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₁-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

¹Department of Pharmacology, University of Oslo; ²Merck, Sharpe & Dohme Cardiovascular Research Center, Rikshospitalet University Hospital; and ³Department of Cardiology, Ullevaal University Hospital, N-0316 Oslo, Norway

Submitted 12 June 2003 ; accepted in final form 19 November 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive ⁸⁶Rb⁺ influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of ₁-adrenoceptors (AR) by phenylephrine (30 µM) increased ⁸⁶Rb⁺ influx. The NKCC inhibitor bumetanide (50 µM) reduced the response to phenylephrine by 45 ± 13% (n = 12, P < 0.01). PD-98059 (10 µM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 ± 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that ₁-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after ₁-AR stimulation was shown by immunoprecipitation of the phosphoprotein from ³²P_i prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by ₁-AR stimulation through phosphorylation of this protein involving the ERK pathway.

⁸⁶Rb⁺ influx; phenylephrine; calyculin A; bumetanide; mitogen-activated protein kinase; ₁-adrenoceptors

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals. Animal care was in accordance with the Norwegian Animal Welfare Act, which conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, Revised 1996). The animals were housed in a temperature-regulated room with a 12:12-h day-night cycle.

Materials and reagents. The T4 antibody developed by C. Lytle and B. Forbush III (17) was obtained from the Developmental Studies Hybridoma bank maintained by The University of Iowa, Department of Biological Sciences, Iowa city, IA 52242. Calyculin A, PD-98059, ouabain, wortmannin, LY-294002, and bisindolylmaleimide I were purchased from Calbiochem-Novabiochem (San Diego, CA). Collagenase type 2 was purchased from Worthington (Lakewood, NJ). Laminin was purchased from Invitrogen (Carlsbad, CA). All other reagents including L-phenylephrine hydrochloride, timolol maleate, prazosin hydrochloride, bumetanide, and choline were purchased from Sigma Chemical (St. Louis, MO). Stock solutions were prepared in double-distilled water and maintained at –20°C. Further dilutions were made fresh daily and kept cool (0–4°C).

Isolated perfused heart model measurement of ⁸⁶Rb⁺ influx. Hearts from deeply ether-anesthetized adult male Wistar rats (200–250 g) were excised and placed into ice-cold saline. The hearts were retrogradely aorta perfused as described in detail previously (3). The hearts were spontaneously beating and perfused in a nonrecirculating system at 31°C and at a constant flow of 10 ml/min. After 10 min of perfusion, the hearts were loaded with the K⁺ analog ⁸⁶Rb⁺ for 10 min. The use of ⁸⁶Rb⁺ as a K⁺ analog has been studied previously in our laboratory in both isolated rat heart and cardiomyocyte preparations (1, 3, 28). After 10 min of washout of extracellular ⁸⁶Rb⁺, the hearts were freeze clamped and pulverized. Determination of ⁸⁶Rb⁺ radioactivity in the hearts was done as previously described (3) by normalizing for perfusate radioactivity and heart weight [ml/g = (cpm heart/g)/(cpm perfusate/ml), where cpm is counts per minute, and the results were thus expressed as ml/g heart weight/10 min].

Isolation of cardiomyocytes. Ventricular cardiomyocytes were isolated from adult rat hearts by enzymatic digestion using trypsin (60 U/ml) and collagenase (90 U/ml) as described previously with some modifications (27). Briefly, the hearts were retrogradely aorta perfused at 6.5 ml/min with a Joklik's-MEM solution containing (in mmol/l) 24 NaHCO₃, 1.6 MgSO₄, 1 DL-carnitine, 10 creatine, 20 taurine, and 1 CaCl₂. After equilibration, calcium was washed out by one-way perfusion by changing to a similar but nominally Ca²⁺-free solution, which included 0.1% essentially fatty acid free BSA and trypsin (60 U/ml). After 5 min of calcium-free perfusion, collagenase (90 U/ml) was introduced, and the hearts were perfused by recirculation. After 17 min with collagenase perfusion, 0.25 mmol/l CaCl₂ was reintroduced, and after 13 min of additional perfusion time, the hearts were cut down in a buffer containing 0.5 mmol/l CaCl₂. The atria were removed and the hearts were cut into small pieces and dispersed in an enzyme-containing solution in a shaker bath for 10 min (110 strokes/min). The tissue was filtered through a nylon mesh (pores 250 µm), and the cardiomyocytes were purified by repeated centrifugation (48 g) and sedimentation by gravity through a solution containing 1% BSA. The cardiomyocyte cell population contained 60–70% elongated cardiomyocytes. Cardiomyocytes were plated on laminin (10 µg/ml)-precoated dishes (7,000 cells/cm²) and incubated in a buffer containing the above with 0.1% BSA, 0.5 mmol/l CaCl₂, and 1% penicillin-streptomycin. The incubation solution was changed after a 3-h incubation period to remove the nonattached cardiomyocytes, and the cells were incubated overnight at 37°C. The remaining cell population contained more than 95% elongated cardiomyocytes.

