Inhibition of amiloride-sensitive apical Na+ conductance by acetylcholine in rabbit cortical collecting duct perfused in vitro

Abstract

We examined effects of acetylcholine (ACh) on the electrical parameters and intracellular Ca2+ concentration ([Ca2+](i)) in the isolated rabbit cortical collecting duct (CCD) perfused in vitro using the conventional microelectrode technique and microscopic fluorescence spectrophotometry. ACh (10-8 to 10-5 M) in the bath caused a positive deflection of the transepithelial voltage (V(T)) and an increase in [Ca2+](i). Carbachol also showed similar but smaller effects. The effects of ACh were antagonized by muscarinic receptor antagonists. ACh at 10-6 M hyperpolarized the apical membrane voltage and increased the fractional resistance of the apical membrane of the collecting duct cells accompanied by a positive deflection of V(T) and an increase in transepithelial resistance, whereas it did not affect these parameters in the β-intercalated cells. In the presence of 10-5 M amiloride in the lumen, the effects of ACh were almost completely abolished. The ACh-induced increase in [Ca2+](i) is accounted for by the release of Ca2+ from intracellular store and Ca2+ entry from the bath. In the absence of Ca2+ in the bath, the ACh-induced changes in electrophysiological parameters were significantly smaller than those observed in the presence of Ca2+. Both phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutylate (PDBu), activators of protein kinase C (PKC), also inhibited the apical Na+ conductance. In the presence of PMA or PDBu in the bath, ACh did not show further inhibitory effect. 1-(5- Isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of PKC, partially attenuated the effect of ACh. These observations indicate that ACh inhibits the apical Na+ conductance partly by both increasing [Ca2+](i) and activating PKC. Such an action of ACh may partially explain its natriuretic effect.