Rapid release of the zymogen granule protein by osmium tetroxide and its retention during fixation by glutaraldehyde.

Fixation by osmium tetroxide and glutaraldehyde of zymogen granules isolated from rat parotid and pancreas was investigated. Protein determinations showed that osmium tetroxide caused rapid release of most of the soluble protein of the granule during fixation in buffered isotonic sucrose. Such granules when examined in the electron microscope after shadow casting appeared quite flat, indicating that most of the contents had indeed been removed. Numerous damaged membranes of the granules were also observed. In contrast, zymogen granules fixed by glutaraldehyde and shadow cast essentially retained the spherical shape and the protein contents. The application of the shadow-casting technique in quantitative studies on the protein content of zymogen granules is discussed.

Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000-dalton protein phosphorylated in situ in response to secretagogues.

In the preceding paper, we demonstrated that the endogenous phosphorylation of a protein with a molecular weight of 29,000 was enhanced by various secretagogues in rat pancreatic and parotid lobules, the phosphorylation of this protein correlating both temporally and in a dose-dependent fashion with secretory protein discharge. In the present study, we established a specific methodology to characterize this phosphoprotein. Once established, this 29,000-dalton phosphoprotein was then followed selectively and quantitatively throughout subcellular fractionation procedures. Analysis of two-dimensional polyacrylamide gels demonstrated that proteins with similar mobilities (Mr 29,000; pl greater than 8.4) were affected by cholecystokinin octapeptide and isoproterenol in rat pancreatic and parotid lobules, respectively, suggesting that the same 29,000-dalton phosphoprotein was covalently modified in both tissues. Cellular fractionation studies using differential velocity and sucrose density gradient centrifugation revealed that the 29,000-dalton phosphoprotein copurified with the rough microsomal fraction of pancreas and was highly enriched in ribosomal fractions of both pancreas and parotid. Electrophoresis in two dimensions confirmed that the 29,000-dalton polypeptide that was resolved directly from stimulated cells and from ribosomal fractions exhibited a common mobility, and apparent identity of the species was strongly suggested when the 29,000-dalton polypeptides from both sources were compared by peptide mapping following limited digestion with Staphylococcus aureus V8 protease. This phosphoprotein was tentatively identified as ribosomal protein S6 after analysis by pH 8.6/4.2 two-dimensional PAGE.

Radioautographic analysis of the secretory process in the parotid acinar cell of the rabbit.

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2-2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-(3)H, followed by a chase incubation, showed that the label is initially located (chase: 1-6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16-36 min), condensing vacuoles (chase: 36-56 min), immature granules (chase: 56-116 min), and finally mature storage granules (chase: 116-356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.

Purification of leukocytosis-promoting substance from bovine parotid gland extract.

A leukocytosis-promoting substance was purified from a crude bovine parotid gland extract. The purified substance was proved to be a single component by polyacrylamide gel disc electrophoresis. It stimulates an increase of peripheral leukocyte numbers in rabbits. The molecular weight of the physiologically active component was estimated to be 4.5 . 10(4), and the component was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be dissociated into two subcomponents.

The protein composition of rat parotid saliva and secretory granules.

Rat parotid saliva was collected by surgical cannulation of the ducts and stimulation with pilocarpine; The secreted salivary proteins were resolved on columns of DEAE-Sephadex into five major Fractions, I-V, which were characterized by polyacrylamide disc gel electrophoresis, amino acid analyses and enzymatic assay. Rat parotid secretory granules were isolated by density gradient centrifugation and lysed in hypotonic buffers. Granule content proteins were resolved and examined by the same techniques as for secreted proteins. In both experiments, Fraction I contained RNAase and a major unidentified protein, M1, Fraction II contained the isoenzymes of amylase; DNAase was present in Fraction III and, to a lesser degree, in Fraction IV. The proportions of the enzyme-containing peaks were the same in saliva and granule contents. Fractions IV and V contain proteins of unknown function; Fraction IV contains exceptionally high levels of glutamic acid, glycine and proline in its protein moieties and approx. 6-8% neutral sugars.