Measurement of ⁸⁶Rb⁺ influx in isolated cardiomyocytes. Each dish (35 mm diameter) was incubated the next day with the appropriate agonists, and antagonists (added 15 min before the agonist) and radiolabeled ⁸⁶Rb⁺ (2 ± 0.2 x 10⁵ cpm). The flux was terminated with three rinses in K⁺ buffer (10 ml of phosphate-buffered saline with 5 mM KCl, 37°C) after 10 min. The cells were precipitated and extracted with 5% cold trichloroacetic acid, and the samples were centrifuged at 3,500 rpm (4°C) for 5 min. Radioactivity in the supernatant was determined by Cerenkov radiation (Packard liquid-scintillation spectrometer, Packard Instrument; Downers Grove, IL). In some experiments, Na⁺ was replaced by choline in the incubation buffer to study ⁸⁶Rb⁺ influx in the absence of extracellular Na⁺.

Experimental design: ⁸⁶Rb⁺ influx studies. The effect of ₁-AR stimulation on ⁸⁶Rb⁺ influx was studied by adding phenylephrine (30 µM) to the incubation buffer together with the radioactive isotope ⁸⁶Rb⁺. The ₁-AR antagonist prazosin (1 µM) was present in the control experiments, and the -AR antagonist timolol (1 µM) was present in all experiments. The effect of the protein phosphatase inhibitor calyculin A (0.1 µM) on ⁸⁶Rb⁺ influx was measured by adding ⁸⁶Rb⁺ after 10 min of pretreatment with calyculin A. The increase in ⁸⁶Rb⁺ influx after phenylephrine or calyculin A stimulation was expressed as the percentage above control. NKCC activity was measured as the bumetanide (50 µM)-sensitive component of the ⁸⁶Rb⁺ influx. The ⁸⁶Rb⁺ influx experiments in isolated cardiomyocytes were performed in the presence of the Na-K pump inhibitor ouabain (100 µM). The effect of kinase inhibitors on ₁-AR-stimulated NKCC activity was studied by comparing ₁-AR-stimulated ⁸⁶Rb⁺ influx in the absence or presence of kinase inhibitors with or without bumetanide. Responses to phenylephrine or calyculin A in the absence or presence of the different inhibitors were compared with and statistically evaluated against corresponding and separate control groups from separate hearts. The results are expressed as means ± SE. The significance levels of differences were expressed by calculating P according to Student's two-sample test. One-way ANOVA test was used when comparing more than two groups. A value of P 0.05 was considered to reflect statistically significant differences.

RT-PCR analysis of NKCC1 mRNA. Purified cardiomyocytes, from both right and left ventricles, were centrifuged at 20 g for 5 min to remove as much cell medium as possible before cell lysis. Total RNA from the cardiomyocytes was isolated with the SV Total RNA Isolation System (Promega; Madison, WI) and eluted in RNase free water. Total RNA quality was assessed by agarose gel electrophoresis, and RNA was quantified spectrophotometrically. First-strand cDNA was synthesized from 200 ng total RNA in 20-µl reactions by use of 200 units SuperScript II (Invitrogen; Carlsbad, CA) with oligo dT_12–18 primers (Invitrogen) and subjected to PCR amplification by using specific primers to rat NKCC1 sequence (19) EMBL accession number AF051561 [GenBank] , designed to give a 798-bp product (upper primer: ON243, 5'-TTGAGGATGGTTTTGCGAATG-3', lower primer ON244, 5'-CTTTGGGTATGGCTGACTGAGG-3', Invitrogen). For PCR analysis, 1 µl of the synthesized cDNA was used as a template in 25-µl reactions containing 0.5 units AmpliTaq Gold (Applied Biosystems; Foster City, CA), PCR buffer with 1.5 mM MgCl₂, 0.2 mM dNTPs, and 0.4 µM of each primer. An initial pre-PCR heat step (2 min at 95°C) was followed by 45 two-step PCR cycles of 30 s at 95°C and 45 s at 68°C and a final elongation step (10 min at 68°C). The PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining, and the identity of the PCR product was confirmed by sequencing.

