Perinatal PTX-sensitive G-protein expression and regulation of conductive 22Na+ transport in lung apical membrane vesicles.
Using apical membrane vesicles (AMV) prepared from mature foetal and early neonatal guinea pig lung we show that pertussis toxin (PTX)-sensitive G-protein regulation of conductive 22Na+ uptake undergoes rapid changes following birth. Thus, G-protein activation by intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl, which in late gestation stimulated uptake, consistently induced inhibition of conductive Na+ uptake into AMV prepared from neonatal lung at 4 days of age (N4) (52+/-9%, n=8, P<0.05). This response was not significantly different in the presence of the relatively impermeant anion isethionate (Ise-) (69+/-9%, n=7, P<0.05). Changes in the regulation of uptake were already detectable on the day of birth (N0) in AMV resuspended in NaCl, with GTPgammaS inducing both stimulatory and inhibitory responses. These data indicate that the processes by which 22Na+ uptake into AMV is regulated by G-proteins undergoes a change at birth and by 4 days of age, G-protein regulation of uptake occurs predominantly via modulation of co-localised Na+ channels. Intravesicular incorporation of GDPbetaS or pre-treatment with PTX did not significantly alter conductive 22Na+ uptake in the presence of NaCl or NaIse suggesting that constitutively active G-proteins are not involved in this process. Pre-treatment of AMV with PTX prevented the inhibition of conductive 22Na+ uptake by GTPgammaS (105+/-16% n=7) indicating that a PTX-sensitive G-protein mediates the inhibition of channels in neonatal AMV. Western blotting demonstrated enrichment of Gialpha1, Gialpha2, Gialpha3 and Goalpha in the apical membrane preparations. We also show that there is a significant rise in the levels of Gialpha3 during the early neonatal period providing a potential candidate for the G-protein mediated changes in regulation of conductive 22Na+ uptake in neonatal AMV.
Lung Membrane Transport Group, Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
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Gambling
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Olver
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Baines
D000818:Animals; D000831:Animals, Newborn; D001692:Biological Transport / Q000187:drug effects; D005314:Embryonic and Fetal Development; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D008168:Lung / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D037342:Pertussis Toxin; D020033:Protein Isoforms / Q000096:biosynthesis; D012964:Sodium / Q000378:metabolism*; D012979:Sodium Radioisotopes; D010566:Virulence Factors, Bordetella
D020033:Protein Isoforms; D012979:Sodium Radioisotopes; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012964:Sodium; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins