Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3

S. Akhter1, S. K. Nath1, C. M. Tse1, J. Williams2, M. Zasloff2, and M. Donowitz1

1 Departments of Medicine and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and 2 Magainin Pharmaceuticals, Inc., Plymouth Meeting, Pennsylvania 19642

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 µg/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

sodium absorption; intestinal brush border

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RECOGNITION THAT CERTAIN animals fail to develop cutaneous or peritoneal infections, despite "dirty" wounds, has provided a fruitful strategy to search for antibiotics that occur endogenously in reptiles and mammals (1, 2, 5, 10, 14, 22). A number of endogenous low-molecular-weight antibiotic substances have recently been discovered in vertebrates, including magainins from frog skin (23), cryptdins from mammalian intestine (14), and beta -defensin-1 from human lung airway surface fluid (1, 5). Endogenous antibiotics in vertebrates are chemically divergent and include peptides (mostly in the defensin family), lipids, and alkaloids. These identified compounds are components of the nonadaptive immune system, which provides essential physical and chemical barriers against infection (10).

One such substance, squalamine, was extracted from the dogfish shark Squalus acanthias, which demonstrates low infection rates, even under conditions where pregnant sharks flush their fallopian tubes with seawater to dispose of fetal waste. Stomach extracts of S. acanthias exhibited potent antimicrobial activity, prompting the isolation, structural determination, and characterization of the antibiotic molecule squalamine (11, 17, 22). Squalamine is a broad-spectrum, water-soluble cationic steroid antibiotic that demonstrates, among other characteristics, potent bactericidal activity against gram-negative and gram-positive bacteria, fungicidal qualities, and the ability to induce osmotic lysis of protozoa. Squalamine is formed by the condensation of squalene, an anionic bile salt intermediate, with spermidine. In S. acanthias, squalamine is found not only in the liver and gallbladder, the sites of bile synthesis, but also, in order of decreasing concentration, in the spleen and testes, stomach, gills, and intestine (11). On the basis of the pharmacological effects of squalamine in model cell systems, which included changes in cell shape (M. Zasloff, unpublished observations), the hypothesis was tested that a target of squalamine included transport proteins that control cell volume or cell shape. In this study we describe a specific effect of squalamine, in that it inhibits one particular mammalian Na+/H+ exchanger isoform, NHE3, the Na+/H+ exchanger isoform present in intestinal and renal brush borders.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture

PS120 fibroblasts, which lack endogenous Na+/H+ exchangers, were stably transfected with cDNAs for full-length rabbit NHE1, NHE2, and NHE3, as well as rabbit NHE3 with the COOH-terminal 76 amino acids deleted (9, 18, 20, 21). These cells were grown in DMEM supplemented with 25 mM NaHCO3, 10 mM HEPES, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum in a 5% CO2-95% O2 incubator at 37°C, as described previously (19). Geneticin (400 µg/ml) was used to maintain selection pressure and was added immediately after each subculturing procedure. In addition, cells were exposed to an acid load consisting of 50 mM NH4Cl-saline solution for 1 h followed by an isotonic 3 mM Na+ solution. Surviving cells were then placed in normal culture medium and allowed to reach 30-50% confluence. The acidification process was initially repeated every 2-3 days until >50% of the cells survived and was then repeated every week to maintain high Na+/H+ exchange activity. The transfected NHE cDNAs have been previously described (9, 18, 20, 21).

Measurement of Na+/H+ Exchange Activity

Fluorometry/2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein experiments. Cells were seeded on glass coverslips, grown overnight in serum-free medium, and studied as described previously (9) when they reached 50-70% confluency. The cells were loaded with 5 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM in "Na+ medium" [containing (in mM) 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 glucose, 20 HEPES, pH 7.4] for 30-60 min at room temperature, then washed with "TMA+ medium" [containing (in mM) 130 tetramethylammonium chloride, 5 KCl, 2 CaCl2, 1 MgSO4, 1 NaH2PO4, 25 glucose, 20 HEPES, pH 7.4] to remove the extracellular dye, and the coverslip was mounted at an angle of 60° in a 100-µl fluorometer cuvette designed for perfusion and thermostated at 37°C. The cells were pulsed with 40 mM NH4Cl in TMA+ medium for 2-10 min; then the NH4Cl was removed and the cells were perfused with TMA+ medium, which resulted in the acidification of the cells. In most of the squalamine experiments the cells were incubated with squalamine (dissolved in water) for 60 min during dye loading.

