Tight junctions in the rat parotid gland

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.     

Effects of radioprotectors on the cAMP and cGMP systems

Summary The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-aminothiazoline, and S-S-3-oxapentane-1,5-diisothiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected cAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fall by 1–3 min after the administration of drug and 1.4-fold rise after 15–20 min); serotonin and dopamine caused a slow rise in the cGMP level; the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin.The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system. The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist.     

Effects of cystic fibrosis serum on the rat parotid gland

Summary Injections of serum from human patients with cystic fibrosis into adult rats caused pronounced structural modifications and increased mitotic rate in the parotid gland. Mitotic rate was increased from a low level of 0.02/1,000 acinar cells in parotid glands of adult rats to 6.5/1,000 acinar cells after 2 or 3 days of serum injection. At the light and electron microscopic levels, significant acinar cell atrophy and degranulation were observed. Cellular necrosis, and increases in quantity of lysosome-like dense bodies, mast cells, and macrophages were also detected. These changes are suggestive of tissue response to injurious foreign protein. Furthermore, the fact that normal sera pronounced the same kind of effects (but greatly reduced in extent) strengthens the view that these effects result from the immunologic response of the host organ to foreign antigen. Since, however, the responses of the rat parotid to cystic fibrosis serum were considerably more marked than those elicited by normal serum, the rat parotid may thus have potential usefulness in assaying for the presence of human cystic fibrosis factor.This work was supported in part by U.S.P.H.S. Grant DE 02110The authors wish to thank Dr. Alexander Spock, Cystic Fibrosis Center, Duke University Medical Center, Durham, North Carolina, and Dr. Ralph Tiller, Children's Hospital, University of Alabama Medical Center, for generously supplying blood from patients with cystic fibrosis. The authors also want to thank Dr. A. Siegel, Department of Pathology, University of Alabama Medical Center, and Mr. R. Siegel, for determinations of serum catecholamine levels     

Lysosomal enzymes in relation to the isoproterenol-induced secretory cycle in rat parotid gland

Two lysosomal enzymes, cathepsin D and acid phosphatase, were detected in significant amounts in the lysosome-containing subcellular fractions of rat parotid tissue and found to have dissimilar distributions in these fractions. The total levels of these enzymes were measured at various times throughout a complete secretory cycle induced synchronously by fasting rats overnight and administering isoproterenol at time zero. The results showed a 30% increase in cathepsin D activity in the glands by 10 h post-stimulation, and a 20% decrease in acid phosphatase activity 7 h after stimulation. These results suggest that there are cyclic changes in lysosomal enzymes during the secretory cycle of this gland, but that these changes are complex ones and cannot be related to specific cellular processes at this time.     

Ontogeny of alpha-amylase circadian rhythms in rat parotid gland

The content of alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) and total soluble proteins of parotid glands (from rats exposed to a photoperiod of 14 hr light: 10 hr dark), have been determined every 2 or 3 hr over 24 hr periods in 15, 25 and 90-day-old rats. In 35-, 45- and 72-day-old rats, determinations were performed only at 0100 and 1400 hr. The alpha-amylase and total soluble protein contents from 90-day-old rats show a circadian variation, with a maximum value at 2200 hr and a minimum at 1400 hr. Parotids from 15- and 25-day-old rats also show a circadian rhythm. The minimum value is recorded at 0100 hr and the maximum at 1400 hr. At day 35 and after, there is an inversion of the amylase rhythm. In immature rats, it appears that alpha-amylase and soluble protein are under the influence of another synchronizer, whose timing is independent of that imposed by mastication of solid food.     

Biochemical and physical analysis of subcellular membranes in rat parotid gland enlarged by chronic isoproterenol administration

1. Chronic administration of isoproterenol caused similar alterations of membrane lipid profile in at least two rat parotid subcellular fractions, secretory granular and microsomal. 2. Typical changes in phospholipid classes and fatty-acyl chain groups were an increase of phosphatidylcholine and a decrease of sphingomyelin, and an increase of octadecadienoyl chain and a decrease of eicosatetraenoyl chain, respectively. 3. Electron spin resonance study showed that the isoproterenol-treatment also affected a membrane physical property, which may be through these compositional changes in membrane constituents.     

Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.     

Uptake and secretion of technetium pertechnetate by the rat parotid gland

The ability of acinar cells of the rat parotid gland to transport technetium pertechnetate (99mTcO-4) was examined. After intravenous injection, 99mTcO-4 was rapidly detected in parotid saliva. There was an excellent correlation between saliva and plasma 99mTcO-4 levels. The saliva to plasma ratio was always less than 1, consistent with the inability of rat parotid gland duct cells to concentrate the anion. Output of 99mTcO-4 by the parotid gland closely mimicked fluctuations in parotid saliva flow rate. In vitro, enzymatically dispersed parotid acinar cells accumulated 99mTcO-4 from the incubation medium in a biphasic manner. This uptake was partially blocked by 10(-4) M NaI. Cells which had accumulated 99mTcO-4 showed increased radionuclide efflux after exposure to 10(-5) M carbachol.     

Neural regulation of compensatory enlargement of the parotid gland of the rat

Summary The role of the innervation in mediating compensatory enlargement of the parotid gland of the rat after partial desalivation was examined. The results of denervation experiments show that full compensatory growth requires both parasympathetic and sympathetic innervation. The presence of the parasympathetic innervation alone results in an increase in the number of cells, but not the size of the cells. The sympathetic innervation alone does not mediate either response. We, therefore, conclude that the two types of innervation have a synergistic action on the parotid to produce the maximal compensatory response, which includes an increase in both number and size of acinar cells.Supported in part by the Veterans Administration and Grant DE 02110 of the U.S. Public Health Service     

Effect of postganglionic sympathectomy on the ultrastructure of the rat parotid gland

Summary Following two weeks of superior cervical ganglionectomy, the parotid glands of adult rats were removed and studied by electron microscopy. Sympathectomy induced striking alterations of acini, resulting in a heterogeneous population of acinar cells, but it had no obvious effect on the duct system. Most of the altered cells could be classified on a cytological basis as dark cells or light cells. Dark cells predominated and contained more secretory granules, less granular endoplasmic reticulum, fewer Golgi membranes, and smaller lumina and intercellular canaliculi than normal acinar cells. The synthesis and extrusion of secretory products appeared to be minimal in these cells. Light cells possessed ultrastructural features, such as dilated cisternae of granular endoplasmic reticulum and prominent Golgi membranes, which were opposite to those of dark cells and indicative of a high degree of secretory activity.The heterogeneous population of cells following sympathectomy indicates that the sympathetic nervous system may play an important role in regulating the secretory synchrony of acinar cells.Supported by U.S.P.H.S. Grant DE 02110.     

Alkaline phosphatase and myoepithelial cells in the parotid gland of the rat

Synopsis Alkaline phosphatase activity has been studied in the parotid glands of rats at the light and electron microscopical levels. Reaction product was found to outline the plasma membranes of myoepithelial cells. It was also found in the walls of many capillaries and on the luminal surface and between apposing cells in some intercallary ducts.The distribution of myoepithelial cells in the rat parotid is unusual. The cells run longitudinally around intercalary ducts and send processes on to the bases of adjoining acini but do not embrace the acini. The possible functions of these myoepithelial cells are discussed.     

Discharge and restitution of secretory material in the rat parotid gland in response to isoproterenol

Summary The sequence of morphological events occurring during discharge and restitution of secretory material in the rat parotid in response to isoproterenol administration has been studied using the electron microscope. With the dose used, discharge of secretory granules began within 5 min following injection and was complete by 40 mim. Intracellular accumulation of normal-appearing secretory material became evident at 6 hours, and restitution of resting quantities of secretory material was achieved between 12 and 18 hours after injection. Cellular events occurring during secretory discharge and restitution are discussed.This project was supported by Training Grant No. 5-Ti-GH-326 and by Predoctoral Research Grant No. 1-F1-GM-32, 528-01, National Institutes of Health. I wish to express my gratitude to Dr. Henry S. di Stefano under whose directorship this project was carried out.