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Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study investigated how liquid diet feeding affects the growth of parotid glands. We weaned 21-day-old rats and thereafter fed them a pellet diet (control group) or a liquid diet (experimental group) for 0, 1, 2, 4, or 8 weeks. Their parotid glands were excised, weighed, examined, and tested for 5-bromo-2'-deoxyuridine (BrdU) and cleaved caspase-3 (Casp-3) as markers of proliferation and apoptosis, respectively. Parotid gland weights were consistently smaller in experimental animals than in controls. Morphometrical analysis showed that control group acinar cells increased in area during the experiment, but experimental group acinar cells were almost unchanged. Labeling indices of BrdU in acinar cells in both groups declined during the experiment, but were consistently lower in the experimental group than in controls. Casp-3-positive acinar cells were rare in both groups, which consistently express significantly similar Casp-3 levels. Ultrastructurally, terminal portions of the experimental parotid glands consisted of a few acinar cells that were smaller than those in controls. Control acinar cells showed mitotic figures within short experimental periods, but not in experimental glands. These observations indicate that liquid diet feeding inhibits growth of parotid glands in growing rats through suppression of growth and proliferation of individual acinar cells, but not through apoptosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Previously, the structure of the adult goat parotid salivary glands (PGs) was studied. However, little information was elucidated of the juvenile ones. This study aimed to clarify the correlations between the structure of goats' PGs and the nature of food intake among milk-suckling kids (MSKs) and diet-fed goats (DFGs). The secretory endpieces of the goats' PGs are of the pure serous type. The serous cells in MSKs showed apical accumulation of numerous secretory granules (SGs) of smaller size and of more intense positive periodic acid-Schiff reaction. Ultrastructurally, most of the SGs in the DFGs contained peripherally located inclusions that showed dense reaction products for acid phosphatase. In MSKs, the PGs showed less-developed basal infoldings, sparseness of the inter-cellular inter-digitations, fewer inter-cellular canaliculi and microvilli and also less-developed myoepithelial cells with fewer and shorter cytoplasmic processes. In conclusion, the less-developed membrane specializations and myoepithelial cells, as well as the accumulated SGs in the PGs of MSKs, suggest that it secretes less saliva with a little secretory activity than that of DFGs, which may be correlated with the reduced masticatory activity., (© 2013 Blackwell Verlag GmbH.)

Tadpoles of the tropical bicolored frog, Clinotarsus curtipes are unique in having parotoid glands secreting a white viscous fluid and are structurally similar to granular glands from other amphibians. To ascertain the involvement of these glands and their secretion in predator deterrence, it was tested against a predatory fish, Clarias gariepinus, using a paired choice behavioral assay. The results showed that the fish avoid eating C. curtipes tadpoles when paired with tadpoles of a sympatric species, Sylvirana temporalis. While the fish fed on C. curtipes tadpoles whose parotoid glands were surgically removed, did not touch those with intact glands, suggesting a role for the parotoid gland secretion in predator deterrence. Histochemical and biochemical analyses of the gland secretion revealed the presence of high concentrations of proteins, lipids, and alkaloids. SDS-PAGE showed the presence of proteins with prominent bands at 17 and 50kDa. The presence of other small molecules (950-2000amu) as detected by LC-MS showed the presence of five major peaks. Peaks 1 and 2 are probably tetrodotoxin and/or its analogs. Peaks 3 and 5 are possibly bufalin and argininosuccinic acid, respectively while peak 4 remains unidentified. Thus, secretion of parotoid glands of larval C. curtipes contains chemicals which, either alone or in combination, might be responsible for deterring predators., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The role of the intrauterine growth retardation (IUGR) in the remodeling of a fetal parotid gland at late pregnancy has been presented in the paper. Thirty fetal parotid glands at 20-22 weeks gestation, including, 20--with IUGR and 10--at physiological pregnancy (control) were studied morphologically and morphometrically. Results have shown violations of gland's growth and differentiation, increased volume fraction of pathologic changes. Above mentioned processes may cause salivary glands' dysfunction, which eventually could result in child's dental caries.

In the article new criteria of diagnostics of chronic and reactive pancreatitis due to sonography data are described. Among symptoms of reactive pancreatitis are as follows: presence of pancreatic edema, appearance of hyper echogenic lineal admixtures (visualization of connective tissues jumpers). Among criteria of reactive pancreatitis due to ultrasonography belong all as follows: local (59.7%) or diffuse hyper echogenic (18.8%) parenchyma comparatively to parenchyma of parotic gland, as well as small dots or triangle-like formations - marker of fibrosis. At the same time among signs of long-lasting pancreatitis these symptoms are also characteristic areas of hypoechogenic parenchyma which mimic areas of hyperechogenic structure of pancreas (44.0% of incidences). Widening of pancreatic duct like prominent symptom of chronic pancreatitis they noticed in 1/3 cases of reactive pancreatitis together with local edema of pancreas.

We examined the cellular signaling pathways involved in parotid gland enlargement induced by repeated isoproterenol administration in rats. Immunoblot analysis revealed early (1h) activation of the mitogen activated protein kinase (MAPK) ERK1/2, and progressive activation of epidermal growth factor receptor (EGFR), p38MAPK and p70S6 kinase (p70S6K) during 72h of isoproterenol treatment. Expression of β-adrenergic receptors (ARs) of the β2, but not β1, subtype increased over time in parallel with increases in the proliferation marker PCNA and parotid gland weight. Levels of β2-AR mRNA, assessed by quantitative RT-PCR and Northern blot analysis, were upregulated in parotid glands of isoproterenol treated rats. cAMP response element binding protein (CREB), a positive regulator of β2-AR transcription, was activated at 1h after isoproterenol administration, as evidenced by increased nuclear translocation and DNA binding using immunohistochemical staining and electrophoretic mobility shift assay. ELISA of NF-κB, also a β2-AR transcriptional regulator, revealed an increase in p65 and p50 subunits in nuclear protein extracts from parotid glands of isoproterenol treated rats. Together, these results demonstrate that β-adrenergic stimulation activates diverse cell survival and progrowth signaling pathways, including cAMP and EGFR linked activation of ERK1/2, p38MAPK, and p70S6K, and also induction of β2-ARs, possibly mediated by CREB and NF-κB, resulting in salivary gland enlargement. We propose that during isoproterenol treatment activation of the β1-AR, the predominant β-AR subtype in unstimulated salivary glands, initiates proliferative signaling cascades, and that upregulation of the β2-AR plays an essential role in later stages of salivary gland growth., (Published by Elsevier B.V.)

In the mature rat parotid gland, myoepithelial cells (MEC) invest intercalated ducts, but not acini. During postnatal development, however, these cells differentiate around both intercalated ducts and acini, then translocate to only intercalated ducts during weaning. Previously, we found that thyroxine (T(4)) accelerates translocation of cells with small secretory granules from acini into intercalated ducts and the number of apoptotic cells increased tremendously with high doses. We present here additional analysis of the effects of T(4) on developing rat parotid gland, namely, the distribution of MEC and the proliferation of parenchymal cells. Beginning at age four days, pups were given daily subcutaneous injections of low, medium, and high doses of T(4) or vehicle or no injection. At ages 4, 7, 10, and 15 days, glands were excised and processed for light microscopy. Sections were double-immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and actin, and counterstained with hematoxylin. Proliferative activity was assessed via PCNA histochemistry and MEC were identified using actin histochemistry. MEC in the T(4) groups invested mostly acini at 15 days in vehicle/normal glands and mostly intercalated ducts after 10 days in the T(4) groups. The proliferative activity of acinar cells and MEC in vehicle/normal glands declined progressively with age and T(4) increased the rate of this decline in the MEC in a dose-dependent manner. We conclude that T(4) accelerates the translocation of MEC from acini to intercalated ducts and that an important mechanism is the more rapid decline in the proliferative activity of MEC than in acinar cells in the T(4) groups. Some of the decline in the proliferative activity of all cells in the high and medium dose T(4) groups after seven days may have been due to dose-related thyroxine toxicity.

The juxta-oral organ is a bilateral organ in the mammalian bucca. It consists of epithelial cords with surrounding mesenchyme. It develops from embryonic oral epithelium, but its macroscopic morphology in mice is less studied and seems to be very different from that of humans. The juxta-oral organ in mice extends more widely from the subcutaneous tissue of the mandible near the lateral fascia of the masseter to the submucosa of the soft palate. In this paper, we report that the mutant mouse allele Bmp7(lacZ) presented intense lacZ expression in the epithelial component of the juxta-oral organ in its homo- and heterozygous states. The main aims of this study were to show that this mutant mouse allele is suitable for observing macroscopic structure of the juxta-oral organ and to describe the development of this organ during embryonic and postnatal stages. Whole-mount beta-gal staining of this strain of mouse showed that the juxta-oral organ in mice appeared at E12.0 from oral epithelium and lost connection with it before E12.5. Then, the juxta-oral organ extended anteriorly to the lateral fascia of the masseter and posteriorly to the submucosal layer of the soft palate via the orbit. The mature juxta-oral organ had no connection to other epithelia such as those of the bucca and parotid duct. It persisted until adulthood and there seemed to be no tendency to regress. Transmission electron microscopy showed that each part of the juxta-oral organ was an epithelial cord surrounded by a basement membrane and mesenchymal tissue.

Unlabelled: Previous studies showed that regulatory subunits of type II cyclic AMP-dependent protein kinase (RII) are present in adult rat parotid acinar cells, and are secreted into saliva. If the synthesis and intracellular distribution of RII exhibit developmental specificity, then RII can be an indicator of secretory and regulatory activity of salivary glands., Objective: To determine the expression and distribution of RII in the rat parotid at specific ages representing defined developmental stages., Methods: Parotid glands of fetal, neonatal and adult rats were prepared for morphologic and immunocytochemical study. The cellular distribution of RII was studied using light microscopic immunogold silver staining with anti-RII, and its intracellular distribution using electron microscopic immunogold labeling., Results: In utero, parotid RII levels were low; 5-18 days after birth, labeling of secretory granules and cytoplasm rose to a peak, followed by a rapid decrease in both compartments at 25 days. At 60 days, granule labeling increased to levels near those at 18 days, whereas cytoplasmic labeling remained low. Nuclear labeling was highest during the first 3 weeks after birth, and then declined., Conclusions: The higher nuclear and cytoplasmic labeling during the neonatal period may reflect RII involvement in acinar cell differentiation. The accumulation of RII in secretory granules is similar to the pattern of the major salivary proteins, amylase and PSP. The redistribution of RII in these compartments during development may reflect changing gene expression patterns, and may be useful for identification of genetic or metabolic abnormalities.

Administration of thyroid hormone has been shown to accelerate the early postnatal development of the rat parotid gland, but these studies have dwelt almost entirely on biochemical changes. The objective of this study was to describe the effects of exogenous thyroid hormone on morphologic aspects of the developing parotid gland, in particular the transient appearance of scattered mucous cells in this otherwise serous gland. Pups were given a daily subcutaneous injection of thyroxine (T(4)) of 0.1, 0.5, or 5.0 microg/g body weight, vehicle only (injection control), or no injection (normal control) beginning at 4 days, and killed for the collection of blood and parotid glands at intervals through 15 days. The serum was analyzed for T(4) and the glands were examined by light and electron microscopy. The results indicated that both serum T(4) and the pace of gland development were proportional to the dose of T(4). In particular, T(4) accelerated decreases in acinar size and gland area occupied by stroma and translocation of a subset of cells with small secretory granules, deeply stained with periodic acid-Schiff, from acini to intercalated ducts. However, the chronology of mucous cell disappearance was indifferent to treatment. In addition, signs of toxicity, including slower gain in body weight and greatly increased apoptosis and vacuoles in the glands, occurred with the higher doses of T(4).

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.

