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Institut de Physiologie (URA 309 CNRS), Université Louis Pasteur, Strasbourg, France.

1. 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 activate selectively GABAA receptor-mediated whole-cell and single-channel Cl- currents. 2. In the whole-cell recording (WCR) configuration inward Ca2+ currents triggered before and/or during the application of isoguvacine (100 microM), 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 Ca2+ 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. 3. At pCa 7, simultaneous application of isoguvacine (100 microM) 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. 4. At pCa 7, when isoguvacine (100 microM) was applied together 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. 5. 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. 6. 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. 7. In the cell-attached (CA) configuration, the presence of isoguvacine (3-5 microM) in the pipette solution triggered the opening of channels displaying multiple current levels.(ABSTRACT TRUNCATED AT 400 WORDS)

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