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      Mechanism of Bradykinin-Induced Ca 2+ Mobilization in MG63 Human Osteosarcoma Cells

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          Background: The effect of bradykinin on intracellular free Ca<sup>2+</sup> levels ([Ca<sup>2+</sup>]<sub>i</sub>) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca<sup>2+</sup> dye. Methods/Results: Bradykinin (0.1 n M–1 µ M) increased [Ca<sup>2+</sup>]<sub>i</sub> in a concentration-dependent manner with an EC<sub>50</sub> value of 0.5 n M. The [Ca<sup>2+</sup>]<sub>i</sub> signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca<sup>2+</sup> removal inhibited the peak [Ca<sup>2+</sup>]<sub>i </sub>signals by 35 ± 3%. Bradykinin (1 n M) failed to increase [Ca<sup>2+</sup>]<sub>i</sub> in the absence of extracellular Ca<sup>2+ </sup>after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca<sup>2+</sup> pump inhibitor; 1 µ M). Bradykinin (1 n M)-induced intracellular Ca<sup>2+</sup> release was nearly abolished by inhibiting phospholipase C with 2 µ M 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca<sup>2+</sup>]<sub>i </sub>increase induced by 1 n M bradykinin in Ca<sup>2+</sup>- free medium was abolished by 1 n M HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 n M Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 p M pertussis toxin for 5 h in Ca<sup>2+</sup> medium inhibited 30 ± 3% of 1 n M bradykinin-induced peak [Ca<sup>2+</sup>]<sub>i</sub> increase. Conclusions: Together, this study shows that bradykinin induced [Ca<sup>2+</sup>]<sub>i</sub> increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca<sup>2+</sup> from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca<sup>2+</sup> influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.

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          Bovine Milk Kininogen Fragment 1·2 Promotes the Proliferation of Osteoblastic MC3T3-E1 Cells

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            Functional characterization of N-methyl-D-aspartic acid-gated channels in bone cells.

            Our recent identification of glutamate receptors in bone cells suggested a novel means of paracrine communication in the skeleton. To determine whether these receptors are functional, we investigated the effects of the excitatory amino acid, glutamate, and the pharmacological ligand, N-methyl-D-aspartic acid (NMDA), on glutamate-like receptors in the human osteoblastic cell lines MG63 and SaOS-2. Glutamate binds to osteoblasts, with a Kd of approximately 10(-4) mol/L and the NMDA receptor antagonist, D(L)-2-amino-5-phosphonovaleric acid (D-APV), inhibits binding. Using the patch-clamp technique, we measured whole-cell currents before and after addition of L-glutamate or NMDA and investigated the effects of the NMDA channel blockers, dizolcipine maleate (MK801), and Mg2+, and the competitive NMDA receptor antagonist, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphoric acid (R-CPP), on agonist-induced currents. Both glutamate and NMDA induced significant increases in membrane currents. Application of Mg2+ (200 micromol/L) and MK801 (100 micromol/L) caused a significant decrease in inward currents elicited in response to agonist stimulation. The competitive NMDA receptor antagonist, R-CPP (100 micromol/L), also partially blocked the NMDA-induced currents in MG63 cells. This effect was reversed by addition of further NMDA (100 micromol/L). In Fura-2-loaded osteoblasts, glutamate induced elevation of intracellular free calcium, which was blocked by MK801. These results support the hypothesis that glutamate plays a role in bone cell signaling and suggest a possible role for glutamate agonists/antagonists in the treatment of bone diseases.
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              Fluoxetine-induced Ca2+ signals in Madin-Darby canine kidney cells.

               K. Y. Tang,  J Cheng,  K. Lee (2001)
              The effect of fluoxetine on Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca2+]i concentration-dependently between 5 microM and 200 microM with an EC50 value of 40 microM. The response was reduced by external Ca2+ removal by 30%40%. In Ca2+-free medium pretreatment with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, abolished 100 microM fluoxetine-induced Ca2+ release. Addition of 3 mM Ca2+ to Ca2+-free medium increased [Ca2+]i when cells were pretreated with 100 microM fluoxetine. Suppression of 1,4,5-trisphosphate (IP3) formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 100 microM fluoxetine-induced Ca2+ release. Fluoxetine (5-100 microM) also increased [Ca2+]i in neutrophils, prostate cancer cells and bladder cancer cells from human and rat glioma cells.

                Author and article information

                Horm Res Paediatr
                Hormone Research in Paediatrics
                S. Karger AG
                18 January 2002
                : 55
                : 6
                : 265-270
                Departments of aOrthopedic Surgery and bNeurology, Chang Gung Memorial Hospital-Kaohsiung; Departments of cPediatrics and dMedicine, Pao-Chien General Hospital, Ping Tung; Departments of eRehabilitation, fDentistry, and gMedical Education and Research, Kaohsiung Veterans General Hospital; hDepartment of Surgery, Ping Tung Christian Hospital; iDepartment of Biology and Institute of Life Sciences, National Sun Yat-Sen University, Kaohsiung, and jSchool of Medicine, National Yang Ming University, Taipei, Taiwan
                50011 Horm Res 2001;55:265–270
                © 2002 S. Karger AG, Basel

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                Page count
                Figures: 4, References: 22, Pages: 6
                Original Paper


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