The growth of the mouse parotid glands during 7 and 35 days of postnatal life was studied by morphometric methods. The mass of the gland, the volume of each morphological compartment, and the cell number in each compartment were evaluated. The data obtained for each evaluated dimension were adjusted by an exponential equation, of the type Y = a.e k.x, thus permitting the calculation of their mean duplication time (T D), i.e., an estimation of their growth rate. Analysis of the results showed a marked 1,424% increase in the gland mass during the whole studied period, with T D = 7.10 days. This growth occurred by increases in absolute volume of acini, intercalated ducts, striated ducts, excretory ducts and stroma, with percentual increases of 3,048%, 417%, 2,662%, 2,594% and 367%, respectively, and T Ds of 5.62, 11.71, 5.55, 5.47 and 14.45 days, respectively. Analysis of the cell number growth in each compartment showed increases of 1,904%, 285%, 1,228%, 1,090% and 286%, respectively, and T Ds of 6.62, 20.40, 7.19, 7.26 and 14.51 days, respectively. Based on the present results, we concluded that the growth of the mouse parotid glands from day 7 to day 35 of age occurred by intense cell accumulation, mainly in the acini, striated ducts and excretory ducts, with a growth rate sensibly higher than that of the intercalated ducts and stroma.

The aim of the present investigation was to study the role of hypothalamic nonapeptides in the regulation of proliferation, growth and cell differentiation processes in glandular epithelia of different developmental origin (parotid gland and pancreas of albino rats) under conditions of in vivo organotypical culture by F.M. Lazarenko's method. Histoautoradiography, light and electron microscopy methods were used. Hypothalamic nonapeptides were shown to cause the dedifferentation of glandular epithelium, intensification of DNA-synthetic activity of epithelial cells, fibroblasts, macrophages, to optimize secondary differentation in the newly formed glandular structures, to stimulate the vasculogenesis and fibrillogenesis in an undifferentiated connective tissue. The humoral factors studied provide the conditions for the realization of histo- and organotypical potentialities by the epithelia of parotid gland and pancreas without interfering with their genetic determination. Adaptogenic role of neurohormones in the control of cell and tissue homeostasis of the cultured organ fragments is discussed.

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.

Carbohydrate metabolism was examined in the developing rat salivary glands by analysing enzymatic activity and glycogen content in the postnatal parotid and submandibular glands. The following enzymes of the carbohydrate metabolism, hexokinase (HK), phosphofructokinase-1 (PFK-1), pyruvate kinase (PK), glucose-6-phosphate dehydrogenase (G6PD), and lactate dehydrogenase (LDH) as well as the content of glycogen were determined in the salivary glands of rats aged 2, 7, 14, 21, 30 and 60 days. The specific activity of HK increased from days 2 to 21 and then it decreased up to 60 days old. The values found for the submandibular glands were from 2.5 to 4.9 times higher than those found for the parotid gland, except for rats aged 60 days. PFK-1 showed a different pattern of variation between the glands. In the submandibular gland there was a statistically significant increase in PFK-1 specific activity from 2 to 30 days of age and then, in the 60 days old group a return to level of the rats aged 2 days. In parotid gland, the specific activity of PFK-1 decreased between 2 and 7 days of age, from 7 to 14 days the specific activity increased markedly and from 14 to 60 days old it gradually decreased. The specific activity of PK followed the same pattern of variation in the submandibular and parotid glands, showing no great variation. The specific activity of LDH decreased from 2 to 60 days old in the submandibular glands. In the parotid glands the mean values for this enzyme were higher for the 2 days old group, and then decreased to remained more or less constant. The potential capacity of the pentose phosphate pathway was greater than that of glycolysis at early ages. The glycogen content showed similar variation in both glands. It was initially high and then decreased. In conclusion, our results on the activities of enzymes involved in carbohydrate metabolism in submandibular and parotid glands may be relevant to the initiation of saliva secretion in these animals.

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.

Information on ductal differentiation in the developing rat parotid gland is sparse. One of the main functions of the striated and excretory ducts in this gland is the selective exchange of electrolytes from the primary fluid secreted by the acini. These ducts are rich in a number of enzymes involved in this task, suggesting that they might be useful as markers of ductal differentiation. The objective of this investigation was to delineate the developmental changes in activity of three of these, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), nicotinamide adenine phosphate dinucleotide (reduced form)-dehydrogenase (NADPH-DH). Histochemical localization of all three enzymes in fresh frozen sections was complemented by biochemical assays of CCO and SDH and cytochemical localization of CCO. Biochemically, CCO- and SDH-specific activity in gland homogenates increased progressively after birth, reaching adult levels at 21-28 days. Histochemically, deposits of reaction products of all three enzymes increased more in the striated and excretory ducts, especially in their basal cytoplasm, than in other glandular structures between 19 days in utero and 28 days after birth. During the same age span, the mitochondria in the striated and excretory ducts increased markedly in both number and size, migrated to a mostly basal location, and increased from many to virtually all showing strong cytochemical CCO reactions. These histochemical and cytochemical patterns of changes in enzyme activity at the cellular level accounted for the overall increases in CCO and SDH seen in the biochemical assays. Only the SDH histochemical reaction was consistently weak in the acini and intercalated ducts, and thus provided the most contrast with the progressively stronger reactions in the larger ducts. We conclude that of the three enzymes evaluated in these experiments, SDH is the best marker of the functional differentiation of the striated and excretory ducts in the developing rat parotid gland.

Mucous cells have been known to occur in the terminal portions of the parotid gland in a few species of mammals during a limited period of their development. The aim of this study was to examine the occurrence and features of mucous cells in the parotid gland of the infant Japanese macaque. Light microscopy revealed that mucous cells in the macaque parotid gland were present in the terminal clusters and acini at postnatal day 15, were less prevalent at day 30, and continued to decrease in number over 3 months. Mucous cells were no longer recognized in the parotid gland in 6-month-old macaques. Electron microscopy showed that the mucous cells contained electron-lucent secretory granules and bipartite or tripartite secretory granules. By 3 months of age, there was a scarcity of mucous cells and a concomitant increase in transitional cells. These transitional cells were intermediate in structure between mucous and serous cells, and contained three types of granules: electron-lucent, bipartite or tripartite, and electron-dense. None of the cells showed apoptotic figures. Lectin histochemistry indicated that the mucous cells in the early postnatal period had sugar residues identical in nature to those seen in the granules from mature serous cells in the glands of 3-month-old macaques. Immunohistochemistry using an antibody against human alpha-amylase showed a weakly positive reactivity in the secretory granules of the mucous cells, starting from day 15. In the transitional cells, the electron-dense granules showed a stronger immunoreactivity than either the electron-lucent granules or the heterogeneously structured granules. These results suggest that the secretory granules of mucous cells have characteristics in common with those of serous cells, and that during the transitional period the mucous granules change from the initial electron-lucent to hetorogenous forms, finally becoming the electron-dense granules. The mucous cells in the parotid gland of the juvenile Japanese macaque are therefore suggested to be converted into serous cells.

