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The linear viscoelastic and rheological properties of high molecular weight ovine submaxillary mucin (OSM) solution have been investigated in terms of the Newtonian steady-flow viscosity [eta(gamma)], the complex oscillatory viscosity [eta*(omega)], and the storage and loss shear moduli [G'(omega) and G"(omega)]. It was observed that tau(gamma), eta*(omega), and G'(omega) are always higher when OSM is dissolved in 0.1M NaCl than when at the same concentration in 6M GdnHCl. This is consistent with previous observations that submaxillary mucins self-associate in 0.1M NaCl to form large aggregates, which are disrupted in 6M GdnHCl. As the OSM concentration increases, the appearance of a plateau shear modulus indicates the formation of a gel network in both solvents. The results suggest gelation involves specific intermolecular interactions, perhaps due to hydrophobic forces between interdigitated oligosaccharide side chains. The viscoelastic behavior of OSM solution at high concentration is thus similar to that reported in the literature for porcine gastric mucin (PGM). However, the OSM gels are mechanically weaker, having moduli that are an order of magnitude lower than those for PGM gels of comparable concentration. The oligosaccharide side chains of OSM consist of only 1-2 sugar units compared to 10-15 for PGM, but it appears that this is sufficient to allow for intermolecular interaction and the formation of weak gels.

The localizations of chromogranins A, B, and C, neuron-specific enolase (NSE, gamma gamma-type) and non-NSE (alpha alpha-type), and different forms of somatostatins were immunocytochemically identified. The localizations were compared with those of epidermal growth factor (EGF) and nerve growth factor (NGF) in the submandibular salivary glands (SMG) of male mice at five to six weeks of age, with use of a variety of antibodies and the peroxidase-antiperoxidase (PAP) and avidin-biotin complex (ABC) detection methods. In the SMG of male mice, the major chromogranin present was chromogranin A, whereas chromogranins B and C were not detected at these ages by either method. Chromogranin A-like immunoreactivity was located in the granular convoluted tubule (GCT) cells of the SMG, whereas non-NSE immunoreactivity was observed throughout the duct system and in some acinar-associated cells. NSE was not detected in any part of the SMG. The distribution of chromogranin A and somatostatins in the GCT cells was similar to that of EGF and NGF. Our results strongly suggest that chromogranin A and somatostatins, but not chromogranin B or C, may be useful as a means of differentiation of the cells in the duct system of the SMG responsible for the production of biologically-active factors.

Essential fatty acid (EFA) deficiency has been proposed as a major pathogenic mechanism for cystic fibrosis (CF) and EFA-deficient animals have been proposed as an animal model for CF. In the present study, the elemental composition and ultrastructure of the acinar cells of the submandibular and parotid gland of the pancreas of EFA-deficient rats were investigated by X-ray microanalysis and electron microscopy. The effects of EFA-deficiency were compared to changes in these exocrine glands in chronically reserpine-treated rats, an established animal model for CF. EFA-deficiency did not cause any significant changes in the elemental composition of the acinar cells of the submandibular or parotid gland, or of the pancreas. The changes in elemental composition induced by reserpine treatment were only slightly modified by EFA-deficiency, mainly towards normalization. EFA-deficiency resulted in the presence of abnormal, electron translucent, zymogen granules in the parotid gland and in a reduction of the number of zymogen granules in pancreatic acinar cells. Since EFA-deficiency in rats only causes minor changes in structure and elemental composition of salivary glands and pancreas, and does not potentiate the effect of chronic reserpine treatment on these tissues, it is concluded that EFA-deficiency is likely to be of minor importance in the exocrine gland disturbances in CF.

Several growth factors have been evaluated for their effects on corneal wound healing but few studies have yet demonstrated an acceleration of endothelial repair in vivo. Mesodermal Growth Factor (MGF) was tested in vivo by making standardized freeze wounds in cat corneas and immediately injecting one of four concentrations of MGF in sterile phosphate buffered saline (PBS), or PBS alone, into the anterior chamber. Seven days later, the animals were sacrificed and the corneas excised and stained. Descemet's membrane and endothelium were dissected and mounted onto glass slides. The wound areas were photographed, measured and compared statistically. Those cats receiving the three lowest doses of MGF had significantly smaller wounds than controls (p less than 0.05).

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.

