Extracellular Clminus modulates shrinkage-induced activation of Na+/H+ exchanger in rat mesangial cells

Yukio Miyata, Shigeaki Muto, Satoru Yanagiba, and Yasushi Asano

Department of Nephrology, Jichi Medical School, Minamikawachi, Kawachi, Tochigi 329-0498, Japan


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To examine the effect of hyperosmolality on Na+/H+ exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pHi) in a single MC from rat glomeruli. All the experiments were performed in CO2/HCO-3-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH2O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 µM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na+. The hyperosmolality shifted the Na+-dependent acid extrusion rate vs. pHi by 0.15-0.3 pH units in the alkaline direction. Removal of extracellular Cl- by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na+-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na+-dependent pHi recovery rate or Na+-dependent acid extrusion rate. The Cl--dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pHi, 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl-, and 4) the Cl--dependent NHE activation under hyperosmotic conditions partly occurs via Cl- channel and microtubule-dependent processes.

intracellular pH; chloride ion; chloride channel; microtubule


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THE SODIUM HYDROGEN EXCHANGER (NHE) is a plasma membrane transport protein found in a broad range of biological systems, including glomerular mesangial cells (MCs) (4, 42). In MCs, entry of Na+ into cells in exchange for an intracellular H+ is the main effect of NHE, which therefore is involved in the regulation of intracellular pH (pHi) and initiation of cell growth and proliferation (12, 13).

Cell volume regulation generally involves the movement of ions and organic osmolytes across the surface membrane and is often mediated by pHi-regulating transporters (reviewed in Refs. 6 and 19). In a variety of cells (8, 10, 15, 16, 20, 30-32, 37, 38), one such transporter, the NHE, is activated by shrinkage in a hyperosmotic solution, resulting in Na+ entry into the cell. Osmotic uptake of water due to this Na+ entry leads to cell swelling, a process termed regulatory volume increase. Activation of NHE also results in H+ extrusion, which causes cell alkalinization. However, it is not yet known whether hyperosmolality activates NHE in MCs.

MCs are rich in contractile fibers and smooth muscle-like cells (1, 21). They are located in the intercapillary space of the glomerulus, and may regulate, through its contraction, the intraglomerular hemodynamics and thus the glomerular filtration rate (GFR) (reviewed in Ref. 23). Some vasoactive peptides, angiotensin II (ANG II) and arginine vasopressin (AVP), cause MC contraction through an increase in intracellular Ca2+ and consequently reduce glomerular ultrafiltration (23). Because the sensitivity of contractile proteins to Ca2+ is reduced at low pHi in skeletal and cardiac muscles (11), pHi change might influence MC contractility, and subsequently GFR. Cultured MCs have also been shown to change morphologically, altering their growth characteristics and metabolic activity during periods of cyclic stretching-relaxation (18). Therefore, the ability to regulate cell volume is especially important for MCs, in which cell volume changes can alter glomerular hemodynamics.

In dog erythrocytes (31, 32) and in barnacle muscle fibers (10), it has been demonstrated that hyperosmolality-induced activation of NHE is inhibited by removing Cl- from the extracellular fluid. In the apical membrane vesicle of rat colonic crypt cells, NHE activity under isosmotic conditions has been reported to be dependent on extracellular Cl- (33). In addition, both increase in intracellular Ca2+ concentration and MC contraction induced by ANG II and AVP are attenuated when the extracellular Cl- concentration is reduced (28). It is also well known that Cl- is an important mediator of tubuloglomerular feedback (TGF) regulation of glomerular filtration by the distal tubular flow (35). These reports suggest the possibility that MC NHE activity may be modified by extracellular Cl- under iso- and/or hyperosmotic conditions.

Therefore, we isolated and cultured MCs from rat glomeruli to address the following issues: 1) whether hyperosmolality activates NHE activity, and 2) whether and how NHE activity under iso- or hyperosmotic conditions is modulated by extracellular Cl-.


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Solutions. The composition of the solutions is given in Table 1. All solutions were nominally CO2/HCO-3 free and were adjusted to pH 7.4 at 37°C. The osmolality of all solutions was measured before the experiment, and was verified to be within a range of 300 ± 5 mosmol/kgH2O. The nigericin calibrating solutions were titrated to different pH values at 37°C with either HCl or N-methyl-D-glucamine (NMDG). For the Na+-free solutions (solutions 2 and 3, Table 1), Na+ was replaced with NMDG titrated with the appropriate acid. In Cl--free (solutions 3 and 4, Table 1) and low-Cl- (solutions 5-8, Table 1) solutions, Cl- was replaced with gluconate. Because of the chelation of Ca2+ in gluconate-containing solutions, we compensated by increasing total extracellular Ca2+ concentrations, as shown in Table 1. 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) was prepared as a 10 mM stock solution and diluted 1:1,000 to a final concentration of 10 µM. Ethylisopropylamiloride (EIPA) was prepared as a 100 mM stock solution in methanol and was diluted 1:1,000 to a final concentration of 100 µM. Nigericin was prepared as a 10 mM stock solution in ethanol and was diluted 1:1,000 into solution 13 of Table 1 to a final concentration of 10 µM.

                              
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Table 1.   Composition of solutions

MC culture. Glomerular MCs were obtained by the methods previously described in our laboratory (27). In brief, kidneys were removed from 150- to 200-g male Sprague-Dawley rats. All subsequent steps were performed with sterile conditions under a laminar flow hood. Renal cortices from two to three rats were pooled and minced with a sterile razor blade. The tissue was collected and suspended in PBS. The sediments were gently poured onto a series of stainless steel sieves of decreasing pore sizes (250, 150, and 75 µm; NBC Kogyo, Tokyo, Japan). The filtrates were collected and resuspended in PBS. The sediments comprised a large number of glomeruli that were generally free of capsular and tubular debris. The glomerular suspension was passed through successive sieves and was resuspended in PBS in a culture tube. After centrifugation at 1,000 rpm for 5 min, the supernatant was discarded and the glomeruli were then plated onto 100-mm culture dishes (Becton Dickinson, Rathway, NJ). The cells were cultured in RPMI 1640, buffered with 10 mM HEPES at pH 7.4, and supplemented with 20% fetal bovine serum (FBS), 2 mg/ml NaHCO3, 100 U/ml penicillin, 100 µg/ml streptomycin, and 15.5 µg/ml insulin-transferrin-selenium in a 5% CO2 incubator at 37°C. Glomerular attachment was 20-30% after 48 h. Under these conditions, epithelial cells started to grow from the glomeruli within 2-3 days. MCs started to grow rapidly after 7-10 days, but the number of epithelial cells continuously decreased. MCs grew to confluence after 21-28 days. By this time, the cultures were virtually free of epithelial cells and showed positive staining for alpha -actin, myosin, and desmin, and showed negative staining for an endothelial cell marker factor VIII. ANG II-induced contraction of MCs was also detected by phase-contrast microscopy.

Confluent cultures were passaged through trypsin-EDTA and seeded at a 1:3 ratio in 100-mm culture dishes (Becton Dickinson). The passages were performed at 5- to 7-day intervals after confluence of the cells. Because major phenotypic changes (e.g., failure to contract in response to AVP) occur in MCs after multiple passages (e.g., Ref. 21), we only studied cells passages 3-5.

