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.     

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.     

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.     

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.     

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.     

X-ray microanalysis of the rat parotid gland after chronic sympathectomimetic stimulation

The effects of chronic treatment with isoproterenol, reserpine, prenalterol, and terbutaline on rat parotid gland were investigated by electron microscopy and X-ray microanalysis. Chronic isoproterenol treatment induced lower potassium and calcium concentrations in the acinar cells. The cells were enlarged and contained more and larger zymogen granules than acinar cells in the controls. The zymogen granules contained markedly less sulfur, potassium, and calcium than in control animals. Prenalterol had effects similar to those of isoproterenol, but less pronounced, whereas terbutaline had no significant effects. Chronic treatment with reserpine caused a decrease in calcium levels but did not affect potassium levels. The changes in elemental composition in parotid acinar cells after chronic treatment with isoproterenol and reserpine differed from those induced by the same treatment in the submandibular gland of the rat.     

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.     

Effects of calcitonin on elemental distribution and ultrastructure of rat submandibular gland acinar cells

The effect of calcitonin on rat submandibular gland acinar cells was investigated by X-ray microanalysis and electron microscopy. Calcitonin caused a transient increase of the cellular calcium and magnesium concentration, but did not affect the intracellular concentration of other electrolytes. The relative volume of intracellular mucus increased from 45% in control glands to 72% at 6 h after administration of calcitonin. Calcitonin caused an inhibition of the cellular response of the acinar cells to beta-adrenergic and cholinergic agonists. The changes in elemental composition and ultrastructure of the gland cells are probably due to inhibition of mucus release from the cells.     

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.     

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.     

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.     

Distribution of elements in the pancreatic exocrine cells determined by electron probe X-ray microanalysis

We measured the intracellular electrolytes of acinar cells by making electron probe X-ray microanalysis of hydrated and dehydrated sections of freshly frozen dog pancreas.The concentrations of electrolytes in the cytoplasm were: Na 4.8±2.1, K 132±15, Cl 14±4.7, P 165±36, S 19±2.8 and in zymogen granules: Na 6±5, K 60±16, Cl 31±20, P 36±8, S 172±25, Ca 7±5 (mean±S.D. mmol/kg wet weight).The cytoplasm, which is rich in rough endoplasmic reticulum, had low Na and high K concentrations, as compared with levels in the acinar cells of other exocrine glands such as the submandibular gland, the bulk of which is occupied by secretory granules. Though the representative feature of secretory granules was a high S content, occasionally low S peaks of spectra from secretory granules were obtained. These findings may reflect the content of mature zymogen granules and immature condensing vacuoles.Pilocarpine stimulation increased cytoplasmic Na, Cl and Ca and decreased K levels in the pancreatic acinar cells. This indicates that secretory stimulation increases the permeability of the cell membrane to Na, Cl and K ions and that there is a simultaneous Ca release from the intracellular Ca stores such as zymogen granules and endoplasmic reticulum, and/or Ca influx from the extracellular space.     

Microliths in the parotid of ferret investigated by electron microscopy and microanalysis

The present investigation is an attempt to determine the occurrence, elemental composition and formation of microliths in the parotid of ferret. Parotids from four normal ferrets were examined by electron microscopy and X-ray microanalysis. Crystalline microliths were found in phagosomes of acinar cells, which occasionally contained secretory material, and in phagosomes situated between mitochondria of striated ductal cells. Crystalline microliths and microliths that consisted of granular material either without crystals or mixed with a component of crystals were found in lumina, where they were often associated with cellular debris. The crystals contained calcium and phosphorus. Phagy and stagnation related to pockets of inefficient secretory activity have been previously found to be features of the parotid of ferret. Thus, possibly persistent degradation of redundant cellular material, particularly secretory granules, in phagosomes results in accumulation of calcium and leads to calcified microliths, whereas consolidation of stagnant debris extracellularly does not involve such accumulation and leads to non-calcified or mixed microliths.     

