Toshimi Hattori

Prostaglandin E2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors.

Prostaglandin E2 (PGE2) has been proposed to be a potent stimulator of bone resorption. However, PGE2 itself has been shown to directly inhibit bone-resorbing activity of osteoclasts. We examined the role of PGE2 in the function of mouse osteoclasts formed in vitro. Bone marrow macrophage osteoclast precursors expressed PGE2 receptors EP1, EP2, EP3β, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by receptor activator of NF-κB ligand and macrophage colony-stimulating factor. In contrast, functional EP1 was continuously expressed in mature osteoclasts. PGE2 as well as calcitonin caused intracellular Ca2+ influx in osteoclasts. However, PGE2 and 17-phenyltrinol-PGE2 (an EP1 agonist) failed to inhibit actin-ring formation and pit formation by osteoclasts cultured on dentine slices. When EP4 was expressed in osteoclasts using an adenovirus carrying EP4 cDNA, both actin-ring and pit-forming activities of osteoclasts were inhibited in an infectious unit-dependent manner. Treatment of EP4-expressing osteoclasts with PGE2 further inhibited their actin-ring and pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are similar to those that are calcitonin receptor-mediated. Thus, osteoclast precursors down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts to escape inhibitory effects of PGE2 on bone resorption.

Macrolide antibiotics like azithromycin increase lipopolysaccharide-induced IL-8 production by human gingival fibroblasts

ObjectiveMacrolide antibiotics are reported to modulate the production of cytokines in various type of cells. We examined the effect of macrolide antibiotics on inflammatory cytokines (IL-6 and IL-8) and chemical mediator (PGE2) and also matrix metalloproteinases (MMPs) productions by human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS).MethodsThe effect of macrolide antibiotics [erythromycin (EM), azithromycin (AZM) and josamycin (JOM)] on HGFs proliferation were examined by MTT assay. HGFs were treated with LPS from Porphyromonas gingivalis (PgLPS) and macrolide antibiotics, and IL-6, IL-8 and PGE2 levels were evaluated by ELISA. MMPs were detected by gelatin zymography.ResultsAZM slightly but significantly decreased HGFs proliferation, while EM and JOM did not affected. AZM increased PgLPS-induced IL-8 production dose-dependently, while AZM did not alter IL-6 and PGE2 productions. EM and JOM did not altered PgLPS-induced IL-6, IL-8 and PGE2 productions. All macrolide antibiotics did not alter MMPs production. These results indicate that macrolide antibiotics have no direct anti-inflammatory effect. However, the use of the inhibitors of cell signaling pathway failed to reveal the mechanism that AZM enhanced PgLPS-induced IL-8 production.ConclusionThese results suggest macrolide antibiotics have an indirect anti-inflammatory effect as a result of their antimicrobial properties. Because AZM increased LPS-induced IL-8 production by HGFs, the possibility is considered that neutrophils may be migrated to periodontal tissue and phagocytize the periodontopathic bacteria more efficiently.

The effects of cigarette exposure on rat salivary proteins and salivary glands

OBJECTIVE Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on saliva and salivary glands (SGs). METHODS Cigarette smoke-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (0 day), and 15 and 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. SGs were collected on 31 days. RESULTS The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rates did not differ at 0, 15 or 30 days after the start of CS exposure. However, the amylase and peroxidase activities and total protein content in the saliva were significantly lower in 15-day CS-exposed rats than in 15-day control rats. Histological examination of the SGs of CS-exposed rats showed vacuolar degeneration, vasodilation and hyperemia. CONCLUSION These results suggest that CS exposure has adverse impacts on salivary composition and SGs, which could aggravate the oral environment.

Facilitation of frog neuromuscular transmission by sodium fluoride

1 The effects of sodium fluoride (NaF, 5 mm) alone or in combination with theophylline (1.5 mm) or imidazole (1.5 mm) on the amplitude of the endplate potential (e.p.p.), frequency of the miniature endplate potential (m.e.p.p.), and the quantal content of the e.p.p. of bullfrog muscle were investigated. The effects of forskolin (1 μm) and papaverine (1 μm) on the m.e.p.p. frequency were also studied. 2 NaF caused an increase of 22% in the amplitude of the e.p.p. This NaF‐induced increase was enhanced by theophylline (to 51%) and reduced by imidazole (to 10%). 3 Papaverine (0.1–3 μm) increased the frequency of m.e.p.ps. Forskolin at 1 μm raised the m.e.p.p. frequency by 13%. The effect was increased to 31% by 1 μm papaverine. NaF also raised the m.e.p.p. frequency by 90%. This action too was increased by theophylline (2.6 fold) and by papaverine (2.1 fold); however, it was reduced by imidazole (1.3 fold). 4 NaF increased the quantal content of the e.p.p. by 28%. This effect was enhanced by theophylline to 44%, while it was diminished by imidazole. 5 These results suggest that an increase in the transmitter release via an elevation of cyclic AMP may be involved in the facilitation of neuromuscular transmission by NaF.