Kinetic studies were performed as described previously (9, 19) and did not include data from cells with an intracellular pH (pHi) >7.1, inasmuch as there exists an endogenous acidification process in PS120 cells above this pH (19). At pHi <7.1, the pH recovery was entirely Na+ dependent and amiloride sensitive. Na+/H+ exchange rate data were calculated as the product of Na+-dependent change in pHi times the buffering capacity at each pHi and were analyzed using a nonlinear regression data analysis program (ENZFITTER, Biosoft), which allowed fitting of data to a general allosteric model described by the Hill equation (v = Vmax · [S]n/K' + [S]n) with estimates for maximal velocity (Vmax) and K'[H+]i [a complex parameter, as previously described (9)] and their respective errors (SE), as well as fitting to a hyperbolic curve, as would be expected with Michaelis-Menten kinetics. The SE was calculated by the computer to reflect variability of the parameters estimated, as described previously (9). Control and test experiments were done on the same day by using similarly acid-selected cells of the same passage, as described previously (19).

To determine whether squalamine altered the K'[H+]i of NHE3, a further analysis of the above Na+-dependent alkalinization was carried out by comparing the H+ efflux rates of control and squalamine-exposed monolayers vs. pHi. Analyses of data generated at intervals of 0.01 pH unit were performed and expressed as a ratio of transport rates (squalamine/control). With this method of analysis, regulation of NHEs by changes in Vmax, but not K'[H+]i, showed no change in the test-to-control ratio with increasing pHi, whereas regulation by changes in K'[H+]i, regardless of whether they were accompanied by changes in Vmax, deviated from the horizontal at high pHi values. A positive control of K'[H+]i regulation was demonstrated by studying the effects of serum on NHE1, and a negative control was shown in experiments in which serum stimulated NHE3 (18, 19).

22Na+ uptake studies. Cells were grown to near confluency in 24-well plates, and growth was arrested in serum-free medium overnight. Cells were then washed twice with 1 ml of TMA+ medium containing 10 mM NH4Cl for 20 min at room temperature. Acidification was induced by aspiration of the NH4Cl solution and rapid washing of the cells with 1 ml of TMA+ medium. 22Na+ uptake was measured at room temperature for 2 min by addition of 0.5 ml of incubation medium [containing (in mM) 2 NaCl (1 µCi/ml 22NaCl; NEN), 128 TMA chloride, 5 KCl, 2 CaCl2, 1 MgSO4, 1 K2PO4, 25 glucose, 20 HEPES, pH 7, and 1 ouabain] to the cells in the presence and absence of 1 mM amiloride. The Na+ uptake rate was linear with time for at least 5 min. At the end of the incubation, cells were rapidly washed five times with 1 ml of ice-cold 0.1 M MgCl2. Intracellular 22Na+ then was released by lysing the cells with 0.5 ml of 0.1 M HNO3, and radioactivity was measured with a gamma counter, with the specific activity of the transport buffer being used to determine the amount of Na+ uptake, which was then normalized per well.