Measurement of pHi. For pHi measurements, MCs were plated on 35-mm petri dishes containing a glass coverslip bottom (MatTec, Ashland, MA) and were used 5-7 days later. Cells were incubated in 0.5% FBS-containing RPMI 1640 for 24 h before use. Before each experiment, the cells grown to subconfluence were incubated for 20 min in 0.5% FBS-containing RPMI 1640 treated with 10 µM BCECF-AM at 37°C. The petri dish was then placed on the stage of an inverted epifluorescence microscope (IMT-2; Olympus, Tokyo, Japan) and was then continuously perfused with HEPES-buffered solutions by gravity. A water jacket was used to maintain temperature in the dish at 37°C.

Single-cell measurement of pHi was performed using a microscopic fluorometer (OSP3; Olympus) as described previously (39). Measurements were made ×100 magnification, and the diameter of the beam of light focused on the MC was ~7.5 µm. The light source was a 75-W xenon lamp. The fluorescent dye was excited alternatively at 440 and 490 nm by spinning the sector mirror at 300 rpm and measured at a wavelength of 530 nm. Because in this apparatus it takes 10 ms to obtain one fluorescence ratio (I490/I440), the MC was exposed to light for 1 s to obtain one mean I490/I440. We used only MCs that had at least 20-fold greater fluorescence intensity than that of the background. To minimize dye bleaching and cell damage, protocols were made as short as possible.

Calibration of pHi. Conversion of the I490/I440 ratios to pHi values was performed according to the high-K+/nigericin technique as described previously (4, 34). At the end of each experiment, the cell was exposed to a high-K+ solution (solution 13, Table 1), titrated to pH 7.0, to which 10 µM nigericin was added. The I490/I440 ratios of the entire experiment were normalized by dividing them by the I490/I440 ratio corresponding to pH 7.0. The pHi was then calculated using the following equation (4)
<FR><NU>I<SUB>490</SUB></NU><DE>I<SUB>440</SUB></DE></FR> = 1 + <IT>b</IT> <FENCE><FR><NU>10<SUP>(pH−p<IT>K</IT>)</SUP></NU><DE>1 + 10<SUP>(pH−p<IT>K</IT>)</SUP></DE></FR> − <FR><NU>10<SUP>(7−p<IT>K</IT>)</SUP></NU><DE>1 + 10<SUP>(7−p<IT>K</IT>)</SUP></DE></FR> </FENCE>
where b is the distance between the upper and lower asymptote of the curve. The values of pK and b were determined from experiments in which MCs were exposed to 10 µM nigericin-containing solutions at different pH, always including pH 7.0. We fitted calibration data to the above equation, using a nonlinear least-squares method, and obtained best-fit values for b and pK of 1.54 ± 0.02 (SE) and 7.12 ± 0.02 (SE), respectively.

Determination of intracellular buffering power. Intrinsic buffering power (beta I) of MCs was determined using the method of Boyarsky et al. (4). As shown in Fig. 1A, acid-loaded MCs were exposed to a series of nominally Na+-free solutions (solution 2) that contained 20, 10, 5, 2.5, 1.0, 0.5, and 0 mM total ammonium (NH+4/NH3). Total NH+4/NH3-containing solutions were prepared by adding NH4Cl with replacement of NMDG in Na+-free solution. With each stepwise decrease in [NH+4/NH3]o, the amount of protons delivered to the cytoplasm (Delta [acid]i) was considered equal to the resultant change in [NH+4]i. If it is assumed that [NH3]i equals [NH3]o, and that the pKa governing the NH3/NH+4 equilibrium (8.9 at 37°C) is the same in the cytoplasm as in the extracellular fluid, [NH+4]i can be calculated from the observed pHi. Delta pHi was taken as the change in pHi produced by the stepwise decrease in [NH3/NH+4]o. beta I was then calculated as -Delta [acid]i/Delta pHi (34). beta I was assigned to the mean of the two pHi values used for its calculation. Figure 1B shows the pHi dependence of beta I. The buffering power data were grouped into pH intervals of 0.2. Each closed circle represents the mean buffering power of 0.2 pH interval, and the line represents the least-squares fit to all 56 individual data points. We used this straight line to determine beta I as a function of pHi in buffering power calculations in the present study. The equation of the best-fit line is beta I = 147.2 - 19.7 × pHi (r = 0.997) at a range of physiological pHi, indicating that beta I decreases with increasing pHi.


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Fig. 1.   Intracellular pH (pHi) dependence of intrinsic intracellular buffering power (beta I). A: typical experiment. Cell was bathed in HEPES solution (solution 1, Table 1). Na+ was removed, and then NMDG Cl was replaced with 20 mM NH4Cl. NH4Cl concentration was then decreased step by step, as indicated concentrations. Restoration of [Na+]o to 142 mM caused pHi to increase, indicating the activity of Na+/H+ exchanger (NHE). From these data, beta I is calculated as described in METHODS. B: beta I vs. pHi. Data are summarized from 11 experiments similar to that of A. , mean of buffering power values calculated for data within pHi intervals of 0.2. Bars, SE for mean pHi values and for mean buffering power in each pH interval. Solid line, results of linear least-squares fit to all 56 individual buffering power values; y-intercept of 147.2 mM/pH and a slope of -19.7 (mM/pH)/pH, r = 0.997.

Computation of net acid extrusion rates. Net acid extrusion rates (JH) in MCs were calculated from rates of pHi increase (dpHi/dt) and beta I in two kinds of experiments. First, Na+ was removed from the extracellular solution, causing pHi to decrease. We analyzed the pHi increase elicited by readdition of Na+. Second, we analyzed pHi increase when, at a steady-state pHi, MCs were exposed to hyperosmotic solutions containing mannitol, sucrose, or urea. For both experiments, we fitted a third or a fourth-degree polynomial to the pHi increase using a least-squares method, as described previously (7). At each pHi, data from four or more experiments were averaged to produce a plot of mean net acid extrusion rate vs. pHi (4, 34).

Drugs and chemicals. All chemicals were obtained from Wako (Osaka, Japan) unless noted as follows: RPMI 1640, penicillin, streptomycin, insulin-transferrin-selenium, and FBS were obtained from GIBCO BRL (Rockville, MD); HEPES and BCECF-AM were from Dojindo (Kumamoto, Japan); EIPA and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) were from Research Biochemicals International (Natick, MA); and NMDG, nigericin, DIDS, colchicine, and cytochalasin B were from Sigma (St. Louis, MO).

Statistical analysis. The data are expressed as means ± SE. Comparisons were performed by paired or nonpaired Student's t-test or one-way ANOVA where appropriate. P values <0.05 were considered significant.


