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.     

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 gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.     

Immunocytochemical mapping of basic fibroblast growth factor in the developing and adult rat adrenal gland

We studied the spatial and temporal pattern of basic fibroblast growth factor (bFGF) immunoreactivity in the rat adrenal gland during postnatal development. In the cortex the glomerulosa zone reveals a strong anti-bFGF immunoreactivity at all developmental ages studied. In the fasciculata zone the high number of anti-bFGF immunoreactive cells in the first week decreases during the second and third week. The late developing reticularis zone shows only few anti-bFGF labeled cells at all postnatal ages. This distributional pattern of bFGF immunoreactivity matches that of mitotic activity in the rat adrenal cortex strengthening the role of bFGF as an autocrine growth factor for adrenocortical cells. In the medulla anti-bFGF positive chromaffin cells become detectable at postnatal day (P) 8 and increase in number during the second and third week. In the adult rat the staining intensity of the chromaffin cells was higher than at P18. In the adult medulla bFGF colocalizes with noradrenaline suggesting its presence in a chromaffin cell subpopulation. In accordance with previous results the role of the chromaffin cell bFGF as a neurotrophic factor for preganglionic sympathetic neurons is discussed.     

Immunocytochemical mapping of basic fibroblast growth factor in the developing and adult rat adrenal gland

Summary We studied the spatial and temporal pattern of basic fibroblast growth factor (bFGF) immunoreactivity in the rat adrenal gland during postnatal development. In the cortex the glomerulosa zone reveals a strong anti-bFGF immunoreactivity at all developmental ages studied. In the fasciculata zone the high number of anti-bFGF immunoreactive cells in the first week decreases during the second and third week. The late developing reticularis zone shows only few anti-bFGF labeled cells at all postnatal ages. This distributional pattern of bFGF immunoreactivity matches that of mitotic activity in the rat adrenal cortex strengthening the role of bFGF as an autocrine growth factor for adrenocortical cells. In the medulla anti-bFGF positive chromaffin cells become detectable at postnatal day (P) 8 and increase in number during the second and third week. In the adult rat the staining intensity of the chromaffin cells was higher than at P18. In the adult medulla bFGF colocalizes with noradrenaline suggesting its presence in a chromaffin cell subpopulation. In accordance with previous results the role of the chromaffin cell bFGF as a neurotrophic factor for preganglionic sympathetic neurons is discussed.     

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

Studies were made on changes in the contents of alpha-amylase (EC 3.2.1.1) in the pancreas and parotid gland of rats during postnatal development, 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 the 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.     

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.     

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.     

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.     

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.     

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.     

Immunocytochemical localization of parathyroid hormone in hamster parathyroid gland

The immunocytochemical localization of parathyroid hormone was examined in the hamster parathyroid gland by using the protein A-gold technique. Protein A-gold particles were concentrated over secretory granules, large secretory granules thought to be storage granules and Golgi vacuoles. No protein A-gold particles were detected over large vacuolar bodies and cisternae of the granular endoplasmic reticulum.     

Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons

The lectin, concanavalin A (Con A), was localized in the cotyledon of developing jack beans (Canavalia ensiformis (L.) DC) by electron-microscope immunocytochemistry. In mature seeds, Con A was present in protein-storage vacuoles (protein bodies) of storage-parenchyma cells. Although protein bodies could be seen in other cell types, only protein bodies in storage-parenchyma cells contained Con A. During seed development, Con A was also localized on the endoplasmic reticulum and Golgi apparatus, presumably en route toward deposition within the protein bodies. The intensity of labeling of the endoplasmic reticulum was much greater during the developmental stage of protein-body filling (66% final seed weight) than in mature seeds.Abbreviations Con A concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G     

Immunocytochemical localization of heparan sulphate proteoglycan in the rat submandibular gland

Summary Heparan sulphate proteoglycan is the predominant proteoglycan synthesized by the parenchymal cells of the rat submandibular gland. A polyclonal antibody was used to localize this proteoglycan in the adult rat submandibular gland. Localization was accomplished by indirect immunoperoxidase cytochemistry at the light and electron microscopic levels. Heparan sulphate proteoglycan was localized in a continuous, linear pattern in the lamina densa of the basement membrane surrounding all of the epithelial components of the gland as well as the basement membrane of the capillaries and small arterioles in the glandular stroma. In addition, heparan sulphate proteoglycan was seen in vesicles and pits along the acinar cell basal plasmalemma adjacent to the basement membrane and in the endoplasmic reticulum and Golgi apparatus of the acinar cells.     

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.     

Immunocytochemical localization of prolactin binding sites in mouse adrenal gland

1. Prolactin (PRL) has been previously associated with adrenal secretion of either corticosterone, progesterone, or aldosterone. 2. PRL binding sites have been previously demonstrated in rat adrenal cellular membrane preparations. 3. No studies have reported specific zonal sites of PRL binding. 4. The current study demonstrates presence of both endogenously bound PRL and free receptor for PRL in zona fasciculata of mouse adrenal cortex. 5. This finding is consistent with a role for PRL in regulating adrenal secretion of either corticosterone or progesterone in the mouse.