The chronically isoproterenol-treated rat in the study of cystic fibrosis: X-ray microanalysis of the submandibular gland

The chronically isoproterenol-treated rat has been proposed as an animal model for cystic fibrosis. Ultrastructural studies showed enlarged cells with abnormally large mucus granules that were more often fused than in control animals. X-ray microanalysis of mucous acinar cells showed a significant increase in calcium levels, but unaffected magnesium levels. Combined treatment with isoproterenol and reserpine caused a very large increase in cellular calcium levels that appeared to be an addition of the single effects and increased magnesium levels (as in glands of rats treated with reserpine only). Chronic treatment with isoproterenol, reserpine, or both substances tended to decrease cellular potassium levels. Chronic exposure to the specific beta 1-agonist prenalterol or the specific beta 2-agonist terbutaline did not affect cellular calcium or potassium levels. It is concluded that chronic isoproterenol treatment affects the elemental composition of mucous acinar cells of rat submandibular gland differently from chronic reserpine treatment. The increase in cellular calcium concentration after chronic isoproterenol treatment does not appear to be due to an effect via beta-adrenergic receptors.     

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 chronic furosemide treatment on rat exocrine glands

It has been suggested that a defective chloride transport is the primary cellular basis for the disease cystic fibrosis (CF). Therefore, the effects of chronic furosemide treatment on the structure and function of rat exocrine glands were investigated. X-ray microanalysis of the submandibular gland showed an increase in the cellular Ca and Mg concentrations, and a decrease in the cellular Cl concentration. Transmission electron microscopy showed intracellular accumulation of mucus and the presence of mucus in acinar and ductal lumina. The volume of saliva secreted by the submandibular gland after pilocarpine stimulation was markedly reduced in furosemide-treated animals; the salivary concentrations of Na and Ca were higher, and that of K was lower, than in control animals. The protein concentration in submandibular saliva was not significantly affected. The response of the submandibular gland to isoproterenol stimulation was reduced in furosemide-treated animals. In the parotid gland, chronic furosemide treatment caused an accumulation of immature zymogen granules in the acinar cells and a decrease in the cellular Cl concentration. In the pancreas, the acinar lumen was dilated and completely filled with secretory material, and the acinar cells contained less Na and somewhat less Cl than in control animals. The chronically furosemide-treated rat shows a number of parallels with other animal models for CF, in particular the chronically reserpinized rat. There is also agreement with the human disease itself.     

Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction

Acinar cell regeneration in the rat parotid gland after atrophy induced by a one week period of duct obstruction was examined using histology, immunohistochemistry and transmission electron microscopy (TEM). For immunohistochemistry, antibodies to 5-bromo-2'-deoxyuridine (BrdU), injected one hour before tissue collection, and cytokeratin were employed. When clips were removed from the duct, only ductal epithelial cells remained; all acinar cells had been deleted. Some duct cells were BrdU positive. After three days, newly-formed acini comprising immature acinar cells had appeared; many of the cells were BrdU positive and mitotic figures were readily identified. Thereafter progressive acinar cell maturation and proliferation occurred, parotid gland weight returning to control levels by 7 days. Peak BrdU labelling indices for duct and acinar cells were on days 0 and 4, respectively. By TEM, cytoplasmic organelles in epithelial cells of transitional duct-acinar structures seen at 2 days were poorly developed. Immature acinar cells seen on day 3 contained zymogen granules and had increased endoplasmic reticulum and mitochondria. By day 5, maturing acinar cells had abundant endoplasmic reticulum and zymogen granules, resembling acinar cells in control glands. These observations indicated origin of acinar cell precursors from duct cells during regeneration of the acinar cell-free atrophic gland. Subsequent expansion of the acinar cell population was dependent on maturation and proliferation of these newly-formed cells.     

