Expression and regulation of the Na+-K+-2Cl- cotransporter NKCC1 in the normal and CFTR-deficient murine colon

Abstract

Defective regulation and/or reduced expression of the Na+-K+-2Cl- cotransporter NKCC1 may contribute to the severe secretory defect that is observed in cystic fibrosis, but data concerning the expression and function of NKCC1 in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient cells are equivocal. We therefore investigated NKCC1 mRNA expression, Na+-K+-2Cl- cotransport activity and regulation by cAMP in crypts isolated from the proximal colon of CFTR-containing (CFTR (+/+)) and CFTR-deficient (CFTR (-/-)) mice. mRNA expression levels were determined by semiquantitative PCR, transport rates were measured fluorometrically in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetomethylester (BCECF)-loaded crypts, cytoplasmic volume changes were assessed by confocal microscopy, and [Cl-]i changes were examined by N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) quenching. NKCC1 mRNA expression levels were not significantly reduced in CFTR (-/-) crypts compared to controls. Azosemide-sensitive NH4+ influx (used as a measure of Na+-K+-2Cl- cotransport) was 2.23 +/- 0.72 vs. 1.56 +/- 0.16 mM min-1, and increased by 63.6 % in (+/+) and 87.3 % in (-/-) crypts upon stimulation for 5 min with forskolin. After 20 min of stimulation with forskolin, the Na+-K+-2Cl- cotransport rates in (-/-) and (+/+) crypts were identical. Crypt cross-sectional area and [Cl-]i decreased only in (+/+) crypts upon stimulation. In conclusion, normal NKCC1 expression levels, somewhat reduced Na+-K+-2Cl- cotransport rates, but preserved activation by cAMP were found in colonic crypts from CFTR (-/-) mice, ruling out a severe dysfunction of the Na+-K+-2Cl- cotransporter in the CF intestine. Furthermore, these studies establish the existence of a direct, cell-volume- and [Cl-]i-independent activation of colonic NKCC1 by an increase in intracellular cAMP.

Figure 1. Intrinsic buffering capacity of colonic crypts from CFTR (+/+) and CFTR (−/−) mice

To calculate proton-base flux rates, β_i was determined in murine colonic crypts from CFTR (+/+) (▪-) and CFTR (−/−) mice (•–) between pH_i 6.6 and 7.8, as described in Methods. The buffering curves with a β_i of 45.1 mm (pH unit)⁻¹ and 49.5 mm (pH unit)⁻¹ at pH_i 6.7, 24.7 mm (pH unit)⁻¹ and 26.0 mm (pH unit)⁻¹ at pH_i 7.1 and 26.7 mm (pH unit)⁻¹ and 24.3 mm (pH unit)⁻¹ at pH_i 7.6 in CFTR (+/+) and CFTR (−/−) mice, respectively, resemble that found in other cell types. Data points were fitted with a second-order polynomial function. (n = 13 crypts from 9 mice for normal mice, n = 11 crypts from 6 mice for knockout animals).

Figure 2. Method of the semiquantitative RT-PCR (A) and expression analysis of NKCC1 in colonic crypts and surface cells from CFTR (+/+) and CFTR (−/−) mice (B)

A, determination of NKCC1 expression in colonic crypts from CFTR (+/+) mice, using 18 s rRNA as an internal control. Amplification curves were created for both the NKCC1 and 18 s rRNA primer pairs, and the virtual relationship optical density integrated (ODI) of the studied gene vs. the ODI of 18 s rRNA was used as a measure for the expression level of the gene of interest after correction of the values for the different PCR products according to their length. B, relative expression levels of NKCC1 in colonic crypts (▪) and surface cells (□) from CFTR (+/+) (left panel) and CFTR (−/−) mice (right panel). NKCC1 was significantly more abundant in crypts than in surface cells (P < 0.01, Student's unpaired t test); the expression levels in CFTR (−/−) tissues were slightly, but not significantly reduced (n = 4–6 mice).

Figure 3. I_SC measurements in Ussing-chamber experiments with isolated colonic mucosa

I_SC measurements in Ussing-chamber experiments with isolated colonic mucosa from normal mice (Fig. 3A–C), from NKCC1 (−/−) mice and from their NKCC1 (+/+) littermates (Fig. 3D and E). cAMP-dependent stimulation of I_SC (A) was inhibited to the same extent by 100 μm (not shown) or 500 μm bumetanide (B) as by 100 μm azosemide (C), and neither compound resulted in further I_SC inhibition after the other one was added (B and C). Furthermore, 100 μm azosemide had no effect on I_SC (D and E) or HCO₃⁻ flux (not shown) in NKCC1 (−/−) colon (n = 3–5).