The proteins of the content of the secretory granules of the rat parotid gland.

The proteins of the secretory granules of the rat parotid gland were characterized by sodium dodecylsulfate gel electrophoresis, by chromatography of [3-H]proline-labeled proteins on DEAE-cellulose and by amino acid analysis. Sodium dodecylsulfate gel electrophoresis of the secretory granule content showed five principal proteins and a limited number of minor components. Only two of the principal bands could be identified as known secretory enzymes of the parotid gland. One was identified as the alpha-amylase and one as deoxyribonuclease. Peroxidase and ribonuclease form minor portions of the secretory proteins. The other three major proteins constitute, together, about 60% by weight, of the secretory granule content proteins. Of these, one which represents more than 30% of the total granule protein was found to contain uniquely high amounts of leucine residues (21 mole%). Another one of these principal proteins was relatively rich in cysteine residues (7 mole%). The fifth principal protein was found to contain high amounts of proline (28 mole%) glutamic acid (17 mole%) and glycine (18 mole%) residues. Its amino acid composition was very similar to that of the proline-se granules. This protein, however, differed from the "membranous" proline-rich proteins by several criteria. Two minor glycoproteins of the secretory granule content were also found to be rich in proline residues (37 mole%). As with the other proline-rich proteins of the granule, they contained no sulphur-containing amino acids, stained faintly pink with Coomassie Blue and were underestimated by the Lowry method. They differ however, from all the other proline-rich proteins of the granule by having a significantly higher content of threonine, less glycine (9 mole%) and much less glutamic acid (3 mole%). Of the principal proteins, only the deoxyribonuclease and the half-cystine-rich proteins were positively stained by periodic acid Schiff staining. The possible functions of the leucine-rich, the half cystine-rich and the various proline-rich proteins are discussed.

Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin.

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity.

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.

Inhibitor of protein synthesis co-isolating with polyribosomal RNA.

Although low concentrations of total polyribosomal RNA from porcine parotid glands or rat pituitary cells in culture (GH3) isolated by standard dodecylsulphate/phenol, chloroform extraction techniques can direct the incorporation of radiolabeled amino acids into proteins using a cell-free protein-synthesizing system derived from wheat germ embryos, higher concentrations inhibit the translation of endogenous wheat germ mRNA, or added rabbit globin mRNA or polyribouridylic acid. This inhibitory activity is separated from poly(A)-rich RNA by oligo(dT)-cellulose chromatography. The inhibitory activity appears to reside in a heat-stable protein since it is inactivated by incubation with various proteases but not by DNAase I, pancreatic ribonuclease, alkaline hydrolysis, or treatment with formamide. Specificity of the inhibition is suggested since the inhibitory fraction prepared from GH3 cells also inhibits protein synthesis in a cell-free protein-synthesizing system derived from porcine parotid gland, while the inhibitory fraction prepared from porcine parotid gland has no inhibitory activity in this homologous system. Radioiodination and dodecylsulphate/polyacrylamide gel electrophoresis reveal several protein bands, the most prominent with an apparent molecular weight of 78 000.

Immunochemical studies of an insulin-like material in the parotid gland of diabetic BB rats.

Immunocytochemical and radioimmunoassay studies were performed on pancreatic and parotid tissues from diabetic BB and control Wistar rats. Compared with those of normoglycemic controls, the pancreata of diabetic BB rats generally lacked insulin-containing B-cells. Extracts from the parotid glands of diabetic rats contained less immunoassayable insulin-like material than was present in parotid extracts of controls. However, the parotid glands of both groups of animals contained numerous cells displaying insulin-like immunoreactivity. These insulin-immunoreactive cells, located mainly in the intercalated portion of the duct system, were comparable to those we reported recently in the parotid glands of normal and streptozocin-diabetic Sprague-Dawley rats. The presence of an insulin-like material in the parotid salivary gland of two types of diabetic animals suggests that such cells may be spared, in part, from the effects of both chemical and hereditary diabetogenic factors.

Insulin-like material in parotid and submaxillary salivary glands of normal and diabetic adult male mice.

