Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells

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 microM) plus bicuculline (20 microM) and (-)baclofen (20-100 microM), 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, 1 micrograms/ml, for 4-6 h at 37 degrees C). Baclofen (50 microM) 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 GTP gamma S (100 microM), 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 GTP gamma S-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+.(ABSTRACT TRUNCATED AT 250 WORDS)