505
Biochimica et Biophysica Acta, 436 (1976) 505--511 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 71256
MODULATION OF 2,3-DIPHOSPHOGLYCERATE 31P-NMR RESONANCE POSITIONS BY RED CELL MEMBRANE SHAPE
E.T. FOSSEL and A.K. SOLOMON
Biophysical Laboratory and Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02115 (U.S.A.) (Received March 19th, 1976)
Summary Na ÷ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading t o a 25-fold increase in apparent Na ÷ flux; the effect is dependent u p o n metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 3~p-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 raM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength~ diamagnetic susceptibility or small changes in the Mg:+/2,3-diphospho glycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest t h a t 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent u p o n the physical condition of the dog red cell membrane.
2,3-Diphosphoglycerate is present in red cells in an unusually high concentration and can readily be observed by 3~P-nuclear magnetic resonance in intact red cells as shown by Moon and l~ichards [1] and Henderson et al.[2] for rabbit and h u m a n red cells. 2,3-Diphosphoglycerate is also present at 5.5 mM concentration in dog red cells which exhibit an interesting anomaly in Na ÷ transport t h a t has been linked to phosphoglycerate metabolism by Elford and Solomon [3]. Na ÷ transport in dog red cells depends markedly on the volume of the cell, a 20% decrease in cell volume leading to a 25-fold increase in apparent Na ÷ flux across the membrane [3,4]. Elford and Solomon [3] have suggested t h a t the volume effect is mediated by the concentration of 3-phosphoglycerate, since agents which increased the concentration of this substrate decrease the dependence of Na ÷ flux on cell volume. This raised a general question: does the state of the dog red cell membrane modulate the interaction of phosphorylated metabolic intermediates with membrane-bound
506 enzymes? Consequently, we have studied the effect of dog red cell volume on the ~1P-NMR spectrum of 2,3-diphosphoglycerate, a direct precursor of 3-phosphoglycerate. Our results indicate an interaction between 2,3-diphosphoglycerate and an u n k n o w n site in the red cell that is dependent upon the physical state of the membrane and is not related to the 2,3-diphosphoglycerate interaction with hemoglobin. The NMR experiments were carried out on a Varian XL-100-15 Fourier transform spectrometer operating at 40.5 MHz for 31p observation. The 16 K Varian 620 computer is interfaced with a Computer Operations tape deck for storage of d a t a . 90 ° rf pulses of 32 ps were routinely used. Acquisition times of 0.3--0:5 s allowed accumulation of 120--200 transients per min; 1000--10 000 transients were required for observation of 2,3-diphosphoglycerate. Digital filtering with time constants of 0.075--0.2 s was applied to the accumulated free induction decay to improve signal-to-noise ratios. Broad band proton decoupling (1500 Hz band width; 3 W) was used. The spectrometer was locked on the deuterium signal of 2 H: O using pulsed lock mode. Samples were observed non-spinning. They contained red cells at 50% hematocrit suspended in buffer of the following composition (mM):150, NaCl/5, KC1/17, T~i~/20% :H2 O, modified by the addition of 200 mM sucrose to shrink the cells, unless otherwise specified. Dog red cells were normally observed within 12 h of their removal from the animals. Both of the 2,3-diphosphoglycerate NMR resonances change position with red cell volume as shown in Fig. 1, in which the resonances are identified according to Moon and Richards [ 1]. The separation we have observed between the two resonances in dog red cells is 32 Hz in reasonable agreement with the 28 Hz found in human red cells by Henderson et al. [2]. The left hand portion of the figure shows the results when sucrose is added to the isosmolar medium. As the cells shrink the resonances move downfield from a phosphoric acid external standard. The right hand portion of the figure shows a similar change in resonance position