Toshihiro Nishiura

Isolation of three forms of cystatin from submandibular saliva of isoproterenol-treated rats, its properties and kinetic data

Three rat salivary cystatins (designated as RSC-1, RSC-2 and RSC-3) induced by chronic isoproterenol (IPR) treatment, but not detected in normal rats, were purified from submandibular saliva of chronically IPR-treated rats by Mono-Q, hydroxyapatite and TSKgel Phenyl-5PW chromatographies. Their molecular weights (Mr) and isoelectric points (pI) differed from each other as follows: RSC-1 (Mr 16,500, pI 4.4), RSC-2 (Mr 15,500, pI 4.4) and RSC-3 (Mr 14,500, pI 4.5). The amino acid compositions of these inhibitors were very similar and the three forms showed complete immunological identity in a double immunodiffusion system. The partial amino acid sequence results showed that these inhibitors belonged to family 2 of the cystatin superfamily. These three forms strongly inhibited the enzyme activities of ficin and papain, but not of cathepsin B and trypsin. The inhibition constants (Ki) of RSC-1, RSC-2 and RSC-3 for ficin were 0.19, 0.50 and 0.012 nM, and for papain were 1.5, 0.93 and 0.03 nM, respectively. RSC-3 inhibited ficin and papain more strongly than did RSC-1 and RSC-2.

Immunocytochemical study of cathepsin L and rat salivary cystatin-3 in rat osteoclasts treated with E-64 in vivo

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomal vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC-3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.

Postnatal changes of gene expression for tissue inhibitors of metalloproteinase-1 and -2 and cystatins S and C, in rat submandibular gland demonstrated by quantitative reverse transcription-polymerase chain reaction

The rat submandibular gland is not fully developed at birth and definitive differentiation takes place postnatally. The steady-state mRNA expression for the four proteinase inhibitor molecules, tissue inhibitors of metalloproteinase (TIMP)-1 and -2, and cystatins S and C, and for a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), in rat submandibular glands was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) at different stages of postnatal development. The gene-expression patterns of TIMP-1 and -2 relative to G3PDH were similar to each other. The TIMP-2 and cystatin C genes were more highly expressed than those of TIMP-1 and cystatin S at all stages. Moreover, the gene expressions of TIMP-1 and -2, and of cystatins S and C, were predominant between 1 and 7, and 7 and 12 weeks of age, respectively, and coincided developmentally with the regression of terminal tubule cells and the differentiation of granular convoluted tubule cells, respectively. Quantitative competitive RT-PCR allowed accurate measurement of small changes in the steady-state concentrations of these proteinase-inhibitor mRNA molecules.

The effects of clonidine and three 2-imidazoline derivatives on the secretion of protein and some electrolytes by rat submandibular and parotid glands

1. Three imidazoline analogues of clonidine were potent secretagogues for the parotid and submandibular glands at relatively high doses. 2. Salivation in response to clonidine was completely abolished by prazosin, phentolamine, phenoxybenzamine and dihydroergotamine. 3. The gamma-type of proteins was secreted in response to three of the analogues, whereas with p-aminoclonidine the alpha-type of proteins was secreted by the submandibular gland. 4. Albumin was specifically secreted by the submandibular gland in response to clonidine but not to isoproterenol or phenylephrine.

Expression and postnatal changes of adrenergic receptor subtype mRNA in rat submandibular glands.

Adrenergic receptors (ARs) are involved in regulating saliva secretion and composition in salivary glands. Nine AR subtypes, including three alpha1-ARs (alpha1a-, alpha1b- and alpha1d-ARs), three alpha2-ARs (alpha2A-, alpha2B- and alpha2C-ARs) and three beta-ARs (beta1,beta2- and beta3-ARs), have been identified through molecular cloning. The five subtype genes, alpha1a-, alpha1b-, alpha2A-, beta1-, and beta2-ARs, were expressed in rat submandibular glands. In contrast, the other four subtype mRNAs, alpha1d-, alpha2B-, alpha2C- and beta3-ARs, were not detected by reverse transcription-polymerase chain reaction (RT-PCR). The steady-state mRNA expression for the five AR subtypes in rat submandibular glands was measured by quantitative competitive RT-PCR using synthetic DNA as internal standard at different stages of postnatal development. The relative rank order of AR subtype mRNA expression was alpha1a>beta2>beta1>alpha2A>alpha1b at all stages except that beta1- and alpha2A-subtypes were reversed at 2 weeks of age. The gene expression of alpha1a-AR subtype relative to total AR was low at 2 weeks of age and increased and reached a maximum at 6 weeks of age, whereas those patterns of alpha2A-, beta1- and beta2-AR subtypes were similar to each other and their gene expressions were high at 2 weeks of age and then decreased. On the other hand, the gene expression of alpha1b-AR subtype did not change over the different stages in relation to that of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and to total AR. Although rat submandibular glands contain the five AR subtype mRNAs, distinct subtype-specific expression is evident.

The effects of m-octopamine on salivary flow rates and protein secretion by rat submandibular glands

1. m-Octopamine given i.v. or i.p. was a potent sialogogue for rat salivary glands. 2. Salivation in response to i.v. m-octopamine was completely abolished by prazosin and phenoxybenzamine. 3. The alpha-type of proteins were secreted in response to all doses of i.v. and i.p. m-octopamine and these were converted into the beta-type with prazosin, but not with yohimbine. 4. m-Octopamine stimulated both alpha- and beta-adrenoceptors and was a much more selective alpha 1-agonist than was the p-isomer.

