Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics.

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.

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.

Growth of rat parotid glands is inhibited by liquid diet feeding.

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.

Occurrence of parotoid glands in tadpoles of the tropical frog, Clinotarsus curtipes and their role in predator deterrence.

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.

Structural changes of goat parotid salivary gland: pre- and post-weaning periods.

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.

[Criteria of diagnostics of pancreatitis at children's age according to ultrasound research].

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.

[Antenatal factors of caries susceptibility formation in children].

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.

Cellular signals underlying ß-adrenergic receptor mediated salivary gland enlargement.

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 ß-adrenergic receptors (ARs) of the ß2, but not ß1, subtype increased over time in parallel with increases in the proliferation marker PCNA and parotid gland weight. Levels of ß2-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 ß2-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 ß2-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 ß-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 ß2-ARs, possibly mediated by CREB and NF-κB, resulting in salivary gland enlargement. We propose that during isoproterenol treatment activation of the ß1-AR, the predominant ß-AR subtype in unstimulated salivary glands, initiates proliferative signaling cascades, and that upregulation of the ß2-AR plays an essential role in later stages of salivary gland growth.

Organogenesis of the juxta-oral organ in mice.

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.

Exogenous thyroid hormone affects myoepithelium and proliferation in the developing rat parotid gland.

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.

Immunocytochemical analysis of cyclic AMP receptor proteins in the developing rat parotid gland.

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.

Effects of exogenous thyroid hormone on the postnatal morphogenesis of the rat parotid gland.

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).

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development.

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.

Morphometric study of the postnatal growth of the parotid gland of the mouse.

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.

Morphometric study of the postnatal growth of the parotid gland of the mouse

O crescimento das glândulas parótidas do camundongo durante 7 e 35 dias de vida pós-natal foi estudado por métodos morfométricos. A massa glandular, o volume de cada compartimento morfológico e o número de células em cada compartimento foram avaliados. Os dados obtidos para cada dimensão avaliada foram ajustados por equação exponencial, tipo Y = a.ek.x, permitindo o cálculo do seu tempo de duplicação médio (TD), ou seja, uma estimativa da sua velocidade de crescimento. A análise dos resultados mostrou a ocorrência de um marcante aumento de massa glandular no período estudado de 1.424%, com TD = 7,10 dias. Esse crescimento glandular ocorreu por aumentos nos volumes absolutos dos ácinos, dos ductos intercalares, dos ductos estriados, dos ductos excretores e do estroma, com aumentos percentuais de, respectivamente, 3.048%, 417%, 2.662%, 2.594% e 367%, e TDs de 5,62, 11,71, 5,55, 5,47 e 14,45 dias. A análise da evolução do número de células em cada compartimento demonstrou aumentos de, respectivamente, 1.904%, 285%, 1.228%, 1.090% e 286% e TDs de 6,62, 20,40, 7,19, 7,26 e 14,51 dias. Baseados nos resultados aqui obtidos, concluímos que o crescimento das glândulas parótidas do camundongo entre os dias 7 e 35 de idade ocorre por intenso acúmulo de células, principalmente nos ácinos e nos ductos estriados e excretores, com uma velocidade de crescimento sensivelmente maior que nos ductos intercalares e no estroma.

[The effect of humoral factors of hypothalamic nonapeptidergic centers on histo- and organotypical potentialites of digestive glands of various developmental origin under conditions of in vivo culture by F.M. Lazarenko's method].

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.

Glycogen content and activities of enzymes involved in the carbohydrate metabolism of the salivary glands of rats during postnatal development.

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.

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.

An enzyme histochemical and biochemical study of the activity of three oxidative enzymes in the developing rat parotid gland.

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.

Effects of nutritional conditions around weaning on carbonic anhydrase activity in the parotid gland and ruminal and abomasal epithelia of Holstein calves.

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.