Hmx3, in postnatally developing rat submandibular glands.

Homeobox-containing (Hox) genes play important roles in development, particularly in the development of neurons and sensory organs, and in specification of body plan. The Hmx gene family is a new class of homeobox-containing genes defined by a conserved homeobox region and a characteristic pattern of expression in the central nervous system that is more rostral than that of the Hox genes. To date, three closely related members of the Hmx family, Hmx1, Hmx2, and Hmx3, have been described. All three Hmx genes are expressed in the craniofacial region of developing embryos. Here we show, for the first time, the expression of the transcription factor Hmx3 in postnatally developing salivary glands. Hmx3 protein is expressed in a cell type-specific manner in rat salivary glands. Hmx3 is present in both the nuclei and cytoplasm of specific groups of duct cells of the submandibular, parotid, and sublingual glands. Hmx3 expression increases during postnatal development of the submandibular gland. The duct cells show increasing concentrations of Hmx3 protein with progressive development of the submandibular gland. In contrast, the acinar cells of the three salivary glands do not exhibit detectable levels of Hmx3 protein.

Development of the rat sublingual gland: a light and electron microscopic immunocytochemical study.

Cell differentiation in the rat sublingual gland occurs rapidly and is largely complete by birth. To study differentiation of the serous and mucous cells of the sublingual gland, we used antibodies to the secretory proteins CSP-1, SMGB, PSP, and SMGD, and sublingual mucin as specific cell markers. Glands from rats at ages 18, 19, and 20 days in utero, and postnatal days 0, 1, 5, 9, 14, 18, 25, 40, and 60 were fixed and prepared for morphological analysis and immunocytochemical labeling. At age 18 days in utero, a few cells in the developing terminal bulbs contained mucous-like apical granules that labeled with anti-mucin. Other cells had mixed granules with a peripheral lucent region and a dense core of variable size that occasionally labeled with anti-SMGD. Additionally, presumptive serous cells with small dense granules that contained CSP-1 and SMGB were present. At age 19 days in utero, the dense granules of these cells also labeled with anti-SMGD. By age 20 days in utero, mucous cells were filled with large, pale granules that labeled with anti-mucin, and serous cells had numerous dense granules containing CSP-1, SMGB, PSP, and SMGD. Fewer cells with mixed granules were seen, but dense regions present in some mucous granules (MGs) labeled with anti-SMGD. After birth, fewer MGs had dense regions, and serous cells were organized into well-formed demilunes. Except for PSP, which was undetectable after the fifth postnatal day, the pattern of immunoreactivity observed in glands of neonatal and adult animals was similar to that seen by age 20 days in utero. These results suggest that mucous and serous cells have separate developmental origins, mucous cells differentiate earlier than serous cells, and cells with mixed granules may become mucous cells.

Sialic acid derivatives and their distribution in rat sublingual gland acini during pre- and post-natal development.

Sialoglycoconjugates in rat sublingual gland acinar cells, at different stages of pre- and post-natal development, were investigated in situ with specific lectins and by the selective removal of terminal sialic acids. Cleavage of acetyl substituents sited in the pyranose ring and/or polyhydroxyl side chain was used as an additional means of characterising the glycoconjugates. The first expression of terminal sialic acid linked to beta-galactose was found at gestational day 17 and progressive different derivatives were observed. The terminal disaccharide sialic acid-N-acetylgalactosamine was constantly visualized in the sublingual gland from gestational day 18. In both terminal disaccharides, sialic acids were characterized by variable degrees of acetylation and were found to be highly packaged and responsible for the hydration coat. The complex data obtained indicated that the sublingual gland is characterized by a marked fluctuation of complex sialoglycoconjugates that differ from those in the submandibular gland of the same species.

Immunohistochemical localization of carbonic anhydrases I, II, and VI in the developing rat sublingual and submandibular glands.

