Toshimi Hattori's research while affiliated with Matsumoto Dental University and other places

To investigate pharmacologically whether CaSRs are involved in the Ca(2+) antagonist-induced [Ca(2+)]i elevation in gingival fibroblasts. Gin-1 cells, normal human gingival fibroblasts, were used as the material. The [Ca(2+)] i was measured with fura-2/AM, a Ca(2+)-sensitive fluorescent dye. At first, we confirmed the existence of CaSRs in these cells by showing that [Ca(2+)] i was elevated by high concentrations of extracellular Ca(2+) and by prototypic agonists of the CaSR such as gentamicin. The action of gentamicin was antagonized by inhibitors of phospholipase C (PLC), inositol trisphosphate (IP(3)) receptors, NSCCs, and, importantly, by the CaSR antagonist, NPS2390. Furthermore, the action of gentamicin was potentiated by activators of PLC and protein kinase C (PKC). This confirmed the pathway components mediating Ca(2+) responses to a known agonist of the CaSR. We then investigated whether nifedipine (an L-type Ca(2+) channel blocker) stimulates CaSRs to elevate [Ca(2+)] i via a similar mechanism. Nifedipine Ca(2+) responses were dose-dependently blocked by NPS2390 and by the same inhibitors of PLC, IP(3) receptors, and NSCCs that disrupted the action of gentamicin. Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca(2+) release from stores) also inhibited the nifedipine-induced [Ca(2+)] i elevation. These findings suggest that CaSRs are involved in the nifedipine-induced [Ca(2+)] i elevation in gingival fibroblasts.

Kampo medicines are clinically used for the treatment of various diseases, and their use for oral diseases is desired; however, only a few kampo medicines are adaptable for diseases of the oral region. Against this background, we carried out an investigation into the application of preexisting kampo medicines to diseases of the oral region. Here, we indicate the effects of kampo medicines on periodontal disease, drug-induced gingival overgrowth, and xerostomia using in vitro or animal models. (i) Shosaikoto has an anti-inflammatory effect and is used for the treatment of pneumonia and bronchitis. Because shosaikoto decreases lipopolysaccharide (LPS)-induced prostaglandin E2 production by gingival fibroblasts, it may be effective to improve inflammation in periodontal tissue. (ii) Saireito has an inhibitory effect on cell proliferation and is used for the treatment of idiopathic retroperitoneal fibrosis. Because saireito suppresses nifedipine-induced gingival fibroblast proliferation, it may be effective for the treatment of drug-induced gingival overgrowth. (iii) Byakkokaninjinto and goreisan are used for the treatment of xerostomia. Because diabetes is accompanied by xerostomia, the effects of these two medicines on salivary secretion have been examined using streptozotocin-induced diabetic mice. Both medicines recover the blood glucose level and secretory rate of saliva to almost normal levels. These results suggest that kampo medicines may be adaptable for periodontal disease, drug-induced gingival overgrowth, and diabetes-mediated xerostomia.

In the present study, we investigated the effects of a Kampo medicine Orento (TJ-120) on the production of prostaglandin E(2) (PGE(2)), interleukin (IL)-6 and IL-8 by human gingival fibroblasts (HGFs) treated with lipopolysaccharide from Porphyromonas gingivalis (PgLPS). HGFs proliferation was dose-dependently decreased with Orento at days 3 and 7. However, treatment with PgLPS (10 ng/ml), Orento (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Orento suppressed PgLPS-induced PGE(2) production in a dose-dependent manner but did not alter basal PGE(2) level. In contrast, Orento did not alter PgLPS-induced IL-6 and IL-8 productions. These alterations by Orento were similar to those by a mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059. A Orento showed no effect on cyclooxygenase (COX)-1 and COX-2 activities, and increased cytoplasmic phospholipase A(2) (cPLA(2)) expression and increased PgLPS-induced COX-2 expression. Orento suppressed PgLPS-induced mobility retardation of cPLA(2) band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, that is cPLA(2) phosphorylation and its activation, while Orento alone did not alter cPLA(2) phosphorylation. Orento suppressed PgLPS-induced extracellular signal-regulated kinase (ERK) phosphorylation, which is known to lead to ERK activation and cPLA(2) phosphorylation. These results suggest that Orento decreased PGE(2) production by inhibition of cPLA(2) phosphorylation and its activation via inhibition of ERK phosphorylation, and also that Orento may be useful to improve gingival inflammation in periodontal disease.

In the present study, we investigated the anti-inflammatory effects of a Kampo medicine Shosaikoto (TJ-9) using in vitro periodontal disease model, in which human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) produce IL-6, IL-8 and prostaglandin E2 (PGE2). Treatment with PgLPS (10 ng/ml), TJ-9 (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Moreover, TJ-9 did not alter LPS-induced IL-6 and IL-8 productions. However, TJ-9 significantly suppressed LPS-induced PGE2 production in a dose-dependent manner but TJ-9 alone did not affect basal PGE2 level. Western blotting demonstrated that TJ-9 decreased cyclooxygenase-2 (COX-2) expression in a dose-dependent manner but not phospholipase A2. Moreover, TJ-9 selectively and dose-dependently inhibited COX-2 activity. These results suggest that TJ-9 decreased PGE2 production by inhibition of both COX-2 expression and activity and that TJ-9 may be useful to improve gingival inflammation in periodontal disease.