Rabbit Ileal Brush-Border Vesicle Studies of Na+/H+ Exchange and Na+-Dependent Glucose Uptake

Na+/H+ exchange was determined in rabbit ileal brush-border vesicles prepared by double magnesium precipitation at 4°C, as previously described (4, 15). Rabbit ileal mucosa was incubated in Ringer-HCO-3 solution (gassed with 95% O2-5% CO2) at 37°C without (control) or with 5 µg/ml squalamine for 60 min before the ileal mucosa was scraped on ice, and then brush-border membrane vesicles were prepared. The final brush-border vesicle pellet was needle homogenized in buffer containing (in mM) 200 mannitol, 40 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO)-11.4 Tris, pH 6.5, and 5 magnesium gluconate, and Na+/H+ exchange was determined as 22Na+ uptake with an outwardly directed pH gradient (pHi 6.5/extracellular pH 8.0) minus uptake with the same transport buffer but also including 1 mM amiloride. Transport buffer contained (in mM) 212 mannitol, 28 Tris-9.6 MES, pH 8.5, 5 magnesium gluconate, and 1 sodium gluconate (22Na+, 33-37 µCi/ml; NEN). Final pH was 8.0 when 30 µl of transport buffer were mixed with 15 µl of membrane buffer to initiate uptake. Transport was determined for 3, 5, and 8 s at room temperature, stopped by the addition of 1 ml of ice-cold stop solution [containing (in mM) 40 mannitol, 90 potassium gluconate, 5.6 MES-20 Tris, pH 8.0], as described previously (15, 16), and the buffer was vacuum filtered through 0.45-µm nitrocellulose filters; radioactivity was determined in a liquid scintillation spectrometer. Normalization for brush-border protein content was carried out using a Coomassie brilliant blue-based assay (Bio-Rad).

Na+-dependent glucose uptake was determined as the difference in D-[3H]glucose uptake in the presence and absence of Na+ (4, 15). Membrane buffer was as described above for the Na+/H+ exchange determination, and transport buffer contained (in mM) 20 mannitol, 40 MOPSO-11.4 Tris, pH 6.5, 5 magnesium gluconate, 90 NaCl, and 0.1 D-[3H]glucose (20 µCi/ml; NEN). Transport buffer final concentrations are given after 30 µl of transport buffer were mixed with 15 µl of membrane buffer. Uptake of glucose was determined for 8 s (linear phase of uptake), 90 s (overshoot), or 90 min (equilibrium) at room temperature, followed by vacuum filtration and scintillation counting, as described above, with stop solution consisting of (in mM) 40 mannitol, 90 potassium gluconate, and 20 MOPSO-5.6 Tris, pH 6.5.

Lactate Dehydrogenase Assay

PS120 cells were grown to near confluence in six-well plates and then incubated with various concentrations of squalamine or aminosterol 1436 for 60 min. The drug-treated and control cell media were removed, and lactate dehydrogenase (LDH) activity was assayed by monitoring the conversion of exogenous pyruvate to lactate by measuring the decrease in 340-nm absorbance resulting from the conversion of added NADH to NAD (7). The cells were then detergent solubilized with 10% Triton X-100 at room temperature for 10 min and scraped with a rubber policeman in 1 ml of fresh medium to assay for total cell LDH levels. Comparison of the two measurements was expressed as the percentage of total cell LDH released.

Materials

Squalamine and the related aminosterol 1436 were isolated by Magainin Pharmaceuticals, as described previously (11). The synthetic aminosterols 1271 and 1272 were also provided by Magainin Pharmaceuticals (7). BCECF and nigericin were obtained from Molecular Probes. All other chemicals were obtained from Sigma Chemical.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Squalamine Inhibition of Basal NHE3 Activity