Carbonic anhydrase has been localized to the acini and ducts of mature rat salivary glands. This enzyme has been associated with ion transport, a prominent function of striated and excretory ducts in salivary glands, suggesting that it might be used as a marker of ductal differentiation. The purpose of this study was to immunohistochemically document developmental changes in carbonic anhydrase in the ducts of the rat sublingual and submandibular glands. Immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II and VI on sections of sublingual and submandibular glands from rats at representative postnatal developmental ages. Reactions were weak in the ducts of both glands at 1 day, then progressively increased. By 42 days, reactions had the adult pattern of virtually none in the mucous or seromucous acini, moderate to strong in the striated and excretory ducts, and none to weak in the intercalated ducts. Weak to moderate reactions were observed in the granular convoluted tubules of the submandibular gland as they became recognizable at age 42 days. Reactions to carbonic anhydrase I and II antibodies also increased from none (1 day) to modest (42 days) in the demilunes of the sublingual gland. The order of reaction intensity of the antibodies was II > I > VI. When localized via these anti-human antibodies, carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat sublingual and submandibular glands.

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats.

S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S10100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0-5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands.

Sex-dependent differences in the concentrations of the principal neurotransmitters, noradrenaline and acetylcholine, in the three major salivary glands of mice.

The concentrations of principal neurotransmitters in the submandibular, parotid and sublingual glands were compared between two pairs of age-matched male and female ddY mice, one pair consisting of 4-week-old and the other 8-week-old animals. Sex-dependent differences in both noradrenaline and acetylcholine concentrations were observed only in the submandibular gland, although each neurotransmitter showed distinct features. The acetylcholine concentration in the submandibular gland was higher in the female at both ages, whereas the noradrenaline concentration was higher in the female at the age of 4 weeks but became higher in the male by the age of 8 weeks. On the other hand, the total amounts of noradrenaline and acetylcholine per submandibular gland were already greater in the male at 4 weeks, and the male parotid and sublingual glands also had a greater noradrenaline content by 4 weeks and 8 weeks, respectively. Each type of gland had similar growth rates over the 4-week period, and the male submandibular and parotid glands were heavier than the female. In addition, each type of gland had its characteristic ratio of noradrenaline to acetylcholine concentration, which did not differ between the sexes and remained in similar basic patterns during the period examined, except for the submandibular gland of 8-week-old male mice, which developed greater amounts of the sympathetic neurotransmitter noradrenaline.

Salivary gland nucleotide receptors. Changes in expression and activity related to development and tissue damage.

Experiments were performed to document the presence of G protein-coupled P2Y nucleotide receptors in rat salivary glands and to examine changes in receptor expression during development and under conditions in which gland architecture is altered. The results indicate that, as opposed to mature rat submandibular gland (SMG), immature glands express functional P2Y1 receptors. P2Y1 receptor activity was highest at birth and declined over the next four weeks to undetectable levels. P2Y1 receptor mRNA levels remained constant over this time course, suggesting that receptor activity is regulated at some point other than transcription. Conversely, short-term culture of cells from the three major salivary glands resulted in upregulation of functional P2Y2 receptors. Responses to the P2Y2-selective agonist, UTP, were obtained after 3 h in culture and were maximal by 72 hours. This increase was paralleled by increased steady-state P2Y2 receptor mRNA levels. Upregulation of P2Y2 receptors also occurred in vivo following ligation of the main excretory duct of the SMG. These studies suggest that nucleotide receptors are dynamically regulated during development and as a result of perturbations to gland architecture.

Postnatal development of the rat sublingual glands. A morphometric and radioautographic study.

