Perinatal PTX-sensitive G-protein expression and regulation of conductive 22Na + transport in lung apical membrane vesicles

Abstract

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 μM GTPγS 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 GTPγS 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 GDPβS 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 GTPγS (105±16% n=7) indicating that a PTX-sensitive G-protein mediates the inhibition of channels in neonatal AMV. Western blotting demonstrated enrichment of G iα1, G iα2, G iα3 and G oα in the apical membrane preparations. We also show that there is a significant rise in the levels of G iα3 during the early neonatal period providing a potential candidate for the G-protein mediated changes in regulation of conductive 22Na + uptake in neonatal AMV.