Calcium (Ca2+) antagonists induce gingival overgrowth as a side effect but the pathogenic mechanism is still unknown. The Ca2+-channel activator Bay K 8644 elevates intracellular Ca2+ concentration ([Ca2+]i) and enhances the cell proliferation of gingival fibroblasts in a dose-dependent manner. Verapamil, an L-type Ca2+-channel blocker, also elevates [Ca2+]i in gingival fibroblasts, but it has no effect on other fibroblasts such as those of the lung, skin, and muscle. Moreover, verapamil enhances the proliferation of fibroblasts of the gingiva but has no effect on the proliferation of those of other tissues. These findings confirm that [Ca2+]i elevation induces the proliferation of gingival fibroblasts.

Ca2+ antagonists cause dry mouth by inhibiting saliva secretion. The present study was undertaken to elucidate the mechanism by which Ca2+ antagonists cause dry mouth. Since the intracellular Ca2+ concentration ([Ca2+]i) is closely related to saliva secretion, [Ca2+]i was measured with a video-imaging analysis system by using human submandibular gland (HSG) cells as the material. The Ca2+ antagonist, nifedipine, inhibited the elevation in [Ca2+]i induced by 1-10 microM carbachol (CCh), but had no inhibitory effect on that induced by 30 and 100 microM CCh. The other kinds of Ca2+ antagonists, verapamil (10 microM), diltiazem (10 microM), and the inorganic Ca2+ channel blocker, CdCl2 (50 microM), also inhibited the [Ca2+]i elevation induced by 10 microM CCh. The Ca2+ channel activator, Bay K 8644 (5 microM), significantly enhanced the CCh (10 microM)-induced [Ca2+]i elevation. Endothelin-1 and norepinephrine also increased the CCh (10 microM)-induced [Ca2+]i elevation. SKF-96365 reversed the enhancement of the CCh (10 microM)-induced [Ca2+]i elevation caused by AlF4- and phenylephrine. The phospholipase Cbeta (PLCbeta) inhibitor, U-73122 (5 microM), significantly inhibited the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh, while the PLCbeta activator, m-3M3FBS (20 microM), significantly increased the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh. We therefore conclude that non-selective cation and voltage-dependent Ca2+ channels are involved in resting salivation and that Ca2+ antagonists depress H2O secretion by blocking the Ca2+ channels and thereby cause dry mouth.

A hypertonic saline containing propylene glycol facilitates calcium (Ca(2+)) influx through voltage-dependent Ca(2+) channels. The present study performed experiments to elucidate the mechanism by which Na(+)-K(+)-2Cl(-) cotransporters participate in the rise in the intracellular calcium concentration ([Ca(2+)]i) under the hypertonic condition. Both furosemide and ethacryonic acid significantly decreased the [Ca(2+)]i raised by hypertonicity. Similarly, Na(+)-, K(+)-, or Cl(-)-free saline also reduced it. Both norepinephrine and dopamine significantly enhanced the rise in [Ca(2+)]i. In conclusion, the findings obtained indicate that the Na+-K+-2Cl- cotransporters evoke cell depolarization and that this depolarization raises the [Ca(2+)]i by activating voltage-dependent Ca(2+) channels.

Saiko is predominantly contained in Saireito, a Chinese herbal medicine. The present study was conducted to determine whether or not Saiko is involved in the inhibition by Saireito of nifedipine-induced proliferation and collagen synthesis in gingival fibroblasts. Nifedipine (10 microM) significantly enhanced the proliferation starting on day 5 of the culture period. When added together with nifedipine, Saiko at concentrations of 0.05%-0.2% (w/v) dose-dependently inhibited the nifedipine-induced proliferation, and at the highest concentration tested (0.2%), Saiko inhibited the nifedipine-induced proliferation by about 40%. Moreover, Saiko (0.2%) also inhibited the normal proliferation at days 11 and 14. Sole application of nifedipine (10 microM) augmented the release of bFGF, and Saiko concentration-dependently reduced the level of bFGF in the nifedipine-containing culture medium. Nifedipine (10 microM) increased the production of type I collagen to almost twice that of the control (normal medium), and Saiko at concentrations above 0.1% significantly reduced the nifedipineinduced production of collagen. In conclusion, the present findings demonstrate that Saiko inhibited the nifedipine-induced proliferation of gingival fibroblasts by reducing the release of bFGF and that Saiko is involved in the Saireito-induced inhibition of nifedipine-stimulated proliferation and collagen synthesis in gingival fibroblasts.

Some kinds of drugs such as calcium (Ca(2+)) channel antagonists, antiepileptics and immunosuppressants cause gingival overgrowth as a side effect, the mechanism of which is still unclear. We have examined the effects of isradipine, one of the dihydropyridine-derivative Ca(2+) channel antagonists, on cultured human gingival fibroblast Gin-1 cells. In the present study, to elucidate the mechanism by which isradipine causes gingival overgrowth, we examined whether tyrosine kinase (TK) and phopholipase Cgamma (PLCgamma) are involved in the isradipine-induced proliferation of gingival fibroblasts. Herbimycin A (1 microM) remarkably inhibited the isradipime (10 microM)-induced proliferation. Both U73122 (5 microM), a PLCgamma inhibitor, and xestospongin C (5 microM), an antagonist of a receptor of inositol 1,4,5-trisphosphate in Ca(2+) stores, significantly reduced the [Ca(2+)]i raised by isradipine (10 microM). Thus, the findings obtained here indicate that TK and PLCgamma are closely involved in the isradipine-induced [Ca(2+)]i rise to elicit gingival overgrowth.