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      Effect of Bradykinin on Cultured Bovine Corneal Endothelial Cells

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          Abstract

          Purpose: To clarify the effect of bradykinin on cytosolic free calcium mobilization and cell proliferation in cultured bovine corneal endothelial cells (BCEC). Methods: The cytosolic free calcium concentration (Ca<sup>2+</sup>]<sub>i</sub>) was measured with the InCa<sup>TM</sup> Imaging System after the treatment of bradykinin (10<sup>–11</sup> to 10<sup>–7</sup>  M) alone or with the pretreatments of EGTA, bradykinin receptor (Bk<sub>1</sub> and Bk<sub>2</sub>) antagonists and an inhibition of phospholipase C (U-73122). Also, the effect of bradykinin on cell proliferation in BCEC was evaluated using cell counts. Results: In BCEC, [Ca<sup>2+</sup>]<sub>i</sub> in the resting state was 87 ± 9 n M. Bradykinin induced an increment of [Ca<sup>2+</sup>]<sub>i</sub> in a concentration-dependent manner and its 50% effective concentration was approximately 5 × 10<sup>–11</sup>  M. A [Ca<sup>2+</sup>]<sub>i</sub> increment at 10<sup>–8</sup>  M bradykinin was inhibited with the pretreatment of EGTA, an extracellular calcium chelator. U-73122 (5 × 10<sup>–6</sup>  M) attenuated the bradykinin-induced [Ca<sup>2+</sup>]<sub>i</sub> increment. The pretreatment of HOE-140 (Bk<sub>2</sub> antagonist) almost attenuated the bradykinin (10<sup>–8</sup>  M)-induced [Ca<sup>2+</sup>]<sub>i</sub> increase, but des-Arg<sup>9</sup>-[Leu<sup>8</sup>]-bradykinin (Bk<sub>1</sub> antagonist) did not suppress it. To investigate the physiological effect of bradykinin, the effect of bradykinin on cell proliferation was studied. 10<sup>–8</sup>  M of bradykinin produced a significant increase in cell numbers. This mitogenic effect of bradykinin was inhibited by the Bk<sub>2</sub> antagonist. Conclusions: Bradykinin-induced stimulation of the signal transduction pathway in BCEC is coupled with the Bk<sub>2</sub> type receptor. Furthermore, bradykinin produces the mitogenic effect in BCEC.

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          Most cited references 6

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          Increased mRNA expression of the B1 and B2 bradykinin receptors and antinociceptive effects of their antagonists in an animal model of neuropathic pain.

          We examined the role of B1 and B2 bradykinin receptors in promoting neuropathic hypersensitivity following peripheral nerve injury. Forty eight-hours following chronic constriction injury to a rat sciatic nerve there was an increased expression of B2 receptor mRNA in the lumbar dorsal root ganglia ipsilateral to the site of nerve injury. At 14 days following surgery there was also an ipsilateral increase of B1 receptor mRNA as well as a contralateral increased expression of B2 receptor mRNA. Increased expression of both receptors also coincided with analgesic effects of their antagonists. While HOE-140, a potent B2 receptor antagonist was analgesic at both time points tested, the B1 receptor antagonist des-Arg(9), [Leu(8)]-BK had an analgesic effect only at 14 days. The results support the concept that peripheral nerve injury is associated with local inflammation and that bradykinin, acting on both of its receptors promotes pain hypersensitivity.
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            Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors.

            To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.
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              Purinoceptor-mediated calcium mobilization and cellular proliferation in cultured bovine corneal endothelial cells.

              In the present study, we investigated the effect of adenosine triphosphate (ATP) on cytosolic free calcium mobilization and mitogenic activity in cultured bovine corneal endothelial cells (BCEC). The [Ca2+]i was determined using a Ca2+ sensitive indicator, Fura-2/AM, and cell proliferation was evaluated by counting the cell number. ATP, its metabolites and analogs caused transient increase in [Ca2+]i in a concentration-dependent manner (10(-7) M-10(-3) M) and the potency of agonists was ordered as follows: 2-methylthio-ATP > uridine triphosphate > ATP > adenosine diphosphate. Adenosine monophosphate and adenosine did not affect [Ca2+]i. ATP (10(-4) M) also promoted the accumulation of inositol trisphosphate (IP3). The ATP-induced transient [Ca2+]i increase and IP3 accumulation were attenuated by pretreatment with a phospholipase C inhibitor, U-73122 (5 microM), for 30 min. ATP (10(-5) M) significantly enhanced the proliferation of BCEC. ATP-induced [Ca2+]i increase and cell proliferation were inhibited by a purinoceptor antagonist, suramin (10(-4) M). Thus, the present study indicates that BCEC contain P2 purinoceptors that regulate their proliferation.
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                Author and article information

                Journal
                OPH
                Ophthalmologica
                10.1159/issn.0030-3755
                Ophthalmologica
                S. Karger AG
                0030-3755
                1423-0267
                2001
                August 2001
                31 May 2001
                : 215
                : 4
                : 303-308
                Affiliations
                aDepartment of Ophthalmology, bDepartment of Pharmacology, cCatholic Neuroscience Center and dDepartment of Industrial Medicine, College of Medicine, Catholic University of Korea, Seoul, Korea
                Article
                50879 Ophthalmologica 2001;215:303–308
                10.1159/000050879
                11399940
                © 2001 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: 6, References: 25, Pages: 6
                Categories
                Original Paper · Travail original · Originalarbeit

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