A variety of modifications of sialic acids have been described in nature. There are currently many difficulties in the detection and quantitation of these modified sialic acids from biological sources. We report here that fast-atom bombardment-mass-spectrometry (FAB-MS) of native sialic acids provides specific detection and quantitation of many previously known compounds. Derivatization of the sialic acids by reduction and peracylation under acidic conditions prior to FAB-MS provides further confirmation of their identity and improves the sensitivity of detection. Samples containing as little as 100 ng of a derivatized sialic acid loaded onto the FAB target allowed accurate identification. Mixtures of sialic acids could be analyzed, and minor components were seen, at levels undetectable by other currently known techniques. Analysis of known mixtures of different sialic acids gave reproducible relative signal intensities, indicating that quantitative data can be derived from the FAB-MS spectra. After reduction and peracylation, each sialic acid gave two major molecular ions, corresponding to the fully derivatized linear species and a lactone form, and a minor ion, corresponding to an anhydro form. Lactone formation was minimal in the case of four substituted sialic acids, indicating that the hydroxyl group at the 4-position is involved in lactonization. Differentiation between different positional isomers of the modified sialic acids could be achieved using controlled degradation with periodate, tagging of the fragments with p-aminobenzoic acid ethyl ester under acid reducing conditions, peracylation, and FAB-MS of the derivatized products. We used this FAB-MS strategy to identify a novel sialic acid, 8-O-methyl-7,9-di-O-acetyl-N-glycolyl-neuraminic acid from the starfish Pisaster brevispinus, and to demonstrate the presence of a previously undetected sialic acid, 4,8-anhydro-N-acetyl-neuraminic acid in acid hydrolysates of horse serum. We also use FAB-MS to show that the alkaline conditions traditionally used for analytical de-O-acetylation of sialic acids causes substantial conversion of 4-O-acetylated sialic acids into the same anhydro compound.

A highly sensitive and specific radioimmunoassay for the determination of rat family 2 cystatins has been developed. Antibodies against cystatin were elicited in rabbits after coupling the protein to thyroglobulin. Antibodies were specific for rat family 2 cystatin and did not cross-react with human salivary cystatin, rat family 3 cystatin (kininogen) or other salivary proteins. Fifty percent precipitation of 125I-cystatin was obtained with a 1:100,000 dilution of antisera. Optimal conditions for each step of the assay were established from a series of initial experiments. Precipitation of the antigen-antibody complex was achieved with 10% polyethylene glycol in 0.05 M phosphate buffer, pH 7.5 in the presence of 0.1% gamma-globulin. A standard curve constructed by plotting the fraction of bound 125I-cystatin against submandibular gland cystatin in a logit-log mode yielded a straight line for cystatin concentrations between 0.2 and 200 ng. The lowest limit of detection of family 2 cystatin by RIA was 0.2 ng, and is 1000 fold more sensitive than enzyme-inhibition assays and Laurell rocket electrophoresis. Only trace amounts of cystatin were detected in submandibular, sublingual and parotid glands from normal rats. After isoproterenol-stimulation cystatin levels increased dramatically in salivary glands and moderately in kidney and pancreas.

Using the hybridoma technique, monoclonal antibodies (Mabs) have been produced against three different types of human salivary proteins: high molecular weight mucin, a 20 kD glycoprotein and a 14 kD protein, identified as a member of the cystatin family. The Mabs appeared to be highly specific to their antigen in Elisa and immunoblotting tests. The Mabs were of the IgG-1 (against 20 kD glycoprotein) and IgM (against 14 kD protein and mucin) type. For the 14 kD protein and the 20 kD glycoprotein it was demonstrated that they are present mainly in submandibular-sublingual saliva. None of the antigens studied could be localized distinctly in the human parotid gland. In the submandibular gland, the three proteins have a different pattern of localization. The mucins have been detected particularly in the apical part of the mucous acinar cells, the 20 kD glycoprotein mainly in the serous acinar cells and the 14 kD protein in both serous acinar cells and striated duct cells.

Substance P is a member of the tachykinin peptide family and participates in the regulation of diverse biological processes. The polymerase chain reaction and conventional library screening were used to isolate a complementary DNA (cDNA) encoding the rat substance P receptor from brain and submandibular gland. By homology analysis, this receptor belongs to the G protein-coupled receptor superfamily. The receptor cDNA was expressed in a mammalian cell line and the ligand binding properties of the encoded receptor were pharmacologically defined by Scatchard analysis and tachykinin peptide displacement as those of a substance P receptor. The distribution of the messenger RNA for this receptor is highest in urinary bladder, submandibular gland, striatum, and spinal cord, which is consistent with the known distribution of substance P receptor binding sites. Thus, this receptor appears to mediate the primary actions of substance P in various brain regions and peripheral tissues.