Immunoblotting of NKCC. Detection of the cotransporter protein in cardiomyocytes was done by Western blotting as described by Lytle et al. (17) with some modifications. Briefly, freshly isolated cardiomyocytes in suspension were homogenized in a buffer containing (in mM) 50 Tris·HCl [pH 7.4 at room temperature (RT)], 10 MgCl₂, and 2 EGTA. An equal volume of 1 M KCl was added to each sample, and after 10 min on ice, the membranes were collected by centrifugation at 48,000 g for 20 min. The pellet was resuspended and denatured in a electrophoresis sample buffer (31 mM Tris·HCl, pH 6.8 at RT, 5% glycerol, and 1% SDS). Protein concentration was determined (BCA assay), and equal amounts of protein were loaded onto SDS-PAGE (6% polyacrylamide) and transferred to nitrocellulose membranes. Blots were coated with blocking buffer containing 6% nonfat dry milk and then incubated overnight with the monoclonal T4 antibody (25 µg in 10 ml of buffer). After samples were washed and incubated with a secondary antibody (goat anti-mouse, Bio-Rad Laboratories) for 2 h at room temperature were completed, enhanced chemiluminescence and X-ray films were used to detect the cotransporter protein. In some experiments, the cell lysate was incubated with glycosidase (N-glycosidase F, 0.02 U/µl buffer, Boehringer Mannheim, now Roche Diagnostics; Basel, Switzerland) overnight before electrophoresis to study the effect of deglycosylation on the molecular weight of the protein.

NKCC phosphorylation studies. Cells attached to laminin-coated dishes (100 mm) were incubated the next day for 3 h in phosphate-free MEM containing ³²P_i (20 µCi/ml). After washout of extracellular ³²P_i, the cells were incubated for 15 min with the appropriate inhibitors and then for 10 min with phenylephrine or 5 min with the phosphatase inhibitor calyculin A. Cells were scraped off with a hot lysis buffer containing 1% SDS, 10 mM Tris·HCl (pH 8.0 at RT), and 50 mM NaF. The samples were heated at 95°C for 5 min and sonicated for 30 s. Aliquots were taken for protein assay (BCA), and then an excess volume (6x) of a buffer containing protease and phosphatase inhibitors 20 mM Tris·HCl (pH 8.0 at RT), 200 mM NaCl, and detergents (2% Triton X-100 and 1% NP-40) was added to reduce the SDS concentration in the buffer before the addition of the antibody (T4). The antibody was added after corrections for variations in protein. After incubation overnight at 4°C, immunomagnetic beads (Dynal; Oslo, Norway) coated with secondary (sheep anti-mouse) antibody were added, and the samples were incubated at 4°C for 4 h. The immune complexes were rinsed five times in the detergent buffer described above, electrophoresis sample buffer was added, and the samples were heated at 95°C for 5 min. The immune complexes were electrophoretically separated on 3–8% Tris-acetate gradient gels (Novex; San Diego, CA). Gels were coomassie blue stained to verify a uniform loading of protein. After washing was completed, the gels were dried with a gel dryer. Incorporation of ³²P_i label into the protein was quantified by measuring radioactivity of the gels by electronic autoradiography using an InstantImager (Packard). Some of the results were also visualized by standard autoradiography using X-ray films.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
₁ -AR-stimulated ⁸⁶Rb⁺ influx in perfused hearts is sensitive to the NKCC inhibitor bumetanide. Basal influx of ⁸⁶Rb⁺ after 10 min perfusion of rat hearts was 2.9 ± 0.12 (n = 7) ml·g heart tissue^–1·10 min^–1. The ₁-AR agonist phenylephrine (30 µM) increased the ⁸⁶Rb⁺ influx to 4.0 ± 0.1 ml·g heart tissue^–1·10 min^–1 (39 ± 2.8% increase, n = 26) (P < 0.0001, Fig. 1). To elucidate the involvement of NKCC in ₁-AR-stimulated ⁸⁶Rb⁺ influx, we studied the effect of the NKCC inhibitor bumetanide on basal and ₁-AR-stimulated ⁸⁶Rb⁺ influx. Bumetanide (50 µM) reduced basal ⁸⁶Rb⁺ influx by 11 ± 1.1%, n = 16, (P < 0.01), and the phenylephrine stimulated ⁸⁶Rb⁺ influx by 55 ± 10.9% (n = 22, P < 0.01) (Fig. 1). These experiments were all done by measuring ⁸⁶Rb⁺ influx at 10 min, but similar results were obtained in separate experiments measuring ⁸⁶Rb⁺ influx at 15 min perfusion (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1. 1-Adrenoceptor (AR)-stimulated 86Rb+ influx in the perfused rat heart is inhibited by bumetanide (BUM) and PD-98059 (PD). Isolated perfused hearts were loaded with 86Rb+ for 10 min, and 86Rb+ radioactivity was measured after a 10-min washout. Separate hearts were stimulated by phenylephrine (PE, 30 µM) in the absence (n = 26) or presence of the Na-K-2Cl cotransporter (NKCC) inhibitor BUM (50 µM, n = 22) and/or the presence of the MAPK kinase/ERK (MEK) inhibitor PD (10 µM) with (n = 5) or without (n = 6) BUM. Separate experiments including the appropriate inhibitors were used as individual control groups (n = 4–16 per group). The data are expressed as PE-stimulated increase in 86Rb+ influx (means ± SE) in a percentage above the appropriate controls. *P < 0.01.