The postnatal development of rat sublingual glands was analyzed by morphometric and radioautographic studies. The absolute number of each cell type was evaluated by the Aherne II morphometric method for cell counting and labeling indices of these cell types were determined in radioautographs from animals injected with 3H-thymidine. The quantitative cell population kinetic studies were accompanied by morphologic analysis of the modifications in each gland structure. The data concerning evolution of number of each cell type were submitted to analysis by least squares fit-exponential curve. The exponential equations duplication times for the acinar, serous demilune, intercalated duct, striated duct and stroma cells from 2 to 30 days of age were 7.5, 9.0, 10.8 and 9.5 days, respectively. On the other hand, the mean labeling indices for the same cell types during the same period were 9.5%, 5.8%, 7.2%, 3.3% and 4.3%, respectively. Thus, the intercalated duct cells exhibited the second highest labeling index and the slowest growth rate, while the striated duct cells showed the lowest labeling index and the third highest duplication time. The fact that the striated duct cell labeling index does not explain the relatively short duplication time of these cells, suggests that cells from other neighboring morphologic compartments, probably from intercalated duct, migrate and differentiate into striated ducts cells.

Allometric study of the postnatal development of the rat sublingual glands.

The authors studied the rat sublingual glands growth in the period of 2 to 40 days of the postnatal life. The allometric coefficients for the gland mass growth and morphometrically evaluated volume of different gland components in relation to body mass growth, and for the parenchymal volume growth in relation to stroma volume growth, were calculated by the Wald non parametric method, modified by Bartlett. The allometric analysis showed that the gland mass, the mucous cells volume, the serous cells volume, the duct volume and the stroma volume exhibited statistically significant allometric growths with monophasic pattern and allometric coefficient of 0.93, 1.11, 0.76, 0.86 and 1.00, respectively. The analysis of the confidence intervals for these various k values, permitted to conclude that the differential growth of the gland mass is isometric, of the mucous cells volume is positive, of the serous cells and duct are negative and of the stroma volume is isometric.

Developmental studies on vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in salivary glands of postnatal rats.

The development of vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in parotid, submandibular and sublingual glands of the male rat was followed by immunochemistry and immunocytochemistry. The total amounts of these peptides increased in surges during the first 8 weeks of the animal's life; one within 2-4 weeks and the other beginning 1-2 weeks later. Nerve fibres containing these peptides were present at birth showing a pattern of distribution similar to that in adults. During the first 4 weeks the nerve fibres increased in number.

Lipid droplet accumulation in the exocrine glands of the newborn rat.

Exocrine glands including the submandibular gland, sublingual gland, exocrine pancreas and exorbital lacrimal gland of newborn rats aged 0 to 14 days were examined morphologically, and the following results on the accumulation of lipid droplets were obtained. Lipid droplets tended to localize in secretory cells, especially in their basal cytoplasm. The degree of lipid droplet accumulation varied with the type of exocrine gland. There were large accumulations in the sublingual gland, submandibular gland, and exocrine pancreas, but accumulations were small in the exorbital lacrimal gland. No difference in lipid droplet accumulation was recognized between the 2 types of secretory cells in the sublingual gland. The accumulation of lipid droplets peaked 24-48 hours after birth in the sublingual gland, submandibular gland and exocrine pancreas, but this peak was not clearly observed in the exorbital lacrimal gland. In the group of newborn rats separated from their mothers and therefore not suckled, no lipid droplets were observed in any gland, suggesting a close relationship between lipid droplet accumulation and suckling.

Secretion of peptidylglycine alpha-amidating monooxygenase (PAM) from rat salivary glands.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a regulating enzyme to synthesize the biologically active hormones having carboxy-terminal amide. In the present study we investigated secretion of the enzyme from rat saliva. Property of PAM in the saliva was similar to that in the submandibular gland. Both enzymes showed similar pH optimum at 5.0 and optimal ascorbic acid concentration at 2.5 mM. But molecular size of PAM in the saliva was 75 kDa in the gel permeation chromatography on Superose 12 column, while the size in the submandibular gland was 25 kDa. After the treatment with trypsin, PAM in the saliva was converted to a small size molecule, which is similar to the size in rat submandibular gland. These and other data indicate that a native molecular size of PAM is secreted into saliva and plays some physiological roles.