when the cells are shrunken by the addition of choline chloride or swollen by the removal of NaC1 from the medium. Results for cell swelling with NaC1 were essentially similar to those with choline chloride as are the sucrose ones w~henconverted :to relative cell volume. Thus, the change in resonance positions is independent of the nature of the solute causing the change of cell volume. Heustis and Raftery [5] have shown that binding of 2,3-diphosphoglycerate to hemoglobin produces a change in resonance position. Thus, it was first necessary to see whether the results in Fig. 1 could be attributed to an interaction of 2,3-diphosphoglycerate with hemoglobin. Since 2,3-diphosphoglycerate binding to the oxygenated and carbon m o n o x y g e n a t e d forms of hemoglobin is minimal, studies were made of the effect of oxygen and carbon monoxide. When additional oxygen was passed through the red cell suspension prior to measurement, the resonance of phosphate-3 was shifted upfield by 1.6 Hz and the resonance of phosphate-2 was shifted upfield by 0.8 Hz. When the red cell was treated with carbon monoxide prior to measurement, the resonances shifted upfield by 0.2 and 0.4 Hz respectively for phosphates-2 and -3 in normal cells and 0.3 and 0.2 Hz for cells shrunken by the addition of 200 mM sucrose. Since these shifts are small compared with those shown
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Fig.1. Shift in ~IP-NMR p e a k s o f 2 , 3 - d i p h o s p h o g l y c e r a t e ( D P G ) w i t h d o g red cell v o l u m e . T h e l e f t h a n d s e c t i o n s h o w s t h e e f f e c t o f a d d i n g s u c r o s e t o a m e d i u m i s o s m o l a r w i t h d o g b l o o d (in o n e e x p e r i m e n t , t y p i c a l o f s e v e n ) and the right hand s e c t i o n s h o w s t h e r e s o n a n c e shifts as a f u n c t i o n o f relative cell v o l u m e . T h e cells w e r e s u s p e n d e d in b u f f e r a n d e i t h e r NaCI w a s r e m o v e d t o sv~ell t h e cells, or c h o l i n e c h l o r i d e w a s a d d e d t o shrink t h e cells.
in Fig. 1, it is co n c l uded t hat t he effect we observe does n o t reflect t he interaction with hemoglobin. F u r t h e r m o r e , Heustis and R a f t e r y [5] have shown t h a t one resonance shifts almost twice as m u c h as the o t h e r when 2,3-diphosphoglycerate is b o u n d to hemoglobin. T o the contrary, changes in cell volume cause b o t h resonances to shift by a b o u t the same a m o u n t , as shown in the difference curve at t he b o t t o m o f the sucrose data in Fig. 1. Moon and Richards [ 1 ] have shown t h a t a pH shift from pH 6.5 t o 7.5 causes a shift o f a b o u t 5--10 Hz in the difference b e t w e e n the 2-phosphate and th e 3-phosphate resonances in carbon m o n o x i d e - t r e a t e d rabbit red cell hemolysates. Our experiments were carried o u t on whole red cells f r o m a d if f er en t species, and we have t h e r e f o r e measured the effect of pH directly. For this purpose the buffer pH was shifted by a b o u t one unit, f r o m pH 7.7 to 6.8. D u h m [6] has shown t hat under normal conditions t he cellular pH in h u m a n red cells tracks t he extracellular pH t o within 0.1--0.2 pH units. Since th e D o n n a n equilibrium for C1- in the dog is the same as in man, an external pH change o f 1.1 units should p r o d u c e an intracellular change of a b o u t 1 unit. This pH change caused t he 2-phosphate resonance t o shift by 0.5 Hz for normal dog red cells and 0.1 Hz f or cells shrunken with 200 mM sucrose. For the 3-phosphate resonance the shifts were 0.1 and 0.3 Hz. Thus, the chemical shifts in Fig. 1 m a y n o t be at t r i b ut ed to changes in red cell pH. When th e dog red cell shrinks or swells, t he c o n c e n t r a t i o n of each of the i m p e r m e a n t solutes also changes; some or all of these c o n c e n t r a t i o n changes could be responsible for the chemical shift. T h e cell water volume is
