DIFFERENTIAL REGULATION OF NA- CONDUCTANCES BY PTX-SENSITIVE G-PROTEINS IN FETAL LUNG APICAL MEMBRANE-VESICLES( AND CL)

In apical membrane vesicles (AMV) prepared from late gestation fetal g uinea pig lung we show that conductive Na-22(+) uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G pro teins. Intravesicular incorporation of 100 mu M GTP gamma S into vesic les resuspended in NaCl caused a significant stimulation (P < 0.05) of conductive Na+ uptake in AMV to 150 +/- 10% (n = 10) of control, wher eas GDP beta S reduced uptake to 65 +/- 9% (n = 4) of control. This co ntrasting response to GTP gamma S and GDP beta S is characteristic of a G protein mediated pathway. GTP gamma S induced a significantly smal ler stimulation, 125 +/- 8% (n = 5) of control, in the presence of the relatively impermeant anion isethionate (Ise(-)). Taken together, the se data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive Na-22(+) uptake to 171 +/- 20% (n = 13) of control in AMV r esuspended in NaCl, but did not have a significant effect, 94 +/- 19% of control, in the presence of NaIse indicating the existence of tonic activation of Cl- channels in these AMV under resting conditions. As the combined effects of PTX and GTP gamma S diminished uptake, we prop ose that the G protein(s) responsible for Na+ channel activation in re sponse to GTP gamma S is PTX-sensitive and that additional PTX-insensi tive G proteins might also modulate Na-22(+) uptake in these AMV. The presence of G(i)alpha(1), G(i)alpha(2), G(i)alpha(3) and G,a in this a pical membrane preparation was confirmed by PTX catalysed [P-32]ADP-de pendent ribosylation and Western blotting. Incubation of AMV with 200 mu M DTT caused an inhibition of conductive Na+ uptake in AMV resuspen ded in NaCl or NaIse to 66 +/- 8% (n = 11) and 64 +/- 8% (n = 6) of co ntrol respectively. Pre-treatment with DTT did not affect the ability of GTP gamma S to stimulate conductive Na+ uptake suggesting that the regulation of Na-22(+) uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein. (C) 1998 Elsevier Science B.V, All rights reserved.