Effects of reserpine and isoproterenol on elemental distribution in submandibular gland of rat

The effects of a single dose of reserpine or isoproterenol on the acinar cells of rat submandibular gland were investigated by electron microscopy and X-ray microanalysis. About 4 h after injection of reserpine, the cellular Ca concentration started to increase and at 24 h a level two to three times the control value was reached. The mucus content of the cells increased in parallel with the Ca concentrations. Isoproterenol stimulation depleted the acinar cells of their mucus. Resynthesis of mucus was visible at 8-12 h after injection. Changes in cellular Ca concentrations followed the mucus content of the cells. After 24 h both the mucus content and the Ca concentration were significantly increased over the control values. High-resolution X-ray microanalysis on thin cryosections showed that the calcium was accumulated in the mucus granules, both after reserpine and after isoproterenol administration. The newly synthesized mucus was also more calcium-rich than the original mucus. The effect of a single isoproterenol stimulation on elemental content of rat submandibular gland acinar cells was not significantly different from that of chronic isoproterenol treatment. Chronic treatment with reserpine resulted in a higher cellular Na/K ratio than a single dose.     

Effects of prior culture or isoproterenol injections on the regeneration of rat submandibular gland autografts

This study compares the acinar cell regnerative response in submandibular gland (SMG) autografts that were cultured before grafting to the rat tongue with the acinar cell regenerative response in direct SMG autografts to the tongue. In addition, the effects of isoproterenol on direct SMG autografts were studied. A portion of the left SMG was excised from each rat and cut into fragments which were autografted either immediately into the middle one-third of the rat's tongue; or were cultured for 1, 4, or 7 days and then autografted to the donor's tongue. After 8 weeks the rats were killed and the tongues were removed and processed for light microscopic study. The histologic preparations showed evidence of cytodifferentiation into acinar cells in four of the previously cultured SMG autografts. Some of the direct SMG autografts did not contain acinar cells, whereas other direct. SMG autografts contained numerous acinar cells and even straited ducts. In the SMG autografts that were cultured for 1 day before autografting and in the direct SMG autografts, that contained ductlike structures that were apparently connected to the epithelial surface of the tongue. Lastly, isoproterenol appeared to accelerate the regenerative responses in some of the direct SMG autografts, and the drug caused acinar cell hypertrophy in two of the direct SMG autografts.     

The effect of 5-bromodeoxyuridine on the postnatal development of the rat cerebellum: Morphologic and radioautographic studies

Effects of a thymidine analog, 5-bromodeoxyridine (BrdU), on the development of rat cerebella were studied. BrdU, when 15 milligram per 100 gram body weight was injected twice daily into 2-day-old rats for three consecutive days, caused a transient inhibition of cell formation and destruction of stem cells in the external granular layer. This was followed by a prolonged period of regeneration with delayed dissolution of the external granular layer; however, the restitution in cell number was incomplete and resulted in a miniature cerebellum with significantly reduced myelination at 35 days of age. The regeneration of the external granular layer appeared to alter the synchronized morphogenetic processes of the cerebellar cortex. The “desynchronization” of morphogenetic events appears to account for the peculiar cytoarchitectonic organization of the cerebellar cortex in the BrdU-injected animals, i.e., irregular stacking of Purkinje cells, formation of granule cell ectopia, and sagittal alignment of parallel fibers in the molecular layer. It is postulated that the misalignment of parallel fibers is the consequence of BrdU substitution in place of thymine in the nuclear DNA of granule cell precursors. The present schedule of BrdU injections also appeared to reveal an additional, early period of oligodendroglial proliferative activity which did not coincide with the onset of myelination.     

Induction of the synthesis of a specific protein in rat submandibular gland by isoproterenol

Prolonged treatment of rats with the beta-adrenergic agonist, isoproterenol (IPR), produces hypertrophy and hyperplasia of the parotid and submandibular glands. The drug induces the synthesis of several secretory proteins that are absent or occur at very low concentrations in the gland or saliva of the untreated rat. We have measured the relative concentrations of one of these proteins (termed "large mobile" (LM) protein, Menaker et al. (1974) Lab. Invest. 30, 341-349) by using a solid phase enzyme-linked immunoabsorption assay. LM protein was not measurable in gland extracts of 20-day-old fetuses or 2-day-old rats. Its concentration was very low in the glands of 6- and 13-day-old and adult rats. Administration of IPR for 4 to 7 days to adult or 6-day old rats increased the levels of the LM protein by 20 to 25-fold. The LM protein was localized immunocytochemically primarily in the acinar cells in the glands of control and IPR-treated adult rats. In vitro translation studies using a mRNA-dependent reticulocyte lysate system and labeling with [35S]methionine showed: little synthesis of pro-LM from poly(A+) RNA from glands of adult rats and none from 13-day-old animals, and that, in comparison, poly(A+) RNA from glands of adult or 13-day-old IPR-treated rats directed the synthesis of a much greater concentration of pro-LM. The in vitro precursor of the LM protein migrated electrophoretically as a single band in anti-LM immunoprecipitates, and had a molecular weight of 14,000. The LM protein, which appears to be a single, unique polypeptide induced by IPR in the submandibular glands of developing and adult rats, will be useful in studies examining the effects of catecholamine beta-agonists on gene expression in an exocrine cell.     

Immunochemical analysis of laminin during postnatal development of the rat submandibular gland.