Smooth muscle responses to kallikrein (EC 3.4.21.8) are generally considered to result from kinin formation. In the present study, this premise was reexamined with respect to the isolated rat uterus. Rat submandibular gland kallikrein produced contractions of the rat uterus but the contractions disappeared after successive additions of the same dose of the enzyme to the preparation. Kallikrein-induced rat uterine contractions as well as bradykinin-induced contractions were enhanced by rat submandibular gland bradykinin potentiating factor. The incubation of kallikrein with rat uterine extract in the presence of a kininogen-depleted rat uterus produced kinin which elicited the uterine contraction. An extract from uterine horns previously depleted of kininogen was prepared. Incubation of this extract with kallikrein in a bath containing a kininogen-depleted rat uterus did not evoke uterine contraction. The incubation of four rat uterine horns with kallikrein in the presence of a uterine horn previously depleted of kininogen elicited contractions of the depleted uterus. These results suggest that the contraction produced by kallikrein involves kinin release from the uterus.

The effects of calcium ions on the solution properties of porcine submaxillary mucin (PSM) have been investigated by static and dynamic light scattering. The weight average molecular weights of PSM fractions are unaffected by the addition of up to 0.5M CaCl2: these data are within experimental error of those for solutions in 0.1M NaCl. The distribution of relaxation frequencies derived from the dynamic data shows the existence of two distinct relaxation modes. The average relaxation times have been interpreted to yield the z-average translational diffusion coefficient and the longest intramolecular relaxation time tau1. A plot of tau1 vs the mean value of 1/Rh-3z is linear, and consistent with plots of such data recorded for PSM in 0.1m NaCl and 6M GdnHCl solutions. However, the tau values and the associated results for the mean value of R-1h-1z in 0.5M CaCl2 are smaller than those determined in 0.1M NaCl. This suggests that the conformation of PSM in CaCl2 solution is more contracted than those in the other two solvents. These results are consistent with the compact packaging of mucin in the secretary granules that have elevated Ca2+ levels.

During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.

The primary alcohol group on the carbon 6 of terminal galactosyl and N-acetylgalactosaminyl moieties of glycoproteins can be oxidized to an aldehyde by treatment with galactose oxidase. By reacting these aldehyde groups with 14C-labeled sodium cyanide, 14C-labeled cyanohydrin derivatives were obtained. Similarly, reduction of these aldehyde groups with tritiated sodium borohydride following standard procedures, yields 3H-labeled glycoproteins. 14C- and 3H-labeled derivatives of asialofetuin and asialo ovine submaxillary mucin with high specific radioactivities were prepared using these procedures. Mixtures containing microgram amounts of 14C- and 3H-labeled glycoproteins were subjected to column chromatography and gradient ultracentrifugation and the position of the individual glycoproteins was determined by simultaneous counting for 14C and 3H. These experiments demonstrate the usefulness of this approach for comparative analytical studies using biological specimens available in minute quantities.

The expression of GRP transcripts was found to be highly specific to the rat submandibular gland. GRP cross-reactive species were detected in the saliva of both inbred and outbred rat strains. There was no evidence of GRP transcripts in RNA prepared from bovine, ovine, porcine or murine submandibular glands. Thus the GRPs differ from the family of PRPs that are expressed in several species. The restriction of GRP expression to the rat suggests a relatively recent origin for a functional GRP gene, presumably after rat-mouse divergence. The ontogeny of the relative steady-state levels of GRP transcripts was assessed by dot blot analysis. Maximal levels of RNA-encoding GRP were detected at 6 months; there was a significant age-related decline at both 12 and 18 months. There was, however, no significant age-related alteration in the size of transcripts which encode this protein family.

A system has been developed for the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK-9) and the gamma-subunit of nerve growth factor (mGK-3), as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factor interactions observed in this family of proteins.

We previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termed IsK protein, is small and different from the conventional potassium channel proteins but induces selective permeation of potassium ions on its expression in Xenopus oocytes. In this investigation, we examined cellular localization of rat IsK protein by preparing three different types of antibody that specifically reacts with a distinct part of rat IsK protein. Immunohistochemical analysis using these antibody preparations demonstrated that rat IsK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of rat IsK protein was consistent with that of the IsK protein mRNA determined by blot hybridization analysis. In epithelial cells, the sodium, potassium-ATPase pump in the basolateral membrane generates a sodium gradient across the epithelial cell and allows sodium ions to enter the cell through the apical membrane. Thus, taking into account the cellular localization of the IsK protein, together with its electrophysiological properties, we discussed a possible function of the IsK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

The water release from the sublingual, parotid and submandibular glands of male and female rats was analyzed by thermal analysis in order to detect the total water content and types. Different types of water, which are increasing from the sublingual to the parotid gland, were found and the relative distribution appeared to be a function of the bond energy of water to glandular components. In addition, evidence of a sexual dimorphism in the rat sublingual gland was demonstrated.

Investigations carried out on the digestive tract of rats submitted to an 18 1/2-day orbital flight aboard the Soviet biosatellite Cosmos 1129 revealed that flight produces a reduction of the glycoproteins (GP) of the submaxillary glands, gastric mucosa and the goblet cells of the small and large intestine, the last being the most nonreactive, as well as an augmented staining for leucine-aminopeptidase (LAP) and acid phosphatase (AP). In lamina propria an accumulation of eosinophils was noticed. These changes were more intense than those produced in the groups of rats maintained "synchronous" under environmental conditions similar to flight. On the basis of previous data obtained by various authors and by ourselves on hypokinetic rats from the biosatellite Cosmos 936, it can be concluded that these reactions have a nonspecific, stress character. A stress of restraint applied daily for 2 1/2 hours during a period of 6 days to the animals immediately after landing produced modifications similar to those following flight but more intense, thus underlying the hypothesis of a stress reaction.

A time-course study was carried out of the levels of submandibular kallikrein, serum insulin and blood glucose of rats rendered diabetic by alloxan or streptozotocin. The permanent hyperglycemia seen one day after treatment and the subsequent relative changes in the levels of blood glucose recorded over the following nine days were in agreement with previous observations. A significant reduction in the concentration of submandibular kallikrein did not become apparent until ten days following the injection of alloxan or streptozotocin. Thus the enzyme would not appear to have played either a causal or a preventative role in the production of the hyperglycemic state. Furthermore, in disagreement with previous speculation, the submandibular gland had not compensated for the deficiency of insulin with an increase in production of kallikrein. The fall in the level of serum insulin had preceded the decrease in submandibular kallikrein. However, administration of exogenous insulin over a period of three days did not bring about an increase in the concentration of submandibular kallikrein of the diabetic rats. Thus insulin would not seem to be involved in controlling the level of kallikrein in the submandibular gland.

Occurrence and growth-promoting effect suggest that epidermal growth factor (EGF) is involved in the maintenance of the gastrointestinal mucosa. The aim of this study was to ascertain whether rats with gastric lesions, induced by cold-water stress, ethanol, or indomethacin, have altered EGF levels in their gastrointestinal tract compared with controls. For this purpose we established a solid-phase enzyme immunoassay and measured the amount of immunoreactive EGF (IR-EGF) in extracts of the submandibular gland, stomach, duodenum, jejunum, ileum, and colon. In the submandibular glands of control rats the IR-EGF content was 100-400 times higher than in the gastrointestinal tract. In animals with experimentally induced ulcer the IR-EGF content was increased in the submandibular glands (1480 versus 423 ng/g), duodenum (11.4 versus 5.8 ng/g), and colon (4.1 versus 1 ng/g), whereas no change was observed in the stomach and jejunum. When the formation of lesions was prevented by omeprazole (stress) or prostaglandin E2 (ethanol and indomethacin), the IR-EGF increase in submandibular glands and duodenum was abolished. This indicates that mechanisms regulating the synthesis and secretion of submandibular EGF might be of importance in the development of gastric and duodenal ulcers.