₁ -AR-stimulated bumetanide-sensitive ⁸⁶Rb⁺ influx in perfused hearts is sensitive to ERK cascade inhibition. The ERK subgroup of the MAPK family has been shown to be involved in various aspects of ₁-AR signaling. The MEK inhibitor PD-98059 (10 µM) reduced phenylephrine-induced ⁸⁶Rb⁺ influx by 54 ± 8.6% (n = 6) (P < 0.01, Fig. 1). The presence of PD-98059 completely eliminated the bumetanide-sensitive component of phenylephrine stimulated influx as revealed by a similar increase by phenylephrine in the absence or presence of bumetanide (18 ± 4.7%, n = 6 and 16 ± 1.5%, n = 5), respectively (Fig. 1). PD-98059 had no efect on basal ⁸⁶Rb⁺ influx (data not shown).

₁ -AR-induced bumetanide-sensitive ⁸⁶Rb⁺ influx in perfused hearts is not sensitive to PKC or phosphatidyl inositol 3-kinase inhibitors. PKC and phosphatidylinositol 3-kinase (PI3K) have also been shown to be involved in ₁-AR signaling in the heart (26). We used inhibitors to elucidate an involvement of these kinases in ₁-AR-stimulated bumetanide-sensitive ⁸⁶Rb⁺ influx. The PKC inhibitor bisindolylmaleimide (BIM, 1 µM) had no effect on basal ⁸⁶Rb⁺ influx (2.8 ± 0.09 ml·g heart tissue^–1·10 min^–1, n = 10, and 2.7 ± 0.05, ml·g heart tissue^–1·10 min^–1 n = 8, in the absence or presence of BIM, respectively). Phenylephrine-stimulated ⁸⁶Rb⁺ influx was not inhibited by the presence of BIM (n = 8, data not shown). The PI3K inhibitor wortmannin (0.1 µM) reduced basal ⁸⁶Rb⁺ influx by 17 ± 2.6% (n = 13, P < 0.001). The effect on basal ⁸⁶Rb⁺ influx was not mediated by a bumetanide-sensitive uptake mechanism because the bumetanide-induced reduction in basal ⁸⁶Rb⁺ influx was unaffected by the presence of wortmannin (12 ± 4.2% reduction, n = 8, nonsignificant vs. the absence of wortmannin). No reduction in phenylephrine-induced ⁸⁶Rb⁺ influx was found in the presence of wortmannin. Furthermore, no reduction in phenylephrine-induced ⁸⁶Rb⁺ influx was found when another PI3K inhibitor (LY-294002, 10 µM) was used (data not shown).