Occurrence of a third type of secretory cell in the acinus of the rat submandibular gland

A new, third type of secretory cell was found in the acini of the submandibular gland in rat. In paraffin sections stained with hematoxylin-eosin or reacted with periodic acid-Schiff, acini containing the third type of secretory cells were found as a mass of darkly stained secretory endpieces at the periphery of the lobule. In semi-thin sections stained with toluidine blue, these cells showed numerous dense secretory granules in their cytoplasm. Some acini consisted entirely of these cells, whereas other acini consisted of an admixture of such cells and typical seromucous cells. Acini containing the third type of secretory cell tended to form clusters. Electron microscopically, the cells had well-developed rough endoplasmic reticulum and Golgi apparatus. The secretory granules showed a wide range of variation in substructure, even within the same cell. Well-developed intercellular interdigitation and intercellular canaliculi also were observed between adjacent third type of secretory cells. Although little is known about the biological significance of these cells and the properties of their secretory granules, the present study shows that the rat submandibular gland possesses three types of secretory cells in their secretory endpieces. © 1993 Wiley-Liss, Inc.     

Calcium redistribution,calcification and stone formation in the parotid gland during experimental stimulation and hypercalcaemia

Summary Distribution and redistribution of intra- and pericellular calcium was investigated in the parotid gland of rats under secretory stimulation and hypercalcaemia. The effects of hypercalcaemia and secretory stimulation and of the combination of both were compared. Calcium content was determined by atomic absorption spectrometry. Calcium distribution within the tissue was demonstrated by light microscopical (GBHA) staining and electron microscopical (pyroantimonate method) cytochemistry in combination with X-ray microanalysis. Typical calcium depot sites were the basal and cellular membranes, the calcium buffer organelles (i.e. mitochondria) the secretory granules and the acinar lumina. After stimulation (by isoprenalin) a decrease of calcium-enriched secretory granules and a depletion of intracellular calcium buffer organelles occurred. During hypercalcaemia (induced by dihydrotachysterol), a calcium overloading of the cell membrane and intracellular buffer organelles without calcification was observed. Combined stimulation and hypercalcaemia induced an excessive calcium overloading of all intra-and extracellular calcium depots with excessive calcium release into the acinar lumina resulting in calcium phosphate aggregates and stone formation. Secretory stimulation and simultaneous hypercalcaemia exert potentiating effects on intracellular and intraluminal calcification proposing an importance for pathogenesis of human sialolithiasis.Supported by Sonderforschungsbereich 34 Endokrinologie     

The effects of 5-bromodeoxyuridine and isoproterenol on the postnatal differentiation of rat submandibular gland

The effects of 5-bromodeoxyuridine (BrdU) on the postnatal differentiation of rat submandibular gland and on the isoproterenol-induced changes of differentiation were studied. The rats were injected with BrdU for three consecutive days, beginning at two days of age. The total dose of BrdU was 0.9 mg/g body weight. BrdU caused a severe retardation of growth up to 15 days of age. A rapid growth of the animals between 15 and 22 days indicated a recovery from the effect of BrdU. The growth of the submandibular gland was retarded similarly with a corresponding decrease in DNA, RNA and protein content. Incorporation of tritiated thymidine into the submandibular gland was not altered in the BrdU-treated animals at one and three days after the last injection of the analog. At days 15 and 22 the rate of thymidine incorporation was greater in the submandibular gland of BrdU-treated rats as compared to littermate controls. Isoproterenol stimulated thymidine incorporation into the submandibular glands of two-week-old rats. This stimulation was not observed in rats which received BrdU at age 7–9 days, prior to the administration of isoproterenol. Electron microscopic observations, including a quantitative analysis of the frequency distribution of the various cell types in the terminal tubules and developing acini, indicated a retardation of acinar cell differentiation in the glands of BrdU-treated rats. In addition, there was an increase in the number and size of the secretory granules in the terminal tubule cells. BrdU treatment, however, caused no obvious pathologic alterations in the submandibular gland. Administration of isoproterenol for five days, beginning at five days of age, caused an apparent acceleration of the differentiation of acinar cells. In the glands of isoproterenol-treated rats the acinar cells were enlarged and were filled with homogenous secretory granules. Pretreatment with BrdU partially inhibited the effects of isoproterenol on differentiation and hypertrophy of the submandibular gland. With the given dose of BrdU, approximately 5% of thymine was replaced by bromouracil in the DNA of the submandibular gland. Such a replacement would be consistent with a direct effect of BrdU on the cytodifferentiation in the submandibular gland. However, because of the severe retardation of growth of the BrdU-treated rats, indirect effects can not be excluded.     