Acid-ethanol extracts of homogenates from the parotid and submaxillary salivary glands of normal and streptozocin-induced diabetic adult male mice were investigated for insulin-like material. Extracts of both the parotid and submaxillary glands contained insulin-like immunoreactivity. The values were 156 +/- 72 ng/g wet tissue in the parotid and 104 +/- 36 ng/g wet tissue in the submaxillary gland. Fractionation of this material on Sephadex G-50 (superfine) columns revealed a single peak corresponding to the elution volume of isotopically labeled insulin. Isolated fat cells were stimulated by these extracts to convert [14C]glucose to 14CO2. This effect was blocked by preincubation with anti-insulin serum. It was observed with the avidin-biotin immunocytochemical technique that both the parotid and submaxillary glands of adult male mice possess a population of cells containing an insulin-like material. After intraperitoneal injection of streptozocin there was a marked decrease of insulin-like material extractable from both the parotid and submaxillary glands. However, this beta-cell cytotoxic agent did not completely destroy the salivary cells containing the insulin-like material. These data suggest that both the parotid and submaxillary salivary glands may be extrapancreatic sources of insulin in mice.

Immunochemical studies of the insulin-like material in the parotid gland of rats.

Through the use of radioimmunoassay and immunocytochemical techniques, it was found that the parotid glands of male rats possess a population of cells that contain an insulin-like substance. These cells were situated mainly in groups along the intercalated ducts of the gland, or less frequently as isolated cells dispersed throughout the acini. No cells displaying insulin-like immunoreactivity were observed in the striated or main excretory ducts of the parotid. After intravenous (i.v.) injections of streptozotocin (STZ) there was a marked depletion of insulin from the pancreatic islets of rats having diabetes for a 3-27-day interval. Although this cytotoxin also reduced the amount of insulin extractable from the parotid, it did not destroy the insulin-like immunoreactive cells found in this gland. The results of this study suggest that the parotid may be an important source of extrapancreatic insulin. Moreover, these findings indicate that insulin-immunoreactive cells of this salivary gland are spared from the cytotoxic action of STZ.

Expression of mouse Amy-2a alpha-amylase genes is regulated by strong pancreas-specific promoters.

Three types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome. DNA sequence analysis reveals that both classes of Amy-2a genes specify the same unique pancreatic alpha-amylase mRNA species, since they share common exon sequences. Four independently cloned Amy-2a II isolates were found to be identical in all regions sequenced. This suggests that most, if not all, chromosomal Amy-2a II copies are identical. Amy-X is presumably a pseudogene, since its exon sequences, which are distinct from those of Amy-2a, are not detected in pancreatic alpha-amylase mRNA. We have determined the transcriptional activities of the Amy-2a genes by mapping in vitro elongated nascent transcripts to Amy-2a restriction fragments. Transcription initiation occurs at or close to the cap site. The expression of Amy-2a in vivo is under control of strong promoters, which are active exclusively in the pancreas. The accumulation of alpha-amylase mRNA in cells of the exocrine pancreas is regulated mainly at the transcriptional level. We have searched for pancreatic transcripts of Amy-1a, which specifies both parotid gland and liver-type alpha-amylase mRNAs. Surprisingly, the weak Amy-1a promoter, which directs the synthesis of the mRNA containing the liver-type leader sequence, also is active in the pancreas and, hence, in all alpha-amylase-producing tissues.

Immunoelectron microscopic localization of catalytic and regulatory subunits of cAMP-dependent protein kinases in the parotid gland.

The subcellular distribution of catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases has been studied by electron microscopy immunocytochemistry. The C-subunit was localized in the inner membrane-matrix space of mitochondria, at the cytoplasmic face of the rough endoplasmic reticulum (rER), in the nucleolus and in peripheral heterochromatin regions. A C-specific immunoreactivity was also found in specific domains of the basal and basolateral plasma membranes of acinar and duct cells, in centrosomes and on keratin filaments which anchor in desmosomes. The RI- and RII-subunits showed a basically similar subcellular distribution. A remarkably high RII-immunoreactivity, in the absence of C-immunoreactivity, was demonstrated in the secretory granules. These results demonstrate the presence of cAMP-dependent protein kinases or their subunits in many subcellular organelles. They also indicate a role for cAMP-dependent protein kinases in the regulation of a number of basal cellular functions as well as their importance in functional and structural cell-cell interactions.

