Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Summary Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained.These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated und treated acinar cells.     

Adenylate cyclase activity in the parotid gland of the mouse after isoproterenol stimulation

Previous studies have described a decrease in the activity of adenylate cyclase in the parotid gland of isoproterenol-treated rats. In the present studies, a similar decrease was observed in mice treated with isoproterenol. Studies on the subcellular distribution of adenylate cyclase after isoproterenol stimulation of the parotid gland showed that enzyme activity was increased in the lysosomal fraction and decreased in the cellular membrane fractions. Cytochemical studies on the localization of adenylate cyclase in stimulated gland showed an increase in vesicles which contained enzyme activity and a decrease in activity at the luminal and plasma membranes. It is suggested, based on the present findings and results reported by other investigators, that after isoproterenol stimulation of the parotid gland, adenylate cyclase (along with excess membrane) is degraded by lysosomes. If this suggestion is true, then the observed decrease in adenylate cyclase would have a molecular explanation.     

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.     

Cholecystokinin modulates isoproterenol induced changes in rat parotid gland

1. Parotid gland secretory function and activity of several enzymes involved in intracellular second messenger signalling were measured in rats receiving 0.5 ml i.p. injections of saline (control), isoproterenol, CCK or both drugs.2. Isoproterenol caused a 2.5-fold increase in parotid gland wet weight compared to control. Chronic administration of CCK alone has no effect on gland weight. A combination of CCK and isoproterenol did not alter the hypertrophy of the gland observed with isoproterenol alone.3. Isoproterenol administration caused a 74% decrease in parotid gland amylase enzyme activity. While CCK alone did not influence the enzyme activity, it depressed amylase mRNA steady state levels and had an additive effect on further decreasing mRNA levels when administered in combination with β-agonist.4. Phospholipase C registered an increase ranging from 22 to 38% in all experimental groups as compared to control.5. Parotid gland protein kinase C and PdtIns 3-kinase activity were not altered in response to CCK alone, but in combination with isoproterenol, appeared to moderate β-agonist signal transduction responses.     

Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation

The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland. Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min. Secretion was induced in some animals by intraperitoneal injection of isoproterenol. In resting glands, the tracers microperoxidase , cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts. In glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase , cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands. Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells. The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution. Freeze- fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight greater than or equal to 1,900; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight less than or equal to 34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights. Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution.     

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.     

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.     

Cell proliferation in the mouse parotid gland after unilateral parotidectomy.

Unilateral parotidectomy with or without total submandibulectomy has been used to induce cell proliferation in mouse parotid gland. Maximum DNA synthesis and mitosis were recorded four and five days after the operation. The double operation increased the proliferative response. Such proliferative stimulii was not accompanied by secretion and was sex independent. On the other hand, the response decreased with age. RNA and protein synthesis inhibitors showed that the stimulation of DNA synthesis depends on early protein synthesis, which seems to be synthesized on a preexisting template.     

Mediation of beta-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced ⁴⁵Ca⁺⁺ efflux and 2) a dependence on the extracellular Na⁺ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on ⁴⁵Ca⁺⁺ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release ⁴⁵Ca⁺⁺. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na⁺ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the ⁴⁵Ca⁺⁺ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na⁺. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na⁺ followed by release of intracellularly stored calcium.     

Visualizing form and function in organotypic slices of the adult mouse parotid gland

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.     

Changes in the polypeptide composition of mouse parotid glands after stimulation of secretion and DNA synthesis by isoproterenol

The polypeptide composition of mouse parotid glands has been analysed by unidimensional SDS-polyacrylamide slab gel electrophoresis and Coomassie blue staining after isoproterenol stimulation of secretion and DNA synthesis. Two polypeptides (polypeptides A and B) are lost within 2 h and their restoration in the glands occurs according to a chronology which is identical to that of the alpha-amylase activity. On the other hand, five clearly defined new bands appear consistently during the late prereplicative period of isoproterenol-stimulated mouse parotid acinar cells (polypeptides C, D, E, F and G). These new polypeptides are induced by doses of isoproterenol which provoke secretion and DNA synthesis, but not by doses which provoke only secretion. Although no function has been assigned to any of the above-described polypeptides, a relation between polypeptides A and B and secretion and between polypeptides C, D, E, F and G and the proliferative response is suggested.