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Steady-state pHi. When MCs were perfused with the HEPES-buffered solution (solution 1, Table 1) at a rate of 4.5-5.5 ml/min at 37°C, their pHi values were gradually decreased and sustained at a steady-state level. In MCs, mean steady-state pHi values were 6.96 ± 0.01 (n = 227).

pHi regulation of MCs under isosmotic conditions. At first we observed pHi recovery after removal of extracellular Na+ (Fig. 2A). Cells were first bathed in the Na+-containing HEPES-buffered solution (solution 1, Table 1) and were then incubated in the Na+-free solution (solution 2, Table 1). The removal of extracellular Na+ caused a substantial and sustained decrease in pHi from 7.01 ± 0.07 to 6.48 ± 0.02 (P < 0.001, n = 5). Subsequent addition of Na+ to the extracellular solution caused pHi to rapidly increase to similar values of the initial steady-state pHi (7.00 ± 0.06, n = 5). When a specific inhibitor of NHE, EIPA (100 µM), was added in the continued absence of external Na+, pHi was not changed at all (Fig. 2B). Readdition of Na+ to the extracellular solution in the continued presence of EIPA also caused no effect on pHi (Fig. 2B). When cells were treated with EIPA after readdition of Na+ to the extracellular solution, no further increase in pHi was observed (Fig. 2C). These results indicate that MCs possess NHE and that these cells exclusively regulate their pHi through a NHE mechanism in the nominal absence of CO2/HCO-3.


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Fig. 2.   Evidence for NHE in mesangial cells (MCs). A: MCs were first bathed in the Na+-containing HEPES-buffered solution (300 mosmol/kgH2O) and were then incubated in the Na+-free solution. The removal of external Na+ caused a substantial and sustained decrease in pHi. Readdition of external Na+ caused pHi to rapidly increase to similar values of the initial steady-state pHi. B: readdition of external Na+ in the presence of ethylisopropylamiloride (EIPA) (100 µM) completely inhibited the Na+-dependent pHi increase. C: when MCs were treated with EIPA (100 µM) after readdition of external Na+, no further increase in pHi was observed. D: removing extracellular Cl- by replacement with gluconate caused no effect on Na+-dependent pHi increase after acid load.

pHi regulation of MCs under hyperosmotic conditions. We next examined whether hyperosmolality affects pHi in MCs. MCs were incubated in HEPES-buffered Na+-containing solution (solution 1, 300 mosmol/kgH2O) and were then exposed to the HEPES-buffered solution treated with 200 mM mannitol (500 mosmol/kgH2O). As shown in Fig. 3A, pHi significantly increased from 6.86 ± 0.03 to 7.26 ± 0.02 (P < 0.001, n = 11). When MCs were treated with EIPA (100 µM) in the continued presence of hyperosmotic mannitol solution, no further increase in pHi was observed (Fig. 3B). When MCs were treated with EIPA (100 µM) in the absence of hyperosmotic mannitol solution, pHi significantly decreased from 6.99 ± 0.06 to 6.91 ± 0.06 (P < 0.001, n = 5; Fig. 3C), indicating that in the nominal absence of CO2/HCO-3, the NHE must be active in the normal steady-state pHi to balance a substantial rate of intracellular acid loading. In the continued presence of EIPA, exposure of MCs to the hyperosmotic mannitol solution did not elicit a pHi increase at all (Fig. 3C). We next examined whether cell alkalinization induced by hyperosmotic mannitol is dependent on external Na+. Exposure of the cells to hyperosmotic mannitol solution in the continued absence of external Na+ caused no increase in pHi (Fig. 4A). However, when external Na+ was readded in the presence of hyperosmotic mannitol, pHi recovery was substantially faster, and the steady-state pHi values (7.35 ± 0.06, P < 0.01, n = 7) were greater than in its absence (6.97 ± 0.07, n = 7; see Figs. 2A and 4A). These findings are consistent with the notion that hyperosmotic mannitol activates NHE to cause cell alkalinization in MCs. From the Na+-dependent pHi recovery rate and beta I, we calculated the relationship between Na+-dependent acid extrusion rate (JH) and pHi under iso- and hyperosmotic conditions (Fig. 5). Under the two conditions, Na+-dependent JH decreased as pHi increased. However, the cells exposed to the hyperosmotic mannitol solution had a significantly greater JH than those exposed to the isosmotic solution at comparable pHi values. Furthermore, hyperosmolality shifted the JH vs. pHi by 0.15-0.3 pH units in the alkaline direction.


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Fig. 3.   Cell shrinkage causes an EIPA-sensitive cell alkalinization in MCs. A: exposure of MCs to hyperosmotic HEPES solution (500 mosmol/kgH2O) containing 200 mM mannitol caused cell alkalinization. B: when MCs were treated with EIPA (100 µM) in the continued presence of hyperosmotic mannitol solution, no further increase in pHi was observed. C: addition of EIPA (100 µM) to the MCs caused pHi to decrease. In the continued presence of EIPA, exposure of MCs to hyperosmotic mannitol solution did not elicit a pHi increase at all.



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Fig. 4.   Effect of cell shrinkage on Na+-dependent pHi increase after acid load with or without extracellular Cl-. A: in the presence of extracellular Cl-, shrinkage-induced cell alkalinization was inhibited by Na+-free solution. When external Na+ was readded, pHi increased markedly. B: removal of extracellular Cl- by replacement with gluconate caused no effect on pHi, but reduced the Na+-dependent pHi increase induced by cell shrinkage.



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Fig. 5.   pHi dependence of the Na+-dependent acid extrusion rates (JH) under iso- and hyperosmotic conditions in the presence and absence of extracellular Cl-. The plots were computed from experiments such as illustrated in Figs. 2, A and D, and 4, A and B. Data represent means ± SE. Isosmo, isosmolality; hyperosmo, hyperosmolality. * P < 0.05, ** P < 0.005 compared with isosmolality in the presence of Cl- at comparable pHi values. dagger  P < 0.05, dagger dagger P < 0.005 compared with hyperosmolality in the presence of Cl- at comparable pHi values. Note that under hyperosmotic conditions, the JH is not only pHi dependent but also Cl- dependent.

To determine the effects of different osmolytes on pHi, we used other osmolytes, sucrose and urea. For this purpose, MCs were incubated in HEPES-buffered solution (solution 1, 300 mosmol/kgH2O, Table 1), and were then exposed to the HEPES-buffered solution treated with 200 mM sucrose or urea (500 mosmol/kgH2O). As shown in Fig. 6A, hyperosmotic sucrose solution caused steady-state pHi to increase from 6.93 ± 0.03 to 7.29 ± 0.04 (P < 0.001, n = 6). EIPA (100 µM) caused pHi to decrease from 7.11 ± 0.12 to 7.05 ± 0.13 (P < 0.001, n = 4), but completely inhibited the hyperosmotic sucrose-induced cell alkalinization (Fig. 6B). By sharp contrast, hyperosmotic urea solution had no effect on pHi (6.86 ± 0.06 to 6.86 ± 0.06, n = 8; Fig. 6C). Hyperosmotic mannitol and sucrose solutions showed significant initial rates of cell alkalinization (initial dpHi/dt), but hyperosmotic urea solution failed to detect the initial dpHi/dt (Fig. 6D).


