Introduction

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"Natriuretic hormone," the putative controller of extracellular fluid volume (de Wardener et al., 1961) has been sought for more than 30 years. This compound was believed to be responsible for the three effects observed during saline-induced volume expansion: prolonged natriuresis, inhibition of sodium transport and increased vasoreactivity. Most search paradigms have focused on the inhibition of sodium transport, specifically inhibition of the Na⁺/K⁺-ATPase (sodium pump) or immunocross-reactivity to antibodies developed to the pump inhibitors ouabain or digoxin, as an assay to detect this compound during isolation (Wechter and Benaksas, 1990; Benaksas et al., 1995). This has led to the isolation of digoxin (Goto et al., 1990) and "iso-ouabain" (Ludens et al., 1991; Mathews et al., 1991; Tymiak et al., 1993; Zhao et al., 1995). Digoxin and ouabain, however, are kaliuretic and not natriuretic (Crabos et al., 1987; Murray et al., 1995; Pamnani et al., 1991; Smyth et al., 1992; Sekihara and Yuzaki, 1993). Therefore, this search tool is inappropriate except for bufalin-like compounds which are natriuretic (Pamnani et al., 1991). Our search has concentrated on the natriuresis attributed to this putative hormone, resulting in detection of several natriuretic components in human uremic urine (Benaksas et al., 1993; Murray et al., 1995).

The isolation, purification and structure determination of the most interesting of these compounds to date, LLU-, has been described (Wechter et al., 1996a). A probable metabolite of -tocopherol, LLU-, was shown to be natriuretic, an inhibitor of the 70 pS ATP-sensitive K⁺ (K_ATP) channel in the TAL and without an effect on hemodynamics as measured by the glomerular filtration rate (Wechter et al., 1996a). It was also demonstrated not to be an inhibitor of the Na⁺/K⁺-ATPase in MDBK cells. Unlike atrial natriuretic peptide, which is also not an inhibitor of the sodium pump (Chiou and Vesely, 1995; Charlton and Baylis, 1990), LLU- exhibits a prolonged natriuresis like that described for "natriuretic hormone" (de Wardener et al., 1961).

In the present study LLU- is further characterized. Its origin from -tocopherol is established. The pharmacokinetics of intravenously infused LLU- is reported and compared with the time course of natriuresis. The specificity of the inhibition of the 70 pS K⁺ channel by the enantiomers of LLU- is determined. The analogous -tocopherol metabolite is also investigated for its ability to inhibit this potassium channel. The inhibition of the Na⁺/K⁺-ATPase from different sources is tested.

	Methods

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LLU- and derivatives. The compounds used in these studies (fig. 1) were synthesized as described by Kantoci et al. (accompanying paper, 1997). The enantiomers of LLU- were purified by preparative chiral HPLC as described by Kantoci et al. (accompanying paper, 1997).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Compounds used in these studies. S-LLU- is the natural enantiomer (Kantoci et al., accompanying paper, 1997).

