Effect of vasopressin on Naþ–Kþ–2Cl— cotransporter (NKCC) and the signaling mechanisms on the murine late distal colon

Abstract

It has been demonstrated that the antidiuretic hormone vasopressin is able to regulate the expression of Na–K–Cl cotransporters (NKCC1 and NKCC2) in the kidney. The present study investigated the effects of long- and short-term administration of vasopressin on NKCC and the possible signaling mechanism of vasopressin in the mouse distal colon using the siRNA, real-time PCR, western blotting and Ussing chambers method. The results showed the presence of NKCC2 expression in the colon, which was ver- ified with a siRNA technique. The mRNA and protein expression level of NKCC2 significantly increased by about 40% and 90% respectively in response to restricting water intake to 1 ml/day/20 g for 7 days. In contrast, the NKCC1 expression level was unchanged in the colon. To determine the short-term activation of NKCC2 by vasopressin in vitro, we found that the administration of vasopressin caused a 3-fold in- crease in mouse colon NKCC2 phosphorylation, which was detected with phosphospecific antibody R5. In addition, the Ussing chamber results showed that NKCC2, cAMP and Ca2þ signaling pathway may be involved in the vasopressin-induced response. Further, adenylate cyclase inhibitor MDL-12330A and PKA inhibitor H89 and Ca2þ chelator BAPTA-AM reversed the vasopressin induced NKCC2 phosphorylation level increase by about 35%, 28% and 42% respectively suggesting vasopressin stimulate NKCC2 phos- phorylation increase mediated by cAMP-PKA and Ca2 þ signaling in the colon. Collectively, these data suggest that the expression and phosphorylation of NKCC2 are increased in the colon by vasopressin stimulation, in association with enhanced activity of the vasopressin/cAMP and Ca2 þ pathways.

1. Introduction

Naþ–Kþ–2Cl— cotransporter (NKCC) mediates 1Naþ, 1K þ and 2Cl— ions across cell membranes in an electroneutral fashion (Haas and Forbush, 2000; Flatman, 2002). Two isoforms of the bumetanide-sensitive Naþ–Kþ–2Cl— cotransporter, which are products of different genes, are currently known (Park and Saier, 1996). NKCC1, the basolateral isoform, is by far the more widely distributed isoform and is especially prominent in the basolateral membranes of secretory epithelial cells (Matthews et al., 1998). In contrast, NKCC2 seems to be specifically located in the apical membranes of the thick ascending limb (TAL) of Henle’s loop (Obermüller et al.,1996). Although the previous studies showed that NKCC2 is also expressed in the apical plasma membrane of the colon (Xue et al., 2007, 2009, 2014; Zhu et al., 2011) many studies have focused on the expression and function of NKCC1, not NKCC2, in the distal colon because of limited information about NKCC2 in areas other than the kidney.

Vasopressin is just one of many hormonal and a paracrine factor that has been shown to acutely up-regulate NaCl transport in the thick ascending limb (Hebert et al., 1984). Most previous studies have focused on the regulation of NKCC2 by vasopressin in the kidney. Phosphorylation is involved in the activation of many membrane transport systems. A previous study demonstrated that phosphorylation of NKCC2 is related to its transport rate (Hanne- mann and Flatman, 2011). Phosphospecific antibody R5 (raised against two of the phosphothreonines Thr-184, Thr-189, generous gift from Prof. B. Forbush, Yale University) of NKCC has proven to be a very useful tool for studying NKCC regulation. Short-term actions of vasopressin administration on the thick ascending limb could cause an increase in phosphorylation of regulatory threo- nine at the amino terminus of NKCC2, which we could detect using the R5 antibody (Giménez and Forbush, 2003; Hannemann and Flatman, 2011). The colon could also be a target for vasopressin (Cristià et al., 2007) because most previous studies showed that vasopressin stimulates NaCl and water absorption in in vitro pre- parations of mouse, rat, and human colons (Aulsebrook, 1961; Grady et al.,1970; Bridges et al., 1983, 1984). Recently, we found that short-term vasopressin administration could regulate NKCC2 redistribution and trafficking in the colon (Xue et al., 2014). In addition, differences in NKCC2 phosphorylation in the short term as well as changes in NKCC2 expression level in the long term may affect vasopressin activity. In the kidney, it has been demonstrated that vasopressin stimulation of NKCC2 is mediated by adenylate cyclase activation and intracellular cAMP (Caceres et al., 2009; Ares and Ortiz, 2012). However, to our knowledge, there is only one report describing the decreases in intracellular Ca2þ may affect vasopressin activity in the colon (Bridges et al., 1983). Ac- cording to the previous study, which showed that NKCC2 expres- sion in the colon could be a target of vasopressin, we hypothesized that cAMP signaling may be involved in the effect of vasopressin on the colon. Here, we test the hypothesis that changes in NKCC2 phosphorylation detected by the R5 antibody after a short-term vasopressin administration accompany a change of NKCC2 ex- pression level after long-term vasopressin administration. In ad- dition, to better understand the signaling mechanisms of vaso- pressin in the colon, we observed the effect of vasopressin using a short-circuit current technique in conjunction with inhibitors.