Presence of NKCC mRNA and protein in rat cardiomyocytes. The presence of NKCC1 mRNA in rat cardiomyocytes from both the right and left ventricle was detected by RT-PCR (Fig. 2A), and the identity of the PCR product was confirmed by sequencing (data not shown). Cells from the left ventricle were also subjected to immunoblotting with a monoclonal antibody (T4) raised against the human colonic NKCC (17). A single distinct band was detected in the cardiomyocytes with a molecular mass of 180 kDa (Fig. 2B). A broader band was found in whole heart homogenates from isolated perfused hearts. Deglycosylation of the sample with N-glycosidase before electrophoresis did not change the band pattern. This is in contrast to observations from epithelial cells (31) but in accordance with results obtained in skeletal muscle (29).

View larger version (76K): [in this window] [in a new window]   Fig. 2. Presence of NKCC in isolated adult rat cardiomyocytes. A: presence of NKCC1 mRNA in rat cardiomyocytes from both the right and left ventricles was determined by RT-PCR analysis on two independent RNA isolations (lanes a and b). GAPDH is used as the control. cDNA obtained by oligo-dT-primed reverse transcription of total RNA (RT+) and controls obtained in the absence of reverse transcriptase (RT–) were subjected to PCR amplification with specific primers (see MATERIALS AND METHODS). PCR products were separated by 1.0% agarose gel electrophoresis and visualized by ethidium bromide staining. The bands representing NKCC1 and GAPDH are indicated. B: Western blot of membrane proteins isolated from rat ventricular cardiomyocytes using the monoclonal antibody T4 (see MATERIALS AND METHODS). The antibody reacts primarily with one protein of molecular mass 180 kDa.

₁ -AR-stimulated ⁸⁶Rb⁺ influx in isolated cardiomyocytes is bumetanide sensitive and dependent on the presence of extracellular Na⁺. To clarify whether the ₁-AR-stimulated, bumetanide-sensitive increase in ⁸⁶Rb⁺ influx in the rat heart was due to uptake in the cardiomyocytes, the effect of phenylephrine on ⁸⁶Rb⁺ influx was studied in isolated cardiomyocytes from adult rat hearts. The experiments were performed in the presence of 100 µM ouabain (Na-K pump inhibitor). Control experiments showed that basal ⁸⁶Rb⁺ influx in isolated cardiomyocytes was not influenced by the presence of bumetanide, ouabain, or bumetanide and ouabain combined. Phenylephrine increased the ⁸⁶Rb⁺ influx from 26 ± 0.6 (n = 11) to 36 ± 0.8 (n = 13) ml·g protein^–1·10 min^–1 in the absence of bumetanide (40 ± 3.9% increase, P < 0.0001, Fig. 3A) and from 26 ± 0.4 (n = 8) to 32 ± 1.1 (n = 12) ml·g protein^–1·10 min^–1 in the presence of 100 µM bumetanide (22 ± 4.2% increase, P < 0.01, vs. in the absence of bumetanide, Fig. 3A). This corresponds to a 45 ± 13.2% reduction in the phenylephrine-stimulated ⁸⁶Rb⁺ influx in the presence of bumetanide. One criterion for the NKCC-mediated ion transport, in addition to bumetanide sensitivity, is its dependence on the presence of all three ions transported on the same side of the cell membrane (24). When Na⁺ was substituted with choline in the incubation buffer (in the presence of 100 µM ouabain), the basal ⁸⁶Rb⁺ influx was reduced from 26 ± 0.6 (n = 11) to 9.4 ± 0.33 ml·g protein^–1·10 min^–1 (n = 8, Fig. 3B). In the absence of Na⁺, the ⁸⁶Rb⁺ influx during phenylephrine stimulation was 10.3 ± 0.45 ml·g protein^–1·10 min^–1 (n = 9, Fig. 3B). Phenylephrine stimulated ⁸⁶Rb⁺ influx from 9.5 ± 0.32 to 11.5 ± 0.73 ml·g protein^–1·10 min^–1 (n = 9) in the presence of bumetanide (100 µM, Fig. 3B). Thus ₁-AR-induced bumetanide-sensitive ⁸⁶Rb⁺ influx was eliminated in the absence of extracellular Na⁺ demonstrating its dependence on extracellular Na⁺.