The growth kinetics of different cell populations in the rat parotid was studied. The evolution of the frequency and absolute number of each cell type was determined morphometrically by a particle-counting method and the evolution of the [(3)H]thymidine labeling indices of the same cell types was determined by autoradiography. The data obtained for the evolution of cell number in each gland compartment, i.e. acini, intercalated ducts, striated ducts and stroma, were adjusted by exponential equations, permitting estimation of the effective cell accumulation rate in the compartment for each population, i.e. the mean population duplication time (T(D)). In addition, the cell production rate in each gland compartment was determined using the mean labeling index for the period studied and a mathematical estimation of the mean cell generation time (T(G)), assuming an exponential growth pattern for the acinar, intercalated duct and striated duct populations during the period from 5 to 20 days of postnatal development. Analysis of the relation between effective cell accumulation (T(D)) and presumed cell production (labeling index and T(G)) for each intralobular parenchymal compartment of the rat parotid during this period suggests that the proliferative activity of the acinar cell population was sufficient to guarantee marked growth of its compartment and provided cells that presumably dedifferentiated into intercalated duct cells, whereas cells produced in the intercalated duct compartment migrated to, and differentiated into, cells of the striated duct compartment.

The present study was designed to clarify the more precise ultrastructural feature of granules, especially mucous granules in the early postnatal rat parotid gland by using rapid freezing followed by freeze-substitution fixation. The parotid gland of Wistar rats (aged 0-10 days) was removed under anesthesia and immediately underwent cryofixation followed by substitution with osmium tetroxide. After fixation, the samples were embedded in Epon-Araldite, cut into ultrathin section, and then examined by transmission electron microscopy. Electron microscopy showed that the mucous granules of samples treated by freeze-substitution fixation had low electron density and were almost spherical in shape with a clear limiting membrane. By Day 5, granules that were a little more electron dense than mucous granules, granules with a more electron dense portion at their periphery, and heterogeneous granules with an internal highly electron dense portion were found. Moreover, cells containing both homogeneous granules with a high electron density similar to that of mature serous granules and heterogeneous granules were observed. These findings demonstrated that the morphology of the transforming-type mucous granules by chemical fixation in the previous study was an artifact and, as a result, strongly suggested that on the sequential morphological changes of transitional mucous/serous granules by rapid freezing method in the present study, the mucous cells in the early postnatal rat parotid gland undergo transformation to serous cells., (Copyright 2000 Wiley-Liss, Inc.)

The distribution of bovine carbonic anhydrase isozyme VI (CA-VI), purified from bovine saliva, was studied immunohistochemically using antiserum against bovine CA-VI in bovine parotid glands during fetal and postnatal development. A weak expression of CA-VI in undifferentiated epithelial cells and ductal cells was observed in a 4- to 5-month-old fetus with a 26-cm crown-rump length. The reaction in both acinar and ductal cells subsequently persisted during late gestation and birth. Although anti-CA-VI reactivity was still seen in both regions immediately following birth, the reactivity had almost completely disappeared from most duct segments by 1 month following birth. Changes in the localization and time-dependent expression of the isozyme in parotid glands may reflect changes in the biological function of structurally closely related isozymes., (Copyright 2000 S. Karger AG, Basel)

Information on ductal differentiation in the developing rat parotid gland is sparse. Striated and excretory ducts are rich in a number of enzymes related to ion movement. The objective of this investigation was to delineate histochemically the chronology of two of these, ouabain-sensitive Na(+),K(+)-ATPase and NADH-DE, in the developing rat parotid gland. Parotid glands were excised from rats at representative ages from 20 days in utero to 42 days. Enzyme histochemistry was performed on air-dried frozen sections. For Na(+), K(+)-ATPase, some sections also were fixed in phosphate-buffered formalin. Ouabain blocked Na(+),K(+)-ATPase activity, and neither enzyme reacted without substrate. Weak Na(+),K(+)-ATPase reactions were initially seen in unfixed sections at 1 day, and increased steadily to the adult pattern of strong (concentrated basolaterally) in striated ducts and excretory ducts, respectively, and weak to modest (diffuse) in acini and intercalated ducts at 28 days. In fixed sections, localization was sharper but the reaction was somewhat reduced. NADH-DE was modest in terminal buds and ducts before birth, then progressively changed to the adult pattern of weak in acini and intercalated ducts and strong (concentrated basally and luminally) in striated and excretory ducts at 28 days. As demonstrated by enzyme histochemistry of Na(+),K(+)-ATPase and NADH-DE, differentiation of rat parotid striated ducts and excretory ducts occurs mainly between birth and 28 days. Anat Rec 256:72-77, 1999. Published 1999 Wiley-Liss, Inc.

Mucous cells have been identified in the terminal portions of the early postnatal parotid gland in human and rat, although mature parotid gland acini are composed of serous cells or seromucous cells. Previously, Ikeda et al. demonstrated that mucous cells are present in the rat parotid gland on days 1 to 8 after birth and that the secretory granules within these mucous cells share some histochemical characteristics with mature serous cells. However, it is still not clear whether the mucous cells change into serous cells as the gland develops. The purpose of this study was to determine whether the mucous cells that appear in the early postnatal rat parotid gland change into serous cells. Parotid glands were obtained from male or female Wistar rats (aged 0-14 days and adults). Fixed tissue sections were reacted with soybean agglutinin (SBA) and wheat germ agglutinin (WGA) to detect glycoconjugates, or were stained using an anti-neonatal submandibular gland protein B1 (SMG-B1) antibody to identify serous acinar cells. The sections were observed by transmission electron microscopy. Electron microscopy revealed that cells with characteristics intermediate between those of mucous and serous cells (transitional cells) appeared around day 8 and that the nuclei of these cells did not show chromatin condensation, a characteristic of apoptotic cells. Lectin histochemistry showed that the mucous cells had the same sugar residues as the serous cells, which appeared after day 10. Immunohistochemistry with an anti-SMG-B1 antibody gave a positive reaction not only in the cells with highly electron-dense granules but also in the electron-dense cores of bipartite or tripartite granules in the transitional cells. Cells with morphological characteristics intermediate between those of mucous and serous cells (transitional cells) appearing in the early postnatal rat parotid gland begin to produce B1-immunoreactive protein common to serous acinar cells during development of the gland.

