Patch-clamp techniques were used in primary cultures of rat lactotropes and the rat pituitary clonal cell line GH3 to determine the population characteristics of spontaneous action potentials and the nature of the currents responsible for repolarization of spontaneous action potentials. Spontaneous action potentials were observed in 75% of lactotropes (74/99) and 80% of GH3 cells (42/51). Lactotropes exhibited broad and shallow action potentials (average duration 460 ms, peak –17 mV, slope of upstroke 0.5 mV/ms) compared to the GH3 cells which displayed narrow and tall action potentials (average duration 177 ms, peak –10 mV, slope of upstroke 1.6 mV/ms). Blockers of potassium currents were used to determine the role of specific potassium currents in the repolarization process. Spontaneous action potentials in lactotropes were largely unaffected by 4-aminopyridine (4AP), charybdotoxin, and apamin. Tetraethylammonium (TEA) caused only an small increase in peak amplitude and, in a subset of cells, a small increase in duration. In contrast, in GH3 cells, TEA, 4AP, charybdotoxin, and apamin all caused a significant increase in duration, while TEA and charybdotoxin also caused an increase in peak amplitude. Further, apamin caused a positive shift in the afterhyperpolarization voltage. In lactotropes, strong buffering of intracellular calcium with calcium chelators (EGTA or BAPTA) caused a profound increase in action potential duration. Thus, repolarization of action potentials in lactotropes is a calcium-dependent process, but unlike GH3 cells, is not mediated by calcium-dependent potassium currents, nor is it strongly influenced by voltage-dependent potassium currents.

Keywords:
GH3 cells, Charybdotoxin, Apamin, Tetraethylammonium, 4-Aminopyridine, Lactotropes, K+ channels, Electrophysiology
1.
Taraskevich PS, Douglas WW: Action potentials occur in cells of normal anterior pituitary gland and are stimulated by the hypophysiotropic peptide thyrotropin-releasing hormone. Proc Natl Acad Sci USA 1977;74:4064–4067.
2.
Lewis DL, Goodman MB, St. John PA, Barker JL: Calcium currents and Fura-2 signals in fluorescence activated cell sorted lactotrophs and somatotrophs in rat anterior pituitary. Endocrinology 1998;123:611–621.
3.
Lledo PM, Legendre P, Zhang J, Israel JM, Vincent JD: Effects of dopamine on voltage-dependent potassium currents in identified rat lactotroph cells. Neuroendocrinology 1990;52:545–555.
4.
Simasko SM: A background sodium conductance is necessary for spontaneous depolarizations in rat pituitary cell line GH3. Am J Physiol 1994;266:C709–C719.
5.
Stojilkovic SS, Izumi S-I, Catt KJ: Participation of voltage-sensitive calcium channels in pituitary hormone release. J Biol Chem 1988;263:13054–13061.
6.
Sankaranarayanan S, Simasko SM: A role for a background sodium current in spontaneous action potentials and secretion from rat lactotrophs. Am J Physiol 1996;271:C1927–C1934.
7.
Lingle CJ, Sombati S, Freeman ME: Membrane currents in identified lactotrophs of rat anterior pituitary. J Neurosci 1986;6:2995–3005.
8.
Simasko SM: Evidence for a delayed rectifier-like potassium current in the clonal rat pituitary cell line GH3. Am J Physiol 1991;261:E66–E75.
9.
Herrington J, Lingle CJ: Multiple components of voltage-dependent potassium current in normal rat anterior pituitary cells. J Neurophysiol 1994;72:719–729.
10.
Ritchie AK: Two distinct calcium-activated potassium currents in a rat anterior pituitary cell line. J Physiol 1987;385:591–609.
11.
Rogawski MA, Inoue K, Suzuki S, Barker JL: A slow calcium-dependent chloride conductance in clonal anterior pituitary cells. J Neurophysiol 1988;59:1854–1870.
12.
Sartor P, Dufy-Barbe L, Vacher P, Dufy B: Calcium-activated chloride conductance of lactotrophs: Comparison of activation in normal and tumoral cells during thyrotropin-releasing hormone stimulation. J Membr Biol 1992;126:39–49.
13.
Dubinsky JM, Oxford GS: Ionic currents in two strains of rat anterior pituitary tumor cells. J Gen Physiol 1984;83:309–339.
14.
