Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles.
In apical membrane vesicles (AMV) prepared from late gestation fetal guinea pig lung we show that conductive 22Na+ uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G proteins. Intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl caused a significant stimulation (P<0. 05) of conductive Na+ uptake in AMV to 150+/-10% (n=10) of control, whereas GDPbetaS reduced uptake to 65+/-9% (n=4) of control. This contrasting response to GTPgammaS and GDPbetaS is characteristic of a G protein mediated pathway. GTPgammaS induced a significantly smaller stimulation, 125+/-8% (n=5) of control, in the presence of the relatively impermeant anion isethionate (Ise-). Taken together, these data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive 22Na+ uptake to 171+/-20% (n=13) of control in AMV resuspended 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 GTPgammaS diminished uptake, we propose that the G protein(s) responsible for Na+ channel activation in response to GTPgammaS is PTX-sensitive and that additional PTX-insensitive G proteins might also modulate 22Na+ uptake in these AMV. The presence of Gialpha1, Gialpha2, Gialpha3 and Goalpha in this apical membrane preparation was confirmed by PTX catalysed [32P]ADP-dependent ribosylation and Western blotting. Incubation of AMV with 200 microM DTT caused an inhibition of conductive Na+ uptake in AMV resuspended in NaCl or NaIse to 66+/-8% (n=11) and 64+/-8% (n=6) of control respectively. Pre-treatment with DTT did not affect the ability of GTPgammaS to stimulate conductive Na+ uptake suggesting that the regulation of 22Na+ uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein.
Gambling
L
L
Lung Membrane Transport Group, Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
Olver
R E
RE
Fyfe
G K
GK
Kemp
P J
PJ
Baines
D L
DL
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000838:Anions; D002462:Cell Membrane / Q000502:physiology*; D002463:Cell Membrane Permeability; D018118:Chloride Channels / Q000502:physiology*; D004229:Dithiothreitol / Q000494:pharmacology; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D007513:Isethionic Acid / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D008168:Lung / Q000196:embryology* / Q000648:ultrastructure*; D037342:Pertussis Toxin; D015222:Sodium Channels / Q000502:physiology*; D012979:Sodium Radioisotopes / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D000838:Anions; D018118:Chloride Channels; D008081:Liposomes; D015222:Sodium Channels; D012979:Sodium Radioisotopes; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D007513:Isethionic Acid; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D004229:Dithiothreitol