The rat parotid gland produces a number of well-characterized secretory proteins. Relatively little is known, however, about the onset of their synthesis and cellular localization during gland development. Secretory protein expression was studied in parotid glands of fetal and postnatal rats using light and electron microscopic immunocytochemistry and Northern blotting. Amylase, parotid secretory protein (PSP), common salivary protein-1 (CSP-1), and SMGB were first detected by immunofluorescence in parotid glands of 18 day fetuses. By 5 days after birth, light and electron microscopic immunolabeling localized all of these proteins to the secretory granules of developing acinar cells. Labeling of acinar cells for DNAse I, however, was not observed until 18 days after birth. Between 9 and 25 days, CSP-1 and SMGB reactivity of acinar cells declined, but increased in intercalated duct cells. After 25 days, CSP-1 and SMGB were found only in intercalated ducts, and amylase, PSP, and DNAse I were restricted to acinar cells. Levels of CSP-1 and SMGB mRNA were relatively constant through 21 postnatal days, but declined significantly after that. Amylase and PSP mRNA increased rapidly and continuously from five days after birth to the adult stage. In contrast, DNAse I mRNA was not detectable until 18 days after birth. The immunocytochemical and molecular analyses define three basic patterns of protein expression in the rat parotid gland: proteins whose synthesis is initiated early in development and is maintained in the acinar cells, such as amylase and PSP; proteins that are initially synthesized by immature acinar cells but are restricted to intercalated ducts in the adult gland, such as CSP-1 and SMGB; and proteins that are synthesized only by mature acinar cells and first appear during the third postnatal week, such as DNAse I. The parotid gland exhibits four distinct developmental stages: prenatal, from initiation of the gland rudiment until birth; neonatal, from 1 day up to about 9 days postnatal; transitional, from 9 days to 25 days of age; and adult, from 25 days on. Although differences exist in timing and in the specific proteins expressed, these developmental stages are similar to those seen in the rat submandibular gland. Additionally, the results support the suggestion that intercalated ducts may differentiate from the neonatal acini.

Wistar strain rats fed low calcium diets (1, 2, 3, 4, 6 and 8 weeks) exhibited changes in secretory function of whole saliva. In particular, there were changes in salivary flow rate, total salivary protein, amylase enzyme activity, salivary amylase content and the level of cyclic AMP in the parotid gland acinar cell. Although there were no changes for the first 3 weeks, the levels increased at week 4 and decreased at week 6. The wet weight of the parotid gland started to decrease at week 4. These results suggest that when fed low calcium diets for long periods of time, rats develop defective salivary secretion.

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 suggest 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.

Background: Carbonic anhydrase has been localized to the acini and ducts of mature rat parotid 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 histochemically to document developmental changes in carbonic anhydrase in the ducts of the rat parotid gland., Methods: Parotid glands were excised from rats at representative developmental ages. Enzyme histochemistry was done on frozen sections fixed in acetone, and immunohistochemistry was performed with antibodies to human carbonic anhydrase isoenzymes I, II, and VI on paraffin sections of glands fixed in Helly's fluid., Results: Carbonic anhydrase activity was weak until age 21 days after birth, when it had increased slightly in the acini and intercalated ducts and moderately in striated and excretory ducts. The adult pattern was attained by 28 days, in which reactions were moderate to strong in the striated and excretory ducts and modest in the acini and intercalated ducts. Immunohistochemical reactions were weak until 14 days, then increased rapidly, and by 28 days approached the adult pattern of virtually none in the acini and modest to moderately strong in the striated and excretory ducts. The order of reaction intensity of the antibodies was II > I > VI., Conclusions: Carbonic anhydrase is a useful marker of the functional differentiation of the striated and excretory ducts of the developing rat parotid gland.

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.

Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands. With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay.

The growth of the rat parotid gland induced by daily treatment with isoproterenol (IPR) for 2 weeks was investigated by stereological methods applied to light microscopy. After 7 days of treatment, the glandular mass presented a 286% growth, with the first 3 days being the period of greatest growth. Total acinar volume exhibited a 363% increase during the period from 0 to 7 days, while acinar-cell volume presented a 468% growth from 0 to 5 days of treatment. On the other hand, total acinar-cell number did not increase during the study period. Thus, under the conditions used, IPR-stimulated gland growth was essentially hypertrophic. However, a significant increase in the number of bipolar and multipolar mitoses was also observed, especially on the third and fifth days of treatment. As no increase in acinar-cell number occurred during growth, the presence of these mitoses suggests that cell death occurred during gland growth. On this basis, bipolar mitoses may occur to replace cells that probably degenerated during treatment, whereas multipolar mitoses may lead to the occurrence of polyploidy.

