Use of Diethyl(2-methylpyrrolidin-2-yl)phosphonate as a Highly Sensitive Extra- and Intracellular 31P NMR pH Indicator in Isolated Organs

DIRECT NMR EVIDENCE OF ACIDIC COMPARTMENTS IN THE ISCHEMIC AND REPERFUSED RAT LIVER*

Sylvia PietriDagger §, Sophie MartelDagger , Marcel CulcasiDagger , Marie-Christine Delmas-Beauvieux, Paul Canioni, and Jean-Louis Gallis

From the Dagger  Structure et Réactivité des Espèces Paramagnétiques, CNRS-UMR 6517 Universités d'Aix-Marseille I et III, F-13397 Marseille Cedex 20 and the  Résonance Magnétique des Systèmes Biologiques, CNRS-UMR 5536, Université Victor Segalen Bordeaux 2, F-33076 Bordeaux Cedex, France

Received for publication, September 1, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The novel phosphorylated pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH) was evaluated as a 31P NMR probe of the pH changes associated with ischemia/reperfusion of rat isolated hearts and livers. In vitro titration curves indicated that DEPMPH exhibited a 4-fold larger amplitude of chemical shift variation than inorganic phosphate yielding an enhanced NMR sensitivity in the pH range of 5.0-7.5 that allowed us to assess pH variations of less than 0.1 pH units. At the non-toxic concentration of 5 mM, DEPMPH distributed into external and cytosolic compartments in both normoxic organs, as assessed by the appearance of two resonance peaks. An additional peak was observed in normoxic and ischemic livers, assigned to DEPMPH in acidic vesicles (pH 5.3-5.6). During severe myocardial ischemia, a third peak corresponding to DEPMPH located in ventricular and atrial cavities appeared (pH 6.9). Mass spectrometry and NMR analyses of perchloric extracts showed that no significant metabolism of DEPMPH occurred in the ischemic liver. Reperfusion with plain buffer resulted in a rapid washout of DEPMPH from both organs. It was concluded that the highly pH-sensitive DEPMPH could be of great interest in noninvasive ex vivo studies of pH gradients that may be involved in many pathological processes.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because of the dependence of the chemical shift of phosphates on pH (1-3), 31P NMR spectroscopy has progressively become the standard method for the measurement of intracellular pH (pHi)1 in biological systems, mostly using inorganic orthophosphate (Pi) as a naturally occurring pH probe (4-6). Although this technique was first applied to measure pH in a variety of biological fluids, the development of pulsed Fourier transform NMR and wide-bore supraconducting magnets has soon allowed the noninvasive study of cell cultures and isolated perfused organs under physiologic or pathologic conditions. In particular, the use of 31P NMR has considerably increased our understanding of the dynamics of pHi changes during ischemia-induced acidosis in the heart (7-9) and liver (10, 11). Although Pi resonance has been successfully used to describe the main pHi-regulatory systems in these organs (12, 13), more subtle trans-sarcolemmal proton movements that could occur in different pathologies may escape investigation if they are related to extra- and intracellular pH values different by less than 0.2-0.3 pH units (4). In addition to this relative lack of resolution, Pi levels vary with cell metabolism, and the chemical shift of Pi has been demonstrated to be affected by ionic strength (4-6).

The search for improved exogenous 31P NMR pH indicators as alternatives to Pi yielded a variety of alkyl- and aminoalkylphosphonic acids having their resonance peak distinct from that of phosphorylated metabolites, and an NMR sensitivity Delta delta ab (defined as the mean difference between the chemical shifts of the protonated delta a and the unprotonated delta b forms) in the range of Pi (i.e. 2-3 ppm). There have been a number of studies on the in vitro properties of synthetic alkylphosphonates as 31P NMR pH probes (14-20), but only a few have focused on more complex systems such as the isolated perfused bladder (21), heart (22, 23), or liver (24). In these studies, phenylphosphonate was found to be a specific extracellular pH and volume marker (21-24), but even though it was reported to allow the monitoring of pHi in the liver where it can reach a suitable degree of cytosolic uptake (24), no information could be obtained on the occurrence of more acidic organelles participating in proton transport mechanisms (25, 26).

By introducing an amino function in the alpha  or beta  position of the alkyl group of the charged phosphonate structure, suitable NMR pH probes of lower pKa values (in the range 5.5-6.3) that entered in Dictyostelium discoideum amoebae were used to study the pH compartmentation and the kinetics of endosomal acidification (27) and the effect of ammonia on cytosolic and vesicular pH (28). In view of these previous results (27, 28), we have recently proposed that uncharged aminophosphonates that protonate at the amine function may show enhanced properties as 31P NMR pH probes in biological systems (29). A large series of such uncharged alpha - and beta -aminophosphonates have been tested as pH markers in standard perfusion buffers and found to give access to a large range of pKa values, with a 4-fold better NMR sensitivity than Pi and other previously reported alkyl- and aminophosphonates (29).

In the present study, the non-toxic alpha -aminophosphonate diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH), having a pKa of 6.8 at 37 °C and an extended Delta delta ab of 9.7 ppm (29), was used to probe the pH changes occurring during ischemia/reperfusion of isolated rat hearts and livers. It was observed that DEPMPH, which did not affect the cellular phosphorylated metabolite content during ischemia in both organs, allowed for the first time the simultaneous and accurate 31P NMR monitoring of extra- and intracellular pH in the normoxic and ischemic heart and liver. In the liver, use of DEPMPH as 31P NMR pH probe offered the possibility to investigate in real time the ischemia-induced pH changes associated with the acidic compartments (pH < 5.5).