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells.

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.

Rapid size determination of mRNAs complementary to cloned DNA sequences: plaque and colony hybrid-selection of cDNAs.

We describe a novel screening method for the rapid size determination of mRNAs. This method has been used successfully to identify the cDNA and genomic DNA clones that had been constructed using the plasmid vector pBR322 or phage vectors M13mp7 and lambda EMBL3. Poly(A) RNA is reverse-transcribed into 32P-labeled cDNA under conditions that yield a high proportion of full-length cDNA copies. This radioactive cDNA is then hybridized in situ to bacterial colonies or phage plaques harboring recombinant DNA molecules. Colonies or plaques containing sequences complementary to abundant or moderately abundant mRNAs are detected by autoradiography and excised from nitrocellulose filters. The hybridized cDNA is eluted from the filter by alkaline denaturation and sized directly by alkaline agarose gel electrophoresis. The mRNAs characterized thus far by this technique measure between 450 and 2100 nucleotides and account for 0.03% to 15% of the mass of cytoplasmic poly(A) RNA.

Zinc, magnesium, copper, and protein concentrations in human saliva: age- and sex-related differences.

Normal concentrations of trace elements in parotid saliva, supernatant- and sediment-mixed saliva, plasma, and hair were determined in 278 healthy adults grouped as young (18-29 y), middle-aged (30-64 y), and elderly (65-93 y). Age-related increases (p less than 0.05) were observed in concentrations of zinc in the supernatant of mixed saliva and parotid saliva, copper in plasma, and protein in all fractions of saliva studied. Concentrations of zinc in salivary sediment and plasma did not vary with age. Age-related decreases (p less than 0.05) were found in concentrations of magnesium in mixed-saliva supernatant, copper in salivary sediment, and zinc and copper in hair. Males had higher concentrations of zinc in plasma (p less than 0.05) and of copper in sediment (p less than 0.01) than did females but lower amounts of copper in plasma and of protein in parotid saliva (p less than 0.05). Concentrations of zinc in saliva were not correlated with those in plasma or hair. Copper in mixed-saliva supernatant was positively associated with concentrations in plasma but negatively related to concentrations in hair.

Isoprenaline-induced and constitutive members of a proline-rich protein sub-group from mouse parotid glands studied with monoclonal antibody NAL1.

Members of the proline-rich protein (PRP) family of mouse parotid glands were analysed before and after stimulation with the beta-agonist isoprenaline by using a monoclonal antibody raised against the induced PRP A3(0) (GP-27). Antibody NAL1 reacted strongly with isoprenaline-induced B-type PRP precursors and their salivary counterparts, but not against the A-type PRPs A1(0) (Gp-66) and A2(0) (GP-45) or human salivary proteins, and it is likely that NAL1 recognizes a proline-rich repeat variant unique to this group of rodent PRPs. PRP-related antigens were observed in the parotid glands (N1(0) and N2(0] and saliva of normal mice. The antigens were located immunocytochemically in secretory granules of parotid acinar cells of both normal and isoprenaline-stimulated mice. The total amount of PRP antigens increased 16-fold from 2.5 to 40% of parotid protein after 10 days of isoprenaline treatment, as estimated by enzyme-linked immunosorbent assay. Immunoblotting showed that new PRP species appeared during the period of increase. After treatment with isoprenaline, B-type PRPs appeared first, followed by A3(0) and another member of the family. These results show that the mouse PRP family is larger than previously thought and can be divided immunologically into sub-groups. That a subset of PRPs are produced in the normal mouse indicates that there is differential beta-adrenergic regulation within the family, and also has implications for the role of PRPs in the normal maintenance of healthy dentition and other processes.

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.

Immunocytochemical localization of insulin- and glucagonlike peptides in rat salivary glands.

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.