The chronically pilocarpine-treated rat in the study of cystic fibrosis: investigations on submandibular gland and pancreas

The chronically pilocarpine-treated rat has been proposed as an animal model for the disease cystic fibrosis, a generalized exocrinopathy. The effect of chronic pilocarpine treatment on structure, composition, and function of the acinar cells of rat submandibular gland and pancreas was investigated by electron microscopy, X-ray microanalysis, and biochemical analysis. The morphological effects of chronic pilocarpine treatment were most pronounced in the pancreas. The number and size of the zymogen granules was increased, and the granules had a less electron-dense appearance. X-ray microanalysis at the cellular level showed in both the submandibular gland and the pancreas a significant increase in calcium and a decrease in sodium. The increase in cellular calcium concentrations can be explained by an increase in the relative volume of secretory material in the cell (assessed by morphometry) and an increase in the local calcium concentration in the secretory material (assessed by X-ray microanalysis at the subcellular level). Chronic pilocarpine treatment caused a disturbance of glycolysis and energy metabolism in the submandibular gland, but no significant effects in this respect were noted in the pancreas. On the other hand, a nearly twofold increase of the pancreatic amylase activity was noted. The pancreas appeared somewhat hyperreactive towards cholinergic stimulation.     

Fine structure of the differentiating acini in submandibular glands of isoproterenoltreted rats

Chronic treatment with isoproterenol, a β-adrenergic drug, accelerates the postnatal differentiation of the rat submandibular gland. This report compares the ultrastructure of submandibular glands of acute and chronically treated, 2, 11 and 17-day-old rats with that of controls. In all cases a greater number of acinar cells were found in the treated glands. The correlation noted between changes in the nature of the contents of the condensing vaculoes and Golgi apparatus of some proacinar cells and the formation of secretory granules intermediate in appearance between those of proacinar and acinar cells, support the hypothesis that proacinar cells are the precursors of acinar cells in the young rat. Differences in the morphology of secretory granules in acinar cells of treated and control animals are described. It is concluded that precocious differentiation of the submandibular gland may be induced by the administration of isoproterenol as early as the first day of postnatal life, that proacinar cells become recognized as acinar cells following a change in the nature of their secretory product, and that the secretory material produced by acinar cells in the treated animals is not identical to that of the controls.     

X-ray microanalysis of resting and stimulated rat pancreas

The elemental distribution in acinar cells of rat pancreas was investigated by X-ray microanalysis of thin, freeze-dried cryosections. In the resting cell, the highest calcium concentrations were found in the basal part of the cell (including the endoplasmic reticulum) and in the zymogen granules. Mitochondrial calcium concentrations were low. Zymogen granules were rich in sulphur, but low in phosphorus, sodium and potassium. Stimulation of the pancreas by perfusion in vivo with the cholinergic agonist carbachol caused a significant decrease of the calcium concentration in the basal part of the cell and an increase in the calcium concentration in the apical part of the cell. The mitochondrial calcium concentration was not significantly altered. In addition, increased sodium and decreased potassium concentrations, giving rise to a significant increase in Na/K ratio were observed in all cell compartments measured, except in the zymogen granules.     

Intercalated duct cells in the rat parotid gland may behave as tissue stem cells

This study was conducted to determine whether intercalated duct cells in the rat parotid gland have the properties of tissue stem cells. After induction of cellular proliferation by repeated administration of isoproterenol (IPR), a β-adrenergic agonist, proliferation activity in acinar, intralobular, and intercalated ductal cells was quantified using Ki-67 immunohistochemistry. The total number of each type of component cell in a gland was also estimated in the course of IPR treatment. IPR was found to induce proliferation of acinar and intercalated duct cells, but not intralobular duct cells. The total number of acinar cells in a gland on day 5 of IPR treatment was 1.6 times of that at day 0 (baseline). In contrast, the total numbers of intercalated and intralobular duct cells did not change from baseline, indicating a high possibility that the proliferated intercalated duct cells differentiated into acinar cells. On days 2 to 3 of IPR treatment, intercalated duct cells with amylase-positive secretory granules were recognized in a region very close to the acini, and were suspected of being transitional cells from intercalated duct to acinar cells. This quantitative study indicates that intercalated duct cells may have the properties of tissue stem cells upon IPR stimulation.     

The effects of reserpine on the ultrastructure and secretory responses of rat exocrine pancreas.