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Fig. 6.   Effects of hyperosmotic mannitol, sucrose, or urea on NHE activity in MCs. A: exposure of MCs to hyperosmotic HEPES solution (500 mosmol/kgH2O) containing 200 mM sucrose caused cell alkalinization. B: EIPA (100 µM) caused steady-state pHi to decrease and completely inhibited the sucrose-induced cell alkalinization. C: exposure of MCs to hyperosmotic HEPES solution (500 mosmol/kgH2O) containing 200 mM urea caused no effect on pHi. D: initial rate of cell alkalinization (initial dpHi/dt) induced by hyperosmotic mannitol, sucrose, and urea. Data represent means ± SE; number of experiments in parentheses. Initial pHi values at which we estimated initial dpHi/dt induced by hyperosmotic mannitol, sucrose, and urea, were 6.86 ± 0.03 (n = 11), 6.93 ± 0.03 (n = 6), and 6.86 ± 0.06 (n = 8), respectively, and were not significantly different among them. Initial dpHi/dt in the hyperosmotic sucrose solution was significantly (P < 0.05) smaller than that of the hyperosmotic mannitol solution.

Glomerular MCs require Cl- for the development of a variety of metabolic and functional properties (22, 28, 29). Also, in canine erythrocytes (31, 32) and barnacle muscle fibers (10), hyperosmolality-induced NHE activation has been reported to require extracellular Cl-. Therefore, we next examined whether NHE activity in MCs under iso- or hyperosmotic conditions is dependent on extracellular Cl-. For this purpose, extracellular Cl- was replaced with a nonpermeant anion, gluconate (solutions 3 and 4, Table 1). Removal of extracellular Cl- by replacement with gluconate did not influence pHi (Fig. 7A). When hyperosmotic mannitol solution was added in the continued absence of extracellular Cl-, pHi was not changed at all (Fig. 7A). However, when extracellular Cl- was readded in the continued presence of hyperosmotic mannitol, pHi significantly increased from 6.92 ± 0.03 to 7.30 ± 0.02 (P < 0.001, n = 12; Fig. 7A and Table 2). EIPA (100 µM) alone caused the steady-state pHi to decrease by 0.08. When extracellular Cl- was readded to the MCs treated with hyperosmotic mannitol in the continued presence of EIPA, the Cl--dependent cell alkalinization was completely inhibited (Fig. 7B). We next examined whether removal of extracellular Cl- affects the Na+-dependent pHi recovery in the presence of hyperosmotic mannitol. As shown in Fig. 4B, the Na+-dependent pHi recovery rate in the absence of extracellular Cl- (56.1 ± 6.4 pH units/s × 104, P < 0.005, n = 9) was significantly smaller than that in its presence (92.7 ± 9.1 pH units/s × 104, n = 7). The relationship between Na+-dependent JH and pHi under hyperosmotic conditions in the absence of extracellular Cl- is shown in Fig. 5. Removing extracellular Cl- under hyperosmotic conditions caused a significant decrease in JH at comparable pHi values. By sharp contrast, when extracellular Cl- was removed under isosmotic conditions, either pHi, Na+-dependent pHi recovery rate (Fig. 2D), or Na+-dependent JH at different pHi values (Fig. 5) were not affected.


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Fig. 7.   Cl--dependent cell alkalinization induced by cell shrinkage. A: removal of extracellular Cl- by replacement with gluconate completely abolished the cell alkalinization induced by hyperosmotic mannitol (500 mosmol/kgH2O). When external Cl- was readded, cell alkalinization occurred. B: EIPA (100 µM) caused steady-state pHi to decrease and completely inhibited the Cl--dependent cell alkalinization induced by cell shrinkage. C: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 µM) caused no effect on steady-state pHi, but partially inhibited the Cl--dependent cell alkalinization induced by cell shrinkage. D: DIDS (100 µM) caused no effect on steady-state pHi, but partially inhibited the Cl--dependent cell alkalinization induced by cell shrinkage. E: colchicine (100 µM) caused no effect on steady-state pHi, but partially inhibited the Cl--dependent cell alkalinization induced by cell shrinkage. F: cytochalasin B (100 µM) caused no effect on steady-state pHi or Cl--dependent cell alkalinization induced by cell shrinkage.


                              
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Table 2.   Effect of various inhibitors on Cl--dependent initial rate of pHi increase after hyperosmotic mannitol solution

To examine the dose-dependent effect of Cl- on the shrinkage-induced NHE activation, MCs were exposed to hyperosmotic mannitol solution (500 mosmol/kgH2O) with different extracellular Cl- concentrations (0, 13, 36, 64, 93, and 132 mM) by replacement with gluconate (solutions 1, 4, and 5-8, Table 1). Figure 8 shows the relationship between extracellular Cl- concentrations and the rate of cell alkalinization (dpHi/dt) induced by the hyperosmotic mannitol at pHi of 6.85. The dpHi/dt decreased in a sigmoidal fashion as extracellular Cl- concentrations decreased. The apparent 50% inhibitory concentration of extracellular Cl- for the dpHi/dt induced by hyperosmotic mannitol was 69.2 mM.


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Fig. 8.   Relationship between extracellular Cl- concentrations and the rate of cell alkalinization (dpHi/dt) induced by the hyperosmotic mannitol at pHi of 6.85. The dpHi/dt decreased in a sigmoidal fashion, as extracellular Cl- concentrations decreased. Data represent means ± SE; number of experiments in parentheses.

We next examined whether the Cl--dependent NHE activation induced by hyperosmotic mannitol occurs through Cl--dependent transport processes, including Cl- channel and Cl-/base exchange. For this purpose, we added NPPB (100 µM), a Cl- channel inhibitor, or DIDS (100 µM), an inhibitor of Cl-/base exchange, to the MCs, and then observed the Cl--dependent cell alkalinization induced by hyperosmotic mannitol. NPPB or DIDS alone caused no effects on pHi values (Fig. 7, C and D). When MCs were treated with hyperosmotic mannitol in the presence of NPPB, pHi significantly increased from 6.94 ± 0.03 to 7.25 ± 0.07 (P < 0.001, n = 8). However, the final pHi values (7.25 ± 0.07, P < 0.05, n = 8) in the presence of NPPB were significantly smaller than those in its absence (7.30 ± 0.02, n = 12; Fig. 7, A and C, Table 2). Furthermore, in the presence of NPPB, the Cl--dependent initial alkalinization rate induced by hyperosmotic mannitol (initial dpHi/dt; 41.4 ± 6.8 pH units/s × 104, P < 0.005, n = 8) was significantly smaller than that in its absence (73.3 ± 8.0 pH units/s × 104, n = 12; Table 2). When MCs were treated with hyperosmotic mannitol in the presence of DIDS, pHi significantly increased from 6.95 ± 0.02 to 7.36 ± 0.05 (P < 0.001, n = 8; Fig. 7D and Table 2). However, in the presence of DIDS, the initial dpHi/dt under hyperosmotic conditions (51.8 ± 7.1 pH units/s × 104, P < 0.05, n = 8) was significantly smaller than that in its absence, although the final pHi values (7.36 ± 0.05, n = 8) in the presence of DIDS were not significantly different from those in its absence (Table 2). It has been reported that the interactions between the cytoskeleton and the plasma membrane regulate the activity of many ion channels and transport proteins, including NHE (8, 42, 43). Thus we examined whether the cytoskeletal elements are involved in the Cl--dependent NHE activation induced by hyperosmotic mannitol. For this purpose, we added colchicine (100 µM), a disruptor of microtubules, or cytochalasin B (100 µM), a disruptor of filamentous actin, to the MCs, and then observed the Cl--dependent cell alkalinization induced by hyperosmotic mannitol. Colchicine or cytochalasin B alone had no effects on pHi values (Fig. 7, E and F, Table 2). When MCs were treated with hyperosmotic mannitol in the presence of colchicine, pHi significantly increased from 6.95 ± 0.02 to 7.12 ± 0.02 (P < 0.001, n = 7). However, the final pHi values (7.12 ± 0.02, P < 0.001, n = 7) in the presence of colchicine were significantly smaller than those in its absence (Fig. 7, A and E, Table 2). Furthermore, in the presence of colchicine, the initial dpHi/dt under hyperosmotic conditions (32.7 ± 9.5 pH units/s × 104, P < 0.001, n = 7) was significantly smaller than that in its absence (Table 2). In sharp contrast, pretreatment with cytochalasin B caused no effect on Cl--dependent pHi increase induced by hyperosmotic mannitol (Fig. 7F and Table 2).

