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Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl(-) secretion and inhibit amiloride-sensitive Na(+) transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na(+) channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl(-) transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N(2),2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl(-).
Fig. 1. Expression of ENaC and CFTR in Xenopus oocytes. (A) Continuous recording of amiloride-sensitive whole cell conductance (GAmiloride) over 90 min does not show any run down of GAmiloride. (B) Original recordings of the whole cell currents obtained in a CFTR/ENaC expressing oocyte. Activation of CFTR by stimulation with IBMX (1 mmol/l) and forskolin (2 μmol/l) inhibits the amiloride-sensitive whole cell current. The inhibition shows a partial recovery of GAmiloride 15 min after omission of IBMX/forskolin. (C) GAmiloride can be inhibited repetitively by activating and deactivating CFTR. * indicate significant difference from control (paired t-test). (Number of experiments).
Fig. 2. Effects of stimulation of luminal purinergic receptors by ATP or UTP on ion transport in mouse trachea and Xenopus oocytes expressing ENaC. (A) Continuous recording of the transepithelial voltage (Vte) in mouse trachea. ATP (100 μmol/l) induced a transient voltage deflection and inhibited amiloride (A) sensitive transport. (B) Summary of the amiloride-sensitive short circuit currents before and after stimulation with ATP. (C) Whole cell currents measured in Xenopus oocytes expressing the epithelial Na+ channel ENaC and effects of amiloride (10 μmol/l) in the absence or presence of ATP (100 μmol/l). (D) Summary of the amiloride-sensitive whole cell conductances (GAmil) measured in ENaC expressing oocytes. Stimulation by ATP or UTP (both 100 μM) has no impact on GAmil. (Number of experiments).
Fig. 3. Effects of stimulation by IBMX (100 μmol/l)/forskolin (2 μmol/l) (I/F) and ATP or UTP (both 100 μmol/l) on transepithelial voltages and summary of short circuit currents measured in mouse trachea. (A) Incubation with hexokinase (5 U/ml) and glucose (15 mM) attenuates ATP-activated transport but has no effect on I/F induced secretion. (B) The P2Y blocker suramin (100 μmol/l) inhibits UTP-activated transport but has no effect on I/F-induced secretion. (C) The Clâ channel blocker DIDS inhibits ATP-activated transport but has no effect on I/F-induced secretion. * indicate significant difference from control (paired t-test). (Number of experiments).
Fig. 4. Summary of whole cell conductances measured in Xenopus oocytes coexpressing ENaC and P2Y2 receptors. A whole cell Clâ conductance is activated by stimulation with ATP (100 μmol/l) (A), which does not affect amiloride-sensitive Na+ conductance (GAmil) (B). (CâE) Summary of whole cell conductances measured in oocytes coexpressing ENaC, P2Y2 receptors and CFTR. A whole cell Clâ conductance is activated by stimulation with ATP (100 μmol/l). Suramin itself has no effects on basal ion conductance (C) or amiloride sensitive conductance (D). However, the effect of ATP is completely suppressed by suramin. Activation of CFTR by IBMX (1 mmol/l) and forskolin (2 μmol/l) (I/F) inhibits GAmil in the absence or presence of suramin (D) or hexokinase (5 U/ml) and glucose (15 mmol/l) (E). * indicate significant difference from control. (Number of experiments).
Fig. 5. (A) Continuous recording of the transepithelial voltage (Vte) in mouse colon and effects of amiloride on Vte in the presence or absence of IBMX/forskolin and hexokinase/glucose. (B) Summary of the amiloride-sensitive short circuit currents (Isc-Amil) obtained before and after stimulation of CFTR. Isc-Amil is inhibited by CFTR, even in the presence of hexokinase/glucose. Inhibition in the presence of hexokinase/glucose is not different to inhibition of Isc-Amil in the absence of hexokinase/glucose. * indicate significant difference from control (paired t-test). (Number of experiments).
Fig. 6. Transepithelial voltages measured in human colonic biopsies. (A) Continuous recording of the transepithelial voltage (Vte) in human colonic epithelia and effects of stimulation by basolateral carbachol (CCH; 100 μmol/l) and luminal UTP (100 μmol/l). Experiments were performed in the continuous presence of indomethacin and amiloride (both 10 μmol/l). ÎVte=voltage deflection induced by pulsed current injection. (B) Summary of short circuit currents induced by basolateral CCH and luminal ATP in the absence of stimulation by IBMX/forskolin (âcAMP). (C) Summary of short circuit currents induced by basolateral CCH and basolateral or luminal ATP in the absence (âcAMP) or after stimulation by IBMX/forskolin (+cAMP). No effect of ATP on short circuit currents could be detected. (D,E) Missing effect of stimulation by luminal ATP on Isc-Amil. * indicate significant difference from control (paired t-test). (Number of experiments).
Fig. 7. Effects of cGMP on ENaC expressed in Xenopus oocytes. (A) Continuous recording of the whole cell currents measured in Xenopus oocytes expressing the epithelial Na+ channel ENaC, and effects of amiloride (10 μmol/l) (upper trace). Incubation with 1 mmol/l of membrane-permeable cGMP inhibits ENaC (middle trace). The inhibitory effect of cGMP on ENaC is suppressed by an inhibitor of protein kinase G (lower trace). (B) Summary of the effects of membrane permeable cGMP on amiloride-sensitive whole cell conductance (GAmil) in the absence or presence of the PKG inhibitor. Note that cGMP has a slight but significant inhibitory effect on GAmil only in the absence of PKG inhibitor. The PKG inhibitor enhanced GAmil. (C) Downregulation of GAmil by activation of CFTR with IBMX (1 mmol/l) and forskolin (2 μmol/l) (I/F) takes place in the absence or presence of the PKG inhibitor. # indicate significantly enhanced GAmil in the presence of PKG inhibitor (unpaired t-test). * indicate significant difference from control (paired t-test). (Number of experiments).
Fig. 8. (A) Continuous recording of the transepithelial voltage (Vte) in mouse trachea and effects of amiloride on Vte in the presence or absence of membrane permeable cGMP. (B) Summary of the amiloride-sensitive short circuit currents (Isc-Amil) obtained before and application of membrane permeable cGMP. (Number of experiments).
Fig. 9. Summary of amiloride-sensitive whole cell conductances (GAmil) measured in Xenopus oocytes coexpressing ENaC and CFTR. (A) Oocytes were injected with the inhibitor of nucleoside diphosphate kinase, naringenin (final oocyte concentration 50 μmol/l), or water. Downregulation of GAmil by activation of CFTR with IBMX (1 mmol/l) and forskolin (2 μmol/l) (I/F) was not suppressed by naringenin. (B) Additional coexpression of NDPK did not affect downregulation of GAmil by activation of CFTR with IBMX and forskolin. Inhibition of GAmil was reversible upon replacement of extracellular Clâ by gluconate. * indicate significant difference from control (paired t-test). (Number of experiments).