Metabolism of -tocopherol in rats. Two rats (Sprague-Dawley, Harlan) were each injected intraperitoneally with 500 µCi/kg of [³H]--tocopherol (9.6 Ci/mmol, Amersham custom synthesis) in 2.5 ml/kg of 10% ethanol in corn oil (Chiku et al., 1984; Simon et al., 1956a, b). The rats were then placed into metabolic cages for urine collection for 4 days. The urine (220 ml) was pooled, solids removed by filtration. The solution was extracted with 2 g Amberlite XAD-2 (Aldrich, Milwaukee, WI) with stirring for 18 h. The XAD was recovered by filtration and extracted with 4 volumes of methanol totaling 60 ml. The methanol was removed under a stream of 5.0 µm filtered air. The dried residue was resuspended in 1.8 ml of 40:60 methanol/0.2 M acetic acid for chromatography. Beckman System Gold (126 pump, 168 diode array detector) controlled by System Gold software v 5.1 on PC was used for all chromatography. A C₁₈ RP-HPLC (Beckman Ultrasphere ODS column; 5 µm; 10 × 250 mm) was eluted at 2 ml/min with a gradient of 0.2 M acetic acid (A), methanol (B) and 70% toluene/30% methanol (C) (60% A:40% B for 5 min, a linear gradient to 50% A:50% B over 5 min, a linear gradient to 30% A:70% B over 55 min, a linear gradient to 100% B over 2 min, 100% B for 3 min, 100% C for 8 min, 100% B for 7 min). The eluant was monitored for absorbance at 265 and 290 nm and ultraviolet spectra were collected (202-390 nm). Seventy-eight fractions of 1-min duration were collected. An aliquot (50 µl) of each fraction was counted in 5 ml of liquid scintillation cocktail (ScintiVerse^TM Bio-HP, Fisher, Tustin, CA) in a LS 3801 Liquid Scintillation Counter (Beckman, San Ramon, CA). Fractions (39-42) eluting in the region known to contain LLU- were pooled, 25 µg of synthetic, racemic LLU- was added and the pooled material was dried under reduced pressure in a centrifugal vacuum concentrator (Jouan, Winchester, VA). The residue was dissolved in 1.8 ml of 15:85 45 mM acetic acid in acetonitrile/50 mM acetic acid (aq.) and chromatographed on C₁₈ RP-HPLC (Beckman Ultrasphere ODS column; 5 µm; 4.6 × 250 mm) eluted at 1 ml/min with a gradient of 50 mM acetic acid (A) and 45 mM acetic acid in acetonitrile (B) (85% A:15% B for 3 min, a linear gradient to 100% B over 42 min, 100% B for 5 min). The eluant was monitored for absorbance at 265 and 295 nm by the diode array detector. Fifty 1-min fractions were collected with aliquots (100 µl) counted as described above. This experiment was performed in duplicate.

In vivo bioassay. The assay for natriuresis in conscious rats has previously been described in detail (Benaksas et al., 1993), but will be briefly reiterated here. The bladder of ether-anesthetized female Sprague-Dawley (Harlan) rats (200-250 g) was catheterized for collection of urine in 10-min periods. The femoral artery and vein were cannulated for monitoring of mean arterial pressure and infusion of saline (0.49 ml/h) and samples, respectively. After recovery from anesthesia, furosemide (100 µg, approximately 0.4 mg/kg b.wt.; 1 mg/ml in 0.17% saline) was infused as a positive control at the beginning of the sixth 10-min urine collection period. The sample was infused at the beginning of the 17th 10-min period. Urine was collected for another 150 min. The volume of the urine was determined gravimetrically. The urine Na⁺ and K⁺ concentrations were determined with a Beckman E2A electrolyte analyzer. From these data the sodium excretion values (UNaV; [UNaV = urine sodium concentration × urine volume per time]) were calculated.

Pharmacokinetics of LLU-. The in vivo bioassay for natriuresis (described above) was adapted for these experiments. At the time of sample infusion, 10 µg of [³H]LLU- (4.2 µCi/nmol, Amersham Custom Synthesis, 0.25 mCi/ml ethanol; 640 µl dried under a stream of argon and resuspended in 100 µl of 47.5% ethanol) was infused, and the saline infusion rate was changed to 1 ml/min. The experiment was performed on two animals. Blood (approximately 0.5 ml) was drawn from each animal into heparinized microfuge tubes at various time points. Blood was drawn from animal 1 at 1, 5, 30 and 120 min and from animal 2 at 2, 15, 60 and 240 min. The blood was centrifuged for 5 min. An aliquot of plasma (100 µl) was withdrawn and placed into a 15-ml centrifuge tube. HCl (2 N, 20 µl) was added to each centrifuge tube and vortexed for 30 sec. Hexane/ether (4:1, 2 ml) was added to each tube and the tube was vortexed for 3 min. The samples were sonicated and centrifuged as necessary and 1.5 ml of the hexane/ether sample was removed and placed in a glass test tube. Unlabeled synthetic, racemic LLU- (50 µg) was added to each sample. The solvent was removed under a stream of filtered air. The sample was resuspended in 1.8 ml of 15:85 45 mM acetic acid in methanol/50 mM acetic acid (aq.) and chromatographed on acetic acid/acetonitrile RP-HPLC as described in "Metabolism of -Tocopherol in Rats." Fifty 1-min fractions were collected, with aliquots (100 µl) counted as above. A pilot study was conducted using one animal infused with 10 µg of [³H]LLU- and blood drawn at 5, 15, 30, 60 and 160 min.