While antibiotics are broadly used in dental and medical therapy, little attention has been directed towards the potential toxic side effects of antibiotics on tissue regeneration. Here we examined the effect of a quinolone antibiotic, pefloxacin (Rhone Poulenc) on rat parotid gland responses to chronic isoproterenol treatment. Groups of rats received injections of isoproterenol to induce glandular growth, saline (controls), pefloxacin, or isoproterenol and pefloxacin in combination. Parotid gland weight decreased significantly after pefloxacin treatment for 7 days as well as inhibiting glandular enlargement provoked by isoproterenol. The same trend was observed for the rates of DNA synthesis, with the incorporation of [3H]-thymidine in isoproterenol/pefloxacin-treated rats reduced to 49% of isoproterenol treatment alone levels. Saline-treated animals were 42% of the rate of [3H]-thymidine incorporation into DNA observed in isoproterenol treated rats. While isoproterenol treatment increased steady-state mRNA levels for fos, jun, myc, src, c-erbB-2, ras and topo II, inclusion of pefloxacin with the isoproterenol regimen blocked these increases. Pefloxacin treatment by itself did not alter proto-oncogene mRNA levels in the parotid gland. Glandular amylase activity was decreased in the pefloxacin treated group, while the combination of isoproterenol with pefloxacin did not decrease glandular amylase levels to the extent of that observed with beta-agonist treatment alone. In acute experiments, pefloxacin significantly decreased the volume of saliva secreted by the parotid gland. These results suggest that quinolone-based antibiotics disturb the secretory function of the parotid gland and can inhibit cell proliferation and regeneration.

We investigated the influence of the streptozocin (STZ)-diabetic state on the dose-response curves for salivary flow and protein content in saliva stimulated by pilocarpine and noradrenaline in mice. The diabetic state increased the relative weights of parotid and sublingual salivary glands but not the weight of submandibular glands, despite body weight loss. In the dose-response curves, (1) the maximal responses to stimulation with pilocarpine and noradrenaline on salivary flow, and with noradrenaline on protein content in saliva, were depressed by the diabetic state, and (2) the value of the 50% effective dose for salivary flow with pilocarpine, but not with noradrenaline, was decreasingly altered by diabetic mice. These results suggest that xerostomia, one of the complications of non-insulin-dependent diabetes mellitus, is caused in part by muscarinic and adrenergic receptor dysfunction in the salivary glands.

The relation of the tests/testosterone with the structure and function of the mammalian parotid gland has so far been poorly investigated. The present study deals with the morphology of the rat parotid gland and its changes after orchiectomy and testosterone substitution. The glands of control and experimental animals (orchiectomized and orchiectomized with testosterone substitution) were analyzed by electron microscopy and stereology. In orchiectomized animals 30-60 days after castration, a significant reduction of the volume of the acini and the duct system as well as a significant increase of the connective tissue volume per cubic millimeter of the gland were noted. The volume and length of the intercalated and the striated ducts per cubic millimeter of parotid tissue are significantly reduced 45-60 days after orchiectomy. Excretory ducts seem to be unaffected by orchiectomy. The structure of the rat parotid acini is also changed by castration, indicating a reduction of acinar-cell activity. In controls, the volume of acinar cells with wide cisternae of rough endoplasmic reticulum is 3 times larger than the volume of acinar cells with regular and narrow cisternae of rough endoplasmic reticulum. After orchiectomy, the volume of acinar cells with wide cisternae of rough endoplasmic reticulum is significantly decreased, while the volume of acinar cells with regular and narrow cisternae of rough endoplasmic reticulum is significantly increased. Exogenously given testosterone can prevent or alleviate the mentioned effects of orchiectomy on the gland. It is concluded that orchiectomy affects the rat parotid gland, demonstrating the existence of an interaction between the testis and the mammalian parotid gland.

Protein deficiency results in an increased susceptibility to dental caries, suggesting that oral host-defense properties are compromised. An important component of oral host defense is salivary gland function, which is affected by both protein deficiency and diet consistency. This study describes the effects on rat parotid gland growth and secretory function induced by feeding rats diets of normal (20%) or moderately low (7%) protein content, provided in either a powdered or solid form. In addition, since protein deficiency may result in a secondary zinc deficiency which, in turn, may affect salivary gland function, the effects of these diets on liver zinc concentration were also measured. From 22 to 47 days of age, rats (18/group) were fed the following diets: normal protein, powdered; normal protein, solid; low protein, powdered; and low protein, solid. With each diet consistency, liver zinc was higher for the normal protein group. Within each protein level, liver zinc was higher for the solid diet. This latter observation suggests that food mastication and the resultant stimulation of salivary gland function may also play a role in zinc metabolism. With the normal-protein diet, parotid gland weight was higher for the solid diet; with the low-protein diet, parotid gland weight was similar for both consistencies and did not differ from that of the group fed the "normal protein, powdered" diet. For both consistencies, parotid saliva protein concentration was greater for malnourished rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: In contrast to the considerable amount of research that has been done on the proliferative activity of the several types of parenchymal cells in the developing submandibular glands of rodents, systematic studies of cellular proliferation in the developing parotid gland have been confined to the acinar cells. The purpose of the present study was to attempt to fill this knowledge gap., Methods: Tritiated thymidine was parenterally administered to Sprague-Dawley rats at ages representative of the pre- and postnatal development of the parotid gland, and glands were harvested for autoradiography 90 min after injection. Mitotic activity among all cell types was verified by electron microscopy., Results: At all ages, the % labeled cells was much greater among the acini than any other cell type, including well-differentiated cells at 25 and 40 days. However, there were only small alterations in the proportions of cells comprised by the major cell types., Conclusions: Current theories on the histogenesis of salivary glands and their neoplasms are based on the renewing population model, in which both normal differentiated cells and neoplastic cells arise from undifferentiated stem cells in the ducts. However, these results suggest that most of the migration and redifferentiation in the developing rat parotid gland must be in the opposite direction, i.e., the acinar cells redifferentiate into ductal cells. They also indicate that until there are precise data on the rates of cell death among the several cell types, it remains more appropriate for salivary glands to be categorized as an expanding, rather than renewing, population.

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.