Ultrastructural changes in exocrine tissues of a DeltaF-508 CFTR mouse model

Cystic fibrosis (CF) is characterized by abnormal secretion from epithelial cells. We wanted to detect changes in the ultrastructural characteristics of cells within a number of exocrine tissues, including the colon, submandibular and parotid salivary glands of DeltaF-508 CFTR animals. Therefore, in the present study a DeltaF-508 CFTR mouse model was compared to control, by applying conventional and complex carbohydrates staining techniques to tissue sections at the electron microscope level. The colon of DeltaF-508 CFTR mice contained thick mucous secretions that harbored many bacteria, along with cytoplasmic fragments and leukocytes. Leukocytes were also seen to infiltrate the cytoplasm of goblet cells. Tissues were taken before, 10 min after isoprenaline, and 30 min after a further injection of methacholine. In the submandibular gland, there is limited secretory activity after isoprenaline treatment, and this increases further with methacholine treatment. Depletion of the secretory granules of acinar cells is observed, following the combined isoprenaline and methacholine treatment, but no significant changes in granule numbers occurred in granular tubule cells. Glycogen, abundant before treatment, is reduced within 10 min of isoprenaline treatment and is completely exhausted by 30 min, especially in the convoluted granular tubule cells. A few secretory granules in acinar and in granular tubule cells of the DeltaF-508 CFTR submandibular glands displayed two electron densities. The secretory responses of the parotid gland cells were similar to those in submandibular gland cells, except that in these DeltaF-508 CFTR cells, secretory granules appeared more polymorphic in structure than those found in control animals.     

Parathyroid Glands in Chronic Aluminum Intoxication

In chronic renal failure, aluminum overload may influence parathyroid function. In a study of possible aluminum-induced parathyroid abnormalities, parathyroid glands from nine parathyroidectomized patients on hemodialysis were examined by light and electron microscopy and by X-ray microanalysis. Aluminum overload was assessed by the presence of stainable aluminum (aluminum surface, 23.3% ± 11% of total surface) in bone biopsy specimens. The mean plasma aluminum concentration was 7.7 ± 1.9 μmol/L. All patients but one had elevated plasma concentrations of immunoreactive parathyroid hormone as well as osteitis fibrosa. The aluminum concentrations in bone and parathyroid gland from these patients were significantly higher than those in tissue from patients on hemodialysis without stainable bone aluminum. Abundant aluminum deposits were present in parathyroid chief cell cytoplasm in lipoid bodies, lipofuscin granules, and mitochondria. These cells exhibited features of active hormonal synthesis and contained numerous secretory granules. The data show that in the parathyroid glands of these aluminum-intoxicated patients the presence of aluminum deposits neither induced cellular damage or chief cell necrosis nor interfered with the production of parathyroid hormone.     

Histamine and calcium secretion in the submandibular gland of the cat evoked by two types of stimulation

The aim of this work was to compare the pools of histamine and calcium in the submandibular gland of the cat during two kinds of stimulation of the gland. Submandibular glands of the cat were stimulated electrically (chorda tympani nerve) and by close arterial infusion of pilocarpine. Each stimulation was adjusted in such a way as to obtain a near maximal secretory response. Concentrations of histamine and calcium were measured in nonstimulated and stimulated glands, in saliva, secreted during stimulation and in the venous outflow from the gland before and during the stimulation. The experiments showed that:

1. pilocarpine stimulation lowered the histamine concentration in the gland more than electrical stimulation of the chorda tympani nerve. Calcium stores in the gland were depleted to the same extent by each type of stimulation,
2. the concentration of histamine and calcium in the saliva produced by close arterial pilocarpine infusion was significantly higher than in the saliva secreted during nerve stimulation,
3. histamine and calcium concentrations were higher in the venous outflow from stimulated glands than before stimulation.

The results indicate a close relationship between histamine and calcium in salivary secretion, although the origins of the two substances are different.