Na⁺/K⁺-ATPase inhibition assays. Inhibition of the Na⁺/K⁺-ATPase by the reduction of ⁸⁶Rb⁺ uptake into human lymphocytes was examined. Lymphocytes were isolated from heparinized blood with a Histopaque-1077 (Sigma, Buchs, Switzerland) density gradient and then washed with buffer by centrifugation similar to previously described methods (Anner et al., 1994). Cell viability was examined by the trypan blue dye exclusion test and was found to be greater than 99%. Lymphocytes (5 × 10⁶ cells) were incubated in the presence or absence of synthetic, racemic LLU- (125 µM, final concentration) in 50 µl of PBS (154 mM NaCl, 10 mM sodium phosphate, pH 7.2-7.4) with 50 nCi ⁸⁶Rb⁺ for 30 min at 36°C with shaking. Cells were washed three times with PBS (once) or D-PBS (twice; 137 mM NaCl, 2.68 mM KCl, 0.90 mM CaCl₂, 0.49 mM MgCl₂, 6.46 mM Na₂HPO₄, 1.47 mM KH₂PO₄) by centrifugation at 0°C at 500 × g. The recovered cells were then counted. Cell viability was checked by trypan blue dye exclusion after incubation under the same conditions but without ⁸⁶Rb⁺. Na⁺/K⁺-ATPase was isolated from lamb renal outer medulla as described previously (Anner et al., 1992). Na⁺/K⁺-ATPase activity was measured by the linked enzyme assay method described (Anner et al., 1992). Synthetic, racemic LLU- in 50 mM sodium phosphate buffer, pH 7.2, was added to a final concentration of 125 µM either during the assay or 30 min. before the assay for preincubation at 37°C. Only buffer was added for control assays.

Patch-clamp experiments. Pathogen-free Sprague-Dawley rats (male or female, 80-120 g) were maintained on normal rat chow before being sacrificed as described previously (Wang, 1994). Kidneys were then removed and thin (1 mm) coronal sections were cut. The cortical and medullary TAL tubules were dissected at room temperature. The TAL tubules were immobilized onto cover glass coated with Cell-Tak (Collaborative Research, Bedford, MA). The cover glass was then placed in a chamber mounted on an inverted microscope and the tubules superfused at 37°C with Hepes-buffered Ringer's solution (135 mM NaCl, 5 mM KCl, 1.5 mM MgCl₂, 1.8 mM CaCl₂, 5 mM glucose, 10 mM Hepes, pH 7.4). The apical membrane was exposed by cutting the tubule with a sharpened micropipette. Single-channel currents were recorded in cell-attached, inside-out configuration with 140 mM KCl in the pipette with a resistance of approximately 4 to 6 megohm. The recordings were collected with an Axon 200A patch-clamp amplifier. Single-channel currents were low-pass filtered at 1 kHz with an eight-pole Bessel filter (Frequency Devices 902LPF) and converted to digitized signals at a sampling rate of 44 kHz by a Sony PCM-501ES pulse code modulator and a videotape recorder. Analysis was done with use of the pCLAMP software (v. 6.0, Axon Instruments, Burlingame, CA) on an IBM-compatible 486 computer. The potency of the inhibition of the K⁺ channel is expressed as IC₅₀, which was calculated by linear regression analysis.

	Results

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70 pS K⁺ channel inhibition. Racemic, synthetic LLU- has been previously shown to inhibit the 70 pS K⁺ channel in the TAL of the loop of Henle with an IC₅₀ of 6 nM (Wechter et al., 1996a and fig. 2). R- and S-LLU- as well as racemic, synthetic 5-methyl-LLU- (the analogous metabolite of -tocopherol) were tested in this system. 5-Methyl-LLU- demonstrated no inhibition of the 70 pS K⁺ channel in the concentration range of 100 to 500 nM (fig. 2), whereas S-LLU- (IC₅₀ = 3 nM) was a more potent inhibitor than the racemate and R-LLU- was about 20-fold less effective, IC₅₀ = 51 nM (fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibition of the 70 pS K+ channels from TAL. S-enantiomer of rac-LLU- (closed squares), R-enantiomer of rac-LLU- (closed triangles), rac-LLU- (closed circles) and rac-5-methyl-LLU- (open circles) were examined for their ability to inhibit the potassium channel as described under "Methods." Data are presented as mean ± S.E.M.

