Premature induction of amylase in pancreas and parotid gland of growing rats by dexamethasone

Studies were made on changes in the contents of α-amylase (EC 3.2.1.1) in the pancreas and parotid gland of rats during postnatal devlopment, on the premature induction of this enzyme by hormones and on the existence of specific glucocorticoid receptors in these tissues.The amylase content in the pancreas increased from the 9th day after birth and reached the adult level on the 28th day, its content in the parotid gland increased rapidly from the 16th to 28th day after birth and then rose more gradually to the adult level.Injection of dexamethasone into rats 6–8 days after birth induced increase in the amylase of the pancreas but not the parotid gland. However, injection of dexamethasone into weanling rats 21–23 days after birth resulted in precocious induction of amylase in both tissues.Specific glucocorticoid receptors were detectable in the parotid gland of rats from 6 days after birth but were almost undetectable in the pancreas until adolescence.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.     

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.     

Changes of salivary amylase in serum and parotid gland during pharmacological and physiological stimulation.

Although serum amylase level is an important diagnostic factor in certain salivary and pancreatic diseases, little information is available regarding the mechanism by which parotid amylase reaches the circulatory system. The present study was carried out to investigate the relationship between parotid isoamylase concentrations in blood serum and in parotid tissue in response to various stimuli. Wistar rats were fed with standard laboratory rodent chow; water was supplied ad libitum. In the first experiment, after a 16-h fasting, rats received either 5 mg/kg pilocarpine or saline (control). In the second study, after fasting, half of the rats were fed for 1 h, the other half received no food. In the third experiment, the changes in serum and tissue enzyme levels were monitored in freely fed animals during the peak-food intake phase, the first 2 h of the dark period. Amylase concentration was determined by using starch as a substrate. Pancreatic and parotid isoamylase levels in serum were separated by gel-electrophoresis utilizing differences in ionic properties of the isoenzymes. As expected, pilocarpine strongly stimulated tissue amylase discharge and serum amylase elevation. Similar, but less pronounced changes were observed not only during refeeding of fasted animals, but also in nonfasted rats during their peak-feeding period. Our data suggest that pharmacological stimulation, such as with pilocarpine or feeding in fasted state, as well as a mild stimulation of parotid function by spontaneous food intake during nonfasted state results in a decrease in parotid tissue amylase activity and a proportional increase in serum levels of parotid isoamylase.     

Identification of amylase crystalloids in cystic lesions of the parotid gland

OBJECTIVE: To identify alpha-amylase crystalloid formations in parotid specimens obtained by fine needle aspiration. STUDY DESIGN: The study concerned three cases of sialadenitis with crystalloid formation observed between 1993 and 1998. In one of these cases, transmission electron microscopy, mass spectrometry and measurement of amylase activity were used to characterize the nature of the crystalloids. RESULTS: Light microscopy revealed the same crystalloid structure in all three cases. In one case, where the material was saved, a biochemical method made it possible to reveal high amylase activity, while protein electrophoresis and mass spectrometry were used to identify salivary alpha-amylase. CONCLUSION: Crystalloids of salivary alpha-amylase can be identified by May-Grünwald-Giemsa and Papanicolaou stain and can be rapidly confirmed through determination of amylase activity.     

In situ localization of amylase mRNA and protein. An investigation of amylase gene activity in normal human parotid gland

The distribution of human salivary amylase mRNA was studied by in situ hybridization to a [32P]-labeled amylase cDNA probe. Amylase mRNA was localized to the apical portion of acinar cells in frozen sections of human parotid salivary gland. No hybridization was noted in ductal cells, skeletal muscle, or in connective tissue. These results were consistent with immunohistochemical localization of amylase. The technique of in situ hybridization was modified to permit localization of amylase mRNA in variously fixed, paraffin-embedded parotid glands. Although the hybridization signal decreased with all fixatives, the pattern of localization paralleled that obtained with frozen sections. No advantage was noted in fixation with ethanol-acetic acid or Bouin solution over routine fixation with formalin. These results have important implications for researchers interested in studies of gene expression. We have demonstrated that routinely fixed paraffin blocks of human tissue can be used for cellular localization of specific mRNA. In coordination with immunocytochemistry, in situ hybridization offers a powerful tool for studies of mRNA and protein expression in individual cells.     

