ATP mediates flow-induced NO production in thick ascending limbs

Abstract

Mechanical stimulation caused by increasing flow induces nucleotide release from many cells. Luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in thick ascending limbs. However, the factors that mediate flow-induced NO production are unknown. We hypothesized that luminal flow stimulates thick ascending limb NO production via ATP. We measured NO in isolated, perfused rat thick ascending limbs using the fluorescent dye DAF FM. The rate of increase in dye fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nl/min stimulated NO production from 17 ± 16 to 130 ± 37 arbitrary units (AU)/min (P < 0.02). Increasing flow from 0 to 20 nl/min raised ATP release from 4 ± 1 to 21 ± 6 AU/min (P < 0.04). Hexokinase (10 U/ml) plus glucose, which consumes ATP, completely prevented the measured increase in ATP. Luminal flow did not increase NO production in the presence of luminal and basolateral hexokinase (10 U/ml). When flow was increased with the ATPase apyrase in both luminal and basolateral solutions (5 U/ml), NO levels did not change significantly. The P2 receptor antagonist suramin (300 μmol/l) reduced flow-induced NO production by 83 ± 25% (P < 0.03) when added to both and basolateral sides. Luminal hexokinase decreased flow-induced NO production from 205.6 ± 85.6 to 36.6 ± 118.6 AU/min (P < 0.02). Basolateral hexokinase also reduced flow-induced NO production. The P2X receptor-selective antagonist NF023 (200 μmol/l) prevented flow-induced NO production when added to the basolateral side but not the luminal side. We conclude that ATP mediates flow-induced NO production in the thick ascending limb likely via activation of P2Y receptors in the luminal and P2X receptors in the basolateral membrane.

Fig. 1.

Effect of increasing luminal flow on nitric oxide (NO) production by isolated thick ascending limbs . There was no difference between 0 and 5 nl/min (n = 5). *P < 0.002 compared with 5 nl/min. #P < 0.02 compared with 0 nl/min (n = 5–7).

Fig. 2.

Effect of increasing luminal flow on luminal ATP release by isolated thick ascending limbs (n = 6).

Fig. 3.

Effect of increasing luminal flow in the presence of hexokinase (10 U/ml) on NO production by isolated thick ascending limbs. Luminal flow did not increase NO production in the presence of hexokinase (n = 6).

Fig. 4.

Effect of increasing luminal flow in the presence of the ATPase apyrase (5 U/ml) on NO production by isolated thick ascending limbs. Luminal flow did not increase NO production significantly in the presence of apyrase (n = 6).

Fig. 5.

Effect of increasing luminal flow in the presence of hexokinase (10 U/ml) on luminal ATP release by isolated thick ascending limbs. The presence of hexokinase in the perfusate completely prevented the measured flow-induced increase in ATP (n = 5).

Fig. 6.

Effect of the P2 receptor antagonist suramin (300 μmol/l) on flow-induced NO production by isolated thick ascending limbs. Suramin reduced flow-induced NO production by 83 ± 25% (n = 5).

Fig. 7.

Effect of increasing luminal flow in the presence of luminal hexokinase (10 U/ml) on NO production by isolated thick ascending limbs. Luminal hexokinase reduced flow-induced NO production (n = 7).

Fig. 8.

Effect of increasing luminal flow in the presence of basolateral hexokinase (10 U/ml) on NO production by isolated thick ascending limbs. Basolateral hexokinase blunted flow-induced NO production (n = 6).

Fig. 9.

Effect of increasing luminal flow in the presence of basolateral NF023 (200 μmol/l) on NO production by isolated thick ascending limbs. Basolateral NF023 prevented flow-induced NO production (n = 6).

Fig. 10.

Effect of increasing luminal flow in the presence of luminal NF023 (200 μmol/l) on NO production by isolated thick ascending limbs. Luminal NF023 did not affect flow-induced NO production (n = 6).