508 markedly different at the two extremes in Fig. 1. Addition of 200 mM sucrose causes the red cell volume to decrease to 77% of control (400 mM, 67%) and the cell water volume to decrease to 67% of control (400 mM, 53%}. A control experiment was carried out to study the effect of ionic strength by examining how KC1 concentration affects the-2,3-diphosphoglycerate 31P-NMR spectrum in free solution. 2,3-Diphosphoglycerate was dissolved in a buffer of the following composition: 5 mM NaC1/25 mM Tris/20% 2H2 0 plus KC1 which varied from 100 to 300 mM. This corresponds to a cell volume excursion from 124 to 72% of normal. The pH was adjusted to 7.4 for each sample. The change in the KC1 concentration from 100 to 250 mM shifted the 3-phosphate resonance by 1.1 Hz; raising the KCI concentration to 300 mM caused an additional shift of 1.1 Hz. Since these shifts are much smaller than the 8--10 Hz shifts in Fig. 1 over the same volume range, it is unlikely that the cell-shrinking effect may be attributed to changes in cellular ionic strength. This conclusion was extended in experiments in which the cell membranes in a 2 ml sample of packed red cells were disrupted with 0.5% Triton X-100. Normal buffer was then added in steps of 0.5 ml up to a total of 2.5 ml. This produced small shifts* in the resonances in the direction opposite to that observed when whole cells swell. Between the first and last addition, the 2-phosphate resonance shifted by 2 Hz and the 3-phosphate resonance by 1.5 Hz. It was also possible that changes in diamagnetic susceptibility could contribute significantly to the effect we have observed. To test this, cells were incubated for 3 h with 25 mM phosphate {plus NaC1) to increase intracellular phosphate. After washing to remove extracellular phosphate, the cells were shrunken with 200, 300 and 400 mosM sucrose. The phosphate resonance shifted only slightly with cell volume; the total excursion between control and 400 mosM sucrose amounted* to 2 Hz. Since this is small compared with the shifts in Fig. 1, the resonance shift produced by cell swelling may not be attributed to changes in diamagnetic susceptibility. The effect of Mg2÷ on the 2,3-diphosphoglycerate spectrum was also measured in free solution. Since both Mg2÷ and 2,3-diphosphoglycerate permeate the red cell membrane slowly, if at all, changes in cell volume should not affect the concentration ratio of these two constituents. For this reason the Mg 2÷ concentration was altered by + 20% for the control experiment. The control solution consisted of: 5 mM MgC12/90 mM NaC1/ 10 mM KCI/5 mM 2,3-diphosphoglycerate/100 mM Tris, pH 7.4/20% ~H20. Reducing the Mg2+ concentration to 4 mM shifted the 3-phosphate resonance upfield by 0.2 Hz and increasing it to 6 mM shifted it 0.2 Hz in the same direction. Hence it seems unlikely that the effect of cell shrinking on the spectrum may be attributed to a change in the Mg2+/2,3-diphosphoglycerate concentration ratio in the intracellular free water volume. The concentration of all the cell solutes varies in a similar manner and it is not possible to make direct measurements such as those above for each solute. Instead, we have altered the physical state of the red cell membrane *These spectra were measured on a JEOL, FX 60 NMR spectrometer (operating at 24.15 MHz for ~P) and the observed shifts have been multiplied by 100/60 to make them comparable with the measurements on the Varian XL-100-15 machine.
509 +5C CHLORPROMAZINE
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~_ +1(
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~6 x w
i
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I
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2x10-6
I
6xlO-6
I
2xlO-5
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6xlO-5
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2xlO 4
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6xlO-4
CHLORPROMAZINE (M)
Fig. 2. Shift in 31P-NMR r e s o n a n c e o f t h e 2 - p h o s p h a t e o f 2 , 3 - d i p h o s p h o g l y c e r a t e w i t h increasing a m o u n t s o f c h l o r p r o m a z i n e in o n e e x p e r i m e n t t y p i c a l o f t w o . T h e b o t t o m c u r v e s h o w s m e m b r a n e a r e a r e d r a w n f r o m S e e m a n et al. [ 7 ] .