    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
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Chemicals-- Ultrapure DEPMPH was obtained by as described previously (30) by using commercial 2-methylpyrroline and diethyl phosphite as starting material (Aldrich). All other reagents were of analytical grade. Doubly distilled deionized water was used throughout. All solutions were filtered through a 0.2-µm filter prior to use.

Isolated Liver and Heart Preparations-- Male Wistar rats weighing 80-100 g (liver studies) or 300-390 g (heart studies) were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg body mass), and the studied organ was immediately perfused at 37 °C in a non-recirculating mode either via the portal vein (liver studies) or the ascending aorta (heart studies). Isolated perfused hearts were instrumented to provide continuous monitoring of hemodynamic function as described below. The perfusion medium (pH 7.35) used in liver (heart) studies was a Krebs-Henseleit buffer consisting of 143 (144) mM Na+, 5.0 (5.8) mM K+, 2.0 (2.5) mM Ca2+, 1.2 (1.2) mM Mg2+, 127 (129.8) mM Cl-, 25 (25) mM HCO3-, 1.2 (0) mM H2PO4-/HPO42-, 2.0 (1.2) mM SO42-, 0 (10) mM glucose, and 0 (0.5) mM EDTA (throughout referred to as "standard" buffer). The buffer was saturated with 95% O2, 5% CO2 by using a membrane oxygenator (liver studies) or by bubbling (heart studies). Perfusion was performed at a flow rate of 3 ml·min-1·g-1 liver wet weight or at a constant pressure of 100 cm of H2O in hearts. For NMR experiments, organs were inserted into a 20-mm NMR sample tube that was placed into the wide-bore 9.4-Tesla magnet of the spectrometer.

Experimental Groups and Perfusion Protocols for NMR Experiments-- In a recent study, it was found that DEPMPH did not affect base-line cardiac function when perfused up to 10 mM in normoxic rat hearts (30). Consequently, the selected DEPMPH concentration for all perfusion experiments was 5 mM. Liver perfusion at 37 °C (n = 6) included a 20-min perfusion with plain standard buffer followed by a 20-min normoxic perfusion with DEPMPH, 60 min of ischemia, and a final 20-min reperfusion with plain buffer. Heart perfusion at 37 °C (n = 6) included a 30-min normoxic control period followed by 30 min of no-flow normothermic ischemia and a final 60-min reperfusion. In these latter experiments, DEPMPH was added to the perfusion medium during the last 15 min of control period and the first 5 min of postischemic reflow. In matched untreated groups, livers (n = 8) or hearts (n = 7) underwent perfusion with plain buffer throughout.

To test whether DEPMPH (5 mM) could be a sensitive marker of extracellular pH variations, an additional NMR experiment was carried out in which the effects of increasing the concentration of Pi or decreasing the pH of the perfusate were assessed. For this purpose, livers (n = 2) were perfused normoxically at 37 °C according to the following protocol: an initial 30-min perfusion with plain standard buffer; a 10-min perfusion with DEPMPH-supplemented standard buffer; a 10-min perfusion with a DEPMPH-supplemented buffer in which the concentration of KH2PO4 was raised to 3 mM; a 12-min perfusion with DEPMPH-, KH2PO4 (3 mM)-, and isobutyric acid (35 mM)-supplemented buffer; a 15-min perfusion with the former buffer in which isobutyric acid was omitted; and a final 20 min of perfusion with plain standard buffer. In this experiment, the liver perfusate was timely collected for pH measurement.

Perchloric Acid Extraction-- To assess whether the different NMR resonances seen in liver experiments with DEPMPH could be related to degradation products of the pyrrolidine, livers were subjected to perfusion as in the untreated and DEPMPH (5 mM) groups described above (n = 2/group), except that organs were freeze-clamped with aluminum tongs precooled in liquid nitrogen at the end of ischemia. The frozen tissue was weighed, grounded in liquid nitrogen, and homogenized with 6 volumes of 0.9 M HClO4 (w/v) at 4 °C. After centrifugation at 15,000 × g for 15 min at 4 °C, the supernatant was neutralized by 9 N KOH as described previously (31). After a second centrifugation at 3,500 × g for 10 min at 4 °C, the supernatant was lyophilized and stored in 2 aliquots at -80 °C. An aliquot was used for gas chromatography-mass spectrometry analysis (see below) after suspension into doubly distilled deionized water. Before use for NMR spectroscopy, the second aliquot was dissolved in 0.5 ml of D2O containing 50 mM EDTA, and methylene diphosphonic acid (MDPA) was added as chemical shift reference. In this latter sample, two NMR measurements were performed by consecutively adjusting its pH at 5.5 and 7.5 with KOH or HCl, respectively.

Mass Spectrometry on Liver Perchloric Acid Extracts-- Gas chromatography-mass spectrometry analysis was performed using a Varian 3400 gas chromatograph (Palo Alto, CA) coupled with a Finnigan Mat TSQ700 mass spectrometer (San Jose, CA) operating in electron impact mode and controlled by a Dec Station 5000/33. Helium was the carrier gas, and the column was a fused silicon 25-m capillary column OV1. Mass spectrometer settings were as follows: electron energy, 70 eV in the range 60-650 m/z, scan speed, 600 m/z. The column temperature was programmed from 100-280 °C (10 °C/min) with the injector temperature fixed at 220 °C.

31P NMR Spectroscopy-- 31P NMR spectra of isolated perfused organs and perchloric extracts were recorded at 161.9 MHz using a Bruker DPX 400 spectrometer (Karlsruhe, Germany) equipped with a 31P/13C dual 20-mm probe.