We have examined the temporal expression and cellular localization of the genes and proteins for the extracellular matrix (ECM) proteins laminin (B1, B2 and A chain), collagen types alpha 1 (IV) and alpha 1 (I) and the integrin receptor complex alpha 6/beta 1, during parotid gland postnatal development. Laminin B1 and B2 isoforms and collagens alpha 1 (IV) and alpha 1 (I) mRNA steady-state levels were highest at ages 0, 7 and 14 days after birth and declined to the adult (90 days) level at 21 days and older. Laminin A chain transcripts were not detected at any age. Collagen alpha 1 (IV) and laminin were localized in the basal membrane of the developing acinar and ductal cells, while collagen alpha 1 (I) was localized in the stroma surrounding the cells. The amounts of these ECM components were high at the early stages of development and lower at later times. The pattern of expression of the alpha 6/beta 1 integrin genes during development was similar to those of laminin and collagens alpha 1 (IV) and alpha 1 (I). Accumulations of mRNA were high at 0, 7 and 14 days after birth and lower at 21 days and older. High levels of beta 1 integrin were localized in the developing acinar and ductal cell membranes at early ages (7 days); lower amounts were present in the same distribution pattern at later stages of gland development.(ABSTRACT TRUNCATED AT 250 WORDS)

Feeding a casein-based diet with either 400 g/kg of tannin-containing faba bean hulls (Vicia faba L.) (1.41% condensed tannins) or 60 g/kg of a tannin-rich hull extract of faba beans (1.99% condensed tannins) to rats over a period of 7 d resulted in a 2.6 and 1.5 fold increase in weight of the parotid glands, respectively, (P < 0.05) and a corresponding 5.5 and 3.7 fold increase in the level of proline-rich proteins in the glands (P < 0.05). In a dose-response experiment, increasing the level of tannin-rich hull extract in the diet (0.0, 3.8, 7.5, 15.0, 30.0 and 60.0 g/kg) resulted in a linear increase in both the relative size of parotid glands (R2 = 0.90; P < 0.05) and the quantity of proline-rich proteins in the glands (R2 = 0.89; P < 0.05). The apparent digestibility of total (R2 = 0.97) and individual amino acids (R2 varied from 0.27 to 0.99) decreased linearly (P < 0.05). The quantity of proline-rich proteins in the cecum of rats was estimated from the decrease in digestibility of proline, glycine and glutamic acid. The estimated secretions of proline-rich proteins, when calculated on the basis of the three respective amino acids, were 3.5, 3.5 and 3.9 mg of proline-rich proteins per 10 mg of additional hull extract (21.8% condensed tannins). The results indicate that tannins from faba beans stimulate the parotid glands to increase the secretion of proline-rich proteins in rats. The proline-rich proteins then interact with dietary condensed tannins to reduce their antinutritional effects.

The chronic daily administration of isoproterenol provokes in mouse parotid glands the induction and progressive accumulation of a family of secretory polypeptides named polypeptides C, D, E, F, and G (polypeptides C-G). These polypeptides, which seem to be part of the family of proline-rich proteins, have been considered as molecular markers of the growth-in-size response in the mouse parotid acinar cells. In the present study, two pharmacological approaches were used to determine whether the induction and the postsecretory reappearance of polypeptides C-G may be distinguished from each other. First, actinomycin D, a transcriptional inhibitor, was found to interfere with the induction by isoproterenol but not with the postsecretory reappearance. Second, pilocarpine, a secretagogue that was found to be a very weak inducer of polypeptides C-G, was able to provoke secretion and then reappearance of the whole group of isoproterenol-induced polypeptides. Accordingly, these data suggest that the induction of polypeptides C-G is dependent on transcriptional activity and that it is unrelated to secretion stimulation. By contrast, the postsecretory reappearance of polypeptides C-G may occur even when transcriptional activity is inhibited and it would be related to the secretory activity.

The appearance and distribution of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibers was studied by indirect immunofluorescence microscopy in the developing salivary glands of the rat at daily age intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 16th, 30th postnatal (p.n.) days. The findings were compared to samples of adult rats. CGRP-IR fibers appeared on day 20 i.u. both in parotid and submandibular glands. In submandibular glands, fibers were first seen surrounding the developing ductal branches, in the mesenchyme and also in association with developing ducts and blood vessels. In the parotid gland CGRP-IR fibers first appeared around ductal branches and blood vessels on day 20 i.u. and on day 21 i.u. CGRP-IR nerve fibers were found in the mesenchyme and around developing ducts. The density of CGRP-IR fibers was highest between the 2nd and 16th postnatal days, after which the amount of CGRP immunoreactivity slowly decreased to the levels of adult glands. The increase in a number of CGRP-IR nerve fibers especially around the ductal and vascular elements in the developing salivary glands indicates that they may play a role in the functional maturation of salivary glands.

The course of development of salivary peroxidase, an enzyme that has an important role in oral defense mechanisms, has been well documented in rat submandibular glands. However, the only report on salivary peroxidase activity in the other major salivary glands of the rat has been a cytochemical study of the adult parotid gland. In the present investigation, the accumulation of salivary peroxidase activity in developing parotid glands of rats was followed both biochemically and cytochemically. Specific activity (units per mg protein) attributable to salivary peroxidase began at 1 day after birth, then rose rapidly but unevenly, with peaks at 21 and 70 days, and no difference between the sexes at any age. Activity per gland increased progressively to 42 days in both sexes and was significantly higher in males at 70 days. The cytochemical observations on peroxidase activity localized to the rough endoplasmic reticulum and secretory granules of the developing acini were well correlated with the biochemical findings. Peroxidase-negative cells occurred in immature acini at 1 and 7 days, but only in the intercalated ducts thereafter. This observation suggests that the acini are a source of some of the ductal cells, at least during early postnatal development. The developmental pattern of specific activity differed from those of other rat parotid secretory enzymes, indicating that control of their synthesis during development is noncoordinate. The patterns of specific activity of the parotid and submandibular glands were complementary, suggesting that their combined secretions may supply biologically significant peroxidase activity to the oral cavities of rats throughout postnatal development.

In parotid slices and membranes from Wistar rats 2, 12 and 24 months old, changes are noted in adrenoceptor-stimulated K+ fluxes, formation of [3H]inositol phosphates ([3H]IPs), cAMP production, and membrane environment. Norepinephrine-stimulated K+ efflux and formation of [3H]IPs in the slices proceed through an alpha 1-adrenergic mechanism and are reduced 20% and 40% during aging, respectively. In beta-adrenoceptor stimulation with isoproterenol, no age changes were observed in K+ influx and cAMP production. The cholesterol content in membranes was reduced with age; concomitantly, the membrane viscosity decreased with age. These results indicate that the alterations in the membrane environment may provide age-dependent modulation of alpha 1-adrenoceptor coupling mechanisms and their functions.

