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Free keywords:
CA2+ CHANNEL; CHROMAFFIN CELL; BACLOFEN; GABA; G-PROTEIN; GTP-GAMMA-S; INTRACELLULAR CA2+; FURA-2
Abstract:
Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABAb) receptor activation on K+-depolarization-induced signals and on voltage-activated Ca2+ currents were investigated. Both GABA (20 μM) plus bicuculline (20 μM) and (-)baclofen (20–100 μM), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10–20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1μg/ml, for 4–6h at 37°C). Baclofen (50 μM) inhibited Ca2+ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5′-triphosphate GTPγ S (100 μM), led to complete inhibition of the Ca2+ conductance and of voltage-evoked intracellular Ca ([CA]i) transients within 2 min. In paired cells intracellularly perfused with GTPγS-free solution, the Ca2+ current amplitude decreased by only about 30% for 5–6 min. It is concluded that bovine chromaffin cells have functional GABAb receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca2+. The small size of the effect on Ca2+ current in whole-cell mode as compared to that on the Ca signal in intact cells may be explained by washout of some regulatory element during cell dialysis, or by a relatively small contribution of the normal voltage-activated Ca2+ current to the Ca signal. Alternatively, it might indicate GABAb effects on mechanisms other than Ca2+ channels.