31P NMR on Perfused Livers-- A one-pulse sequence was used to acquire spectra in 2-min blocks corresponding to 240 free induction decays obtained using a 70° flip angle, a 0.5-s repetition time, 4K data points, and a 10-kHz spectral width. A Lorentzian line broadening of 15 Hz was applied prior to Fourier transformation. All chemical shifts were expressed with respect to the resonance from a capillary of MDPA at 18.4 ppm. The peak area of the beta -NTP resonance was measured by integrating the peak from the spectrum and by comparing the area with that of MDPA (13 µmol of 31P nuclei). Variations in beta -NTP content were expressed as percentage of the control beta -NTP level obtained during the initial perfusion period with plain buffer. pHi was estimated from the position of the intracellular Pi resonance.

31P NMR on Isolated Hearts-- A one-pulse sequence was used to acquire spectra in 5-min blocks corresponding to 140 summed free induction decays obtained using a 60° pulse, a 2.151-s interpulse delay, a 8K data table, and a 9.4-kHz spectral width. A Lorentzian line broadening of 15 Hz was applied prior to Fourier transformation. The peak areas of the beta -ATP and creatine phosphate (CrP) resonances were analyzed by both planimetry and an automatic integration procedure. Variations in beta -ATP and CrP contents were expressed as percentage of the base-line levels obtained during the initial phase of perfusion with DEPMPH-free buffer. pHi was measured from the chemical shift of the Pi peak relative to CrP (32).

31P NMR Spectra of Perchloric Acid Extracts-- 31P NMR spectra were recorded in a 5-mm tube using a reverse probe tuned at 161.9 MHz. Instrument settings were as described in the isolated liver experiments except that acquisition time was extended to improve the signal-to-noise ratio.

Titration Curves-- The aim of these studies was to compare the effects of pH on the 31P NMR chemical shift of the resonance peak of DEPMPH in three environments having the ionic strength representative of either the extracellular medium (i.e. the standard Krebs- Henseleit medium described in perfusion studies), or the protein-free, or complete cytosolic medium (i.e. aqueous 125 mM KCl or a supernatant solution of a rat heart homogenate (see below) diluted in aqueous 125 mM KCl, respectively). Each of these three solutions was supplemented with KH2PO4 (1.2 mM) and DEPMPH (5 mM) and was allowed to equilibrate at 22 °C or 37 °C. The pH was then adjusted to 30 different values in the range 4.0-10.0 with 6 N solutions of HCl or NaOH and transferred into a 10-mm NMR tube for 31P NMR analysis (Bruker AMX 400 NMR spectrometer) at its corresponding equilibration temperature. The pKa values were calculated by iterative fitting of the chemical shift delta  and pH data according to the standard Henderson-Hasselbalch Equation 1,
<UP>pH</UP>=<UP>p</UP>K<SUB>a</SUB>+<UP>log</UP> [(&dgr;−&dgr;<SUB><UP>a</UP></SUB>)/(&dgr;<SUB><UP>b</UP></SUB>−&dgr;)] (Eq. 1)
using a nonlinear regression (delta a and delta b are the protonated and unprotonated DEPMPH chemical shifts, respectively).

To assess the influence of metal ions on DEPMPH resonance chemical shift, increasing concentrations of Fe3+ (5 µM to 1 mM) or Fe2+ (1 µM to 1 mM) as sulfate salts were added to a test 5 mM DEPMPH solution adjusted to pH 7.35, and 31P NMR spectra were obtained at 37 °C.

Preparation of Rat Heart Homogenates-- Heart homogenates were obtained by a modification of the method described in Ref. 32 as follows: a total of 25 g of freshly excised rat heart was minced in 35 ml of 125 mM KCl at 2 °C, homogenized in a blender for 10 min, and centrifuged at 10,000 × g for 15 min at 2-3 °C, and the clear supernatant solution was used in titration experiments.

Measurements of Spin Lattice Relaxation Times (T1)-- 31P NMR spectra of rat livers (n = 3) perfused normoxically for 80 min with standard Krebs-Henseleit buffer containing 5 mM DEPMPH were acquired at 37 °C as described above. T1 calculation was achieved using a progressive saturation experiment consisting of a pulse sequence, (90° - Ta - tau ]nn, with an acquisition time (Ta) of 0.18 s and tau  values varying from 0.1 to 16 s. For each spectrum a total of 100 transients were acquired. A 5-Hz line broadening was applied before Fourier transformation. To allow correct T1 estimation, spectra were first deconvoluted into Lorentzian lines. Relaxation parameters were evaluated using the same non-linear least squares fitting method to the repetition time dependence of the resonance area.

Experimental Groups and Protocol for Hemodynamic Experiments in Rat Hearts-- To assess the effect of DEPMPH on cardiac function, additional untreated (n = 13) or DEPMPH (5 mM)-treated (n = 11) rat hearts were placed into a small water-jacketed chamber at a controlled temperature of 37 °C and underwent the ischemia-reperfusion protocol described above. Contractile function was assessed by means of a saline-filled latex balloon inserted into the left ventricle and connected to a pressure transducer (Gould Statham P23); the filling pressure was adjusted until left ventricular end diastolic pressure (LVEDP) was in the range 8-12 mm Hg. Throughout perfusion, LVEDP, left ventricular developed pressure (LVDP), and its first derivative with time (dp/dt), and heart rate were monitored at 5-min intervals (Gould 8000S recorder). Rate pressure product, an index of overall cardiac performance, was calculated as the product of heart rate and LVDP. Coronary flow was measured by timely collecting the coronary perfusate.

Statistical Analysis-- Metabolic and hemodynamic data were expressed as mean ± S.E. Evaluation of statistical significance was performed with two-way analysis of variance with repeated measures over the different phases of the experimental protocols. If a difference was established, the values in the DEPMPH-treated groups were compared with that of the corresponding untreated groups using the Newman-Keuls test. One-way analysis of variance was then carried out to test for any differences among the mean values of the different groups at every time, and this was followed by the Duncan test. Significance was accepted at the p < 0.05 level. When necessary, significance values were adjusted for multiple comparisons with use of the Bonferroni correction. Animal care and handling conformed to the Guide for the Care and Use of Laboratory Animals (33).