Effects on amiloride-sensitive Na+-dependent intracellular alkalinization in PS120/NHE3 cells (Na+/H+ exchange). The effects of squalamine on Na+/H+ exchange in PS120/NHE3 cells, measured as changes in pHi with BCECF, are shown in Fig. 1. When PS120/NHE3 cells of the same passage and density were compared at similar pHi, Na+-dependent alkalinization was inhibited by 1 h of exposure to 5 µg/ml squalamine (Fig. 1A, right). In contrast, acute exposure to the same concentration of squalamine did not affect Na+/H+ exchange (Fig. 1A, left). On the basis of kinetic analysis of the squalamine effect, a 1-h preincubation with 5 µg/ml squalamine reduced the NHE3 Vmax by 47%: from 1,509 to 783 µM/s (Fig. 1B). The squalamine-induced inhibition of NHE3 occurred in a concentration-dependent manner (Table 1). PS120/NHE3 cells treated for 1 h with 1 µg/ml squalamine demonstrated a 13% inhibition, whereas a similar 1-h treatment with 7 µg/ml squalamine caused 57% inhibition. The effect of squalamine was also time dependent: a 1-h exposure was sufficient to merit a maximal response. No effect occurred with acute addition, less-than-1-h exposures showed less inhibition, and there was no further inhibition after 12 h of treatment with squalamine (Fig. 1C). Furthermore, the squalamine effect was reversible. PS120/NHE3 cells regained full Na+/H+ exchange activity after a 1-h exposure to squalamine and then washout, with Na+/H+ exchange activity determined 3 h later (Fig. 1D).


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Fig. 1.   Squalamine inhibits Na+/H+ exchange in PS120/NHE3 cells. Cells were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in Na+ medium alone () or Na+ medium containing 5 µg/ml squalamine (open circle ) for 1 h. Cells were then acidified by incubation with NH4Cl, perfused in TMA+ solution, and allowed to recover to steady-state pH in Na+ medium. Intracellular H+ concentration ([H+]) was monitored using BCECF in a spectrofluorometer. A: representative traces of Na+-dependent pH recovery of PS120/NHE3 cells in HCO-3-free medium as a function of time. Traces on left show intracellular pH (pHi) recovery in control cells and cells on acute addition of squalamine; traces on right show inhibition of pH recovery with 1-h preincubation with squalamine. Differences in control rates are due to different passages of cells, but extent of squalamine inhibition was constant through at least 10 different passages of PS120/NHE3 cells. Traces are shown offset in time to allow demonstration of individual results. B: kinetic studies. [H+] efflux rates, equivalent to Na+/H+ exchange, were plotted against intracellular [H+]. Na+/H+ efflux rates were calculated at various pHi values, and lines were fit to data by using an allosteric model. Maximal velocity (Vmax) was 47% lower in squalamine-treated cells (783 µM/s) than in untreated control cells (1,509 µM/s). C: 12-h treatment with 5 µg/ml squalamine inhibits PS120/NHE3 by 47% (from 823 to 427 µM/s), demonstrating no greater inhibition than 53% inhibition seen with a 1-h treatment with 5 µg/ml squalamine. D: squalamine-mediated inhibition of NHE3 is reversed on 3 h of removal of compound. PS120/NHE3 cells were exposed to 5 µg/ml squalamine for 60 min, medium was removed and replaced with medium without squalamine for 3 h, and Na+/H+ exchange was determined. Na+/H+ exchange rates in PS120/NHE3 cells 3 h after removal of squalamine are indistinguishable from those in time control cells, which were treated similarly except without squalamine.

                              
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Table 1.   Concentration dependence of squalamine inhibition of NHE3

Previous studies demonstrated that NHE3 is usually regulated by changes in the Vmax (19), with no accompanying changes in the K'[H+]i. In contrast, squalamine-treated cells not only showed a decrease in the NHE3 Vmax, but also an accompanying change in the K'[H+]i (Fig. 2A). The K'[H+]i changes were determined by an analysis of data in which ratios of squalamine to control transport rates were compared in paired experiments at intervals of 0.01 pH unit. This analysis of regulation of NHE3 used two conditions of NHE regulation as controls (Fig. 2, B and C): a negative control involved NHE3 stimulation by serum through a Vmax shift (12, 18, 19), and a positive control was the K'[H+]i shift in the serum stimulation of NHE1 (21). Figure 2B shows no change in the serum-to-control ratio with increasing pHi, whereas Fig. 2C shows an increase in this ratio at lower intracellular H+ concentration ([H+]i). In the case of squalamine-treated PS120/NHE3 cells, the ratio of Na+/H+ exchange (squalamine/control) also changed at low [H+]i (Fig. 2A), indicating that squalamine alters K'[H+]i. The shift of the squalamine-to-control ratio for NHE3 is in the opposite direction from that of the serum-to-control ratio for NHE1.