The effects of chronic reserpine administration on rats mimic in several respects the exocrine dysfunction observed in cystic fibrosis. This drug treatment has been proposed as a model for the human disease. In cystic fibrosis, the pancreas is usually a major organ involved pathologically. The present study was designed to investigate the fine-structural morphology and secretory responses of the pancreas of rats treated chronically with reserpine. Reserpine treatment resulted in increased storage of zymogen granules in pancreatic acinar cells and an apparently reduced ability to discharge these granules following stimulation with cholecystokinin. In addition, granule storage may have produced feedback inhibition on the protein synthesizing machinery as manifested by a slight reduction of rough endoplasmic reticulum. Many acinar cells also had autophagic bodies, suggesting the degradation of excess secretory material. Cholecystokinin stimulation of both control and reserpine-treated rats resulted in the appearance of large vacuoles containing myelin figures and granular material, numerous autophagic bodies (cytosegresomes), and a slight decrease of rough endoplasmic reticulum in pancreatic acinar cells. Secretin stimulation did not cause large vacuole formation, but did cause cytosegresome formation in acinar cells. Large vacuoles in intralobular ducts were noted after reserpine treatment, but were also present after stimulation of untreated pancreas with cholecystokinin.     

Effects of chronic and in utero treatment with diuretics on mouse exocrine cells studied by X-ray microanalysis.

In an attempt to develop an animal model for the disease cystic fibrosis, mice were chronically treated with diuretics. In addition, pregnant mice were treated with diuretics and the effect of this treatment in utero on the newborn mice was studied. Pancreas and submandibular gland acinar cells were investigated by X-ray microanalysis and transmission electron microscopy. Long-term treatment with furosemide (up to 13 months) caused transient changes in the elemental content of the pancreatic acinar cells: a decrease in chloride and sulfur, and an increase in phosphorus, potassium and magnesium. All changes normalized with prolonged treatment. Some morphological changes were found in the zymogen granules. Treatment with amiloride or furosemide in utero caused a decrease in cellular sodium and chloride levels, indicative of inhibition of transepithelial ion and fluid transport. Also treatment of adult animals for two months with amiloride caused lower intracellular sodium and chloride levels. In adult animals only minor effects of diuretic treatment on submandibular gland acinar cells were noted. In utero treatment with amiloride caused an increase in sodium and chloride content indicative of cell damage.     

Effects of continuous light on rat parotid gland structure and reactivity

Summary The effects of continuous light on ultrastructural organization and sympathetic secretory responses of the rat parotid gland are reported.After 50 days of continuous light exposure, the fine structure of the parotid gland exhibited features of enhanced secretory activity as judged by the striking development of rough endoplasmic reticulum and Golgi complexes, the depletion of secretory granules and the increased turnover of secretory cells. The secretory responses of parotid gland to isoproterenol revealed that continuous light induced a 30% increase in amylase release. This secretory hyperactivity appears to be related to a postsynaptic supersensitivity of sympathetic fibers of the autonomic nervous system.     

The Reserpinized Rat in the Study of Cystic Fibrosis: X-ray Microanalysis of Submandibular Gland and Pancreas

The chronically reserpinized rat has been suggested as an animal model for cystic fibrosis. X-ray microanalysis of thick and thin cryosections was carried out to assess elemental redistribution in the submandibular glands and the pancreas of reserpinized rats at the cellular and subcellular level. In the submandibular gland of reserpinized rats, calcium and magnesium concentrations were significantly elevated. Mucus globules, secretory granules, and endoplasmic reticulum were the primary sites of the localization of excess calcium and magnesium. A significant potassium loss from the gland had occurred, particularly from the serous cells. Electron microscopy of conventionally prepared tissue showed marked swelling of the endoplasmic reticulum, especially in mucous cells.

The elemental changes in the pancreatic acinar cells of reserpinized rats were reminiscent of elemental redistribution connected with cell death: increased levels of sodium, chlorine, and calcium and decreased levels of magnesium and potassium. Ultrastructural changes included swelling of the endoplasmic reticulum and obstruction of the acinar lumen. It is concluded that elemental redistribution in chronically reserpinized rats presents interesting parallels with cystic fibrosis.     