Stannous chloride-induced increase in calcium entry into motor nerve terminals of the frog

The effects of SnCl2 on potentials recorded extracellularly from motor nerve terminals of the bullfrog were studied to elucidate the mechanism of the SnCl2-induced facilitation of evoked transmitter release. Under conditions in which the muscle preparations were pretreated with d-tubocurarine and tetraethylammonium in a K+-free medium, SnCl2 (50 microM) augmented the prolonged positive deflection ascribed to the inward Ca2+ current, an effect which was reduced by addition of Cd2+. The results suggest that SnCl2 could increase Ca2+ entry into the nerve terminals.

Pharmacological evidences for the stimulation of calcium-sensing receptors by nifedipine in gingival fibroblasts

Objective: To investigate pharmacologically whether CaSRs are involved in the Ca2+ antagonist-induced [Ca2+]i elevation in gingival fibroblasts. Materials and Methods: Gin-1 cells, normal human gingival fibroblasts, were used as the material. The [Ca2+] i was measured with fura-2/AM, a Ca2+-sensitive fluorescent dye. Results: At first, we confirmed the existence of CaSRs in these cells by showing that [Ca2+] i was elevated by high concentrations of extracellular Ca2+ and by prototypic agonists of the CaSR such as gentamicin. The action of gentamicin was antagonized by inhibitors of phospholipase C (PLC), inositol trisphosphate (IP3) 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 Ca2+ responses to a known agonist of the CaSR. We then investigated whether nifedipine (an L-type Ca2+ channel blocker) stimulates CaSRs to elevate [Ca2+] i via a similar mechanism. Nifedipine Ca2+ responses were dose-dependently blocked by NPS2390 and by the same inhibitors of PLC, IP3 receptors, and NSCCs that disrupted the action of gentamicin. Calphostin C (a PKC inhibitor) and TMB-8 (an inhibitor of Ca2+ release from stores) also inhibited the nifedipine-induced [Ca2+] i elevation. Conclusion: These findings suggest that CaSRs are involved in the nifedipine-induced [Ca2+] i elevation in gingival fibroblasts.

Dental Caries Area of Rat Molar Expanded by Cigarette Smoke Exposure

Objectives: Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on caries risk and experimental dental caries. Methods: Experimental dental caries was induced in rat maxillary molars which were inoculated orally with Streptococcus mutans MT8148 and maintained on a cariogenic diet (diet 2000) and high sucrose water during the experimental period. CS-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (day 0) and for 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. Maxillary molars were harvested on day 31. Results: The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rate, concentration of S. mutans in the stimulated saliva and caries activity score did not significantly differ between 0 and 30 days after the start of CS exposure. Histological examination of the caries-affected area on maxillary molars 30 days after CS exposure showed expansion compared to control rats. In the electron probe microanalysis, no differences were observed between the mineral components of the CS-exposed teeth and the control teeth. Conclusion: These results suggest that CS exposure expands the caries-affected area in the maxillary molars of the rat.

Acceleration by stannous ion of the evoked release of transmitter from motor nerve endings in the frog.

Stannous ion (Sn2+, 30 microM) increased the amplitude of endplate potential (EEP) in the frog sartorius muscle, although the amplitude of miniature endplate potential (MEPP) or acetylcholine potential evoked by iontophoretic application of acetylcholine was unchanged. Sn2+ (10-100 microM) dose-dependently increased the quantal content of the EPP. MEPP frequency was not altered by 30 microM Sn2+. These findings indicate that Sn2+ may increase the EPP as a result of acceleration of the transmitter release evoked by nerve impulses.

Development of Novel Therapy for Oral Diseases Using Kampo Medicines

Abstract 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 E 2 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.

Involvement of Na(+)-K(+)-2Cl(-) cotransporters in hypertonicity-induced rise in intracellular calcium concentration.

A hypertonic saline containing propylene glycol facilitates calcium (Ca2+) influx through voltage-dependent Ca2+ 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 ([Ca2+]i) under the hypertonic condition. Both furosemide and ethacryonic acid significantly decreased the [Ca2+]i raised by hypertonicity. Similarly, Na+-, K+-, or Cl−-free saline also reduced it. Both norepinephrine and dopamine significantly enhanced the rise in [Ca2+]i. In conclusion, the findings obtained indicate that the Na+-K+-2Cl− cotransporters evoke cell depolarization and that this depolarization raises the [Ca2+]i by activating voltage-dependent Ca2+ channels.