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Fig. 2.   Squalamine alters K'[H+]i of NHE3. Potential effects of squalamine on K'[H+]i of NHE3 were tested by comparing test/control Na+/H+ exchange rates at intervals of 0.01 pH unit. As a positive control for this analysis in which K'[H+]i changes were previously shown to occur, dialyzed serum (10%) stimulation of NHE1 was analyzed (C), and as an expected negative control, dialyzed serum (10%) stimulation of NHE3 was analyzed (B). Similar to change in test/control at low intracellular [H+] ([H+]i) seen with serum-NHE3 (C), there is also a significant change in test/control with squalamine-NHE3 (A).

Effects on amiloride-sensitive 22Na+ uptake in PS120/NHE3 cells. To confirm that squalamine inhibits Na+/H+ exchange and does not alter another mechanism of pHi homeostasis (including a process found only in PS120/NHE3 cells), 22Na+ uptake was studied. Amiloride-sensitive 22Na+ uptake in cells studied in HCO-3-free media can be attributed to Na+/H+ exchange. As previously reported, 22Na+ uptake, which is amiloride-sensitive, occurs linearly for at least 5 min under the conditions studied in these PS120 cells (19). Preincubation for 1 h with 7 µg/ml squalamine inhibited NHE3-dependent 22Na+ uptake activity by 53%: 4,611 cpm for squalamine vs. 9,810 cpm for control. The effect was also concentration dependent between 0.5 and 7 µg/ml squalamine (data not shown). Again, there was no effect on 22Na+ uptake on acute addition of squalamine to PS120/NHE3 cells.

Squalamine Inhibits Na+/H+ Exchange in Rabbit Ileal Brush-Border Vesicles

Squalamine was also evaluated as to whether it regulated Na+/H+ exchange in another NHE3-containing model system, the brush border of ileal Na+-absorbing cells. Squalamine pretreatment (5 µg/ml) inhibited rabbit ileal brush-border Na+/H+ exchange. The mean of three separate vesicle Na+/H+ exchange experiments is shown in Fig. 3. Squalamine reduced Na+/H+ exchange (slope of 3-, 5-, and 8-s uptake) from 15.2 ± 2.8 to 7.9 ± 0.4 pmol · mg protein-1 · s-1 (P < 0.0001, n = 3). The specificity of this effect was checked by determining whether squalamine affected Na+-dependent D-glucose uptake. Preincubation with squalamine for 1 h did not alter Na+-dependent or Na+-independent glucose uptake during the period of linear uptake at 8 s, uptake at time of overshoot (90 s), or equilibrium uptakes (90 min; Table 2). This further demonstrates specificity of the squalamine effect on Na+/H+ exchange in the ileal brush border.


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Fig. 3.   Squalamine inhibits rabbit ileal brush-border Na+/H+ exchange. 22Na+ uptake was determined in brush-border vesicles from villus cells of control and squalamine-exposed ileum over period of linear Na+ uptake. Results represent mean amiloride-sensitive Na+ uptake at 3, 5, and 8 s from 3 separate vesicle preparations. Slopes of 3-, 5-, and 8-s uptakes were calculated from each experiment for control and squalamine, and means ± SE were determined. Control slope is 15.2 ± 2.8 pmol/mg protein, and slope for squalamine-exposed tissue is 7.9 ± 0.4 pmol/mg protein. P < 0.0001, control vs. squalamine.

