cGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2)

Abstract

NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal=100%; db-cGMP 100 microM=77+/-7%; 500 microM=54+/-10% and 1,000 microM=61+/-8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal=100%; 8-Br-cGMP=75+/-5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48+/-4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.

Fig. 1.

Dibutyryl cGMP (db-cGMP) decreases surface apical Na/K/2Cl cotransporter (NKCC2) levels in thick ascending limb of the loop of Henle (THALs). A: dose-response curve showing maximum inhibition by db-cGMP at 500 μM (n = 5 for each concentration, *P < 0.05 vs. basal). B: representative Western blotting showing surface NKCC2 levels in THALs treated with vehicle (basal, lane 1) and db-cGMP 500 μM (lane 2) and intracellular NKCC2 in the same samples vehicle, (basal, lane 3) and db-cGMP 500 μM (lane 4). The intracellular protein GAPDH is absent from the surface fraction (lanes 1 and 2) but strongly expressed in the intracellular fraction (lanes 3 and 4).

Fig. 2.

8-Br-cGMP decreases surface NKCC2 levels in THALs. A: 20-min incubation with 8-Br-cGMP (500 μM) decreased surface NKCC2 by 25% (n = 5, *P < 0.02 vs. basal). B: representative Western blot showing surface NKCC2 levels in THALs treated with vehicle (basal; lane 1) and 8-Br-GMP (500 μM; lane 2) and intracellular NKCC2 in the same samples [vehicle, basal (lane 3) and 8-Br-GMP (500 μM; lane 4)]. The intracellular protein GAPDH is absent from the surface fraction (lanes 1 and 2) but strongly expressed in the intracellular proteins (lanes 3 and 4).

Fig. 3.

Characterization of luminal (extracellular) NKCC2-directed antiserum. A: representative confocal image showing apical immunofluorescent labeling in medullary THAL cells after incubation of intact nonfixed perfused tubules with antiserum targeting an extracellular (luminal) sequence between transmembrane domains 5 and 6 of NKCC2. B: representative confocal image showing complete absence of immunofluorescent labeling after preadsorption of the luminal NKCC2 antiserum with excess immunizing peptide under the same conditions as in A. C: representative confocal image showing complete absence of immunofluorescent labeling after incubation of intact nonfixed THALs with preimmune serum from the same rabbit. D: representative confocal images showing dual fluorescent labeling in a medullary THAL with luminal NKCC2 antiserum (top; green) and with the apical surface marker peanut agglutinin (middle; red). The merged image (bottom) shows colocalization of staining in the apical surface of THAL cells.

Fig. 4.

db-cGMP decreases apical surface NKCC2 staining in perfused medullary THALs. A: representative confocal micrographs showing apical surface NKCC2 staining in isolated, perfused medullary THALs under basal (unstimulated) conditions (top) and in a THAL treated with db-cGMP (500 μM) for 20 min (bottom). B: cumulative data for apical NKCC2 immunofluorescence staining under basal conditions (n = 5) and in THALs treated with db-cGMP (500 μM) (n = 6). The mean fluorescent intensity in the apical membrane was measured in 15–20 THAL cells in each tubule and then averaged.

Fig. 5.

A–C: PDE2 inhibition blocks the cGMP-induced decrease in surface NKCC2 levels in THALs. A: representative Western blots showing the expression of PDE2 in medullary THALs from 2 different rats (25 μg of proteins THAL lysate/lane; n = 4). B: cumulative data showing the effect of db-cGMP and the PDE2 inhibitor BAY 60-7550 (BAY), alone and combined, on surface NKCC2 levels in the THAL (n = 6, *P < 0.05 vs. basal). C: clamping cAMP levels blocks the cGMP-induced decrease in surface NKCC2 levels in THALs. Cumulative data showing the effect of db-cGMP and a nonstimulatory concentration of db-cAMP (10 μM), alone and combined, on surface NKCC2 levels in the THAL (n = 6, *P < 0.05 vs. basal).

Fig. 6.

PDE2 inhibition blocks cGMP-induced decrease in net Cl absorption by isolated, perfused THALs. A: cumulative data showing the effect of db-cGMP (500 μM) on THAL net Cl absorption (n = 6, *P < 0.05). B: cumulative data showing the effect of db-cGMP (500 μM) on THAL net Cl absorption in tubules preincubated with the PDE2 inhibitor BAY 60-7550 (100 nM) (n = 6). Addition of the PDE2 inhibitor alone to the bath did not affect net Cl absorption (see results).