To further examine the specificity of extracellular Cl- for the shrinkage-induced NHE activation, 125 mM Cl- in the solution was substituted with equimolar gluconate, I-, Br-, SCN-, or F- (solutions 5 and 9-12, Table 1). Effects of substitution of extracellular Cl- with I-, Br-, SCN-, or F- on pHi under iso- and hyperosmotic conditions are illustrated in Fig. 9, A-D, respectively. Substitution of Cl- with I-, Br-, or SCN- under isosmotic conditions had no effect on pHi. However, when the cells were exposed to hyperosmotic mannitol (500 mosmol/kgH2O) in the presence of I-, Br-, and SCN-, the pHi significantly increased from 6.82 ± 0.01 to 7.35 ± 0.03 (P < 0.001, n = 9), from 6.82 ± 0.04 to 7.26 ± 0.02 (P < 0.001, n = 6), and from 6.79 ± 0.02 to 6.95 ± 0.05 (P < 0.001, n = 6), respectively. Pretreatment with EIPA (100 µM) completely inhibited the pHi increase induced by hyperosmotic mannitol in the presence of I-, Br-, and SCN- (Fig. 9, E-G, respectively). By sharp contrast, substitution of Cl- with F- under isosmotic conditions caused a significant decrease in pHi from 6.83 ± 0.02 to 6.61 ± 0.03 (P < 0.001, n = 9). When hyperosmotic mannitol was added in the presence of F-, pHi was increased to 6.81 ± 0.02 (P < 0.001, n = 9). However, these pHi values (6.81 ± 0.02) were not significantly different from basal pHi values (6.83 ± 0.02). When MCs were treated with EIPA under isosmotic conditions, pHi significantly decreased from 6.89 ± 0.03 to 6.75 ± 0.02 (P < 0.001, n = 5; Fig. 9H). When MCs were exposed to the solution containing F- in the continued presence of EIPA, pHi further decreased to 6.62 ± 0.02 (P < 0.001, n = 5). However, the EIPA-sensitive pHi decrease in the presence of F- (Delta pHi: 0.13 ± 0.02, P < 0.05, n = 5) was significantly smaller than that in its absence (Delta pHi: 0.22 ± 0.03, n = 9). Pretreatment with EIPA completely inhibited the pHi increase induced by hyperosmotic mannitol in the presence of F- (Fig. 9H). We calculated acid extrusion rates (initial JH) at the initial point of cell alkalinization induced by hyperosmotic mannitol solution with different anions. As shown in Fig. 10, these studies yielded an apparent specificity of Cl- >=  Br- >=  I- > SCN- > F- = gluconate for the initial JH induced by hyperosmolality. When 125 mM Cl- in the solution was substituted with equimolar cyclamate, the initial JH induced by hyperosmolality were similar to those observed in the gluconate solution (data not shown).


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Fig. 9.   Effects of substitution of extracellular Cl- with different anions on pHi under iso- and hyperosmotic conditions. 125 mM Cl- was replaced by equimolar I-, Br-, SCN-, or F-. Substitution of extracellular Cl- with I- (A), Br- (B), or SCN- (C) in the isosmotic solution had no effect on pHi, whereas it caused cell alkalinization in the hyperosmotic mannitol solution (500 mosmol/kgH2O). Substitution of extracellular Cl- with F- (D) under isosmotic conditions caused cell acidification. When hyperosmotic mannitol (500 mosmol/kgH2O) was added in the presence of F-, pHi returned to basal pHi level. EIPA (100 µM) caused steady-state pHi to decrease, but completely inhibited the pHi increase induced by hyperosmotic mannitol in the presence of I- (E), Br- (F), SCN- (G), or F- (H).



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Fig. 10.   Initial JH at the initial point of cell alkalinization induced by hyperosmotic mannitol solution with different anions. Data represent means ± SE; number of experiments in parentheses.

Figure 9, D and H, showed that, under isosmotic conditions, F- caused pHi to decrease and that the decreased pHi induced by F- was partially inhibited by EIPA. These data suggest the possibility that, under isosmotic conditions, F- may partially inhibit NHE activity. To demonstrate this possibility, we examined Na+-dependent pHi increase after acid load in the absence and presence of F-. When MCs were first bathed in the Na+-containing HEPES solution (control, solution 1, Table 1) and were then incubated in the Na+-free solution (solution 2, Table 1), pHi was decreased to ~6.50. Thereafter, MCs were exposed to the Na+-containing control solution (solution 1, Table 1) or the Na+- and F--containing solution (solution 12, Table 1). As shown in Fig. 11A, subsequent addition of Na+ to the extracellular solution caused a rapid increase in pHi in MCs exposed to both control and F--containing solutions, but the rise in pHi in the presence of F- (19.2 ± 4.7 pH units/s × 104, P < 0.005, n = 9) was significantly smaller than that in its absence (56.2 ± 11.1 pH units/s × 104, n = 11). Furthermore, the Na+-dependent JH at pHi of 6.60 after acid load in the presence of F- (36.6 ± 9.7 µM/s, P < 0.05, n = 9) was significantly smaller than that in its absence (110.6 ± 22.6 µM/s, n = 11; Fig. 11B). In MCs exposed to both control and F--containing solutions, the Na+-dependent pHi increase after acid load was completely inhibited by EIPA (100 µM; Figs. 2B and 11A, respectively). The control solution contained 132 mM Cl-, whereas the F--containing solution contained 13 mM Cl- (Table 1). Because removing extracellular Cl- had no effect on NHE activity under isosmotic conditions (Fig. 5), it is unlikely that the decreased JH in the presence of F- is due to decreased Cl- concentration of the solution. Rather, the above data indicate that F- decreased the Na+-dependent JH via an NHE process.