2. Materials and methods

2.1. Animal handling and tissue preparation for immunoblotting

Male C57BL/6 mice weighing 20–25 g were used in the present study. All procedures employed in the present work involving animals were approved by guidelines and practices established by the Beijing Administration Office Committee of Laboratory Animal. The mice were killed by cervical dislocation. The distal colon and kidney were removed by opening the abdominal cavity. Late distal colonic segment away from the anus about 2 cm was quickly re- moved (Xue et al., 2007; Zhang et al., 2007) rinsed with ice-cold Krebs–Henseleit (KH) solution. The serosa, muscularis and sub- mucosa layers were stripped away with fine forceps to prepare a mucosa sample. Dissected tissues were homogenized in lysis buffer containing Tris–HCl, 2% SDS, glycerol, phenylmethylsulfonyl fluoride, and protease inhibitors. Protein concentrations were measured using a BCA protein assay. Whole homogenates from the colon was used to study the expression level and phosphorylation of NKCC.

2.2. Water restriction study

A 7-day water restriction was employed to produce a physio- logical increase in circulating vasopressin levels. Control mice and treated mice were randomly chosen, and the two groups were provided with the same amount of mouse chow. The chow for each group contained a different amount of water. The protocol is described in a previous study (Kim et al., 1999).

2.3. Vasopressin treatment in vitro

Fresh mucosa dissected from the mice colon, being placed in Krebs–Henseleit (KH) solution or in Krebs solution including va- sopressin (AVP, 5 ~ 10—8 mol/l), or H89 (20 μM), MDL-12330A (10 μM), BAPTA-AM (50 μM) respectively. The solution was gassed with 95% O2 and 5% CO2 to maintain tissue activity at 37 °C for 3 h or 30 min. After incubation, tissues were collected for protein preparation.

2.4. Hormone analysis

For serum AVP, mice were decapitated, blood was collected and serum AVP was determined by enzyme immunoassay (Cayman chemical, Ann Arbor, Michigan USA, 583951). For each animal, determination of serum AVP concentration was performed in tri- plicate with independent standard curves.

2.5. siRNA and western blotting

The small interfering RNA (siRNA) used in this study was siS- table-modified, in vivo-purified siRNA from Thermo Scientific Dharmacon (Lafayette, CO, USA). The siRNA sequence used for si- lencing NKCC2 was: sense 5′-GGAUAUAACCCACGCCUUUUU-3′ and antisense 5′-AAAGGCGUGGGUUAUAUCCUU-3′. Non-targeting siR- NA (catalog #D-001700-01) was used as a negative control. An Entranster™–in vivo (in vivo transfection reagent)/siRNA complex was prepared following the manufacturer’s protocol (Engreen Biosystem Co, Ltd., Beijing, China). In brief, diluted siRNA and Entranster™-in vivo were each added to a separate 10% glucose solution. Equal volumes of the two solutions were then combined and incubated for 15 min at room temperature. The complex containing 75 μg NKCC2 or non-targeting siRNA was injected into the caudal vein of mice. To establish the specificity of a NKCC2 antibody, we used a siRNA technique. P-NKCC (generous gift from Prof. B. Forbush) was only detected in the presence of phosphatase inhibitors. The proteins (20 mg) was separated by 8% SDS/PAGE, and the separated proteins were electroblotted onto a PVDF membrane (Millipore), which was then washed for 10 min with TBST and immersed in blocking buffer containing 5% non-fat dry milk in TBST for 1 h at 25 °C. The blot was washed with TBST and then incubated with a polyclonal primary antibody against NKCC2 or NKCC1 (Santa Cruz sc-133823, 1:1000; Fitzgerald 70 R-3806, 1:1000; sc-21574, 1:2000) overnight at 4 °C. After washing in TBST, the blot was incubated with a secondary antibody against rabbit IgG (Santa Cruz 1:2000) for 1 h at 25 °C. The blot was finally wa- shed with TBST, and the protein bands were visualized with a chemiluminescence system (ECL Plus, Applygen Technologies Inc.). The resulting image was analyzed using Image J software.

2.8. Solutions and chemicals

Krebs–Henseleit solution (KHS) (mmol/L): NaCl, 117; KCl, 4.7; MgCl2, 1.2; KH2PO4, 1.2; NaHCO3, 24.8; CaCl2, 2.5; and glucose, 11.1.
The solution was bubbled with 95% O2 to 5% CO2 to maintain a pH value of 7.4. The indomethacin, tetrodotoxin (TTX), vasopressin, MDL-12330A, H89, BAPTM-AM were purchased from Sigma (St Louis, MO, United States). Stock solutions of all the above chemi- cals were dissolved in DMSO. The final DMSO concentrations never exceeded 0.1% (v/v). Preliminary experiments indicated that the vehicle did not alter any baseline electrophysiological parameters.