Figure 4. NKCC1 activity in colonic crypts, measured as the azosemide-sensitive fraction of pH_i recovery from a NH₃/NH₄⁺ load

A, C and F, time course of changes in pH_i; B, D and G, corresponding base efflux rates; E and H, relative NKCC1 activity. After demonstrating equal base efflux rates during multiple NH₄⁺ pulses (A, B; -3.65 ± 0.5 mm min⁻¹), NKCC1 activity was measured using azosemide, which decreased the ΔpH_i/Δt (C) and the flux rate (D; -1.4 ± 0.3 mm min⁻¹). Cotransport activity is expressed as a percentage of the initial flux(es) in each experiment as an internal control, to better standardize the results (E; 42 ± 7 %). When stimulated, the total and azosemide-sensitive ΔpH_i/Δt (F) as well as the base efflux rates (G; -4.54 ± 0.67 and -2.15 ± 0.45 mm min⁻¹) and the relative NKCC1 activity (H; 70 ± 14 %) was higher (Ctrl = control, Azo = azosemide, F/Forsk = forskolin; n = 8–10 crypts from 5–7 mice).

Figure 5. Base efflux rates in murine colonic crypts in the absence and presence of Ba²⁺

Total (A) and azosemide-sensitive (B and C) base efflux rates in murine colonic crypts. In C, the azosemide-sensitive flux rates are expressed in relation to the first NH₄ pulse as an internal control (see Fig. 4A). Ba²⁺ slightly decreased total flux rates (A, P < 0.05) as well as azosemide-sensitive base efflux (n.s.; B and C), indicating an inhibitory effect of 1 mm Ba²⁺ on both non-NKCC1-related base efflux and on basal NKCC1 activity. However, forskolin stimulation increased the azosemide-sensitive base efflux rates to a similar extent both in the presence and absence of Ba²⁺ (C; 42 ± 7 % vs. 69 ± 14 % and 40 ± 6 % vs. 69 ± 16 %, n = 6–8 crypts from 4–5 mice, *P < 0.05: § Student's unpaired t test, # Student's paired t test). When values are given as the flux rate as a percentage of the control measured within the same experiment (C), the variability is somewhat smaller, and therefore forskolin stimulation was significant at a relatively low value of n.

Figure 6. NKCC activity in colonic crypts from CFTR (+/+) and (−/−) mice, in the presence and absence of forskolin stimulation

cAMP-dependent stimulation with forskolin markedly enhanced the relative azosemide-sensitive flux rate in CFTR (+/+) crypts (42 ± 4 % to 69 ± 14 %, P < 0.05: Student's paired t test; n = 8 crypts from 5 mice) as well as in CFTR (−/−) crypts (24 ± 2 % to 45 ± 9 %, P < 0.05: Student's paired t test, n = 8 crypts from 6 mice). Interestingly, a longer duration of stimulation with forskolin yielded a much more pronounced stimulation on top of the brief forskolin stimulation in CF mice (72 ± 12 %, n = 10 crypts from 8 mice) than in normal mice (76 ± 11 %, n = 9 crypts from 6 mice; *P < 0.05: § Student's unpaired t test, # Student's paired t test).

Figure 7. Changes in crypt volume and [Cl⁻]_i in response to forskolin stimulation in CFTR (+/+) and (−/−) crypts

A, time course of changes in the crypt cross-sectional area. B, maximal change upon forskolin stimulation or incubation with non-isotonic solutions. The volume change after a hyperosmotic challenge was almost equal in CF and normal mice. Hyposmotic challenge produced a stronger change in crypt cross-section area in CFTR (+/+) mice. Upon forskolin stimulation, crypt cross-section area decreased by 9.4 ± 1.2 % in CFTR (+/+) crypts, while there was no change in CFTR (−/−) crypts (n = 10 crypts from 6–7 mice in each group, P < 0.01). C shows MQAE fluorescence at t = 10 min, which was used to follow changes in [Cl⁻]_i in CFTR (+/+) and CFTR (−/−) crypts. Azosemide alone caused no change in MQAE fluorescence in CF or normal mice. Subsequent forskolin stimulation caused a substantial increase in MQAE fluorescence (13.8 ± 0.34 %) in CFTR (+/+) crypts, and no change in CFTR (−/−) crypts (n = 5–7 crypts from 3–5 mice in each group; **P < 0.01: Student's unpaired t test). While a portion of the increase in MQAE fluorescence in the normal crypts could be due to the fact that they shrink, the complete lack of change in signal intensity rules out a forskolin-induced change in [Cl⁻]_i in the CF crypts.

Figure 8. Effect of Hoe 293b, an inhibitor of cAMP-activated K⁺ channels, on cotransporter activity

A, the volume effect of Hoe 293b alone, which is a slight swelling of the crypt (2.03 ± 0.3 %, n = 5 crypts from 3 mice, n.s.: Student's t test), measured confocally as the change of cross-section area. The basal NKCC activity is decreased in the presence of Hoe 293b (B), but NKCC1 (B) is activated by a rise in intracellular cAMP upon forskolin stimulation in both cases (n = 6–8 crypts from 3–5 mice in each group; *P < 0.05: §Student's unpaired t test, # Student's paired t test).