The transcriptional activity of renin genes in the mouse kidney and submandibular gland (SMG) was examined by measurement of renin messenger RNA (mRNA) (nanograms per g tissue) and compared with renin activity (micromoles angiotensin I per h/g tissue). In control adult mice renin mRNA and renin activity (in parentheses) were 1.8 +/- 0.24 (11 +/- 1.1) in male kidney, 3.6 +/- 0.66 (18 +/- 2.8) in female kidney, 230 +/- 34 (903 +/- 59) in male SMG, and 31 +/- 6 (188 +/- 47) in female SMG (mean +/- SE, n = 6). The ratio of renin mRNA among these four tissues was similar to that of renin activity (1:2:100:17, respectively). Although the values in male kidney were one one-hundredth those in SMG, 1000 to 10000 times more SMG cells are involved per gram of tissue so that, per renin-synthesizing cell, kidney values would be 10 to 100 times SMG values. Expression of renin gene(s) in a renal juxtaglomerular cell may thus be higher than in a SMG granular duct cell. Values in adult male SMG were a consequence of a 40-fold rise at puberty, were decreased to 16% (+/- 3.8) by castration, but were not significantly influenced by treatment with testosterone, T4, propylthiouracil, sodium depletion, or spironolactone. Renin mRNA in adult female SMG was 18-fold higher than juvenile values, was increased 10-fold (+/- 1.6) by testosterone (to adult male levels) and 5.5-fold (+/- 0.81) by T4 (P less than 0.005), but was decreased to 42% (+/- 29) of normal by propylthiouracil (P less than 0.05). Propylthiouracil caused a small but significant decrease in testosterone-treated female values. In the kidney renin mRNA was increased 3.0-fold (+/- 0.30) by captopril, 2.3-fold (+/- 0.23) by a low sodium diet, and 1.7-fold (+/- 0.13) by spironolactone after 1 week, whereas T4, testosterone, or propylthiouracil had little or no effect. Sialoadenectomy increased renin mRNA and renin in male but not in female kidney, suggesting that a SMG factor, possibly renin, may have a role in suppression of renin synthesis in male kidney. In conclusion, measurement of renin mRNA suggests that testosterone regulates renin gene expression by a direct effect in male mouse SMG, whereas in the female regulation is by thyroid hormone. In the kidney conditions which increase renin content are accompanied by parallel (5-fold higher) increases in renin mRNA, suggesting enhanced expression of renin gene(s) in renal juxtaglomerular cells in chronic low sodium states.

Fast atom bombardment mass spectrometry has been used to characterize epidermal growth factor isolated from mouse submaxillary glands. The preparation is found to consist of two peptides, one of which has the average molecular weight predicted for the familiar gene product. The molecular weight of the second component is found to be reduced by the mass of one asparagine residue. These observations are discussed in light of previous reports of heterogeneity.

Whilst the content of epidermal growth factor in the submandibular salivary glands has been considered to be controlled primarily by the hormonal secretions of the testes, our previous studies indicated that the adrenal glands also affect the cellular content of epidermal growth factor in these glands. In the present study, the influence of the thyroid gland was investigated. One hundred and ten males Swiss White mice were subjected to orchidectomy and/or bilateral adrenalectomy, or thyroidectomy, and their submandibular salivary glands examined at 90 days of age. Some of these received specific hormone replacement prior to examination of the glands. The presence of epidermal growth factor was detected by indirect immunofluorescence, and adjacent sections of tissue were stained with alcian blue and acid fuchsin to demonstrate the convoluted granular tubules, which are the site of storage of the factor in the glands. In the glands of normal males, these tubules were prominent and showed strong factor-positive immunofluorescence. Administration of thyroxine reversed orchidectomy-induced atrophy of the tubules, and restored factor content, but did not restore normal tubular morphology or factor content in orchidectomised-adrenalectomised animals. In these latter animals, combined administration of thyroxine and cortisone resulted in a return to normal structure and growth factor content in the cells of the convoluted granular tubules. The glands from thyroidectomised males had atrophied tubules, which did not contain demonstrable epidermal growth factor. These effects were reversed by the administration of thyroxine, testosterone, thyroxine and testosterone, or thyroxine and cortisone. The results show that the content of epidermal growth factor-positive cells in the convoluted granular tubules of the submandibular salivary glands of male mice is under the control of the thyroid as well as the testes and adrenal glands.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

A morphological and histochemical study was carried out on the submandibular gland of the hare (Lepus europaeus). The results indicated that it is a characteristic tubulo-acinar gland formed by terminal tracts, preterminal tracts and excretory ducts of various calibre. The striated ducts are formed by dark and pale cells with the pale cells predominating. The results of histochemical staining show that the preterminal tracts did not react to any of the methods used, while the terminal tracts reacted intensely to high or low iron diamine-thiocarbohydrazide-silver proteinate and tannic acid-uranyl acetate, indicating the presence of sulphated, acidic and neutral glycoconjugates. In particular in the terminal tracts the different structural appearance of the electron-lucent granules corresponded to different reactivity. The morphological and histochemical data were compared to those of the rabbit submandibular gland and the functional significance of the cells in the terminal and preterminal tracts was discussed.

