Kazunari Ishibashi

Roles of CLCA and CFTR in Electrolyte Re-absorption from Rat Saliva

A molecular basis for Cl− re-absorption has not been well-characterized in salivary ductal cells. Previously, we found strong expression of a rat homologue proposed to be Ca2+-dependent Cl− channels (rCLCA) in the intralobular ducts of the rat submandibular gland. To address the question as to whether rCLCA and cystic fibrosis transmembrane conductance regulator (CFTR) are involved in Cl− re-absorption, we evaluated the electrolyte content of saliva from glands pre-treated with a small interfering RNA (siRNA). Retrograde injection into a given submandibular duct of an siRNA designed to knock down either rCLCA or CFTR reduced the expression of each of the proteins. rCLCA and CFTR siRNAs significantly increased Cl− concentration in the final saliva during pilocarpine stimulation. These results represent the first in vivo evidence for a physiological significance of rCLCA, along with CFTR, in transepithelial Cl− transport in the ductal system of the rat submandibular gland.

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.

The Effects of p-Octopamine on Salivary Flow Rates and Protein Secretion by Rat Submandibular Glands

The effects of different doses of p-tyramine injected i.v. and i.p. on salivary flow rates and proteins secreted by the submandibular glands of rats were studied with and without various types of autonomic blockers and two enzyme inhibitors. The salivary flow rates and the amounts of protein secreted progressively increased with increasing doses injected both i.v. and i.p., whereas they were dramatically reduced with all autonomic blockers except the lowest doses of β-blockers, atropine, and yohimbine. Salivation in response to p-tyramine injected i.v. and i.p. was completely abolished by simultaneous injections of both prazosin and propranolol. The concentration of protein was not dose-dependent and was not reduced by yohimbine and phenoxybenzamine at almost all doses used. However, prazosin significantly increased the protein concentration. Protease activities were dose-dependent but were significantly reduced with a-blockers other than yohimbine, and with most β-blockers. The proteins secreted in response to p-tyramine at all doses injected i.v. and i.p. were of the a-type except with the lowest dose injected i.p. However, the a-type was completely replaced by the β-type in the presence of all a-blockers except yohimbine, but not with β-blockers, atropine, or two enzyme inhibitors. Pargyline, a monoamine-oxidase inhibitor, but not disulfiram, a dopamine-β-hydroxylase inhibitor, affected all parameters except the type of protein. Thus, p-tyramine may activate both the α1- and β1adrenoceptors in the submandibular glands of rats directly or indirectly.

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 Cholinergic and Peptidergic Agonists at Various Doses

Fluid and protein secretion by the submandibular glands of 25‐day‐old rats were examined and compared in response to three cholinergic and four peptidergic sialogogues at various doses.