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

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          Abstract

          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.
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                Author and article information

                Journal
                HRE
                Horm Res Paediatr
                10.1159/issn.1663-2818
                Hormone Research in Paediatrics
                S. Karger AG
                1663-2818
                1663-2826
                2001
                2001
                18 January 2002
                : 55
                : 6
                : 265-270
                Affiliations
                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
                Article
                50011 Horm Res 2001;55:265–270
                10.1159/000050011
                11805429
                © 2002 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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

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