                              
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Table 2.   Effect of squalamine on D-[3H]glucose uptake with Na+ or K+ in the transport buffer

Specificity of Squalamine Inhibition of NHE3: Studies With NHE1 and NHE2 Expressed in PS120 Cells

To further examine the specificity of the squalamine inhibition of NHE3, its effects on other NHE isoforms expressed in PS120 cells were also determined. PS120/NHE1 and PS120/NHE2 cells were studied spectrofluorometrically with BCECF. Preincubation with 5 µg/ml squalamine for 1 h did not affect NHE1 or NHE2 activity. Figure 4A shows that squalamine does not alter the kinetics of Na+/H+ exchange of rabbit NHE1 expressed in PS120 cells. Figure 4B demonstrates that rabbit NHE2 expressed in PS120 cells is similarly unaffected by squalamine treatment. This further indicates the specificity of squalamine inhibition of NHE3 and not other NHE isoforms or non-NHE-related acid-base regulatory processes in PS120 cells.


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Fig. 4.   Effect of squalamine on NHE1 and NHE2. Squalamine does not affect Na+/H+ exchange of PS120/NHE1 or PS120/NHE2 cells, indicating that it specifically inhibits Na+/H+ exchange of NHE3. Cells were loaded with BCECF in Na+ medium alone () or Na+ medium containing 5 µg/ml squalamine (open circle ) for 1 h. They were then acidified by incubation with NH4Cl, perfused with TMA+ solution, and allowed to recover to steady-state pH in Na+ medium. A: Vmax and K'[H+]i of NHE1 cells treated with squalamine (2,148 µM/s) did not differ significantly from untreated controls (1,945 µM/s). B: Vmax of NHE2 cells treated with squalamine (775 µM/s) was not significantly different from that of untreated controls (875 µM /s).

Effects of the Aminosterols 1436, 1271, and 1272 on NHE1, NHE2, and NHE3

Three squalamine-derived compounds, aminosterols 1271 and 1272 and the natural aminosterol 1436, with closely related chemical structures (Fig. 5A), were studied to define structural requirements for NHE3 inhibition. Functional differences between the compounds in their effects on the NHE isoforms expressed in PS120 cells could yield information on the active portions of the compounds or potential-specific regulation of other NHE isoforms. Measurements of pHi showed that aminosterol 1436 inhibited NHE3 activity (Fig. 5B). A 1-h exposure of PS120/NHE3 to 10 µg/ml aminosterol 1436 reduced the Vmax by 65%: from 1,034 to 364 µM/s. Similar studies showed that aminosterol 1436 did not affect PS120/NHE1 or PS120/NHE2 cells, whereas <= 10 µg/ml aminosterols 1271 and 1272 failed to inhibit NHE1, NHE2, or NHE3 (data not shown).


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Fig. 5.   Effect of squalamine analogs aminosterol 1436, 1271, and 1272 on NHE3. A: chemical structures of squalamine and its analogs aminosterol 1436, 1271, and 1272 (7, 18). B: 1436 inhibits Na+/H+ exchange in PS120/NHE3 cells. Experiments were conducted with BCECF spectrofluorometry. Treatment for 1 h with 10 µg/ml aminosterol 1436 lowered Vmax (364 µM/s) by 65% compared with control cells (1,034 µM/s). , Control; open circle , 1436.

Mechanism of Squalamine Inhibition of NHE3

Squalamine inhibition of PS120/NHE3 cells requires the COOH-terminal 76 amino acids of NHE3. The specificity of the squalamine effect on NHE3 could result from an effect on a signaling pathway that culminates in the regulation of NHE3. Previous studies have shown that NHE3 is regulated through distinct COOH-terminal cytoplasmic domains that mediate the effects of various signal transduction intermediaries such as protein kinase C, calmodulin, tyrosine kinases, fibroblast growth factor, serum, and serine/threonine phosphatases 1 and 2A (8, 9). To determine what portion of the NHE3 COOH terminus was responsible for the squalamine inhibition of Na+/H+ exchange, a previously described truncation mutant of the NHE3 COOH terminus, E3756 (which refers to a cDNA in which NHE3 is truncated at amino acid 756), was expressed in PS120 cells. Spectrofluorometric studies with mixed populations of this COOH-terminal truncation mutant of NHE3 transfected into PS120 cells showed that squalamine inhibition required the last 76 amino acids, since Na+/H+ exchange in PS120/E3756 cells was not affected by 5 µg/ml squalamine (Fig. 6). As a positive control, we previously reported that the same mixed populations of PS120/E3756 cells are inhibited by the phorbol ester phorbol 12-myristate 13-acetate (1 µM) and stimulated by 10% serum (9).


