cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues.

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

[Effects of cernitin pollen extract (CN-009) on the isolated bladder smooth muscles and the intravesical pressure].

Cernitin pollen extract (CN-009), extract from several pollen species, has been used for urinary dysfunction. As its mode of action has not been clarified, we investigated the action of CN-009 on the isolated bladder smooth muscles of rats, guinea pigs and cats and the intravesical pressure in female rats. CN-009 contracted isolated detrusor muscles of rats, guinea pigs and cats in a concentration-dependent manner. In the guinea pig detrusor muscle, the contractile effect of CN-009 was depressed by atropine, diphenhydramine and increased by cimetidine. In the rat detrusor muscle, the CN-009-induced contraction was depressed by atropine. In adult rats (11-23 weeks old) and aged rats (2 years old), CN-009 showed a dose-dependent increase of intravesical pressure to the same extent in spite of the fact that the aged rats had a lower responsiveness to acetylcholine. In adult rats, the CN-009-induced increase of intravesical pressure was reduced completely by atropine and partly reduced by phentolamine and guanethidine. Three weeks consecutive oral administration of CN-009 tended to increase the basal intravesical pressure and tended to elevate the isoproterenol-induced decrease and serotonin-induced increase in the intravesical pressure. These results suggest that CN-009 contracts the detrusor muscle, a process that is mainly mediated by muscarinic receptor activation. The contraction induced by CN-009 of detrusor muscle causes the increase of intravesical pressure.

[Inhibitory effect and synergism of cernitin pollen extract on the urethral smooth muscle and diaphragm of the rat].

Inhibitory effects of cernitin pollen extract (CN-009), consisting of T-60 (a water-soluble extract) and GBX (an acetone-soluble extract) at a ratio of 20:1, were investigated in rat urethral smooth muscle and diaphragm. In the urethral smooth muscle, CN-009 (3.0 x 10(-4) approximately 3.0 x 10(-3) g/ml), T-60 and GBX (corresponding to CN-009) concentration-dependently inhibited the noradrenaline (NA, 10(-5) g/ml)-induced contraction. According to Bürgi's calculation, the inhibition by T-60 and GBX was synergistic. On the other hand, GBX inhibited the NA-contraction non-competitively and also inhibited the K+-contraction. In contrast, T-60 tended to inhibit competitively, but did not affect the K+-contraction. In the diaphragm, CN-009 (5.25 x 10(-3) approximately 2.1 x 10(-2) g/ml) concentration-dependently inhibited the nerve stimulation-induced twitch response. T-60 (corresponding to CN-009) showed no effect, while GBX slightly inhibited the twitch response. The effects of T-60 and GBX were synergistic. The inhibitory effects of CN-009 (2.1 x 10(-2) g/ml) and GBX (1.0 x 10(-2) g/ml) were specific against the nerve stimulation and were not antagonized by neostigmine (1.0 x 10(-5) g/ml). These results suggested that these effects were neither musculotropic nor competitive against ACh. In conclusion, CN-009 concentration-dependently inhibited the urethral contraction and the skeletal muscular twitch response. It was suggested that T-60 and GBX had different mechanisms and inhibited the response synergistically.