Developmental changes of sugar residues and secretory protein in mucous cells of the early postnatal rat parotid gland.

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 Reserpinized Rat in the Study of Cystic Fibrosis: X-ray Microanalysis of Submandibular Gland and Pancreas

The chronically reserpinized rat has been suggested as an animal model for cystic fibrosis. X-ray microanalysis of thick and thin cryosections was carried out to assess elemental redistribution in the submandibular glands and the pancreas of reserpinized rats at the cellular and subcellular level. In the submandibular gland of reserpinized rats, calcium and magnesium concentrations were significantly elevated. Mucus globules, secretory granules, and endoplasmic reticulum were the primary sites of the localization of excess calcium and magnesium. A significant potassium loss from the gland had occurred, particularly from the serous cells. Electron microscopy of conventionally prepared tissue showed marked swelling of the endoplasmic reticulum, especially in mucous cells.

The elemental changes in the pancreatic acinar cells of reserpinized rats were reminiscent of elemental redistribution connected with cell death: increased levels of sodium, chlorine, and calcium and decreased levels of magnesium and potassium. Ultrastructural changes included swelling of the endoplasmic reticulum and obstruction of the acinar lumen. It is concluded that elemental redistribution in chronically reserpinized rats presents interesting parallels with cystic fibrosis.     

Dispersed rat parotid acinar cells. I. Morphological and functional characterization.

Morphologically and functionally intact acinar cells have been obtained from the rat parotid gland through enzymatic dispersion with pure collagenase, hyaluronidase, and trypsin as well as mild mechanical forces. Cell yields of 30-50% of the original tissue weight with over 95% acinar cells were accomplished. The cells in suspension assumed a more or less spherical shape but the intracellular polarity of organelle distribution was maintained. The cells in suspension at 37 degrees C maintained stable monovalent cationic composition but lost potassium and gained sodium rapidly upon exposure to ouabain, 10(-5) M. The intracellular amylase concentration and the patterns of secretion of amylase and of synthesis of cyclic AMP by the cells in response to adrenergic stimulation with epinephrine or isoproterenol were comparable to those of the intact gland in situ. In addition, the cells showed good O2 consumption and maintained it constant for periods up to 8 h. These cells could be used as experimental tools for in vitro studies of receptor physiology and biochemistry, cell membrane function, cellular secretory mechanisms, and other parameters of exocrine gland cell physiology.     

The effects of hypophysectomy on the digestive glands of the mouse

The objective was to observe the influence of hypophysectomy on the secretory cells of digestive glands in the mouse and to compare these findings with those reported for other mammalian species. Hypophysectomy of the mouse induced involutional changes in glandular cells concerned with secretion of proteins and mucins, and in those involved in ion transport or release. Gastric chief cells, pancreatic acinar cells, and serous cells of submandibular tubules were markedly involuted; acinar cells of the parotid gland were changed less significantly. Gastric mucous neck cells and mucous acinar cells of the submandibular and sublingual glands contained less secretory material. A colloidal iron-positive variety of mucous neck cell became dominant. These changes were less profound than those which occurred in most types of zymogenic cells. Parietal cells became atrophic and contained a greatly contracted intracellular canalicular system. Salivary ducts of the parotid and sublingual glands showed partial involution. It was concluded that, as in the rat and man, protein-secreting cells of the digestive tract of the mouse depend on the support of pituitary hormones. Those cells which secrete mucopolysaccharides or are involved in ion transport also require pituitary hormones for complete maintenance of their structure and function.     