Cyclic Mechanical Strain Induces Interleukin-6 Expression via Prostaglandin E2 Production by Cyclooxygenase-2 in MC3T3-E1 Osteoblast-like Cells

Abstract Interleukin (IL)-6, produced by osteoblasts, is one of the key cytokines that promote bone resorption. This study examined the effect of cyclic mechanical strain on the expression of IL-6 mRNA and protein, and the induction mechanism of IL-6 in MC3T3-E1 osteoblast-like cells using RT-PCR and ELISA. MC3T3-E1 cells were cultured in α -MEM with 10% FBS on flexible, collagen I-coated membranes and subjected to cyclic mechanical strain at 6 cycles/min. The production of IL-6 protein was increased at 3 h, reached a peak at 6 h, and was maintained up to 24 h by the cyclic mechanical strain. The strain increased the production of IL-6 protein in a force-dependent manner. The expression of IL-6 mRNA was increased by the cyclic mechanical strain. Prostaglandin(PG)E 2 production was induced at 1 h, dramatically increased to 3 h, and was gradually increased from 3 to 24 h by the cyclic mechanical strain. The strain increased PGE 2 production in a force-dependent manner. Treatment with PGE 2 increased the production of IL-6 protein from MC3T3-E1 cells. The cyclic mechanical strain promoted cyclooxygenase (COX)-2 but not COX-1 mRNA expression. Pretreatment with indomethacin and NS-398 prevented PGE 2 production and partly inhibited the production of IL-6 protein induced by the cyclic mechanical strain. These findings suggest that cyclic mechanical strain increases IL-6 expression in osteoblasts, the induction of which is in part mediated via PGE 2 production by COX-2.

Abnormally High Levels of Cystatin S in Submandibular Glands, Saliva, and Gingiva of Plaque-resistant Rats

To identify salivary biomarkers of periodontal diseases, we used plaque-resistant and -susceptible rats as animal models. The levels of salivary cystatin S in saliva, salivary glands, and gingiva were tested in Nembutal-anesthetized young and adult plaque-resistant and -susceptible rats of both sexes with and without chronic treatment with isoproterenol. Isoproterenol was injected i.p. once a day for 4 or 6 consecutive days. Isoelectric focusing electrophoresis by the PhastSystem and the Western blotting method were used to separate different proteins and to identify a salivary cystatin S band in these samples. The expression of salivary cystatin S mRNA was also determined by the Northern blotting method. Depending upon the types of agonists, a few differences were observed in secretory functions between both strains of rats in both sexes, but the levels of salivary cystatin S in saliva elicited from the submandibular gland and in the extracts of the submandibular glands and gingiva were significantly higher in plaque-resistant rats when compared with those of plaque-susceptible rats in both sexes. However, no significant difference was seen between the strains after chronic treatment with isoproterenol. The N-terminal 26-amino-acid sequence of salivary cystatin S purified from submandibular saliva of plaque-resistant rats was identical with that purified from submandibular saliva of Sprague-Dawley rats subjected to chronic treatment with isoproterenol. The expression of salivary cystatin S mRNA was dramatic in the submandibular glands of the plaque-resistant rats and in the submandibular glands of Wistar rats subjected to chronic treatment with isoproterenol, but not in those of plaque-susceptible rats. These results suggest that salivary cystatin S might be a good biomarker in distinguishing between the two strains of rats and that its concentration is correlated with plaque resistance.

Translocation of orally administered rat salivary cystatin into serum and kidney

This translocation was followed by means of a sensitive enzyme immunoassay. Salivary cystatin could be detected in serum, oesophagus, stomach, small intestine and kidney 15 min after oral administration. The salivary cystatin level in serum and kidney extract was maximal 15-60 and 30-60 min, respectively, after its administration and increased dose-dependently. Chromatography showed that the cystatin molecule in serum was identical to that in the submandibular saliva used for oral administration, but differed from that in kidney extract. These results suggest that salivary cystatin is absorbed intact by the gastrointestinal tract and reaches the bloodstream, from which it is taken up by the kidney and modified.

Fluid and protein secretion by the submandibular glands of weanling rats in response to different doses of various adrenergic agonists.

The present study was designed to determine whether the submandibular glands of weanling rats respond to α1-and β-adrenoceptor agonists in the same manner as glands from adult animals, to determine the doses of these agonists, which give the maximum responses, and using isoelectric focusing and twodimensional electrophoreses, to determine whether proteins in submandibular saliva are modified posttranslationally as they pass through the duct systems. All of the agonists used were potent sialogogues and were given i.p. over a wide range of doses. The volumes of saliva, the total output of protein and the relative activity of protease progressively increased with increasing doses of each agonist. The optimal dose of each agonist was neither low nor excessively high. The submandibular glands of weanling rats seem to be as fully developed for fluid and protein secretion as those of adult rats. In addition, no special protein was involved in saliva secreted by the submandibular glands of weanling rats at any of the doses of the various agonists used here. However, some proteins in submandibular saliva of 25-or 35-day-old rats, when compared with extracts of the submandibular gland, appeared to have been modified posttranslationally or degraded by proteases while passing through the duct systems of the submandibular glands of weanling rats.