Rogawski MA: Transient outward current (IA) in clonal anterior pituitary cells: Blockade by aminopyridine analogs. Arch Pharmacol 1988;338:125–132.
15.
Sankaranarayanan S, Simasko SM: Characterization of an M-like current modulated by thyrotropin-releasing hormone in normal rat lactotrophs. J Neurosci 1996;16:1668–1678.
16.
Lang DG, Ritchie AK: Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line. Pflügers Arch 1987;410:614–622.
17.
Lang DG, Ritchie AK: Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone. Pflügers Arch 1990;416:704–709.
18.
Ritchie AK: Thyrotropin-releasing hormone stimulates a calcium-activated potassium current in a rat anterior pituitary cell line. J Physiol 1987;385:611–625.
19.
Simasko SM: Effect of calcium on membrane potential behavior in a rat pituitary cell line (GH3). Mol Cell Endocrinol 1991;78:79–86.
20.
Taraskevich PS, Douglas WW: Electrical behaviour in a line of anterior pituitary cells (GH cells) and the influence of the hypothalamic peptide, thyrotrophin releasing factor. Neuroscience 1980;4:421–431.
21.
Ritchie AK, Lang DG: Activity of single ion channels that regulate membrane excitability in GH3 anterior pituitary cells; in Lakoski JM, Perez-Polo JR, Rassin DK (eds): Neural Control of Reproductive Function. New York, Liss, 1989, pp 463–479.
22.
Sand EH, Gautvik KM: Effects of thyroliberin and 4-aminopyridine on action potentials and prolactin release and synthesis in rat pituitary cells in culture. Acta Physiol Scand 1980;108:247–252.
23.
Israel JM, Kirk C, Vincent JD: Electrophysiological response to dopamine of rat hypophysial cells in lactotroph-enriched primary cultures. J Physiol 1987;390:1–22.
24.
Mason WT, Ingram CD: Techniques for studying the role of electrical activity in control of secretion by normal anterior pituitary cells. Methods Enzymol 1986;124:207–242.
25.
Sartor P, Dufy-Barbe L, Corcuff JB, Taupignon A, Dufy B: Electrophysiological response to thyrotropin-releasing hormone of rat lactotrophs in primary culture. Am J Physiol 1990;258:E311–E319.
26.
Burris TP, Freeman ME: Low concentrations of dopamine increase cytosolic calcium in lactotrophs. Endocrinology 1993;133:63–68.
27.
Tashjian AH Jr: Clonal strains of hormone-producing pituitary cells. Methods Enzymol 1979;57:527–535.
28.
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved technique for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981;391:85–100.
29.
Horn R, Marty A: Muscarinic activation of ionic current measured by a new whole-cell recording method. J Gen Physiol 1988;92:145–159.
30.
Wagoner PK, Oxford GS: Aminopyridines block an inactivating potassium current having slow recovery kinetics. Biophys J 1990;58:1481–1489.
31.
Moczydlowski E, Lucchesi K, Ravindran A: An emerging pharmacology of peptide toxins targeted against potassium channels. J Membr Biol 1988;105:95–111.
32.
Magistretti J, Mantegazza M, Guatteo E, Wanke E: Action potentials recorded with patch-clamp amplifiers: Are they genuine? Trends Neurosci 1996;19:530–534.
33.
Korn SJ, Bolden A, Horn R: Control of action potentials and Ca2+ influx by the Ca2+-dependent chloride current in mouse pituitary cells. J Physiol 1991;439:423–437.
34.
Garcia L, Rigoulet M, Georgescauld D, Dufy B, Sartor P: Regulation of intracellular chloride concentration in rat lactotrophs: Possible role of mitochondria. FEBS Lett 1997;400:113–118.
35.
Vergara L, Rojas E, Stojilkovic SS: A novel calcium-activated apamin-insensitive potassium current in pituitary gonadotrophs. Endocrinology 1997;138:2658–2664.
36.
Marrion NV: Control of M-current. Annu Rev Physiol 1997;59:483–504.
37.
Kalman D, O’Lague PH, Erxleben C, Armstrong DL: Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells. J Gen Physiol 1988;92:531–548.
38.
Rendt J, Oxford GS: Absence of coupling between D2 dopamine receptors and calcium channels in lactotrophs from cycling female rats. Endocrinology 1994;135:501–508.
1998
Copyright / Drug Dosage / Disclaimer
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.
You do not currently have access to this content.