2.9. Statistical analysis

Data are expressed as mean 7S.E.M.; statistical significance between two individual measurement groups was determined using an unpaired t test. Differences among groups were determined using one-way ANOVA and the Tukey’s post hoc method of multiple comparisons. The level of significance was set at P o0.05.

3. Results

3.1. Chronic water restriction in mice

Restriction of water intake normally triggers an increase in the circulating vasopressin level, which results in a homeostatic de- crease of renal water excretion. The serum AVP concentration was markedly higher in water-restricted mice compared with controls, as expected (83.274.91 vs. 220.8718.67 pg/ml n 5 P o0.001).The body weights of mice in both two groups of mice changed from 22.77 0.68 g to 26.370.52 g in control group and from 23.471 g to 21.971 g in the water-restricted mice, respectively. There is no obvious weight-loss in the water-restricted mice sug- gesting their well-being is only mildly affected.

3.2. Effect of chronic water restriction on NKCC expression

To further prove NKCC2 expression in the colon, we introduced NKCC2 RNA interference oligonucleotides using an in vivo trans- fection. As shown in Fig. 1, the western blot results showed a specific band at 150 kDa and confirmed NKCC2 expression in mouse colonic mucosa and kidney. The siRNA significantly de- creased the NKCC2 protein level 3 days after the siRNA transfec- tion. A quantitative analysis revealed a 63% suppression of NKCC2 protein expression of kidney and colon in the siRNA group com- pared to the non-targeting siRNA treatment group (Fig. 1 n 3 P o0.01). Real-time PCR results verified the knock-down of NKCC2 mRNA. The results showed that the NKCC2 mRNA expression was knocked down by about 60%. To assess the effect of chronic water restriction on mouse colonic NKCC cotransporter expression, we ran real-time PCR and semi-quantitative immunoblots using colonic mucosa homogenates from the control mice receiving 5 ml/20 g body wt of water per day and from the water-restricted mice receiving 1 ml/20 g body wt of water per day for 7 days. As shown in Fig. 2A, the mRNA expression of NKCC2 in the colon of water-restricted mice, when compared with the normal mice, was increase by 47%. (n 5, P o0.05) Water restriction for 7 days also resulted in a large increase in NKCC2 protein expression in the colon. The normalized band densities for the water-restricted and control mice were 194 711.44 and 10070.47%, respectively (Fig. 2B and D, n 4, P o0.001). In contrast to NKCC2, the 7-day water restriction protocol didn’t increase NKCC1 expression in the colonic mucosa as shown in Fig. 2C. (95712% for the water-re- stricted mice vs. 100 70.5% for the control mice, n ¼ 4, P 40.05).

3.3. NKCC phosphorylation is stimulated by short-term vasopressin stimulation in vitro

To determine if vasopressin activates NKCC in the colon, phos- phorylation of NKCC2 was measured with the phosphospecific an- tibody R5 (generous gift from B. Forbush, Yale University, New Haven, CT). We examined the effect of vasopressin on the total NKCC2 expression in vitro for 3 h as well as P-NKCC in the colonic mucosa. As shown in Fig. 3A, an in vitro short-term incubation with vasopressin induces an increase by 3-fold in overall NKCC2 phos- phorylation determined by the ratio of R5 to NKCC2 signal in par- allel blots. Importantly, this short-term administration of vaso- pressin does not increase the expression level of NKCC1 (Fig. 3B).

3.4. NKCC involved in the signaling mechanism of vasopressin-in- duced response in the late distal colon

In a previous study (Xue et al., 2014), we demonstrated that vasopressin induced an immediate and sustained decrease in the isolated mice colonic mucosa in the short circuit current. In this experiment, serosal addition of vasopressin (5 ~ 10—8 mol/l) also induced an immediate decrease of — 16.5374.05 μA/cm2 in the ISC.

3.5. cAMP-PKA and Ca2 þ signaling involved in the vasopressin-in- duced NKCC2 phosphorylation increase

To further demonstrate whether the NKCC2 was involved in the cAMP-PKA signaling under the vasopressin-stimulation in the colon in vitro, treatment with MDL-12330A (10 μM), H89 (20 μM ), BAPTA-AM(50 μM) and vasopressin (5 10—8 mol/l) for 30 min, we detected the P-NKCC level under different condition. The result showed that MDL-12330A or H89 or BAPTA-AM indeed reversed the vasopressin induced NKCC2 phosphorylation level increase by about 35%, 28% and 42% respectively (Fig. 4C n 3, P o0.05) sug- gesting vasopressin stimulates NKCC2 phosphorylation increase mediated by cAMP-PKA and Ca2þ signaling in the colon.