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pH Dependence of the DEPMPH 31P NMR Chemical Shift-- The variation of the 31P NMR chemical shift of DEPMPH (5 mM) was determined as a function of pH at 22 and 37 °C in the standard Krebs-Henseleit medium used for heart studies which contained 1.2 mM Pi (ionic strength 164 mM). The results in Table I show that, in this medium, the apparent pKa of DEPMPH is in the physiological range and that the sensitivity Delta delta ab of the pyrrolidine as a pH marker is about 3.6 times higher than that of Pi at both tested temperatures. These variables did not significantly vary when titrations were performed either in an aqueous solution of physiological cellular ionic strength (i.e. aqueous 125 mM KCl; ionic strength 125 mM) or in a KCl (125 mM)-supplemented rat heart homogenate taken as a complete intracellular medium (Table I). These latter data showed that pH measurement from DEPMPH 31P NMR chemical shift is rather insensitive to the release of cytosolic proteins or to changes in ionic concentrations within physiological limits. Since phosphates are good ion chelators and since ischemia/reperfusion of isolated organs has been shown to trigger the release of metal ions, including Fe2+ or Fe3+ ions (34), the effect on the chemical shift of DEPMPH of adding iron ions to the Krebs medium has been examined. Supplementing the Krebs medium with concentrations of Fe2+ or Fe3+ ions in large excess of the physiological range (i.e. 1 µM to 1 mM) at pH 7.35 had no displacing effect on the chemical shift of DEPMPH (data not shown).


                              
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Table I
Effect of milieu and temperature on pH-dependent 31P NMR parameters of DEPMPH (5 mM) and Pi (1.2 mM)
The composition of Krebs-Henseleit medium used in heart studies, the preparation of rat heart homogenates, and the protocol for titrations were described under "Materials and Methods." Values are means ± S.D. from three independent experiments. delta a, limiting chemical shift in acidic medium; delta b, limiting chemical shift in basic medium; Delta delta ab = delta a - delta b. Chemical shifts are expressed relative to 85% H3PO4 as external reference.

31P NMR Studies on the Ischemic Reperfused Liver Using DEPMPH as pH Probe-- Fig. 1 shows typical 31P NMR spectra recorded during normoxic perfusion of the same liver with plain buffer (Fig. 1A), after 20 min of normoxic perfusion with DEPMPH (5 mM)-supplemented buffer (Fig. 1B), after 25 min of ischemia (Fig. 1C), and after 20 min of reperfusion with DEPMPH-free buffer (Fig. 1D). Resonances in Fig. 1A have been assigned as previously reported (13). During normoxia, when DEPMPH was added to the perfusion medium at 5 mM, no significant alteration of the base-line metabolic variables or pHi was observed as compared with livers perfused with plain buffer (Table II). This pre-ischemic perfusion of DEPMPH had also no effect on ischemia-induced decrease of the levels of phosphorylated metabolites and pHi, and on post-ischemic recovery of metabolic variables of livers reperfused with plain buffer (Table II and Fig. 1, C and D).



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Fig. 1.   31P NMR spectra recorded during the ischemia-reperfusion of the same isolated rat liver. Spectra were acquired after 20 min of initial normoxic perfusion at 37 °C with Krebs-Henseleit buffer (A), after 20 min of normoxic perfusion with DEPMPH (5 mM)-supplemented buffer (B), after 25 min of total ischemia in the presence of DEPMPH (C), and after 20 min of reperfusion with plain buffer (D). The expanded regions show the peaks assigned to DEPMPH in extracellular (a), cytosolic (b), and acidic (c) liver compartments. Spectra were acquired as described under "Materials and Methods," and chemical shifts are given relative to external MDPA resonance. Peak assignments are as follows: PME, phosphomonoesters; GPE, glycerol 3-phosphorylethanolamine; GPC, glycerol 3-phosphorylcholine.


                              
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Table II
Effect of DEPMPH on metabolic variables in perfused rat livers
DEPMPH (5 mM) was added to the Krebs-Henseleit buffer during the last 20 min of the 40-min normoxic period. Untreated group received plain buffer throughout. The protocols for perfusion and 31P NMR experiments are described under "Materials and Methods." Data are presented as mean ± S.E. For beta -NTP, units are percent of base-line control values (set at 100%) taken after 20 min of normoxic perfusion. NM, not measurable.

Owing to the close similarity of the pH titration curves obtained in standard buffer and in rat heart homogenate-containing buffer, the pH values in all perfusion experiments were determined from the DEPMPH calibration curve in the standard buffer at 37 °C (Table I). Although the NMR spectrum of DEPMPH in the perfusate at 37 °C consisted of a single peak at 30.63 ppm, corresponding to a pH of 7.35, three resonances (termed as a, b and c) were observed in the low field portion of the NMR spectra of normoxic livers perfused in the presence of 5 mM DEPMPH, resonance peak b appearing as a shoulder of the large resonance peak a (Fig. 1B). The chemical shifts (mean ± S.E., n = 6) of these new resonances were 30.49 ± 0.14, 29.57 ± 0.16, and 24.02 ± 0.08 ppm, corresponding to pH values of 7.36 ± 0.03, 7.12 ± 0.04, and 5.65 ± 0.03 for peaks a, b, and c, respectively. The three peaks shifted to high field during ischemia as a consequence of acidosis, the shift of peaks a and c being less pronounced than that of peak b, which became fully separable from peak a (Fig. 1C). After 25 min of ischemia, the pH values derived from the chemical shifts of peaks a-c were 7.00 ± 0.03, 6.28 ± 0.05, and 5.48 ± 0.08, respectively. Given that the pH changes for peak b and Pi peak were parallel (Fig. 1), it was assumed that peak b was relative to cytosolic DEPMPH. Analysis of the time course of pH variation during the entire perfusion protocol for peaks a-c confirmed that peak b was significantly correlated to the Pi peak (r = 0.995; Fig. 2) and that it could be reasonably attributed to DEPMPH located in intracellular cytosolic medium.



