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      Patch-Clamp-Induced Perturbations of [Ca 2+] i Activity in Somatotropes

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          Abstract

          Somatotropes and GC cells, a GH-producing cell line, exhibit [Ca<sup>2+</sup>]<sub>i</sub> oscillations that result from rhythmic Ca<sup>2+</sup> action potentials. Determination of this operating mode required simultaneous recording of both parameters by fura-2 imaging and patch-clamp techniques. In order to test whether patch recording induces artificial alteration of the [Ca<sup>2+</sup>]<sub>i</sub> oscillatory pattern, we recorded separately or simultaneously [Ca<sup>2+</sup>]<sub>i</sub> and membrane potential. In the absence of any other stimulation, seal formation in patch-clamp recording evoked by itself a 2.5- to 4-fold persistent increase in basal [Ca<sup>2+</sup>]<sub>i</sub>, speeded up their frequency (from 0.03–0.17 to 0.4 Hz) and changed their pattern to a tonic mode. Patch-induced [Ca<sup>2+</sup>]<sub>i</sub> increase was reproduced by mechanical contact between the pipette and the membrane. It was reduced by nifedipine, a blocker of L-type Ca<sup>2+</sup> channels, as well as by removal of external Na<sup>+</sup>. It was fully blocked by external Ca<sup>2+</sup> removal or gadolinium. All patch-clamp-induced perturbations were reversed by membrane hyperpolarization. We propose that patch-clamp recording evokes Ca<sup>2+</sup> entry through L-type Ca<sup>2+</sup> channels either directly, or indirectly via membrane depolarization. This shows that patch recordings in endocrine cells showing mechanosensitivity have to be interpreted with caution, and explains why long-lasting patch recordings are so difficult to obtain.

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          Most cited references 3

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          Mechanically activated currents in chick heart cells.

          As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na+ and K+ but not Cl-, and were independent of external Ca2+. The currents had linear I/V curves reversing at -16 mV and were completely blocked by Gd3+ >/= 30 microM and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations-the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at -2 mV and a 90 pS K+ selective reversing at -70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd3+ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd3+. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels.
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            Differential Management of Ca 2+ Oscillations by Anterior Pituitary Cells: A Comparative Overview

            Most electrical and ionic properties of anterior pituitary cells are common to all pituitary cell types; only gonadotropes exhibit a few cell specific features. Under basal conditions, the majority of pituitary cells in vitro, irrespective of their cell type, display spontaneous action potentials and [Ca 2+ ] i transients that result from rhythmic Ca 2+ entry through L-type Ca 2+ channels. The main function of these action potentials is to maintain cells in a readily activable responsive state. We propose to call this state a ‘pacemaker mode’, since it persists in the absence of extrinsic stimulation. When challenged by hypothalamic releasing hormones, cells exhibit two distinct response patterns: amplification of pacemaker activity or shift to internal Ca 2+ release mode. In the internal Ca 2+ release mode, [Ca 2+ ] i oscillations are not initiated by entry of external Ca 2+ , but by release of Ca 2+ from intracellular stores. In somatotropes and corticotropes, GHRH or CRH triggers the pacemaker mode in silent cells and amplifies it in spontaneously active cells. In contrast, in gonadotropes GnRH activates the internal Ca 2+ release mode in silent cells and switches already active cells from the pacemaker to the internal Ca 2+ release mode. Interestingly, homologous normal and tumoral cells display the same type of activity in vitro, in the absence or presence of hypothalamic hormones. Pacemaker and internal Ca 2+ release modes are likely to serve different purposes. Pacemaker activity allows long-lasting sequences of [Ca 2+ ] i oscillations (and thus sustained periods of secretion) that stop under the influence of hypothalamic inhibitory peptides. In contrast, the time during which cells can maintain internal Ca 2+ release mode depends upon the importance of intracellular Ca 2+ stores. This mode is thus more adapted to trigger secretory peaks of large amplitude and short duration. On the basis of these observations, theoretical models of pituitary cell activity can be proposed.
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              Calcium signalling in single growth hormone-releasing factor-responsive pituitary cells

               L Cuttler (1992)
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                Author and article information

                Journal
                NEN
                Neuroendocrinology
                10.1159/issn.0028-3835
                Neuroendocrinology
                S. Karger AG
                0028-3835
                1423-0194
                1999
                November 1999
                17 November 1999
                : 70
                : 5
                : 343-352
                Affiliations
                Dynamique des Systèmes Neuroendocriniens, INSERM U159, Paris, France
                Article
                54495 Neuroendocrinology 1999;70:343–352
                10.1159/000054495
                10567860
                © 1999 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: 5, Tables: 2, References: 29, Pages: 10
                Categories
                Pituitary Transduction

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