A new transplantable murine salivary gland carcinoma (myoepithelioma) was further characterized by comparing total protein patterns with normal submaxillary gland proteins. Analysis by isoelectric focussing or by labelling studies with radioactive leucine showed considerable differences between tumour and normal proteins. In contrast, analysis of staining patterns after electrophoresis in sodium dodecyl sulphate containing gels revealed a striking similarity between normal proteins, tumour proteins and proteins obtained from metastatic growths. It thus appears that tumour proteins closely resemble the normal proteins from the tissue of origin with respect to molecular size as determined by electrophoretic mobility, while significant differences occur in charge and labelling kinetics.

Immunoreactive vasoactive intestinal peptide (VIP) and substance P (SP) were studied in parotid, submaxillary and sublingual glands of the rat. The concentration of VIP was highest in the submaxillary gland and lowest in the parotid gland. The concentration of SP was highest in the parotid gland; it was at, or below the limit of detection in the sublingual gland. In the parotid gland the total amounts of VIP and SP were reduced by 95% after parasympathetic denervation (section of the auriculo-temporal nerve). In the submaxillary gland the total amounts of the peptides were unchanged after parasympathetic decentralization (section of the chorda-lingual nerve). In this gland the total amount of SP was reduced by 92% and that of VIP by 50%, when the chorda tympani nerve fibres were cut deep into the hilum. Cutting the nerve fibres at the hilum left the total amounts of the peptides unchanged in the submaxillary gland, whereas in the sublingual gland the total amount of VIP was reduced by 70%. Sympathetic denervation did not reduce the total amounts of the peptides. Duct ligation caused gland atrophy. In the parotid gland the total amounts of VIP and SP were reduced by 40%. In the submaxillary gland the same percentage reduction occurred with regard to SP; however, the total amount of VIP was reduced by 99%. The VIP- and SP-containing nerve fibres reach the salivary glands by the parasympathetic nerves. In both submaxillary and sublingual glands a certain fraction of VIP originates within the glands.

Tonin was purified from rat submaxillary gland homogenates by affinity chromatography on soybean trypsin inhibitor coupled CH-Sepharose 4B and two additional steps of conventional chromatography. The use of affinity chromatography by soybean trypsin inhibitor coupled CH-Sepharose 4B permits a new approach in the purification of tonin, since it can completely separate in one step troublesome contamination of the enzymes which showed tosyl-L-arginine methyl ester hydrochloride esterase activity. Tonin is purified 11-fold to a homogeneous state on polyacrylamide gel electrophoresis at a yield of 21%.

The physicochemical property and immunohistochemical localization of progestin (P) and estrogen (E) receptors (PR and ER) were examined in the submandibular gland (SMG) of 5-8-week-old castrated rats. The localization of epidermal growth factor (EGF) was simultaneously examined in the same tissue. The tissue cytosols from male and female rats specifically bound 3H-promegestone (3H-R5020) and 3H-estradiol-17 beta with high affinity and low capacity; the values were within the range of those reported for other tissues. However, E-treatment suppressed the specific P-binding in the female, whereas it did not in the male. On the contrary, E-treatment did not at all suppress specific E-binding in both sexes. Monoclonal antibodies against PR and ER were mainly located in the epithelium of the excretory duct and granular convoluted tubule, but not in the acinus. The monoclonal antibodies were also located in the large polygonal cell with irregular cell border, probably macrophage in the tissue. The EGF-immunoreactivity was observed in the epithelium of the same tissue region as that in which the monoclonal antibodies were located. The present results clearly suggest that the rat SMG tissue contains specific PR and ER that are mainly located in the epithelium of the duct system where EGF-producing cells are also located. The possibility that P and E may influence EGF-production through their receptors in this tissue was discussed.

