K+ secretion activated by luminal P2Y2 and P2Y4 receptors in mouse colon

Abstract

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K+ secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP-stimulated K+ secretion was still present in P2Y2 receptor knock-out mice, but significantly reduced (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 2.08 +/- 0.52 mV, n = 9). In P2Y4 receptor knock-out mice the UTP-induced K+ secretion was similarly reduced. Luminal UTP-stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.

Figure 1. Effect of luminal ATP and UTP on ion transport in mouse distal colonic mucosa (NMRI mice)

Original recording of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te). The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te, see Methods). Application of luminal ATP or UTP to the luminal side led to a transient deflection of V_te to lumen-positive values and a decrease of R_te.

Figure 2. Concentration response relationship of luminal UTP- and ATP-induced transepithelial voltage deflections

Both nucleotides showed a similar potency with an EC₅₀ value of 13 μm for ATP and 10 μm for UTP.

Figure 3. Effect of luminal Ba²⁺ on luminal ATP-induced transepithelial voltage effect in mouse distal colonic mucosa

Original recording of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te). The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te). Luminal Ba²⁺ completely inhibited the ATP-induced V_te deflections. After wash-out this effect was fully reconstituted.

Figure 4. RT-PCR identification of different P2Y receptors mRNA transcripts from isolated distal colonic crypts of NMRI mice

Positive results were found for the P2Y₂, the P2Y₄ and the P2Y₆ receptors.

Figure 5. Effect of luminal UTP in P2Y₂ receptor KO and WT littermates

Two original recordings of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te) in KO and WT mice. The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te). The right panel shows the summary of all experiments.

Figure 6. RT-PCR identification of different mouse P2Y receptors mRNA transcripts from isolated distal colonic crypts from P2Y₂ KO and WT mice

Positive results were found for the P2Y₄ and the P2Y₆ receptors in WT and KO. P2Y₂ receptor transcripts were absent in the KO mouse and present in the WT mouse.

Figure 7. Effect of luminal UTP in P2Y₄ receptor knock-out mice in comparison to wild-type littermates

Two original recordings of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te) in KO and WT mice. The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te). The right panel shows the summary of all experiments.

Figure 8. RT-PCR identification of different mouse P2Y receptor mRNA transcripts from isolated distal colonic crypts of P2Y₄ KO and WT mice

Positive results were found for the P2Y₂ and the P2Y₆ receptors in WT and KO. P2Y₄ receptor transcripts were absent in the KO and present in the WT mice.

Figure 9. Effect of luminal UTP in P2Y₄ receptor knock-out mice in comparison to wild-type littermates treated on a low Na⁺ diet for 2 weeks

Two original recordings of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te) in KO and WT mice. The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te). Note the significantly more lumen negative V_te values in both original traces as compared to those in Figs 1, 3, 5 and 7. Importantly, in all P2Y₄ KO mice, luminal UTP always induced the rapid and transient V_te deflection. The right panel shows the summary of 13 mouse pairs.

Figure 10. Summary of all UTP-stimulated K⁺ secretory effects (rapid and transient V_te deflections) in P2Y₂ and P2Y₄ WT and KO mice on normal and low Na⁺ diet

Note the significant up-regulation of this effect in all tested animals after the treatment on a low Na⁺ diet.

Figure 11. Absence of luminal UTP-stimulated K⁺ secretion in P2Y₂/P2Y₄ double receptor KO mice in comparison to wild-type littermates

Two original recordings of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te) in DKO and WT mice. The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te). The right panel shows the summary of 7 mouse pairs.

Figure 12. No effect of basolateral UTP in a P2Y₄ receptor WT mouse

An original recording of transepithelial voltage (V_te) and transepithelial voltage changes (ΔV_te). The upper line shows V_te and the bandwidth of voltage deflections reflects the transepithelial resistance (R_te). The dashed line represents the baseline before addition of basolateral UTP.