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Fig. 2.   Time course of the changes in the intra- and extracellular pH during rat liver ischemia-reperfusion in the presence of DEPMPH (5 mM). pH values are calculated from the corresponding 31P NMR resonances of Pi () and the resonances a (open circle ), b (black-square), and c () of DEPMPH. For conditions of perfusion and for 31P NMR measurements, see "Materials and Methods." Each point corresponds to the mean ± S.E. (n = 6).

Since peaks a and c could be related to DEPMPH metabolites that could have been formed during normoxia and ischemia and further washed out at reperfusion (Fig. 1D), additional perfusion experiments were performed in which livers (n = 2) undergoing a normoxic perfusion with DEPMPH (5 mM)-supplemented buffer and a total ischemia were subjected to perchloric acid extraction followed by 31P NMR and gas chromatography-mass spectrometry analyses. Fig. 3 shows a typical 31P NMR spectrum of a DEPMPH-treated liver submitted to perchloric acid extraction after 60 min of ischemia and recorded at pH 5.90. This signal was similar to that obtained from an untreated ischemic liver (with a high level of Pi and dramatically decreased NTP levels), except that the supplementary resonance peak of DEPMPH was evident at 24.2 ppm. Raising the pH of the extract to 7.34 caused an expected downfield shift of the DEPMPH peak to 30.53 ppm (data not shown). Gas chromatography-mass spectrometry analysis of perchloric extracts from DEPMPH-treated livers allowed the detection of an m/z = 221 peak similar to that recorded from a DEPMPH (5 mM) solution in standard buffer. No additional peak was observed in the 31P NMR and gas chromatography-mass spectrometry spectra of extracts obtained from control livers perfused with plain buffer. Taken together, these data suggest that the three peaks a-c observed in DEPMPH-treated normoxic and ischemic livers (Fig. 1) are likely related to DEPMPH located in different extra- or intracellular environments. Interestingly, pH values during the normoxic period and at the end of ischemia, when calculated from the peak a chemical shift (Fig. 2), accurately match the mean pH values directly measured in the bath surrounding the liver during these two phases of the perfusion protocol (i.e. 7.36 ± 0.03 and 6.81 ± 0.06, respectively). These data suggest that peak a may be related to DEPMPH located in extracellular medium (i.e. the perfusate itself and the liver vascular volume), the pH of which decreased during ischemia due to a slow removing of acid equivalents from the cytosol.



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Fig. 3.   31P NMR spectrum at pH 5.90 of the perchloric acid extract from a normothermic 60-min ischemic rat liver receiving DEPMPH (5 mM). The perfusion protocol, 31P NMR spectroscopy and perchloric acid extraction are given under "Material and Methods." Peak assignments of phosphorylated metabolites are as indicated in the legend of Fig. 1 except for phosphodiesters (PDE).

A quite unexpected feature of the 31P NMR spectra of normoxic livers perfused with DEPMPH-containing buffer was the presence of the high field peak c (Fig. 1B). Based on the calibration data of Table I, this peak c was related to some DEPMPH located in an intracellular compartment having the very low pH of 5.65 ± 0.07 during normoxia. During ischemia, a slight acidosis of this acidic compartment was evidenced (Fig. 1C), the pH of which reached 5.37 ± 0.07 after 60 min of ischemia (Fig. 2).

In a further attempt to assign peak a seen in Fig. 1, B and C, to DEPMPH in the extracellular environment, a load of 35 mM isobutyric acid (pKa 4.84) was added to a DEPMPH (5 mM)- and KH2PO4 (3 mM)-supplemented Krebs buffer that was used as a perfusion medium for normoxically perfused livers. Isobutyric acid has been shown, in the presence of HCO3-, to decrease selectively the extracellular pH without significantly affecting pHi in the normoxically perfused rat liver (13). Addition of isobutyric acid led to a rapid decrease of the pH of the perfusate to 7.10, which recovered its initial pH value of 7.35 when isobutyric acid was removed from the perfusion buffer. Under these conditions, very similar profiles of pH variation were obtained whether pH was directly measured or calculated from the 31P NMR resonance peaks of either extracellular KH2PO4 or peak a (Fig. 4), confirming that peak a is related to extracellular pH. The 31P NMR resonance of peak b remained unaffected by the presence of isobutyric acid (initially at 29.92 ± 0.09 versus 29.98 ± 0.06 ppm during isobutyric acid treatment, n = 2), corresponding to a stable pH of 7.18-7.20. As expected from the data of Ref. 13, this result confirms that peak b was related to cytosolic DEPMPH. During isobutyric acid infusion, a decrease of high field peak c chemical shift was observed (initially at pH 5.60 ± 0.04 versus 5.46 ± 0.09 during isobutyric acid treatment, n = 2).



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Fig. 4.   Typical time course of the changes in intra- and extracellular pH during normoxic perfusion of an isolated rat liver in the presence of DEPMPH (5 mM), Pi (3 mM), and isobutyric acid (isoBu, 35 mM). pH values are calculated from the corresponding 31P NMR resonances of external Pi (black-triangle) and the resonances a (open circle ) and c () of DEPMPH or directly measured into the perfusion bath surrounding the liver (down-triangle). For conditions of perfusion and for 31P NMR measurements, see "Materials and Methods."