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.     

Electrophoretic behavior of amylase in mouse: the parotid gland is major source of serum amylase

Amylase activity in the saliva, salivary glands, serum, liver (perfused and non perfused) and pancreas was assayed and isoamylases were separated by electrophoresis in these organs using C57BR/cdJ and M. m. molossinus (Kor) mice. Amylase isozymes in the saliva, parotid gland, serum and liver were identical in both strains, respectively. Amylase activity in the liver was lower than that in the serum and liver amylase disappeared almost by perfusion. Major serum amylase was released from the parotid gland in intact animals.     

Control of amylase biosynthesis and release in the parotid gland of the rat

1. Amylase biosynthesis and release in the rat parotid were studied under various conditions. Incorporation of [(3)H]leucine into amylase, extracted from the tissue by immunoadsorbent, was measured and found to be time-dependent and totally inhibited by the protein synthesis inhibitor puromycin. 2. Adrenaline, at a concentration (10mum) that gave maximum stimulation of release, inhibited [(3)H]leucine incorporation into both total protein and amylase. This effect was reversed by phentolamine. 3. Adrenaline (1mum) and isoproterenol (10mum) stimulated biosynthesis of total protein and amylase. These effects were blocked by propranolol, as were the effects on release. Dibutyryl cyclic AMP (2mm) mimicked the effects of isoproterenol and adrenaline (1mum) on both amylase biosynthesis and release. All the above stimulatory effects on amylase biosynthesis were only observed if the tissue was pretreated with effector before pulse-labelling with [(3)H]leucine. 4. Insulin (625muunits/ml initial concentration, 150muunits/ml final concentration) stimulated incorporation of [(3)H]leucine into total protein and amylase when added to the tissue at the same time as the leucine. 5. Carbamoylcholine (10mum) decreased [(3)H]leucine incorporation into total protein and amylase when both were added to the tissue simultaneously, but this effect was prevented by removal of effector and washing the tissue before addition of [(3)H]leucine. 6. Stimulation of beta-adrenergic receptors increased both amylase release and biosynthesis, but stimulation of alpha-receptors can inhibit biosynthesis without inhibiting release. Cholinergic agents can also inhibit amylase biosynthesis, but stimulate release. Insulin at approximately physiological concentration can increase incorporation of leucine into amylase without stimulating release. The system described therefore provides an excellent model for the further investigation of the mechanisms of these diverse effects.     

Protease-activated receptor-2 (PAR-2) in the pancreas and parotid gland: Immunolocalization and involvement of nitric oxide in the evoked amylase secretion

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH2, administered intraperitoneally (i.p.) at 1-10 micromol/kg and 1.5-15 micromol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 micromol/kg, but not at a smaller dose, 5 micromol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.     

The effect of calcium and cyclic AMP on amylase release in digitonin-permeabilized parotid gland cells

Rat parotid cells were permeabilized with digitonin to examine their secretory dynamics. Cells were isolated by a modification of the method previously described by Hootman [1985). J. Biol. Chem. 260, 4186-4194) in which alpha-chymotrypsin was included. The final preparation consisted of approx. 40-60% single cells. The cells were 85-90% viable by trypan blue exclusion and secreted amylase when stimulated with isoproterenol. Digitonin (2 or 5 microM) was sufficient for permeabilization while 2 microM digitonin was somewhat more effective in maintaining cell integrity as indicated by lactate dehydrogenase release. Digitonin had minimal effects on intracellular granules in the whole cell and was, thus, relatively selective. The response of digitonin-permeabilized cells to calcium (without secretagogues) in the incubation medium was monitored by amylase release. For a wide range of applied free calcium concentrations (1 X 10(-7) M to 10(-4) M) a statistically significant increase in amylase secretion was observed. Control cells did not release amylase to a similar extent without secretagogue. Cyclic AMP (50 microM) significantly enhanced amylase secretion from digitonin-treated cells at all concentrations of free calcium tested. Neither calcium nor cyclic AMP alone was sufficient to stimulate maximal amylase release. Our results provide direct evidence for a model in which calcium and cyclic AMP work on separate pathways as interacting regulators of exocytosis.     