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Fig. 6.   Squalamine inhibition of NHE3 requires COOH-terminal 76 amino acids of NHE3. PS120 cells transfected with a COOH-terminal truncation mutant of NHE3 lacking COOH-terminal 76 amino acids were loaded with BCECF in Na+ medium alone () or Na+ medium containing 5 µg/ml squalamine (open circle ) for 1 h. They were then acidified by incubation with NH4Cl, perfused with TMA+ solution, and allowed to recover to steady-state pHi in Na+ medium. Without last 76 amino acids of its cytoplasmic tail, NHE3 truncation mutant demonstrates no inhibition of Na+-dependent pH recovery with squalamine treatment.

Cytotoxicity of Squalamine and Aminosterol 1436

To determine whether the inhibitory effects of squalamine and aminosterol 1436 were due to their toxic effects on PS120/NHE3 cells, the cytotoxicity of the compounds was studied by determining the percent release of the cytoplasmic enzyme LDH (7). LDH release was measured to ensure that cytotoxicity was not interpreted as inhibition of NHE3 exchange rate. Cytotoxicity was expressed as the percentage of total cell LDH, which was determined by Triton X-100 solubilization of cells. Although aminosterol 1436 and squalamine caused a small amount of cell death in a concentration-dependent manner, the amount of LDH released with the experimental concentrations that altered NHE3 was <5% of the total cell LDH and was not significantly greater than that released by untreated controls (Fig. 7). This shows that the inhibitory effect of squalamine on NHE3 is not secondary to toxicity, as defined by LDH release.


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Fig. 7.   Cytotoxicity of squalamine and aminosterol 1436 assessed by release of lactate dehydrogenase (LDH) activity. Cells were grown to near confluence in 6-well plates and then treated with different concentrations of squalamine or aminosterol 1436 for 1 h. Drug-treated and control cell media were removed, and LDH activity was assayed by monitoring conversion of exogenous pyruvate to lactate and simultaneously measuring decrease in 340-nm absorbance resulting from conversion of added NADH to NAD. Cells were then detergent solubilized and scraped to assay for total cell LDH levels. Comparison of 2 measurements showed percentage of total cell LDH released due to treatment with squalamine or aminosterol 1436.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

These studies demonstrate that squalamine is a unique Na+/H+ exchange inhibitor. This effect of squalamine is different from that of previously described Na+/H+ exchanger inhibitors, including amiloride and its 5'-amino-substituted analogs HOE-694, cimetidine, and harmaline, in that all previously described inhibitors affected multiple NHE isoforms, had a sensitivity for NHE1 > NHE2 > NHE3, and act very quickly (9, 12, 13). Squalamine seems to inhibit only NHE3, and not NHE1 or NHE2, when all isoforms were studied in the same cell type, and inhibitory effects took ~1 h to develop.

Is the effect of squalamine specific for NHE3? The model systems studied thus far demonstrate that squalamine affects a limited number of transport processes. In addition to only inhibiting NHE3 in PS120 cells, and not NHE1 or NHE2 expressed in the same cells, squalamine inhibited ileal brush-border Na+/H+ exchange and did not alter Na+-dependent D-glucose uptake, Na+-independent D-glucose uptake, or glucose equilibrium values in the same vesicles. However, preliminary reports have described other effects of squalamine on transport processes in different cells and on other signaling pathways. For instance, squalamine has been shown to inhibit vascular endothelial growth factor-induced growth of endothelial cells, which do not contain NHE3 (16). Thus the specificity of squalamine, as currently recognized, is partially defined by cell type.