The appearance of three NMR peaks during DEPMPH perfusion in normoxic livers (Fig. 1B) allowed the calculation of individual T1 values, which were found quite different as follows: 3.94 ± 0.4, 3.5 ± 0.2, and 4.16 ± 0.3 s for peaks a, b, and c, respectively. The T1 for cytosolic Pi was 0.62 ± 0.2 s, in agreement with previously published data (35).

31P NMR and Hemodynamic Studies on Ischemic Reperfused Hearts Using DEPMPH as pH Probe-- When DEPMPH (5 mM) was added to the perfusate of isolated normoxic rat hearts (n = 7), base-line hemodynamic variables did not significantly differ from that recorded in untreated hearts (n = 12), although developed pressure was not significantly increased (Table III). Contractile activity rapidly ceased in both groups immediately following induction of ischemia, and only 2 hearts in each group showed systolic oscillations of less than 2 mm Hg in amplitude at 10 min of ischemia; after 15 min of ischemia all hearts remained asystolic. Diastolic ventricular contracture, as assessed by LVEDP measurements, progressively increased in both groups during ischemia, and no significant intergroup difference was found with respect to contracture development over the entire period of ischemia (Table III). Due to the severity of ischemia, LVEDP values peaked within the first 5 min of post-ischemic reflow in both groups, DEPMPH appearing significantly protective on the development of contracture. At the end of reperfusion, mechanical performance recovery was similar in both groups, coronary flow and diastolic pressure being significantly improved in the DEPMPH-treated group (Table III).


                              
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Table III
Effect of DEPMPH on hemodynamic variables during ischemia-reperfusion of isolated rat hearts
DEPMPH (5 mM) was added to the Krebs-Henseleit buffer during the last 15 min of equilibration period and the first 5 min of reperfusion (n = 7). Untreated hearts received buffer throughout perfusion (n = 12). The protocol for heart perfusion is described under "Materials and Methods." Data are presented as mean ± S.E. The abbreviations used are: RPP, rate pressure product; CF, coronary flow. Statistics (two-way analysis of variance followed by Student's t test).

Fig. 5 shows representative 31P NMR spectra from the same heart at the end of normoxic perfusion in presence of 5 mM DEPMPH (Fig. 5A), after 10 min of global ischemia (Fig. 5B), after 30 min of global ischemia (Fig. 5C), and after 5 min of reflow (Fig. 5D). As for hemodynamic variables, pre-ischemic application of DEPMPH did not affect metabolic variables and pHi values, when compared with untreated hearts (Table IV). DEPMPH had also no effect on ischemia-induced decrease of the levels of phosphorylated metabolites and pHi. Although DEPMPH induced no effect on post-ischemic pHi values, an improved recovery of metabolic variables was obtained (Table IV). Two resonances (termed as a and b) were observed in the low field portion of the 31P NMR spectra of normoxic hearts treated with DEPMPH (Fig. 5A). Due to the relatively low catabolic activity of the heart, it was considered that both peaks a and b were relative to DEPMPH in two different compartments having the respective pH values of 7.36 ± 0.03 and 7.12 ± 0.04 (n = 6). The two peaks shifted to high field during ischemia as a consequence of acidosis, the shift of peak a being less pronounced than that of peak b (Fig. 5, B and C). After 30 min of ischemia, the pH values derived from the chemical shifts of peaks a and b were decreased to 7.23 ± 0.03 and 6.41 ± 0.02, respectively. Again, the pH values calculated from peaks b and Pi chemical shifts were similar (Table IV). Analysis of the time course of pH variation during the entire perfusion protocol for peaks a and b confirmed that peak b was significantly correlated to the Pi peak (r = 0.997; Fig. 6) and that it could be reasonably attributed to DEPMPH internalized into the cytosol. Direct measurements of the pH of the external perfusate (data not shown) showed that peak a could be attributed to external medium, which slightly acidified during ischemia (Fig. 6). A third peak (termed as peak d in Fig. 5B) appeared as a shoulder of peak a after 10 min of ischemia, becoming fully separable from peak a when the duration of ischemia increased to 30 min, corresponding to a mean pH of 7.05 (Fig. 5C). A similar pH value was directly measured from the coronary effluent driven from the atrial cavity during ischemia. With the onset of reperfusion, peaks a, b, and d returned back to downfield (Fig. 5D and Fig. 6) and rapidly disappeared as DEPMPH perfusion stopped.



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Fig. 5.   31P NMR spectra recorded during the ischemia-reperfusion of the same isolated rat heart. Spectra were acquired after 30 min of initial normoxic perfusion at 37 °C with DEPMPH (5 mM)-supplemented Krebs-Henseleit buffer (A), after 10 min of total ischemia (B), after 30 min of total ischemia (C), and after 5 min of reperfusion with DEPMPH (5 mM)-supplemented Krebs-Henseleit buffer (D). The expanded spectra B-D show the peaks assigned to DEPMPH in extracellular (a and c) and cytosolic heart compartments. Spectra were acquired as described under "Materials and Methods," and chemical shifts are given relative to CrP. Other peak assignments are follows: phosphomonoesters (PME); Pi; and ATP.


                              
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Table IV
Effect of DEPMPH on metabolic variables in perfused rat hearts
DEPMPH (5 mM) was added to the Krebs-Henseleit buffer during the last 15 min of equilibration period and the first 5 min of reperfusion (n = 6). Untreated group received buffer throughout perfusion (n = 7). The protocols for perfusion and 31P NMR experiments are described under "Materials and Methods." Data are presented as mean ± S.E. Units are percent of base-line control values after 10 min of normoxic perfusion (set at 100%), except for pHi. The abbreviation used is: NM, not measurable. Statistics (two-way analysis of variance followed by Student's t test).