The role of protein kinase C on amylase secretion from rat parotid gland

The activities of Ca2+.phospholipid-dependent protein kinase (protein kinase C) in rat salivary gland were assayed using synthetic peptide syntide-2(Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys- Lys) as substrate. Levels of the protein kinase C were less than 0.05 units/g in the parotid and submandibular glands. The protein kinase C inhibitor, H-7, inhibited amylase secretion from rat parotid gland stimulated by PMA or the combination of phosphatidylserine and 1,2-diolein. The results supported the hypothesis of the secretory mechanism that protein kinase C mediates amylase secretion in rat parotid glands.     

The functions of cyclic AMP and calcium as alternative second messengers in parotid gland and pancreas.

Biochemical and ultrastructural studies of rat parotid gland slices have led to the identification of alpha- and beta-adrenergic receptors and a cholinergic receptor, all operating within the same secretory cell. While cyclic AMP serves as the second messenger in the beta-adrenergic response of enzyme secretion, Ca++ serves as the second messenger in the alpha-adrenergic and in the cholinergic responses which both lead to K+ release and water secretion. Ca++ also serves as a second messenger for the muscarinic cholinergic receptor in rat pancreas slices in which it causes enzyme secretion. Analysis of this information leads to the conclusion that neither the neurotransmitter, nor the receptor, nor the second messenger are unique for a certain type of response. The latter seems to be dictated by a component of the specific response pathway which is affected by the second or a subsequent messenger. By having different neurotransmitters operate the same response and a single neurotransmitter operate different responses diversity of control is achieved.     

Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland

Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP)-mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTPS in PT-treated membranes was much larger than that in normal membranes. ANP dose-dependently inhibited adenylate cyclase stimulated by GTPS in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10^–7M) inhibited cAMP accumulation stimulated by forskolin (10^–6M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells, amylase release stimulated by isoproterenol (10^–6M) and forskolin (10^–6M) were also depressed by ANP (10^–7M) by 27 and 30%, respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP-ribosylated 41-KDa protein by incubation of parotid membranes with PT and [-³²P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi. (Mol Cell Biochem)139: 53–58, 1994)     

Cholecystokinin- and gastrin-induced protein and amylase secretion from the parotid gland of the anaesthetized rat

I.V. infusion of pentagastrin (20 microg/kg/h) or cholecystokinin (CCK)-8 (1 microg/kg/h) for 10 min caused secretion of salivary proteins from the parotid gland in the anaesthetized rat without any accompanying overt fluid secretion. This "occult" response was revealed by a subsequent wash-out injection of methacholine (5 microg/kg, I.V.) 10 min after the end of the infusion period (aiming at avoiding synergistic interactions). While the fluid response to methacholine was unaffected by the preceding infusion of pentagastrin and CCK-8, the output of protein increased by 147% (pentagastrin) and 74% (CCK-8) and that of amylase by 45% (CCK-8) compared to the responses to methacholine upon saline infusion. Those increases were abolished by the CCK-A receptor blocker (lorglumide), but not by the CCK-B receptor blocker (itriglumide). Evisceration, combined sympathetic and parasympathetic denervation of the glands and assay under adrenoceptor blockade excluded contribution from the gastro-intestinal tract, central or ganglionic mechanisms and circulating catecholamines to the increase in protein/amylase. Furthermore, Western blot demonstrated CCK receptors for both A and B subtypes in normal and chronically denervated glands. In the submandibular gland, both pentagastrin and CCK-8 evoked a trace secretion of saliva but, under the present experimental set-up, no statistically significant increase in protein output. Thus, in addition to the autonomic nervous system, gastrointestinal hormones may, in some types of glands, be involved in the secretion of salivary gland proteins.     

New genetic variants of parotid salivary amylase.

Isoelectric focusing can reveal at least two heterozygotes of the Amy1R class of variants. Family data on these variants show co-dominant inheritance for both alleles with respect to the normal Amy1A allele. The frequency of the alleles Amy1R1 and Amy1R2 in the Dutch population (n = 144) amounts to 0.006 and 0.03, respectively. Electrophoretic analysis of whole parotid saliva from different Amy1R phenotypes and of purified normal and Amy1R1 gene products indicates that the variant proteins differ from the normal protein by enhanced deamidation of asparagine and/or glutamine residues.