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Fig. 11.   Effect of F- on NHE activity under isosomotic conditions. A: Na+-dependent pHi increase after acid load in the absence and presence of F-. MCs were first bathed in the Na+-containing HEPES-buffered solution, and were then incubated in the Na+-free solution. The removal of external Na+ caused a substantial and sustained decrease in pHi. Readdition of external Na+ in the absence and presence of F- (125 mM) caused pHi to rapidly increase, but the pHi increase in the presence of F- was slower and smaller than that in its absence. The pHi increase in the presence of F- was completely inhibited by addition of EIPA (100 µM). B: The Na+-dependent JH in the absence and presence of F- at the initial point of cell alkalinization after acid load. JH was obtained from the product of dpHi/dt and beta I at pHi of 6.60. Data represent means ± SE; number of experiments in parentheses. * P < 0.05 compared with control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was designed to determine whether hyperosmolality activates NHE in MCs and, if so, to determine the underlying mechanisms, especially the role of extracellular Cl-. We demonstrate that hyperosmotic mannitol and sucrose activate NHE to cause cell alkalinization, but hyperosmotic urea causes no effect. The acid extrusion rate via a NHE process is greater under hyperosmotic conditions than under isosmotic conditions. Furthermore, NHE activation under hyperosmotic conditions requires the presence of extracellular Cl-, whereas NHE activity under isosmotic conditions is independent of extracellular Cl-. The Cl--dependent NHE activation under hyperosmotic conditions occurs, at least in part, via Cl- channel- and microtubule-dependent processes. Substitution of extracellular Cl- with different anions modulates NHE activation induced by hyperosmolality.

Evidence for NHE in MCs

The present study demonstrates that, in the nominal absence of CO2/HCO-3, pHi recovery rate from an acid load was dependent on external Na+ and was completely inhibited by a specific NHE inhibitor (EIPA; see Fig. 2, A-C). Thus MCs exclusively regulate their pHi through a NHE mechanism in the nominal absence of CO2/HCO-3. These findings confirm previous studies (4, 13).

pHi Regulation of MCs Under Iso- and Hyperosmotic Conditions

pHi dependence of NHE. Our results show that cell alkalinization induced by hyperosmotic mannitol is due to activation of NHE because it is dependent on external Na+ and is inhibited by EIPA (see Figs. 3 and 4A). As shown in Fig. 5, under both iso- and hyperosmotic conditions, Na+-dependent JH through a NHE process decreased as pHi increased from the acidic to the normal range. However, the cells exposed to the hyperosmotic mannitol solution had a significantly greater JH than those exposed to the isosmotic solution at comparable pHi values. In other words, hyperosmolality shifted the JH vs. pHi by 0.15-0.3 pH units in the alkaline direction. Similarly, in Na+-depleted glial cells, Jean et al. (20) reported that shrinkage shifted the flux vs. pHi profile by 0.3-0.4 pH units in the alkaline direction. In addition, Grinstein et al. (15) studied the Na+-dependent component of a pHi recovery from an acid load in lymphocytes, finding that shrinkage shifts the flux vs. pHi relationship by 0.2-0.3 pH units in the alkaline direction. Similar findings have been reported in barnacle muscle fibers exposed to hyperosmolality (10) and in C6 glioma cells treated with hyperosmotic mannitol solution (37). Because the pHi sensitivity of the exchange system appears to be largely determined by an allosteric modifier site (2) located on the cytoplasmic face of the membrane, the mechanisms for the shrinkage-induced NHE activation seem to be a shift in the pHi dependence of the antiport.

Effects of different osmolytes. In the present study, hyperosmotic mannitol and sucrose solutions showed significant initial rate of cell alkalinization, whereas hyperosmotic urea solution failed to detect it (see Figs. 3 and 6). In addition, the cell alkalinization induced by hyperosmotic mannitol and sucrose was completely inhibited by pretreatment with EIPA (see Figs. 3C and 6B). Therefore, these findings indicate that, at steady-state pHi, both hyperosmotic mannitol and sucrose activate NHE, but hyperosmotic urea does not. Because mannitol and sucrose are relatively impermeant, it is most likely that they make the solution hypertonic. In contrast, urea is freely permeant and raises the osmolality but does not affect the solution tonicity (40). Accordingly, solution tonicity rather than absolute osmolality is the important factor for the NHE activation at steady-state pHi. Similarly, Muto et al. (24) reported that, in vascular smooth muscle cells, hyperosmotic glucose or mannitol media stimulated Na+-K+-ATPase alpha 1- and beta 1-mRNA accumulation, alpha 1- and beta 1-subunit protein accumulation, and Na+-K+-ATPase activity, whereas hyperosmotic urea medium caused no effect. Similar actions of hyperosmotic NaCl, raffinose, or urea on accumulation of organic osmolytes have been reported in Madin-Darby canine kidney cells (25, 26): NaCl- or raffinose-induced hyperosmotic stress resulted in the accumulation of betaine, glycerophosphorylcholine, and myo-inositol, but urea-induced hyperosmotic stress failed to induce osmolyte accumulation.

Effects of removing extracellular Cl-. NHE activity in the apical membrane of the colonic crypt cell under isosmotic conditions is dependent on extracellular Cl- (33). In dog erythrocytes (31, 32) and in barnacle muscle fibers (10), NHE activation under hyperosmotic conditions is inhibited by removing extracellular Cl-. Therefore, the present study examined whether the MC NHE activity under iso- or hyperosmotic conditions is dependent on extracellular Cl-. The MCs exposed to the hyperosmotic mannitol solution in the absence of extracellular Cl- had a significantly smaller JH than that in its presence at comparable pHi values (see Fig. 5). Furthermore, extracellular Cl- modulates the shrinkage-induced NHE activation in a dose-dependent manner (Figs. 2 and 9), whereas, under isosmotic conditions, it causes no effect on NHE activity (Fig. 2, A and D, and Fig. 5). Therefore, the NHE activation under hyperosmotic conditions is not only pHi dependent but also Cl- dependent. Also, NHE in MCs is distinct from that of the apical membrane in the colonic crypt cell.

The mechanisms by which extracellular Cl- affects the shrinkage-induced NHE activation are not clear presently. The effects of extracellular Cl- may be through changes in intracellular Cl-, as reported in barnacle muscle fibers treated with hyperosmotic mannitol solution (10). Several mechanisms would be proposed for the Cl--dependent NHE activation induced by hyperosmolality. Removal of extracellular Cl- by replacement with gluconate did not influence NHE activity under isosmotic conditions (see Fig. 2, A and D, and Fig. 5), whereas it inhibited NHE activity under hyperosmotic conditions (Figs. 4 and 5). On the contrary, when external Cl- was readded in the continued presence of hyperosmotic mannitol, NHE activation occurred (Fig. 7A). Therefore, it is unlikely that this inhibition was due to the introduction of gluconate as the extracellular Cl- replacement. Rather, Cl- might be required for some aspects of the shrinkage-induced activation process, the sensor or signal transduction system, as Davis et al. (10) suggested. Regarding the latter, the activation of G proteins has been reported to be involved in the shrinkage-induced activation of NHE in barnacle muscle fibers (9). Regarding the former, we found that the Cl--dependent NHE activation induced by hyperosmolality was partially inhibited by pretreatment with NPPB (the Cl- channel inhibitor; see Fig. 7, A and C, Table 2). These results indicate that the shrinkage-induced NHE activation partly occurs through Cl- channel activation. Pretreatment with DIDS (the inhibitor of Cl-/base exchange) also partially inhibited the Cl--dependent NHE activation induced by hyperosmolality (see Fig. 7, A and D, Table 2). In the nominal absence of CO2/HCO-3, removing extracellular Cl- caused no increase in pHi, as shown in Fig. 7A. Thus, under these conditions, the Cl-/base exchange process is indeed inoperative, although, in the presence of CO2/HCO-3, the MC anion exchanger has been reported to transport base (5). Rather, in the nominal absence of CO2/HCO-3, DIDS may act on MCs as a Cl- channel inhibitor. In other cell systems, DIDS has been reported to be one of the Cl- channel inhibitors (17, 36). As shown in Fig. 7, E and F, and Table 2, colchicine, but not cytochalasin B, partially inhibited the Cl--dependent NHE activation induced by hyperosmolality. These findings indicate that microtubule, but not filamentous actin, is involved in the Cl--dependent NHE activation induced by hyperosmolality, and that under these conditions, a colchicine-sensitive process may be activated, and microtubule may regulate the exocytic insertion of the NHE protein-containing vesicles into the plasma membrane. Similarly, Wagner et al. (41) reported in rat proximal tubules that ANG II stimulated H+-ATPase activity and that this stimulation was inhibited by removing external Cl-, NPPB, and colchicine. Their report suggests that ANG II stimulates proton extrusion via H+-ATPase by a Cl--dependent process involving brush-border insertion of vesicles. The mechanisms by which the Cl--dependent NHE activation induced by hyperosmolality occurs via a colchicine-sensitive process are still unclear.