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Fig. 6.   Time course of the changes in the intra- and extracellular pH during rat heart ischemia-reperfusion in the presence of DEPMPH (5 mM). pH values are calculated from the corresponding 31P NMR resonances of Pi () and the resonances a (open circle ), b (black-square) and d (box-plus ) of DEPMPH. For conditions of perfusion and for 31P NMR measurements, see "Materials and Methods." Each point corresponds to the mean ± S.E. (n = 6).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The widespread use of 31P NMR for the noninvasive measurement of pH in biological systems suggests a need to control better the requirements that have to be met by any compound intended to be used as an improved auxiliary pH indicator. The possibility of measuring pH by 31P NMR has been first demonstrated using endogenous Pi as a probe (1-3), but although it is actually an accurate indicator of the cytosolic pH, some metabolic conditions may exist where the signal/noise ratio of cytosolic Pi can be too low to allow a good pH determination (4-6). The NMR visibility of the cytosolic Pi resonance can also be reduced by the external Pi signal arising from KH2PO4-containing buffers commonly used in perfusion experiments. The Delta delta ab value for Pi is only of 2.6 ppm, making cellular compartments not distinguishable if their pH values differ by less than 0.3 pH units (4-6). This has been for long an important limitation for the use of Pi as a probe of trans-sarcolemmal proton movements in ischemic situations. By using phenylphosphonate as an extracellular NMR probe, it was shown that the pH gradient across the sarcolemma collapses in early ischemia in isolated perfused hearts (22). Although these data (22) and other results from perfusion studies using phenylphosphonate (21, 24) or, in a lesser extent, methylphosphonate and 3-aminopropylphosphonate (24) have shown the usefulness of charged phosphonates as external pH markers, none of these molecules could be used to probe simultaneously pHi due to their weak Delta delta ab range of 2.1-3.9 ppm (27).

The data of the present study provide experimental support to our assumption (29) that using the uncharged pyrrolidine DEPMPH as 31P NMR pH marker in the perfused heart and liver, simultaneous information on ischemia-induced or chemically induced weak intra- and extracellular pH modifications can be directly obtained which previously escaped from investigation with other charged alkyl- and aminophosphonates. This outstanding property of DEPMPH clearly appears as a conjunction of all the basic requirements that have to be fulfilled by a potential NMR pH probe (5). Table I shows that the pKa value of DEPMPH is in the physiological range and exhibited a low sensitivity to changes in composition and ionic strength in several biologically relevant milieu. In standard Krebs buffer, a satisfactory correlation between the experimental pKa values and the structure (i.e. the nature of substituents linked to the nitrogen atom) has been obtained for a wide series of cyclic alpha -aminophosphonates such as DEPMPH (29), suggesting that this pyrrolidine has an ideal structure for applications in biological studies at a pKa close to that of Pi. The use of DEPMPH as an NMR pH probe also takes advantage of the low variation of the pKa with ionic strength for compounds having a non-dissociable protonation site such as the amino group of pyrrolidines (29). Another important feature of DEPMPH is that it did not affect the main metabolic parameters such as ATP and pHi of hearts and livers during normoxic perfusion and ischemia (Tables II-IV) when added to the perfusate at a concentration (5 mM) that permitted a good NMR detection. As pHi profiles were not modified by the presence of DEPMPH during heart and liver ischemia, the DEPMPH resonance peak b can therefore be considered as a reliable index for intracellular pH monitoring in these organs (Figs. 1 and 5). In agreement with other heart studies on phosphorylated compounds (30, 36, 37), a slight cardioprotection was exerted by DEPMPH on reperfused hearts (Table III), whereas this aspect has not been investigated in detail in previous studies on alkylphosphonate NMR probes (21-24). As a tentative explanation for the cardioprotective effect of DEPMPH on the reperfused heart, it has been hypothesized that pro-oxidant transition metal ions and/or Ca2+ released from the post-ischemic heart may have been inactivated by chelation on the diethoxyphosphoryl group of the pyrrolidine (30). The data from Figs. 1 and 5 show that DEPMPH, even perfused at the lowest concentration (5 mM) among those used in previous NMR studies with charged alkylphosphonates (21-24), reasonably penetrates heart and liver tissue, distributing into extra- and intracellular compartments to provide a satisfactory NMR detection. In cell studies where aminoalkylphosphonates have been used as 31P NMR pH probes, it was found that compounds having a low dipole moment (i.e. aminomethylphosphonate and 2-aminoethylphosphonate), by easily entering the cell, behave as intra- and extracellular pH reporters (27, 28), whereas 3-aminopropylphosphonate, which is more polar, is restricted to the extracellular compartment (38, 39). In recent 31P NMR study on rat isolated livers, 2-aminoethylphosphonate was found to penetrate the cytosol, but although its pKa was of 6.45, it did not provided simultaneous information on the acidic and extracellular compartments (40). In light of these results (27, 28, 38-40), it is therefore likely that the greatest ability of DEPMPH to allow simultaneous NMR investigation of extra- and intracellular compartments results from the low polarity of its diethoxyphosphoryl moiety. This assumption appears in accord with the results of mass spectrometry studies on liver perchloric extracts (Fig. 3) showing that DEPMPH was internalized into several intracellular compartments, with no significant metabolism or degradation. In agreement was the finding, in normoxic livers perfused with DEPMPH, of three different T1 values having the same order of magnitude as those previously reported for charged phosphonates in heart or liver perfusion (23, 24) and which are short enough to render this pyrrolidine suitable for use in in vivo NMR experiments.