b y an o th er mechanism, Seeman and his colleagues have studied the effects o f a wide variety of anesthetics on red cell properties. In the course of these studies t h e y have shown [7] t hat the dissolution of anesthetics in the red cell m e m b r a n e f r e q u e n t l y casuses an increase in m e m b r a n e area. In t he case o f c h l o r p r o m a z i n e the h u m a n red cell m e m b r a n e area passes t h r o u g h a m a x i m u m at a b o u t 6% expansion, as shown in the b o t t o m section o f Fig. 2. The t o p p o r t i o n of Fig. 2 shows t ha t t he addition of chl orprom azi ne also caused t h e 2-phosphate resonance to shift t h r o u g h a m axi m um , analogous t o t h e ef f ect on m e m b r a n e area. Since t he area data were obtained on h u m a n red cells and th e NMR data on dog red cells, t he difference in m a x i m a is p ro bab ly n o t significant, particularly because the red cell m e m b r a n e lipids in the t w o species differ appreciably [ 8 ] . Fig. 2 shows t hat t he 2-phosphate resonance in the shrunken red cells also shifts t h r o u g h a similar m axi m um , even f u r t h e r displaced f r o m t he m a x i m u m in m e m b r a n e area. A similar b u t smaller shift was observed in the 3-phosphate resonance. To make sure t hat the shifts in Fig. 2 were of a general nature rather t h a n specific to chlorpromazine, similar experiments were carried o u t with a n o t h er anesthetic, 1-pentanol, whose effect on red cell area had also been measured b y Seeman et al. T he results o f these experiments, shown in Fig. 3, are similar to those with c h l o r p r o m a z i n e for normal cells t h o u g h t here are differences in th e behavior of shrunken cells. Th e measurements o f cell area were made on spherical h u m a n red cell ghosts so t h a t t h e cell volume also increases with t he area. In the case of the biconcave disc which t he nor m a l red cell approximates, Jacobs [9] has
510
1-PENTANOL o shrunken
x °---°-
/0~
=
f3~
~F37
uJ 136 o_
r3~ i x ! O -2
/ I
L
1
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4xlO 2
6xlO 2
~___
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Fig,3. Shift in 31P-NMR resonance of t h e 2 - p h o s p h a t e o f 2 , 3 - d i p h o s p h o g l y c e r a t e w i t h increasing a m o u n t s o f 1 - p e n t a n o l in o n e e x p e r i m e n t ~ typical of two. The b o t t o m curve s h o w s m e m b r a n e area r e d r a w n f r o m S e e m a n et al. [7].
shown that red cell area is independent of cell volume in the range between the biconcave disc and the sphere and Sha'afi et al. [10] have argued t h a t this also applies when cells are shrunken. Thus we should not expect that the changes in membrane area produced in the biconcave disc by chlorpromazine and 1-pentanol would necessarily be accompanied by any significant change in volume in the range around the normal volume. Furthermore, Weed and Chailley [11] have pointed out that chlorpromazine causes cups to form in human red cell membranes, whereas alcohols such as ethanol and butanol cause spikes to form. With low drug concentrations, the surface modifications of either kind do not produce a change in volume. Hence the changes in resonance positions in Figs. 2 and 3 appear to be due to physical changes in the cell membrane rather than being mediated through changes in cell volume. The results with the anesthetics, taken together with those in which red cell volume was changed by osmotic pressure, lead us to conclude that there is an interaction between 2,3-diphosphoglycerate and a binding site that is dependent upon the physical condition of the dog red cell membrane. This finding is of particular relevance to cation transport since in this species similar alterations in osmotic pressure cause profound changes in Na ÷ transport which appear to be closely related to phosphoglycerate metabolism. This p r o j e c t has b e e n s u p p o r t e d in part by the I n s t i t u t e of General Medical Sciences, National I n s t i t u t e s o f Health, U.S.A., t h r o u g h grant 5 RO1 G M 1 5 6 9 2 - 0 8 and also in part by United States Public Health Service grant AM 07300. We a c k n o w l e d g e w i t h t h a n k s the use o f the Varian XL-100-15 NMR i n s t r u m e n t ( p r o v i d e d by the National Science F o u n d a t i o n u n d e r grant GP-32317) at t h e D e p a r t m e n t o f Chemistry. O n e o f us ( E . T . F . ) t h a n k s the Muscular D y s t r o p h y A s s o c i a t i o n s o f A m e r i c a for a fellowship. We s h o u l d like
511
t o e x p r e s s o u r t h a n k s t o Dr. J. H e r z f e l d f o r s t i m u l a t i n g d i s c u s s i o n , t o M i c h a e l R. T o o n a n d M u r r a y Welt f o r t h e i r t e c h n i c a l a s s i s t a n c e a n d t o Drs. E. K i r k a n d P e t e r A. M a r o k o f o r g e n e r o u s l y m a k i n g s u p p l i e s o f d o g b l o o d available.