View larger version (15K): [in this window] [in a new window]   Fig. 3. 1-AR-stimulated 86Rb+ influx in isolated cardiomyocytes is inhibited by BUM and dependent on extracellular Na+. Isolated cardiomyocytes from adult rat hearts were plated on laminin-coated dishes and kept overnight. The next day each dish was incubated with the appropriate agonist and antagonist and radiolabeled 86Rb+ for 10 min, and 86Rb+ radioactivity was measured after washing was completed. A: separate groups of cells were stimulated by the 1-AR agonist PE (30 µM) in the absence (n = 13) or presence (n = 12) of the NKCC inhibitor BUM (50 µM). The data are expressed as PE-stimulated increase in 86Rb+ influx (means ± SE). Separate experiments including the appropriate inhibitors were used as individual control groups (n = 9–12 per group). *P < 0.01. B: extracellular Na+ was substituted with choline, and 86Rb+ influx in cardiomyocytes was measured as described above. 86Rb+ influx was measured in the absence (n = 8) or presence of PE (n = 9) in the absence (n = 8) or presence (n = 9) of BUM. The data are expressed as 86Rb+ influx (means ± SE; in ml·g protein–1·10 min–1).

Myocardial NKCC protein is phosphorylated by ₁-AR stimulation. To study whether the cotransporter protein is regulated by phosphorylation, phosphate content was measured in immuoprecipitates of NKCC from ³²P_i-labeled adult cardiomyocytes with or without ₁-AR stimulation by phenylephrine. Phenylephrine (30 µM) increased phosphate incorporation into the cotransporter protein by 143 ± 17% above control (n = 10, P < 0.0001) at 10-min stimulation (Fig. 4, A and B). Inhibition of the ERK cascade by PD-98059 (50 µM) almost eliminated the ₁-AR-induced phosphorylation. Phenylephrine increased phosphate incorporation into the NKCC by 15 ± 10% above control (n = 4) in the presence of PD-98059, corresponding to a 90 ± 14% reduction of maximal response to phenylephrine (P < 0.001 vs. without PD-98059) (Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Phosphorylation and activation of the rat myocardial NKCC in response to 1-AR stimulation or phosphatase inhibition. A and B: phosphorylation studies. Cardiomyocytes isolated from adult rat hearts were plated on laminin-coated dishes and kept overnight. The next day each dish was incubated with 32Pi for 3 h followed by washout and incubation with the appropriate inhibitors and then for 10 min with PE (30 µM, n = 10) or the phosphatase inhibitor calyculin A (CalyA, 0.1 µM, n = 7). The NKCC protein was immunoprecipitated using the T4 antibody and separated on 3–8% Tris-acetate gels. The 32Pi content was measured by electronic autoradiography (see MATERIALS AND METHODS). A: representative autoradiogram. B: data are expressed as increase (means ± SE) in NKCC phosphorylation in percent above the control. C: 86Rb+ influx studies. 86Rb+ influx was measured after phosphatase inhibition by CalyA (0.1 µM, n = 8) as described in Fig. 3 except that CalyA was added 10 min before 86Rb+. The data are expressed as BUM-sensitive 86Rb+ influx. The data from Fig. 3A expressed as PE-stimulated BUM-sensitive 86Rb+ influx are included for comparison. **P < 0.0001; *P < 0.05.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Effect of ERK cascade inhibition on phosphorylation of the rat myocardial NKCC in response to 1-AR stimulation. Cardiomyocytes from adult rat hearts were isolated and labeled with 32Pi as described in Fig. 4. NKCC was immunoprecipitated from cardiomyocytes incubated for 10 min in the absence or presence of PE (30 µM, n = 10) following preincubation for 15 min in the absence (–) (n = 4) or presence (+) (n = 4) of the MEK inhibitor PD (50 µM). Separate experiments including the appropriate inhibitors were used as individual control groups. A: representative autoradiogram. B: data are expressed as PE-stimulated increase (means ± SE) in NKCC phosphorylation in percentage above the appropriate control. *P = 0.001.