Anti-laminin serum was used to investigate distribution patterns and chain composition of laminin during postnatal development of rat submandibular gland. Immunohistochemical analysis showed that in glands of newborn rats laminin was not uniformly present around growing acini. Staining was frequently weak or absent in clefts formed between adjacent cells. This irregular staining pattern decreased progressively over the subsequent periods, and in 30-day-old animals immunoreactivity was observed only at the periphery of glands. Immunoblot analysis showed that laminin was composed of bands corresponding to the alpha 1, beta 1 and gamma 1 polypeptides. The correlation between the pattern of laminin expression and gland maturation suggests a role of laminin in the functional maturation of acinar cells.     

Selective effects of testosterone and isoproterenol upon regenerating submandibular gland isografts in BALB/c mice

Single submandibular glands were isografted intraperitoneally to normal BALB/c mice. From the day following transplantation, 20 mg testosterone enanthate fortnightly, or daily intraperitoneal injections of 0.25 mg per gram body weight of isoproterenol-HCl were given. Control mice were untreated. The grafts were removed from five mice in each group 1, 2, 4, or 8 weeks after grafting. Regardless of treatments, only ductal cells were observed one week after transplantation. With testosterone treatment for longer than two weeks, their remaining ductal cells tended to accumulate secretory granules. Some appeared to be secretory tubules resembling those in the glands of normal adult male mice. When the glands were transplanted from females to male hosts, surviving grafts responded to endogenous androgen of the hosts, and some secretory tubules reappeared without testosterone treatment. Contrarily, with isoproterenol treatment for longer than two weeks, acinar cells reappeared in the grafts, but no secretory tubules were observed. The remaining ductal cells often underwent hyperplastic changes with reappearing tubules or acinar cells amongst them corresponding to post-transplantational treatment. Mechanisms of differentiation of these three different components of submandibular glands were found to be different and each had specific affinity to testosterone or isoproterenol. Grafts were removed from hosts treated with testosterone for two months, and when 4 or 8 of these grafts were retransplanted into each new host, they demonstrated a lethal effect.     

Effect of glucocorticoid on the differentiation and development of terminal tubules in the fetal rat submandibular gland

Glucocorticoids (CORT) are known to promote branching of the epithelial cords during the development of the rat submandibular gland. The aim of this study was to examine the effect of CORT (triamcinolone) on the differentiation of cells forming the terminal tubules in the developing fetal rat submandibular gland and the properties of the secretory granules. Light and electron microscopy showed that the terminal tubules of the glands in the experimental group contained more type III cells, which have been identified as proacinar cells, than those in the control group, whereas the relative number of type I cells, which have been identified as terminal tubule cells, was reduced. Immunoelectron microscopy using an antibody against neonatal submandibular gland secretory protein B (SMGB) revealed the presence of more gold particles over type III cell granules in the experimental group than in the control group. Lectin histochemistry demonstrated more wheat germ agglutinin (WGA)-labeled gold particles over type III cell granules in the experimental group than in the control group. These findings suggest that CORT promote the differentiation of type III cells, and moreover stimulate the production of secretory granules reactive for SMGB and WGA by acting on the terminal tubules of the developing rat submandibular gland.     

Cell differentiation in the terminal tubule of fetal rat submandibular gland in organ culture

Submandibular glands from 17-day-old rat fetuses were maintained in organ culture for five days in a medium consisting of Eagle's MEM (87%), horse serum (10%), and chick embryo extract (3%). Each day of the culture period explants were incubated for the demonstration of peroxidase activity and processed for light and electron microscopic observations. In some experiments cultures were exposed to ³H-thymidine one hour prior to fixation and incubation for the demonstration of peroxidase activity. Labelling index was determined using radioautographs of 1 μ Epon-embedded sections. At the time of explantation the submandibular gland rudiment consisted of undifferentiated epithelial cells arranged in cords. On day 3 of culture two additional cell types could be distinguished: terminal tubule cells and proacinar cells. The proacinar cells were characterized by peroxidase activity in their granules and cytoplasm. By day 4 acinar cells begin to appear. On the fifth day of culture the four cell types of the terminal tubule were present in the following proportions: undifferentiated cells, 44%; terminal tubule cells, 19%; proacinar cells, 31%; acinar cells, 6%. These results indicate that the cytodifferentiation of the secretory unit of rat submandibular gland in vitro is comparable to the differentiation in vivo.     

Effects of irradiation on the submandibular gland of the rat

Summary Single-dose cervical irradiation by cobalt 60 in rats induced lasting functional disturbances of the submandibular gland which were excessive when compaired with the relative integrity of the gland as seen under the light microscope. Enzyme histochemical and ultrastructural studies revealed severe damage shortly after exposure with appearance of karyolytic bodies and autophagosomes accompanied by increased hydrolase activity. Mitochondrial alterations were concomitant with diminished ductal oxidative enzyme activity. Although most of these alterations resolved rapidly as a result of acinar and ductal cell repair and regeneration originating in the intercalated ducts, secretory abnormalities were still observed two months after exposure as evidenced by the accumulation of granules in acinar cells and the heterogeneity of ductal cell granules. These anomalies, comparable to those observed in sialadenoses, probably result from persistent alterations of intralobular nerve endings.The authors wish to thank M.F. Baucher, A. Lesot and M. Tacnet for their technical assistance     

Development of dipeptidyl peptidase IV (DPP IV) activity in postnatal differentiation of the submandibular gland in mice

The localization of a serine exopeptidase--DPP IV--in the male and female submandibular gland of the mouse during gland postnatal development was studied histochemically in the light microscopic level. The present results suggest that localization of DPP IV is closely related to the postnatal differentiation and maturation of acini and male granular convoluted tubules. Remarkable sex differences of DPP IV activity were detected in the submandibular gland of pubescent and adult animals. The possible physiological role of DPP IV in major salivary glands is also discussed in the paper.