Effects of substitution of Cl- with different anions. The present study further examined the Cl- specificity for the shrinkage-induced NHE activation. When extracellular Cl- under isosmotic conditions was replaced with Br-, I-, or SCN-, basal pHi values were not affected (see Fig. 9, A-C). Substitution of extracellular Cl- with different anions yielded an apparent specificity of Cl- >=  Br- >=  I- > SCN- > gluconate for the shrinkage-induced acid extrusion rates through a NHE process (Fig. 10). The hyperosmolality-induced cell alkalinization in the presence of Cl-, Br-, I-, SCN-, and gluconate was completely inhibited by pretreatment with EIPA (Figs. 3C and 9, E-G). These results indicate that both Br- and I-, like Cl-, modulate the shrinkage-induced NHE activation. Therefore, these three anions may share common metabolic and/or transport pathway(s), as discussed above. On the other hand, when Cl- was replaced with SCN-, the acid extrusion rate induced by hyperosmolality was 38.9% of that of Cl-. These findings indicate that SCN- partially inhibits the Cl--dependent NHE activation induced by hyperosmolality. When external Cl- under isosmotic conditions was substituted with F-, basal pHi values were significantly decreased (Fig. 9D). Pretreatment with EIPA partially inhibits the F--induced cell acidification (Fig. 9H). Furthermore, as shown in Fig. 11, the Na+-dependent JH at pHi of 6.60 after acid load in the presence of F- was significantly smaller than that in its absence, and the Na+-dependent pHi recovery was completely inhibited by EIPA. Thus, at steady-state pHi, F- inhibits NHE activity, and consequently causes cell acidification, although the possibility that the F--induced cell acidification may occur via mechanisms other than inhibition of the NHE cannot be excluded. NaF (more specifically AlF4-) at a concentration of 10 mM is a well-known pharmacological probe for establishing the significance of G protein activation in cellular systems (14, 30). The concentration of NaF used in the present study was 125 mM. It is not known whether this concentration of NaF actually acts on MC NHE as a G protein activator. We found that the hyperosmolality-induced cell alkalinization in the presence of F- was completely inhibited by pretreatment with EIPA (Fig. 9H) and that the initial JH in the hyperosmotic F- solution was similar to that in the hyperosmotic gluconate solution (Fig. 10). These findings indicate that the hyperosmolality-induced cell alkalinization in the presence of F- is due to NHE activation and that F- does not influence the Cl--dependent NHE activation induced by hyperosmolality. In contrast to MCs, in vascular smooth muscle cells, NaF has been shown to stimulate NHE activity under both iso- and hyperosmotic conditions (30). From our present findings, different anions modulate Cl--dependent NHE activation induced by hyperosmolality. However, the underlying mechanisms will be required to be elucidated.

In conclusion, the present study clearly demonstrates that, at steady-state pHi, hyperosmolality by the poorly permeating solutes (mannitol and sucrose) activates NHE to cause cell alkalinization, whereas the rapidly permeating solute (urea) has no effect, and shrinkage-induced NHE activation requires extracellular Cl- and is modulated by substitution of Cl- with different anions. The Cl--dependent NHE activation induced by hyperosmolality partly occurs via Cl- channel- and microtubule-dependent processes. The Cl--dependent NHE activation under hyperosmotic conditions may be important for cell volume regulation, intraglumerular hemodynamics, and/or TGF mechanisms.


    ACKNOWLEDGEMENTS

We thank H. Kasakura for expert secretarial assistance in preparing the manuscript.


    FOOTNOTES

This work was supported in part by a grant from the Japanese Kidney Foundation (Jinkenkyukai), by a grant from the Salt Science Foundation, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

A portion of this work was presented at the 1998 Annual Meeting of the American Society of Nephrology in Philadelphia, PA, and has been published in abstract form (23a).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Muto, Dept. of Nephrology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan (E-mail: smuto{at}jichi.ac.jp).

Received 25 February 1999; accepted in final form 7 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andrews, PM, and Coffey AK. Cytoplasmic contractile elements in glomerular cells. Federation Proc 42: 3046-3052, 1983[ISI][Medline].

2.   Aronson, PS, Nee J, and Suhm MA. Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature 299: 161-163, 1982[ISI][Medline].

3.   Boron, WF, Boyarsky G, and Ganz M. Regulation of intracellular pH in renal mesangial cells. Ann NY Acad Sci 574: 321-332, 1989[ISI][Medline].

4.   Boyarsky, G, Ganz MB, Sterzel RB, and Boron WF. pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO-3. Am J Physiol Cell Physiol 255: C844-C856, 1988[Abstract/Free Full Text].

5.   Boyarsky, G, Ganz MB, Sterzel RB, and Boron WF. pH regulation in single glomerular mesangial cells. II. Na+-dependent and -independent Cl--HCO-3 exchangers. Am J Physiol Cell Physiol 255: C857-C869, 1988[Abstract/Free Full Text].

6.   Chamberlin, ME, and Strange K. Anisosmotic cell volume regulation: a comparative review. Am J Physiol Cell Physiol 257: C159-C173, 1989[Abstract/Free Full Text].

7.   Chen, LK, and Boron WF. Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO-3. Am J Physiol Renal Fluid Electrolyte Physiol 268: F179-F192, 1995[Abstract/Free Full Text].

8.   Dascalu, A, Nevo Z, and Korenstein R. Hyperosmotic activation of the Na+-H+ exchanger in a rat bone cell line: temperature dependence and activation pathways. J Physiol (Lond) 456: 503-518, 1992[Abstract].

9.   Davis, BA, Hogan EM, and Boron WF. Role of G proteins in stimulation of Na-H exchange by cell shrinkage. Am J Physiol Cell Physiol 262: C533-C536, 1992[Abstract/Free Full Text].

10.   Davis, BA, Hogan EM, and Boron WF. Shrinkage-induced activation of Na+-H+ exchange in barnacle muscle fibers. Am J Physiol Cell Physiol 266: C1744-C1753, 1994[Abstract/Free Full Text].

11.   Fabiato, A, and Fabiato F. Effects of pH on the myofilments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscle. J Physiol (Lond) 276: 233-255, 1978[Abstract].