Isolation of [³H]LLU- from [³H]-tocopherol-treated rats. To test the hypothesis that LLU- originates as a result of side-chain degradation of -tocopherol, rats were administered [³H]-tocopherol. Urine collected from these rats was extracted and chromatographed as described under "Methods." The final chromatographic step is shown in figure 3. A peak of radioactivity (3800 cpm total) coelutes with added nonlabeled, synthetic racemic LLU- standard. The approximate overall yield from administered [³H]-tocopherol was 2 × 10³%.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Last step in isolation of [3H]LLU- from [3H] -tocopherol-treated rats. Absorbance at 295 nm of eluant collected from the acetic acid/acetonitrile chromatography of urine from rats administered [3H]-tocopherol is shown (solid line). The programmed acetonitrile gradient is plotted (- - -). Total cpm in each fraction (corrected for background cpm) is represented by the open bars. The arrow indicates the elution time of the synthetic, racemic LLU- standard.

Pharmacokinetics of LLU-. [³H]LLU- infused intravenously demonstrates biphasic pharmacokinetics (fig. 4). The data were fit to the equation: and the data were weighted (1/y [cpm]). The half-life for the first phase is 12.5 min. whereas that for the second phase is 6.3 h. Complete clearance is estimated to be 3 days.²

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Natriuretic/diuretic response and pharmacokinetics of LLU-. Sodium excretion (UNaV, solid bars) and water excretion (UV, open bars) as a result of infusion of 10 µg synthetic, racemic LLU- are measured for 150 min. The five 10-min periods before infusion were used to determine the background excretions. RN = .54 for this experiment. (Inset) Levels of [3H]LLU- in 100 µl plasma determined by extraction and RP-HPLC as described under "Methods." Each point represents a single determination.

Inhibition of Na⁺/K⁺-ATPase. Inhibition of the Na⁺/K⁺-ATPase by synthetic, racemic LLU- was examined with two different methodologies. Synthetic, racemic LLU- did not reduce uptake of ⁸⁶Rb⁺ into human lymphocytes at 125 µM (data not shown). Isolated Na⁺/K⁺-ATPase, from lamb renal outer medulla, examined by a linked enzyme assay was not inhibited by 125 µM synthetic, racemic LLU- (data not shown).

	Discussion

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Biology of LLU-. We previously showed that synthetic, racemic LLU- does not inhibit the sodium pump in MDBK cells (Wechter et al., 1996a). In the present study we demonstrate that it also does not inhibit the Na⁺/K⁺-ATPase in lymphocytes nor from renal outer medulla (data not shown). Thus LLU- does not inhibit the ₁ (present in the three sources tested [Anner et al., 1992; Vereninov et al., 1993]) and ₃ (present in outer medulla [Barlet-Bas et al., 1993]) isoforms of the sodium pump. This lends additional support to the hypothesis that sodium pump inhibition is a poor search tool for isolation of natriuretic substances with the exception of bufalin-like compounds (Wechter and Benaksas, 1990; Benaksas et al., 1995; Pamnani et al., 1991). This is the second compound isolated and characterized by our laboratory that is natriuretic but is not a sodium pump inhibitor (Wechter et al., 1996b), and other compounds that fit this description are yet to be characterized (Benaksas et al., 1993; Murray et al., 1995). The compounds isolated by Na⁺/K⁺-ATPase inhibition as a search tool, digoxin (Goto et al., 1990) and iso-ouabain (Ludens et al., 1991; Matthews et al., 1991; Tymiak et al., 1993; Zhao et al., 1995), may have a role in volume or blood pressure homeostasis; however, we believe this function is not likely to occur via natriuresis (Pamnani et al., 1991; Sekihara and Yuzaki, 1993; Wechter and Benaksas, 1990; Benaksas et al., 1995).

Production of LLU- from -tocopherol. When LLU- was first identified it was assumed that it originated from metabolism of -tocopherol, most likely by side-chain oxidation. In this study it is demonstrated that [³H]-LLU- can be obtained from urine of rats administered [³H]-tocopherol, thus providing evidence that LLU- is a metabolite of -tocopherol. This is in agreement with previous studies of -tocopherol in humans and -tocopherol in rats (Chiku et al., 1984; Schönfeld et al., 1993; Schultz et al., 1995). That LLU- originates from side-chain degradation without oxidation of the chroman ring is confirmed by the study of Kantoci et al. (accompanying paper, 1997) demonstrating that the stereochemistry at C-2 is S or the same configuration as that of the parent -tocopherol (Burton and Ingold, 1989).