Histochemical and biochemical determination of calcium in salivary glands with particular reference to chronic submandibular sialadenitis

Although salivary calcification is relatively common, little is known about the localization and content of the calcium of normal and diseased human salivary glands. We investigated this in chronic submandibular sialadenitis with a variable mixture of relatively normal and extremely atrophic parenchyma and in normal submandibular, parotid and palatal glands. Calcium was localized histochemically in mucous acinar cells of submandibular and palatal glands at moderate to high levels, in serous acinar cells of submandibular and parotid glands at low to moderate or occasionally high levels, in mucous ductal cells at moderate to high levels, and in extremely atrophic parenchyma at low levels or not at all. Calcium was determined biochemically at relatively high levels in the different glands in the order palatal, submandibular, sialadenitis and parotid. However, the differences were small. The results indicate that most salivary calcium is associated with secretory granules; this is the likely source of the calcium involved in salivary calcification     

Effect of chronic treatment with cystic fibrosis fibroblast medium on rat submandibular gland acinar cells

The effect of chronic treatment with cystic fibrosis (CF) fibroblast medium on rat submandibular gland and pancreas was investigated. Rats were injected for 8 days with conditioned medium from normal or CF fibroblasts. The elemental content of the acinar cells was measured by X-ray microanalysis of cryosections. A significant increase in cellular calcium, and a decrease in cellular sodium concentrations were found after treatment with CF medium. The ultrastructure of the submandibular acinar cells was not affected by the conditioned CF fibroblast culture medium. No effect of treatment with CF medium on ultrastructure and elemental content of pancreatic acinar cells could be demonstrated. The response to alpha-adrenergic, beta-adrenergic, cholinergic, and peptidergic stimulation in submandibular gland acinar cells of rats injected with normal or CF medium was investigated in vitro. With regard to changes in elemental composition after stimulation, no significant differences in response between the two groups could be found. Apparently, a factor in conditioned medium from cultured CF fibroblasts induces a net increase in calcium content of rat submandibular gland acinar cells. Possibly, this factor acts in a similar way in CF patients and may cause elevated calcium levels in CF cells.     

Filamentous Inclusions in Acinar Cell Carcinoma of the Pancreas

Acinar cell carcinoma of the pancreas exhibits a spectrum of histologic appearances. Some tumors can be readily identified by light microscopy, but others resemble endocrine/neuroen-docrine neoplasms. Ultrastructurally, though large zymogen granules of acinar cells are usually distinctive, the zymogen granules of neoplastic acinar cells are sometimes abnormally small, overlapping in size with the granules of endocrine/neu-roendocrine neoplasms. Six cases of acinar cell carcinoma, two with a typical histologic appearance and four that resembled endocrine/neuroendocrine tumors, were studied ultrastructurally. In addition to zymogen granules and abundant rough endoplasmic reticulum, all cases of acinar cell carcinoma exhibited pleomorphic, membrane bound inclusions that contained filaments. Similar inclusions were not identified in islet cell or carcinoid tumors, and several findings indicate that the inclusions represent deranged zymogen granules. In the ultrastructural study of a pancreatic neoplasm with granules, these inclusions may provide a clue for the diagnosis of acinar cell carcinoma.     

Ultrastructural responses of rat exocrine pancreas to cholecystokinin octapeptide and secretin

The study examines the ultrastructural changes in the rat pancreas stimulated in vivo to secrete zymogen and fluid by the hormones cholecystokinin and secretin, administered either separately or in combination. The octapeptide of cholecystokinin (CCK-OP) (2.5 X 10(-7) g/kg) 5 min after injection produced discharge of electron-dense zymogen into the acinar lumen and intercellular canaliculi (ICC), leaving misshapen, collapsed zymogen granule profiles around the lumen. Five minutes after secretin (7.5 clinical units/kg), acinar cells were distended, rough endoplasmic reticulum was dilated, acinar lumina and ICC were expanded and filled by electron-lucent and flocculent contents, and there were "halo" zymogen granules and pale "vacuoles." Electron-lucent zones surrounding acinar and duct cell microvilli indicated transcellular fluid secretion. When secretin was administered with CCK-OP, the picture was a composite between zymogen and fluid secretory patterns. Zymogen granules took up fluid producing a halo appearance, pale vacuoles formed in acinar cells, and acinar lumina and discharging zymogen granules were of intermediate electron density. The results demonstrated that, although fluid is secreted by duct cells in response to secretin, a major site of secretin-stimulated fluid secretion is acinar cells. Fluid is transported across both cell types by transcellular routes, and the acinar cell fluid secretion is integrated with zymogen discharge. CCK-OP produces partial discharge of undiluted zymogen by exocytosis.