12.   Ganz, MB, Pekar SK, Perfetto MC, and Sterzel RB. Arginine vasopressin promotes growth of rat glomerular mesangial cells in culture. Am J Physiol Renal Fluid Electrolyte Physiol 255: F898-F906, 1988[Abstract/Free Full Text].

13.   Ganz, MB, Perfetto MC, and Boron WF. Effects of mitogens and other agents on rat mesangial cell proliferation, pH, and Ca2+. Am J Physiol Renal Fluid Electrolyte Physiol 259: F269-F278, 1990[Abstract/Free Full Text].

14.   Gilman, AG. G proteins: transducers of receptor generated signals. Annu Rev Biochem 56: 615-645, 1987[ISI][Medline].

15.   Grinstein, S, Rothestein A, and Cohen S. Mechanisms of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes. J Gen Physiol 85: 765-787, 1985[Abstract].

16.   Grinstein, S, Woodside M, Sardet C, Pouyssegur J, and Rotin D. Activation of the Na+/H+ antiporter during cell volume regulation. J Biol Chem 267: 23823-23828, 1992[Abstract/Free Full Text].

17.   Hall, JA, Kirk J, Potts JR, Rae C, and Kirk K. Anion channel blockers inhibit swelling-activated anion, cation, and nonelectrolyte transport in HeLa cells. Am J Physiol Cell Physiol 271: C579-C588, 1996[Abstract/Free Full Text].

18.   Harris, RC, Haralson MA, and Badr KF. Continuous stretch-relaxation in culture alters mesangial cell morphology, growth characteristics, and metabolic activity. Lab Invest 66: 548-554, 1992[ISI][Medline].

19.   Hoffmann, EK, and Simonsen LO. Membrane mechanisms in volume and pH regulation. Physiol Rev 69: 315-382, 1989[Free Full Text].

20.   Jean, T, Frelin C, Vigne P, and Lazdunski M. The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock. Eur J Biochem 160: 211-219, 1986[Abstract].

21.   Kreisberg, JI, Venkatachalam M, and Troyer D. Contractile properties of cultured glomerular mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 249: F457-F463, 1985[ISI][Medline].

22.   Kremer, SG, Zeng W, Sridhara S, and Skorecki KL. Multiple signaling pathways for Cl--dependent depolarization of mesangial cells: role of Ca2+, PKC, and G proteins. Am J Physiol Renal Fluid Electrolyte Physiol 262: F668-F678, 1992[Abstract/Free Full Text].

23.   Maddox, DA, and Brenner BM. Glomerular ultrafiltration. In: Brenner & Rector's The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 1996, vol. V, p. 286-333.

23a.   Miyata, Y, Muto S, Yanagiba S, Ebata S, and Asano Y. Hyperosmolality stimulates Na-H exchange activity (NHE) in cultured mesangial cells (MC) (Abstract). J Am Soc Nephrol 9: 9A, 1998.

24.   Muto, S, Ohtaka A, Nemoto J, Kawakami K, and Asano Y. Effect of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells. J Membr Biol 162: 233-245, 1998[ISI][Medline].

25.   Nakanishi, T, Turner RJ, and Burg MB. Osmoregulation of betaine transporters in mammalian renal medullary cells. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1061-F1067, 1990[Abstract/Free Full Text].

26.   Nakanishi, T, Turner RJ, and Burg MB. Osmoregulatory changes in myo-inositol transport by renal cells. Proc Natl Acad Sci USA 86: 6002-6006, 1990[ISI].

27.   Ohhara, T, Ikeda U, Muto S, Oguchi A, Tsuruya Y, Yamamoto K, Kawakami K, Shimada K, and Asano Y. Thyroid hormone stimulates Na+-K+ ATPase gene expression in cultured rat mesangial cells. Am J Physiol Renal Fluid Electrolyte Physiol 265: F370-F376, 1993[Abstract/Free Full Text].

28.   Okuda, T, Kojima I, Ogata E, and Kurokawa K. Ambient Cl- ions modify rat mesangial cell contraction by modulating cell inositol triphosphate and Ca2+ via enhanced prostaglandin E2. J Clin Invest 84: 1866-1872, 1989[ISI][Medline].

29.   Okuda, T, Yamashita N, and Kurokawa K. Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat mesangial cells. J Clin Invest 78: 1443-1448, 1986[ISI][Medline].

30.   Orlov, SN, Resink TJ, Bernhardt J, and Buhler FR. Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signaling systems. J Membr Biol 129: 199-210, 1992[ISI][Medline].

31.   Parker, JC. Volume-responsive sodium movements in dog red blood cells. Am J Physiol Cell Physiol 244: C324-C330, 1983[Abstract].

32.   Parker, JC, and Castranova V. Volume-responsive sodium and proton movements in dog red blood cells. J Gen Physiol 84: 379-401, 1984[Abstract].

33.   Rajendran, VM, Geibal J, and Binder HJ. Chloride-dependent Na-H exchange. A novel mechanism of sodium transport in colonic crypts. J Biol Chem 270: 11051-11054, 1995[Abstract/Free Full Text].

34.   Roos, A, and Boron WF. Intracellular pH. Physiol Rev 61: 296-434, 1981[Free Full Text].

35.   Schnermann, J, Ploth DW, and Hermle M. Activation of tubulo-glomerular feedback by chloride transport. Pflügers Arch 362: 229-240, 1976[ISI][Medline].

36.   Schwiebert, EM, Morales MM, Devidas S, Egan ME, and Guggino WB. Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 95: 2674-2679, 1998[Abstract/Free Full Text].

37.   Shrode, LD, Klein JD, Douglas PB, O'Neill WC, and Putnam RW. Shrinkage-induced activation of Na+/H+ exchange: role of cell density and myosin light chain phosphorylation. Am J Physiol Cell Physiol 272: C1968-C1979, 1997[Abstract/Free Full Text].

38.   Shrode, LD, Klein JD, O'Neill WC, and Putnam RW. Shrinkage-induced activation of Na+/H+ exchange in primary rat astrocytes: role of myosin light-chain kinase. Am J Physiol Cell Physiol 269: C257-C266, 1995[Abstract/Free Full Text].

39.   Takeda, M, Yoshitomi K, and Imai M. Regulation of Na+-3HCO-3 cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor. Am J Physiol Renal Fluid Electrolyte Physiol 265: F511-F519, 1993[Abstract/Free Full Text].

40.   Uchida, S, Garcia-Perez A, Murphy H, and Burg MB. Signal for induction of aldose reductase in renal medullary cells by high external NaCl. Am J Physiol Cell Physiol 256: C614-C620, 1989[Abstract/Free Full Text].

41.   Wagner, CA, Giebisch G, Lang F, and Geibel JP. Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells. Proc Natl Acad Sci USA 95: 9665-9668, 1998[Abstract/Free Full Text].

42.   Wakabayashi, S, Shigekawa M, and Pouyssegur J. Molecular physiology of vertebrate Na+/H+ exchanger. Physiol Rev 77: 51-74, 1997[Abstract/Free Full Text].

43.   Watson, AJM, Levine S, Donowitz M, and Montrose MH. Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin. J Biol Chem 267: 956-962, 1992[Abstract/Free Full Text].


Am J Physiol Cell Physiol 278(6):C1218-C1229
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