Calyculin A increased NKCC phosphorylation and stimulated bumetanide-sensitive ⁸⁶Rb⁺ influx in isolated cardiomyocytes. The phosphatase inhibitor calyculin A, which is known to stimulate NKCC activity in epithelial tissue (15), was used as a positive control. Calyculin A (0.1 µM) increased the phosphate content of the NKCC by 660 ± 224% (n = 7, P < 0.05) (Fig. 4, A and B). ⁸⁶Rb⁺ influx in isolated cardiomyocytes was measured in the absence and presence of pretreatment for 10 min with calyculin A with or without bumetanide. These experiments were performed to elucidate whether increased NKCC phosphorylation by calyculin A also resulted in increased ⁸⁶Rb⁺ influx. Calyculin A (0.1 µM) increased ⁸⁶Rb⁺ influx by 16 ± 2.0 (n = 8) (50% increase) and 8 ± 1.2 (n = 7) ml·g protein^–1·10 min^–1 in the absence or presence of bumetanide (100 µM, Fig. 4C), demonstrating a bumetanide-sensitive influx component of 50%.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study demonstrates that a functionally active NKCC is present in rat cardiomyocytes and that it is activated by ₁-AR stimulation through phosphorylation of the protein involving the ERK cascade. Evidences of the presence of an NKCC in the heart have mainly rested on functional studies on the effect of loop diuretics on ion fluxes (2, 3, 8, 12–14). The presence of the NKCC protein and mRNA has also been demonstrated in the whole heart homogenate (6, 22), but the present study demonstrates that mRNA encoding the NKCC1 isoform as well as the NKCC protein is present in cardiomyocytes from the rat heart. The observed molecular mass of 180 kDa is in the expected range (24).

The NKCC1 is a member of a superfamily of structurally and functionally related membrane proteins termed the cationchloride cotransporter superfamily. The main function of NKCC in the nonepithelial tissue is probably regulation of cell volume and to maintain intracellular Cl^– concentration at a level higher than equilibrium values (9, 10, 16). Regulation of cell volume by the NKCC has also been indicated by studies of heart preparations (4, 7). The NKCC is a electroneutral secondary active transport process, and in most cells the Na⁺ and K⁺ gradients are created by the Na-K-ATPase (24). The NKCC is capable of mediating ion transport both into and out of the cells, and the net direction of the transport over time is determined by the sum of the three ion gradients. Although the Na-K pump is the driving force for NKCC, the chemical gradient for Cl^– will determine the net direction of the cotransporter in most cells including cardiomyocytes. The gradient in cardiomyocytes will be in favor of net mass influx (24), which is a resultant of both inward and outward directed flux components taking place simultaneously.

We have previously demonstrated a bumetanide-sensitive component of ₁-AR-induced ⁸⁶Rb⁺ efflux from perfused rat hearts (3). The present data show that ₁-AR stimulation increases ⁸⁶Rb⁺ influx in the perfused rat heart by a bumetanide-sensitive uptake mechanism, strongly suggesting activation of the NKCC in the rat heart. Separate studies were performed on isolated cardiomyocytes to investigate the possibility that the bumetanide-sensitive ⁸⁶Rb⁺ influx measured in perfused hearts was due to uptake in other heart cells than the cardiomyocytes. ₁-AR stimulation mediated a similar activation of the NKCC in cardiomyocytes as in the perfused heart preparation. To further ensure that the uptake was mediated by the NKCC, additional experiments were done with Na⁺ substituted by choline. Our results demonstrate that when one of the transported ions (Na⁺) is absent, bumetanide-sensitive transport of another ion (⁸⁶Rb⁺) is abolished, confirming the presence of NKCC-mediated ion transport in the cardiomyocytes (24). This also shows that the nonbumetanide-sensitive influx component is reduced in the absence of extracellular Na⁺, demonstrating other Na⁺-dependent ⁸⁶Rb⁺ influx mechanisms.

Very few studies have focused on the regulation of the NKCC in the heart. One study showed that elevation of intracellular Ca²⁺ concentration activated furosemide-sensitive K⁺ fluxes (12), and another study showed increased bumetanide-sensitive Na⁺ influx after aldosterone stimulation (18). The present results extend our previously reported results on bumetanide-sensitive ⁸⁶Rb⁺ efflux in perfused hearts (3) by showing an inhibitory effect of the ERK cascade inhibitor PD-98059 also on bumetanide-sensitive ⁸⁶Rb⁺ influx. This was shown in both isolated hearts and isolated cardiomyocytes confirming the presence of functionally active NKCC in the cardiomyocytes. We have previously shown that PD-98059 is able to inhibit phenylephrine-stimulated ERK activity in perfused rat hearts, validating this tool (3). Thus activation of bumetanide-sensitive fluxes in both directions involves the ERK subgroup of the MAP kinase family of protein kinases. The lack of effect of other kinase inhibitors tested indicates the lack of involvement of other kinases like protein kinase C and PI3K, which are mediators known to participate in ₁-AR signaling (25).

