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.
Lung Membrane Transport Group, Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
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L
Gambling
R E
RE
Olver
G K
GK
Fyfe
P J
PJ
Kemp
D L
DL
Baines
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