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Modulation of gaba gated chloride currents by intracellular calcium ion in cultured porcine melanotrophs

Modulation of gaba gated chloride currents by intracellular calcium ion in cultured porcine melanotrophs

Journal of Physiology (Cambridge) 437: 109-132

The modulatory role of intracellular Ca2+ concentration ([Ca2+]i) on .gamma.-aminobutyric acid type A (GABAA) receptor-gated Cl- currents was investigated in dialysed and intact cells of cultured porcine pituitary intermediate lobe (IL) cells using the patch-clamp technique. In order to isolate Ca2+ and Cl- currents all other membrane currents were blocked pharmacologically. Isoguvacine, a specific GABAA receptor agonist, was used to active selectively GABAA receptor-mediated whole-cell and single-channel Cl- currents. In the whole-cell recording (WCR) configuration inward Ca2+ currents triggered before and/or during the application of isoguvacine (100 .mu.M), did not inhibit the GABAA receptor-mediated response. This lack of effect of calcium currents was obtained in all situations tested, i.e. when the intracellular Ca+ concentration was only weakly buffered (0.5 mM-EGTA in the pipette solution), not buffered at all (no EGTA added to the pipette solution) or when the resting [Ca2+]i was buffered at 10-7 M (pCa 7) with internal EGTA. At pCa 7, simultaneous application of isoguvacine (100 .mu.M) and caffeine (10 mM) resulted in a 47 .+-. 15% reduction of the whole-cell GABAA response. In the same conditions, a ten times lower concentration of caffeine (1 mM), induced a transient increase of the GABAA response which turned into a steady-state inhibition during the subsequent applications. At pCa 7, when isoguvacine (100 .mu.M) was applied togeheter with 3Me-His2-TRH (50 nM), a potent analogue of the calcium-recruiting thyrotrophin-releasing hormone, the GABAA receptor-gated Cl- current was increased by 40 .+-. 8%. In the absence of the Ca2+ chelator EGTA in the pipette solution, either potentiating or inhibitory effects of 3Me-His2-TRH on the GABAA response were observed. If a high concentration (18 mM) of the calcium chelator EGTA was included in the pipette solution, caffeine and 3Me-His2-TRH had markedly lower effects on the GABAA response than those observed at pCa 7, suggesting that the effect of both substances was mediated by an increase in [Ca2+]i. In the absence of extracellular Ca2+, the effects of caffeine and 3Me-His2-TRH were not significantly different from those obtained in the presence of Ca2+ (5 mM), suggesting that Ca2+ influx was not the major route for increasing [Ca2+]i. In the cell-attached (CA) configuration, the presence of isoguvacine (3-5 .mu.M) in the pipette solution triggered the opening of channels displaying multiple current levels. The three main current levels observed at a membrane potential hyperpolarized by 80 mV with respect to the resting potential were 0.55 .+-. 0.04, 1.01 .+-. 0.02 and 2.09 .+-. 0.13 pA. In the cell-attached configuration of caffeine (10 mM) or 3Me-His2-TRH (50 mM) to the cell resulted in a reversible inhibition of single GABAA-gated Cl- channel activity. Thus 3Me-His2-TRH had opposite effects on the GABAA receptor-gated Cl- currents under whole-cell recording at pCa 7 and in the CA mode. It is concluded that a rise in [Ca2+]i triggered by Ca2+ release from intracellular stores but not by Ca2+ influx through voltage-dependent Ca2+ channels affects the activity of the GABAA receptor-Cl- channel complex in cultured porcine IL cells. The functional implications of this effect on the control of excitation-secretion coupling by GABA are discussed.

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Accession: 007560192

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