Epidermal growth factor (EGF)-like activity was isolated for the first time from the submaxillary gland (SMG) and the velvet antler of red deer (Cervus elaphus) by a combination of Sephadex gel or DEAE-Sephacel and IMAC columns in succession. The semipurified cervine EGF-like activity (cEGF), with specific activity of 4.7 ng/micrograms protein from the velvet tissues, can generate a completely parallel competitive binding curve against mouse EGF in both radioreceptor assay (RRA) and radioimmunoassay (RIA). Mitogenic activity of EGF from both tissues was demonstrated by stimulating the incorporation of [3H]thymidine in two different cell lines of fibroblast culture in a dose-dependent manner. The velvet layer may be the site of EGF synthesis outside the SMG.

Human milk beta-N-acetylglucosaminide beta 1 leads to 4-galactosyltransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [14C]galactosylated product chain was obtained as a reduced oligosaccharide by beta-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is Gal beta 1 leads to 3([14C]Gal beta 1 leads to 4GlcNAc beta 1 leads to 6)GalNAcol. Thus it appears that Gal beta 1 leads to 3(GlcNAc beta 1 leads to 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of alpha-N-acetylgalactosaminylprotein beta 1 leads to 3-galactosyltransferase activity using ovine submaxillary asialomucin as an acceptor can be counteracted by the addition of N-acetylglucosamine.

The aim of this study was to investigate the effects of glycoconjugate components extracted from the submandibular glands of four mammal species (rat, mouse, rabbit, hare) on human hemostatic system. The following analyses were performed: thromboelastography and hemocoagulation screening tests (Thrombin Time, Prothrombin Time, Partial Thromboplastin Time). Findings showed that all glycoconjugates induce modifications of fibrin clot formation time, modulus of elasticity and some hemocoagulation tests (TT, PTT). The anticoagulant effects were of inhibitory type.

Epidermal growth factor (EGF) has been measured in extracts of submandibular glands from mice with hereditary muscular dystrophy. RIA results show that adult male and female dystrophic mice have significantly less submandibular gland EGF than do unafflicted controls. Despite the differences in gland content of the protein, serum levels of EGF are similar in both dystrophic and control animals. Furthermore, submandibular gland concentrations of amylase are normal in the dystrophic mice, indicating that not all proteins synthesized by the glands are affected. Gel filtration studied reveal that the elution properties of EGF in extracts of glands from dystrophic and control animals are indistinguishable. Unexpectedly, the chromatographic profiles indicate that most of the EGF in gland extracts elutes as a low molecular weight protein when the molecule is studied at low, biologically active concentrations; only a small portion of the protein is associated with a high molecular weight complex. Under the same experimental conditions, submandibular gland nerve growth factor maintains its association with other components in a high molecular weight form.

Stainings were effected on an ultrastructural histochemical level to localize sulphomucins in the submandibular and sublingual glands of growing mice and rabbits. Sulphates behave in an entirely different way in the salivary glands of Lagomorpha and in those of Rodents. In growing rabbits sulphates can be demonstrated in both glands; at maturity they can be demonstrated only in the sublingual gland and no longer in the submandibular gland. In the salivary glands of Rodents, sulphates cannot be demonstrated histochemically in new born subjects or in adults. The histochemical results are compared to the biochemical ones, and discrepancies, where present, are discussed.

The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied; After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver straining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.

The effects of a single dose of reserpine or isoproterenol on the acinar cells of rat submandibular gland were investigated by electron microscopy and X-ray microanalysis. About 4 h after injection of reserpine, the cellular Ca concentration started to increase and at 24 h a level two to three times the control value was reached. The mucus content of the cells increased in parallel with the Ca concentrations. Isoproterenol stimulation depleted the acinar cells of their mucus. Resynthesis of mucus was visible at 8-12 h after injection. Changes in cellular Ca concentrations followed the mucus content of the cells. After 24 h both the mucus content and the Ca concentration were significantly increased over the control values. High-resolution X-ray microanalysis on thin cryosections showed that the calcium was accumulated in the mucus granules, both after reserpine and after isoproterenol administration. The newly synthesized mucus was also more calcium-rich than the original mucus. The effect of a single isoproterenol stimulation on elemental content of rat submandibular gland acinar cells was not significantly different from that of chronic isoproterenol treatment. Chronic treatment with reserpine resulted in a higher cellular Na/K ratio than a single dose.