The activation of myocardial NKCC-mediated ion transport by ₁-AR stimulation could occur via phosphorylation of NKCC itself or other regulatory proteins. To study whether the NKCC itself is phosphorylated in the cardiomyocytes after ₁-AR stimulation, we used a specific monoclonal antibody (17) to immunoprecipitate the NKCC protein after loading of cardiomyocytes with ³²P_i. Both ₁-AR stimulation and phosphatase inhibition increased the phosphate content of the NKCC, showing that this protein is highly regulated by phosphorylation. This is a novel finding in cardiomyocytes and is in agreement with observations in epithelial tissue and red blood cells (11, 15). ₁-AR stimulation might increase the phosphate content of the NKCC protein by either activating a kinase or inhibiting a phosphatase. The effects of the type 1 phosphatase (PP1) inhibitor calyculin A indicate that the myocardial cotransporter is maintained in a dephosphorylated and nonactivated state in its resting condition by high PP1 activity (Fig. 4). Corresponding observations are reported in epithelial tissue (16). The results from the functional ion flux studies indicating a role of the ERK cascade in the activation of the NKCC were supported by the inhibitory effect of PD-98059 on ₁-AR-induced NKCC phosphorylation. A very recent study identified three threonine residues on the NH₂-terminus of NKCC1 as phosphoacceptor sites on secretory NKCC (5). Phosphorylation at T189 was necessary for activation of the protein by forskolin and calyculin A. The kinase involved is currently not identified (5). Many protein kinases have been proposed as mediators of NKCC phosphorylation, but the data have so far been inconclusive (15, 24). It is possible that different kinases mediate this effect in different cell types. Although the ERK cascade is implicated in this process in the heart, it could still be an indirect effect of the ERK cascade on an unknown kinase that phosphorylates the NKCC protein or even ERK cascade-mediated inhibition of NKCC dephosphorylation. The presence of an active NKCC in striated muscle, which is regulated by adrenergic stimulation through ERK activation as suggested by our earlier work (3), has lately been supported by studies on NKCC activity in skeletal muscle (30). Both ₁-AR and -AR stimulation activated bumetanide-sensitive ⁸⁶Rb⁺ influx in an ERK-dependent way in both the soleus and plantaris muscle (9, 30).

Activation of a myocardial NKCC by ₁-AR stimulation is a novel aspect of sympathetic control of the heart. The physiological and pathophysiological implications of increased NKCC activity are so far unknown, but phosphorylation of this protein must be added to other well-known effects of ₁-AR signaling in the cardiomyocytes, which include a positive inotropic response, a hypertrophic response, increased gene expression, and alterations in ionic currents (23, 26). Because ion transport by the NKCC is by definition electroneutral, it has not been detected in the large number of experiments studying effects of ₁-AR stimulation on ionic currents (26). A role of the NKCC in cell volume regulation and hypertrophic growth mediated by ₁-AR stimulation is possible but has yet to be proven. We showed previously that angiotensin II, another hypertrophic agent, also activated the NKCC in the perfused rat heart model (2).

In summary, we present molecular and functional evidence for expression of the NKCC in the cardiomyocytes of the adult rat heart. In addition, the NKCC in the cardiomyocytes is subjected to regulation by hormones through protein phosphorylation. The data demonstrate that activation of the NKCC by ₁-AR stimulation is mediated by protein phosphorylation of the cotransporter protein involving the ERK subgroup of the MAP kinase family of protein kinases.

	ACKNOWLEDGMENTS

 
GRANTS

This work was supported by The Norwegian Council on Cardiovascular Diseases, the Norwegian Research Council, and the EU Biomed II Concerted action Alpha-1 heart project (Contract PL 950287). Parts of the results of this study have appeared previously in abstract form: Andersen GO, Enger M, Skomedal T. and Osnes J-B. Circulation 102, Suppl.: 484, 2000.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. Ø. Andersen, Dept. of Pharmacology, Univ. of Oslo, PO Box 1057, Blindern, N-0316 Oslo, Norway (E-mail: g.o.andersen{at}labmed.uio.no).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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