4. Discussion

The present study focused on the expression, phosphorylation and signaling mechanism of NKCC in a colon that was stimulated by vasopressin. First, a siRNA technique was performed to demonstrate NKCC2 expression in the colon. It has been reported that vasopressin is able to regulate the expression of NKCC1 and NKCC2 in the outer medullar collecting duct or TAL (Wakamatsu et al., 2009). Changes in NKCC2 protein levels may play an important role in long-term ion transport regulation. To determine if NKCC2 affects ion transport by altering its protein synthesis, we examined the expression level of NKCC after 7 days of dehydration. We demonstrated a large increase in the level of the NKCC2 transporter protein and mRNA in the colon in response to increases in endogenous vasopressin levels resulting from restriction of fluid intake. Because the expression level of NKCC1 was not similarly increased, vasopressin-mediated regulation appears to be selective for the NKCC2. Further studies are needed to determine the molecular mechanisms involved in vasopressin-mediated regulation of NKCC cotransporter expression. Short-term regulation of the NKCC family is mediated by phosphorylation/de- phosphorylation events (Gimenez and Forbush, 2005). We used R5, a phosphospecific antibody that has been proven to be a very useful tool for the study of NKCC regulation, to study this short-term reg- ulation mechanism. Vasopressin is well known for its acute effects on TAL NaCl absorption through NKCC2 activation. This short-term stimulation involves increased translocation of NKCC2 to the apical membrane in addition to protein phosphorylation (Giménez and Forbush, 2003; Flemmer et al., 2002). In the previous study, we demonstrated that vasopressin induced NKCC2 membrane translo- cation. In the present study, the treatment of colonic tissue in vitro by vasopressin produced similar patterns of phosphorylation, which was determined by the ratio of R5 to NKCC2 signal in parallel blots. Meanwhile, we also detected the translocation of NKCC2 after va- sopressin stimulation in vitro, but we did not find the obvious translocation (data not shown). We think it is different from our previous results due to different way of simulation.

Virtually all cell-signaling processes are dependent on protein phosphorylation and dephosphorylation. Therefore, we next in- vestigated a possible signaling mechanism mediating the effect of vasopressin on the distal colon. Most recently, researchers have reported that cAMP can increase surface expression of NKCC2 and that insertion of NKCC2 into the plasma membrane is part of a mechanism by which cAMP enhances NaCl absorption in the TAL (Ortiz, 2006; Haque et al., 2012). A recent study demonstrated that vasopressin increases NKCC2 phosphorylation in cAMP-PKA- mediated signaling (Gunaratne et al., 2010). However, thus far, only one report has shown that vasopressin has an effect on the colon through a decrease in intracellular Ca2þ (Bridges et al., 1983). In the present study, by using a short-circuit current method combined with treatment with adenylate cyclase (AC) inhibitor MDL-12330A and PKA inhibitor H89, we found that pretreatment with MDL-12330A or H89 inhibited the vasopressin- induced ISC by 60% and 66%, respectively. This showed that cAMP was involved in the vasopressin-induced response. Further ex- periment demonstrated that MDL-12330A and H89 could reverse vasopressin-induced NKCC2 phosphorylation level increase sug- gesting cAMP-PKA signaling was involved in the vasopressin sti- mulated NKCC2 phosphorylation. Pretreatment with BAPTA-AM inhibited the ISC by 65% showing that ISC was mitigated by the Ca2þ sensitivity to the treatment with BAPTA-AM, which prevented intracellular Ca2þ mobilization. Moreover, pretreatment with BAPTA-AM also could reverse vasopressin-induced NKCC2 phosphorylation level increase. These results differ from those of an earlier report, due possibly to the different species and location of the colon used in the present experiments.

Our previous data showed NKCC2 expression in the colon, and the present study confirmed the previous results by a siRNA technique. In the present study, our data indicated that when exposed to vasopressin in an in vitro preparation, NKCC2 expres- sion and phosphorylation levels are increased but NKCC1 levels are not. Vasopressin strongly upregulates NKCC2 expression levels in the colonic mucosa of dehydrated mice with marked water restriction, but it does not affect NKCC1 expression levels. The results of the short-circuit current show that cAMP and Ca2þ are involved in the vasopressin-induced response.

5. Conclusion

Our data suggest that hormonal control of NKCC2 phosphor- ylation levels or NKCC2 expression levels are increased in short- and long-term vasopressin regulation but that NKCC1 expression levels are not changed. Additionally, we demonstrated that cAMP and Ca2þ are involved in the effect of vasopressin which stimulates NKCC2 phosphorylation increase mediated by cAMP-PKA and Ca2þ signaling in the colon.