The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.

We have investigated the temporal expression and cellular localization of the c-jun proto-oncogene and two major rat parotid gland secretory protein genes, PRP (proline-rich protein) and amylase, during postnatal development. c-jun mRNA steady-state levels increased at days 1, 7 and 14 after birth and decreased to basal levels at 21 days and older. PRP mRNA was first detected at 14 days and abruptly increased to adult levels at day 21. Amylase transcripts were first seen at day 7 and progressively increased to adult levels by 28 days. In situ hybridization demonstrated c-jun mRNA accumulation in the differentiating acinar cells and the ducts. The c-jun mRNA accumulation with time corresponds with the proliferative activity reported to occur in these two cellular populations. PRP transcripts were present exclusively in the well differentiated acinar cells while the accumulation of amylase mRNA corresponded to the progressive commitment of parotid cells to acinar differentiation. Our data suggest that during the postnatal development of the rat parotid gland: (a) c-jun expression associates with parotid gland proliferation and precedes the expression of PRP and amylase genes, and (b) activation of PRP and amylase genes is not concomitant and apparently occurs only in differentiating acinar cells.

A dietary change from all liquid to solid food is followed by an average increase of 200% in [3H]thymidine uptake into the parotid gland of rat. However, removal of either the parasympathetic (Px) or the sympathetic (Sx) innervation to the parotid gland prior to the dietary change resulted in a partial inhibition of the increase; values for the parasympathectomized gland were 51% of those of the innervated gland, and values of the sympathectomized parotid gland were 42% of those of the innervated gland. Removal of both autonomic nerves caused a complete inhibition. Initiation of nerve growth factor (NGF) injection (1 microgram/kg body wt, two times daily for the 2 days of chow refeeding) at the time of chow refeeding had no effect on completely or partially denervated glands, and thymidine values for the denervated parotid gland of rats given NGF did not differ statistically from those of rats not given NGF. With parasympathectomy, sympathectomy, and complete denervation, weight of parotid gland was decreased from that of innervated glands, and administration of NGF had no effect on the denervation-induced decreases. The data show that both branches of the innervation to parotid gland must be intact to ensure a maximal increase in thymidine uptake with the dietary change from liquid to solid food. The level of the enzyme, beta 1-4 galactosyltransferase, involved in proliferation, also depended on the presence of intact nerves. Enzyme activity of innervated parotid gland showed an average increase of 200% with chow refeeding of rats previously on liquid diet, but with Px, the average increase was 51% of that of the innervated parotid, and with Sx, it was 41%. NGF administration did not cause any change in levels of this enzyme in any Px or PxSx parotid gland and only a small change in Sx parotid; it did increase levels of this enzyme in parotid of rats without submandibular-sublingual glands.

Present data confirm our earlier report that an increase in [3H]thymidine incorporation into DNA of parotid gland occurs with reintroduction of solid chow to rats previously maintained on liquid diet; if, however, the submandibular-sublingual glands are removed prior to the dietary substitution, the increase is prevented (present data and [19]). In addition, present data show that administration of nerve growth factor (NGF) to partially sialoadenectomized rats during the 2-day period of dietary change from liquid to solid diet restores thymidine values to the high levels observed following the dietary change in intact animals. However, the increase in gland size that accompanied the change in dietary consistency was not prevented by prior submandibular gland ablation, and administration of NGF had no influence on gland size or cell size. The removal of both the sympathetic and parasympathetic nerves to the parotid gland prior to the dietary manipulation also suppressed [3H]thymidine incorporation into parotid, and values did not differ from chow controls. The data show that NGF, a submandibular growth factor, has a prominent role in regulation of the autonomically-mediated hyperplastic response. However, neither the submandibular gland nor NGF has an important role in regulation of the increase in gland size that also accompanies the dietary change.

When solid chow was reintroduced for 2 days to rats previously maintained on an all-liquid diet, [3H]thymidine incorporation into rat parotid gland was 2.4-4.4 times that of chow-fed or liquid-fed animals. When the submandibular-sublingual glands were removed before the change in diet from liquid to solid food, values for [3H]thymidine incorporation of parotid gland were not statistically different from those of chow-fed rats: the increase was completely prevented. The increase in gland size that accompanied the change in dietary consistency was not prevented by prior submandibular gland ablation; [3H]uridine incorporation was also not different from that of chow controls. The removal of both the sympathetic and parasympathetic nerves to the parotid gland before the dietary manipulation also suppressed [3H]thymidine incorporation into parotid, and values did not differ from chow controls. Moreover, the return to normal gland size (chow level) also was prevented by autonomic denervation. The data show an important role of the submandibular gland in regulation of the thymidine increase associated with the enhanced parotid activity that accompanies the change from liquid to solid diet. However, the submandibular gland does not have an important role in regulation of the increase in gland size that also accompanies the dietary change, whereas the autonomic nerves have an important role in regulation of hypertrophy and hyperplasia of parotid gland.

The chronic administration of isoproterenol ((+-)-1-(3,4)-dihydroxyphenyl)-2-isopropylaminoethanol) induces both the accumulation of a family of secretory polypeptides (polypeptides C, D, E, F and G) and growth in size in mouse parotid glands. Eleven isoproterenol analogs including minor structural modifications either at the aromatic ring, at the ethanol-derived residue or at the end group bonded to the amino of the side chain, were analysed regarding their ability to produce those two responses. Analogs were distributed into two groups, namely inducers and noninducers. Inducer isoproterenol analogs provoked a massive accumulation of polypeptides C, D, E, F and G and were active in producing parotid gland enlargement. Noninducer isoproterenol analogs produced neither changes in the polypeptide composition nor growth response in these glands. Thus, a correlation between accumulation of polypeptides C, D, E, F and G and the growth in size response in parotid glands was more firmly established. In considering the chemical structure of the isoproterenol analogs with regard to their inducer or noninducer character, the three main domains taken into account appeared to participate in the inductive process. However, while an intact ethanol-derived domain was found to be absolutely necessary for the inductive ability of the analogs, both the aromatic ring as well as the substituent on the side-chain amino group could experience several modifications without resulting in loss of the inductive character.