Regulation of Ca2+-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca2+ current

doi:10.1016/S0006-8993(98)00964-0

Brain Research

Volume 812, Issues 1–2, 23 November 1998, Pages 133-141

https://doi.org/10.1016/S0006-8993(98)00964-0 Get rights and content

Abstract

Ionic currents were investigated by a patch clamp technique in a clonal strain of pituitary (GH₃) cells, using the whole cell configuration with Cs⁺ internal solution. Depolarizing pulses positive to 0 mV from a holding potential of −50 mV activated the voltage-dependent L-type Ca²⁺ current (I_Ca,L) and late outward current. Upon repolarization to the holding potential, a slowly decaying inward tail current was also observed. This inward tail current upon repolarization following a depolarizing pulse was found to be enhanced by Bay K 8644, but blocked by nifedipine or tetrandrine. This current was eliminated by Ba²⁺ replacement of external Ca²⁺ as the charge carrier through Ca²⁺ channels, removal of Ca²⁺ from the bath solution, or buffering intracellular Ca²⁺ with EGTA (10 mM). The reversal potential of inward tail current was approximately −25 mV. When intracellular Cl⁻ was changed, the reversal potential of the Ca²⁺-activated currents was not shifted. Thus, this current is elicited by depolarizing pulses that activate I_Ca,L and allow Ca²⁺ influx, and is referred to as Ca²⁺-activated nonselective cationic current (I_CAN). Without including EGTA in the patch pipette, the slowly decaying inward current underlying the long-lasting depolarizing potential after Ca²⁺ spike was also observed with a hybrid current–voltage protocol. Thus, the present studies clearly indicate that Ca²⁺-activated nonselective cationic channels are expressed in GH₃ cells, and can be elicited by the depolarizing stimuli that lead to the activation of I_Ca,L.

Introduction

Ca²⁺-activated nonselective cationic currents (I_CAN) are recognized in a wide variety of cell types, including cardiac myocytes, thyroid follicular cells, neurons, neutrophils, pancreatic duct cells and pituitary cells 1, 5, 6, 10, 11, 13, 17, 19, 20. The activation of these channels may: (1) maintain the cell at depolarized state, and hence affect the firing pattern of neuroendocrine cells 1, 3, 10, (2) induce an after-depolarization following an action potential [6], or (3) affect the action potential configuration and consequently alter Ca²⁺ influx [20]. In rat lactotrophs like GH₃ cells, repetitive firing in the membrane potential, as found in neurons, has been observed and was thought to induce spontaneous release of prolactin 2, 8. It has been found that the firing pattern in GH₃ cells is potential and calcium dependent 8, 15. Furthermore, the change in Na⁺ conductance may affect spontaneous depolarization in pituitary cells 1, 15. However, it is unclear whether the alterations in electrical activity of GH₃ cells related to I_CAN which can be elicited by depolarizing pulses that activate Ca²⁺ current play an important role in regulating hormonal secretion.

The purpose of the present study was to clarify the characteristics and physiological significance of I_CAN in GH₃ cells with the aid of the whole-cell clamp techniques. The present study clearly demonstrates that in these cells, the Ca²⁺-activated inward current is elicited if Ca²⁺ entry is provoked through voltage-dependent L-type Ca²⁺ channels by applying the depolarizing pulses. Moreover, the change in the pattern of Ca²⁺ spikes caused by the activation of I_CAN which is elicited by depolarizing pulses that readily activate Ca²⁺ current is believed to play an important role in regulating hormonal secretion in GH₃ cells.

Section snippets

Cell culture

The clonal strain GH₃ cell line, originally derived from a rat anterior pituitary adenoma, was obtained from American Type Culture Collection (ATCC; Rockville, MD) [16]. GH₃ cells were grown in monolayer culture in 50 ml plastic culture flasks in a humidified environment of 5% CO₂/95% air at 37°C. Cells were maintained at a density of 10⁶/ml in 5 ml Ham's F-12 nutrient media (Gibco, Grand Island, NY) supplemented with 15% heat-inactivated horse serum, 2.5% fetal calf serum, and 2 mM l-glutamine

Characterization of L-type Ca²⁺ currents and Ca²⁺-activated currents in GH₃ cells

It has been shown that GH₃ cells have at least two types of the voltage-dependent Ca²⁺ channels, one of which is high threshold, dihydropyridine-sensitive, and classified as L-type [14]. The present experiments were conducted in cells bathed in normal Tyrode's solution containing 1.8 mM CaCl₂, tetrodotoxin (1 μM) and tetraethylammonium chloride (10 mM). As shown in Fig. 1, when pipette solution contained Cs⁺ ions without addition of EGTA, the cell was held at −50 mV, and the depolarizing

Discussion

The major findings of the present study are (1) Ca²⁺-activated nonselective cationic current (I_CAN) which was activated by Ca²⁺ influx due to the voltage-dependent L-type Ca²⁺ currents (I_Ca,L) was identified in anterior pituitary GH₃ cells, and (2) the regulation of I_CAN may play an essential role in maintaining the depolarizing state and thus determining firing properties of these cells in response to depolarizing stimuli, particularly under the conditions in which K⁺ channels are blocked.

Acknowledgements

This work was partly supported by grants from National Science Council (NSC-87-2341-B075B-013) and Veterans General Hospital-Kaohsiung (VGHKS-87-52 and VGHNSU-87-06), Taiwan, ROC.

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Research reportRegulation of Ca2+-activated nonselective cationic currents in rat pituitary GH3 cells: involvement in L-type Ca2+ current

Abstract

Introduction

Section snippets

Cell culture

Characterization of L-type Ca2+ currents and Ca2+-activated currents in GH3 cells

Discussion

Acknowledgements

Biochim. Biophys. Acta

J. Biol. Chem.

Biochim. Biophys. Acta

Trends Neurosci.

Biochem. Biophys. Res. Comm.

Methods Enzymol.

Eur. J. Pharmacol.

Action potentials and membrane ion channels in clonal anterior pituitary cells

Proc. Natl. Acad. Sci.

Alterations in the frequency and shape of Ca2+ fluctuations in GH4Cl cells induced by thyrotropin-releasing hormone and Bay K 8644

Biochem. J.

A long-lasting calcium-activated nonselective cationic current is generated by synaptic stimulation or exogenous activation of group I metabotropic glutamate receptors in CA1 pyramidal neurons

J. Neurosci.

Research report
Regulation of Ca²⁺-activated nonselective cationic currents in rat pituitary GH₃ cells: involvement in L-type Ca²⁺ current

Characterization of L-type Ca²⁺ currents and Ca²⁺-activated currents in GH₃ cells

Alterations in the frequency and shape of Ca²⁺ fluctuations in GH₄Cl cells induced by thyrotropin-releasing hormone and Bay K 8644