Kimio Abe

The Effects of Epinephrine, Norepinephrine, and Phenylephrine on the Types of Proteins Secreted by Rat Salivary Glands

The types of proteins secreted by rat submandibular glands in response to secretory stimulation with epinephrine, norepinephrine, or phenylephrine were dose-dependent. At relatively high doses the proteins secreted were characteristic of α-adrenergic stimulation, whereas at low doses they were characteristic of β-adrenergic stimulation.

A NEW METHOD FOR THE STUDY OF THE FORMATION AND TRANSFORMATION OF CALCIUM PHOSPHATE PRECIPITATES : EFFECTS OF SEVERAL CHEMICAL AGENTS AND CHINESE FOLK MEDICINES

A simple method of assaying the formation of amorphous calcium phosphate and its transformation to hydroxyapatite using a conventional pH meter and recorder is described. Its validity was confirmed by direct assay of calcium consumption with atomic absorption spectrophotometry. The method was used to study substances which influence the formation of amorphous calcium phosphate and its transformation to hydroxyapatite, such as albumin, casein, chondroitin sulphate, phospholipid, ATP, Mg2+, Sr2+, pyrophosphate and several Chinese folk medicines.

Effects of Parasympathomimetic and Sympathomimetic Drugs on the Secretion and Composition of Rat Sublingual Saliva

The sublingual glands of adult, male rats secreted considerable volumes of saliva in response to several cholinergic agonists, but very little in response to α-adrenergic stimuli and none in response to a β-adrenergic agonist. The types of proteins secreted were quite different from those in submandibular and parotid saliva or serum, and were largely independent of the nature of the stimulus. However, some differences in staining intensity of electrophoretic patterns occurred both between stimuli and with the same stimulus used on different rats.

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.

Changes in protein secretion by rat submandibular salivary glands after enlargement caused by repeated amputation of the lower incisor teeth

Abstract The lower incisors of male rats were repeatedly amputated at 3- or 4-day intervals for about 1 month. Four days after the last amputation, submandibular saliva was collected from the anaesthetized rats by intra-oral cannulation, using methoxamine (6 mg/kg), an α-adrenomimetic drug, as an acute secretory stimulus. The rats showed enlarged submandibular and sublingual glands and the protein concentration was significantly less in submandibular saliva than in controls. The types of protein secreted by the rat submandibular gland in response to methoxamine normally differ from those secreted in response to isoprenaline. In the enlarged glands, methoxamine stimulation caused the secretion of proteins similar, by polyacrylamide gel electrophoresis, to those normally secreted in response to isoprenaline, suggesting that repeated lower incisor amputation had caused the glands to lose their normal responsiveness to α-adrenergic agonists. An abnormal protein was also secreted, identical electrophoretically and immunologically with one secreted by the enlarged submandibular glands of rats exposed to chronic isoprenaline administration.

Possible regulatory roles of silicic acid, silica and clay minerals in the formation of calcium phosphate precipitates

The effects of silicic acid, silica and clay minerals on the conversion of amorphous calcium phosphate to hydroxyapatite (HAP) were studied in vitro by a pH drop method. At a concentration range of 0.01-0.1 mM. silicic acid stimulated the rate of HAP transformation by about 30-40%. Silica stimulated the rate of HAP transformation by 33-43% at a concentration range of 0.05-1.5 mg/ml. The clay minerals, i.e. kaolin and talc, also stimulated the rate of HAP transformation by 40-90% at a concentration range of 0.4-10 mg/ml, but mica inhibited the reaction markedly at 10 mg/ml. The distribution of silicon in human supragingival dental calculus was studied by an electron-probe microanalyser. We found localized silicon distribution on the oral surface of the calculus. Such silicon-rich areas always contained silicon either alone or together with magnesium, aluminium, potassium, calcium and iron. This implies that the silicon-rich area may be opal and mica. Because silicic acid, silica, kaolin and talc stimulated and mica inhibited the in vitro calcium phosphate precipitation, it is possible that these silicon-rich areas may regulate the formation of the dental calculus.

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.

Changes in Protein Secretion by Rat Submandibular Glands in Response to Isoproterenol, a-Methylnoradrenaline, and Clonidine during Post-natal Development

We studied developmental changes in salivary volumes and proteins secreted by the submandibular glands of male rats at weekly intervals from two to ten weeks of age in response to the β1-, α1-, and α2- adrenoceptor agonists, isoproterenol (IPR), a-methylnoradrenaline (a-mNA), and clonidine (Clonid). The types of proteins in saliva samples were determined and compared by isoelectric-focusing electrophoresis with the Phast system in both the gradient pH -3.5-to-5 and pH-3.5-to-9 gels by means of silver staining. Salivary volume and protein concentration in saliva samples elicited by IPR and a-mNA were positively related to the weight of the submandibular glands up to six or seven weeks of age, whereas in saliva elicited by Clonid, no relation was found. The isoelectric-focusing electrophoretic patterns of proteins secreted by the glands in response to three stimuli were different from each other during post-natal development. Within one stimulation, differences were also observed at two and three weeks of age for Clonid, and from seven weeks of age for the three stimuli, respectively. The a-type proteins, but not the β-type proteins, were very similar to those in extracts from glands of rats at seven weeks of age. Almost all of the a-type proteins, but not the β-type proteins, reacted with antibodies to two proteases. We conclude that functional maturation precedes morphological maturation in the submandibular glands of rats.

Localization of Chromogranins, Non-neuron-specific Enolase, and Different Forms of Somatostatins in the Submandibular Salivary Glands of Mice

The localizations of chromogranins A, B, and C, neuron-specific enolase (NSE, γγ-type) and non-NSE (αα-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 Alike 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.

Autonomic nerve endings in the odontoblast/predentin border and predentin of the canine teeth of dogs

Autonomic nerve endings in the odontoblast/predentin border and the predentin were observed by electron microscopy in the canine teeth of dogs. Adrenergic nerve endings with a number of very electron-dense granular vesicles and a minority of agranular vesicles of different sizes were clearly observed, but seen rarely, at the odontoblast/predentin border, in the predentin adjacent to the odontoblast processes, and as free endings in the middle part of the predentin. Cholinergic nerve endings were also observed with a number of agranular vesicles of different sizes without any visible exocytosis, in the odontoblast/predentin border, and in the predentin. Near the areas of collagen formation, extensive exocytosis of granular vesicles of different sizes was observed in adrenergic nerve endings located as free nerve endings in the predentin.