What is the mechanism of the squalamine inhibition of NHE3? Although it inhibits NHE3, squalamine does not act in a manner similar to amiloride. The squalamine effect is far slower (suggesting that the effect is not from the outside of the cell, as occurs with amiloride). Toxicity does not appear to be involved in the squalamine-induced NHE3 inhibition, as demonstrated by the lack of cell LDH release over the concentration range that inhibits NHE3, by the failure to affect NHE1 or NHE2 transfected in the same PS120 cells over the same concentration range, and by the failure of squalamine to affect other transport processes in the same brush-border vesicles in which Na+/H+ exchange was inhibited. Far more difficult to comment on is whether the effect of squalamine is directly on NHE3 or represents an indirect effect by interfering with some aspect of signal transduction. The long time frame required for the squalamine effect is consistent with the need for uptake of squalamine to occur for a sufficient time until the intracellular concentration needed to inhibit NHE3 accumulates or involves some aspect of signal transduction.

Squalamine and aminosterol 1436 inhibit NHE3, but not NHE1 or NHE2, whereas the structurally related analogs aminosterols 1271 and 1272 had no effect on NHE1, NHE2, or NHE3. These results suggest that the C24/C25 sulfate (Fig. 5A) and/or the 7alpha -hydroxyl group are essential parts of the molecule for squalamine inhibition, whereas the polyamine tail is not similarly limiting. Some specific antibacterial properties of squalamine also require the C24/C25 sulfate rather than the polyamine tail (6).

Squalamine inhibition of NHE3 requires the COOH-terminal 76 amino acids of NHE3. The COOH-terminal ~300 amino acids of NHE3 are thought to be cytoplasmic and are required for regulation by growth factors and protein kinases. The COOH-terminal 76 amino acids are a rich part of the regulatory domain, with regulation by calmodulin, calmodulin kinase II, and tyrosine kinases, as well as squalamine. Although Wakabayashi et al. (21a) found no evidence that elevating intracellular Ca2+ acts by calmodulin to stimulate NHE3, the inhibitory effect of calmodulin on NHE3 at basal intracellular Ca2+ concentration appears to be mediated through these 76 amino acids (9). All these agents are entirely dependent on these COOH-terminal amino acids, whereas protein kinase C inhibition of NHE3, which is absolutely dependent on amino acids 585-689, is modified by removing the COOH-terminal 76 amino acids (9). Whether the squalamine inhibition of NHE3 requires interaction with any of these other regulatory molecules that act via the COOH-terminal 76 amino acids of NHE3 is not known (3).

In S. acanthias, squalamine occurs endogenously in multiple tissues, including the liver and gastrointestinal tract; whether it regulates Na+/H+ exchange and, specifically, NHE3 in shark tissues is unknown. Given the up- and downregulation of NHE3 that occur in the small intestine as part of digestion, it is worth speculating that there may exist endogenous regulators of NHE3, such as squalamine and aminosterol 1436 in mammals, that may be released as part of digestion to act locally to inhibit NHE3 and thus locally regulate intestinal Na+ absorption. The presence in shark intestine of squalamine and related compounds indicates the presence of a specific endogenous inhibitory regulator of NHE3, which works in a unique manner in terms of isoform specificity and kinetics of inhibition.

    ACKNOWLEDGEMENTS

We acknowledge the expert editorial and secretarial assistance of H. McCann and the insightful review of this manuscript by Seth Alper.

    FOOTNOTES

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1 DK-26523, R01 DK-51116, and PO1 DK-44484, the Meyerhoff Digestive Diseases Center, the Hopkins Center for Epithelial Disorders, and a grant-in-aid from Magainin Pharmaceuticals, Inc.

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: M. Donowitz, GI Div., Ross 925, Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205.

Received 9 July 1998; accepted in final form 14 October 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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