Because of the very important Delta delta ab value of 9.6 ppm observed with DEPMPH between pH 4 and 10 (Table I), probing pH using 31P NMR on intact organs perfused with this pyrrolidine appears considerably easier than with other alkyl- and aminophosphonates since several peaks were clearly separated in the normoxic and ischemic liver and heart (Figs. 1 and 5) under conditions where phenylphosphonate yielded only one peak (22-24). Moreover, due to the large pH window of 5.0-8.5 where the DEPMPH titration curve is steeper (Table I), a very accurate investigation of acidic compartments can be considered, and it was one major aim of the present work to use DEPMPH to study acidic compartments having pH values below 6.

During the last recent years, much work has been performed on the characterization of the pHi-regulatory systems in hepatocytes (25, 41). At plasma membrane sites, the Na+-HCO3 - symport was found to play a major role in maintaining hepatocytic pH homeostasis over a large pH range (7.5-6.5), whereas the Na+-H+ antiport becomes preferentially activated during systemic acidosis (13, 42, 43). Moreover, the presence of acidic cytoplasmic organelles, including endocytic and secretory vesicles, lysosomes, and portions of the trans-Golgi complex and of the endoplasmic reticulum, has also been demonstrated (25, 26). In hepatocytes, acidic organelles were found to contribute to pHi regulation via active vacuolar ATP-driven proton pumps, which induced pHi acidification as follows: (i) ATP depletion due to exposure to metabolic inhibitors, (ii) hypotonic stress in K+-free buffer that resulted in proton leakage from acidic compartments without significantly affecting cellular ATP content (44). In both situations, a concomitant alkalinization of cytosolic vesicles resulting in a near equilibration of cytosolic and vesicular pH was evidenced (44). However, since (a) the characterization of acidic compartments in the liver has exclusively derived from studies on cellular systems exposed to external agents that either stimulated or inhibited the activity of vacuolar ATP-driven proton pumps, and (b) the analytical method used (i.e. fluorescence) did not allow the simultaneous study of all cytosolic and organellar compartments (44, 45), the pertinence of the conclusions to the situation encountered in the whole organ may be questioned.

The present study provides the first direct evidence of the involvement of acidic compartments in pHi regulation during ischemia and reperfusion of the intact liver. If the DEPMPH resonance peak c in the different hepatic cytosolic organelles cannot be precisely assigned, it however corresponds to a pH range of 5.2-5.6 (Fig. 2) similar to that attributed to acidic vesicles (44-46). During the normoxic period, the pH determined from the peak c resonance remained stable, and thus a pH gradient of 1.47 pH units between acidic vesicles and cytosol could be determined. Upon ischemia, the cytosol and the acidic vesicles acidified by about 0.9 and 0.3 pH units, respectively, leading to a decrease of the pH gradient reaching a minimum at 0.70-0.80 pH units from 35 to 60 min of ischemia (Fig. 2). This demonstrates that, in liver ischemia, acidic vesicles participate in pHi regulation possibly by removing protons from the cytosol, an opposite mechanism to that reported in the case of chemical hypoxia of hepatocytes where protons are released from acidic vesicles to the cytosol (44). The exact stimuli (i.e. decrease in cytosolic pH or in ATP) responsible for this acidification of liver vesicles during ischemia remains to be determined since the membranes of the acidic vesicles are highly permeable to protons (25) and vacuolar ATP-driven proton pumps are sensitive to ATP changes (44).

Another interesting finding of the present study was that liver vesicles acidified upon exposure to isobutyric acid (Fig. 4). This acid-load technique, which results in a selective acidification of the extracellular compartment by the freely membrane-permeable isobutyric acid, has been developed to study the mechanisms of proton transport from the cytosol (47). In a recent study using this technique in isolated rat livers, the role of the Na+-HCO3 - symport has been found predominant in maintaining cytosolic pH homeostasis (13). The present data suggest that vacuolar proton pumps could also actively remove protons from the cytosol during acid load, therefore explaining, at least in part, the maintenance of the constant pHi observed in Ref. 13.

In perfused hearts and livers, a peak a assigned to DEPMPH located in the extracellular environment (i.e. the interstitial and vascular fluids, and the bath surrounding the organ) was observed (Figs. 1 and 5). When ischemia was induced, the environment directly surrounding the liver and the heart acidified (Figs. 2 and 6), indicating, as expected, the occurrence of mechanisms that actively remove protons from the cytosol to the extracellular space. In heart experiments, however, another interesting property of DEPMPH as NMR pH probe was to permit the monitoring of pH changes within the ventricular and atrial cavities using resonance peak d (Fig. 6).

In conclusion, DEPMPH was found to be an outstanding 31P NMR pH marker in two ex vivo models of ischemia and reperfusion, particularly able to specifically allow intracellular pH monitoring that was not possible with any of all previously proposed phosphonate pH markers. In liver perfusion experiments, the use of DEPMPH has provided several new insights into potential mechanisms of pH regulation during ischemia involving acidic vesicles. It is therefore reasonable to speculate that DEPMPH will be a tool of considerable interest in further studies on pH gradient changes between vesicles and cytosol aiming at a better understanding of pH homeostasis in various ischemic- or non-ischemic-induced physiopathological situations.


    FOOTNOTES

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

§ To whom correspondence should be addressed: SREP-CNRS UMR 6517 (Case 521), Université de Provence, Avenue Escadrille Normandie Niemen, F-13397 Marseille Cedex 20, France. Tel.: 33 4 91 28 85 79; Fax: 33 4 91 98 85 12; E-mail: pietri@srepir1.univ-mrs.fr.

Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M008023200


    ABBREVIATIONS

The abbreviations used are: pHi, intracellular pH; DEPMPH, diethyl(2-methylpyrrolidin-2-yl)phosphonate; delta a, limiting chemical shift in acidic medium; delta b, limiting chemical shift in basic medium; MDPA, methylene diphosphonic acid; CrP, creatine phosphate; LVEDP, left ventricular end diastolic pressure; LVDP, left ventricular developed pressure.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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