References 1 2 3 4 5 6 7 8 9 10 11
Moon, R.B. and Richards, J.H. (1973) J. Biol. Chem. 248, 7276--7278 Henderson, T.O., Costello, A.J.R. and Omachi, A. (1974) Proc. Natl. Acad, Sci. U.S. 71, 2 4 8 7 - 2 4 9 0 Elford, B.C. and Solomon, A.K. (1974) Biochim. Biophys. Acta 373, 253--264 Parker, J.C. and Hoffman, J.F. (1964) Fed. Proc. 24, 589 Heustis, W.H. and Raftery, M.A. (1972) Biochem. Biophys. Res. Commun. 49, 428--433 Duhm, J. (1972) Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (It~rth, M. and Astrup, P., eds.) pp. 583--594, Munksgaard, Copenhagen and Academic Press, New York Seeman, P., Kwant, W.O. and Sauks, T. (1969) Biochim. Biophys. Acta 183, 499--511 Rouser, G., Nelson, G.J., Fleisher, S. and Simon, G. (1968) Biological Membranes, Physical Fact and Function (Chapman, D., ed.), pp. 5--69, Academic Press, London Jacohs, M.H. (1932) Biol. Bull 62, 178--194 Sha'afi, R.I., Rich, G.T., Sidel, V.W., Bossert, W. and Solomon, A.K. (1967) J. Gen. Physiol. 50, 1377--1399 Weed, R.I. and Chailley, B. (1973) in Red Cell Shape, Physiology, Pathology, Ultrastructure (Bessis, M., Weed, R.I. and LeBlond, P.F., eds.), pp. 55--68, Springer Verlag, New York
Biochimica et Biophysica Acta, 436 (1976) 505--511 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA Report BBA 71256
MODULATION OF 2,3-DIPHOSPHOGLYCERATE 31P-NMR RESONANCE POSITIONS BY RED CELL MEMBRANE SHAPE
E.T. FOSSEL and A.K. SOLOMON
Biophysical Laboratory and Department of Biological Chemistry, Harvard Medical School, Boston, Mass. 02115 (U.S.A.) (Received March 19th, 1976)
Summary Na ÷ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading t o a 25-fold increase in apparent Na ÷ flux; the effect is dependent u p o n metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 3~p-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 raM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength~ diamagnetic susceptibility or small changes in the Mg:+/2,3-diphospho glycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest t h a t 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent u p o n the physical condition of the dog red cell membrane.
2,3-Diphosphoglycerate is present in red cells in an unusually high concentration and can readily be observed by 3~P-nuclear magnetic resonance in intact red cells as shown by Moon and l~ichards [1] and Henderson et al.[2] for rabbit and h u m a n red cells. 2,3-Diphosphoglycerate is also present at 5.5 mM concentration in dog red cells which exhibit an interesting anomaly in Na ÷ transport t h a t has been linked to phosphoglycerate metabolism by Elford and Solomon [3]. Na ÷ transport in dog red cells depends markedly on the volume of the cell, a 20% decrease in cell volume leading to a 25-fold increase in apparent Na ÷ flux across the membrane [3,4]. Elford and Solomon [3] have suggested t h a t the volume effect is mediated by the concentration of 3-phosphoglycerate, since agents which increased the concentration of this substrate decrease the dependence of Na ÷ flux on cell volume. This raised a general question: does the state of the dog red cell membrane modulate the interaction of phosphorylated metabolic intermediates with membrane-bound
506 enzymes? Consequently, we have studied the effect of dog red cell volume on the ~1P-NMR spectrum of 2,3-diphosphoglycerate, a direct precursor of 3-phosphoglycerate. Our results indicate an interaction between 2,3-diphosphoglycerate and an u n k n o w n site in the red cell that is dependent upon the physical state of the membrane and is not related to the 2,3-diphosphoglycerate interaction with hemoglobin. The NMR experiments were carried out on a Varian XL-100-15 Fourier transform spectrometer operating at 40.5 MHz for 31p observation. The 16 K Varian 620 computer is interfaced with a Computer Operations tape deck for storage of d a t a . 90 ° rf pulses of 32 ps were routinely used. Acquisition times of 0.3--0:5 s allowed accumulation of 120--200 transients per min; 1000--10 000 transients were required for observation of 2,3-diphosphoglycerate. Digital filtering with time constants of 0.075--0.2 s was applied to the accumulated free induction decay to improve signal-to-noise ratios. Broad band proton decoupling (1500 Hz band width; 3 W) was used. The spectrometer was locked on the deuterium signal of 2 H: O using pulsed lock mode. Samples were observed non-spinning. They contained red cells at 50% hematocrit suspended in buffer of the following composition (mM):150, NaCl/5, KC1/17, T~i~/20% :H2 O, modified by the addition of 200 mM sucrose to shrink the cells, unless otherwise specified. Dog red cells were normally observed within 12 h of their removal from the animals. Both of the 2,3-diphosphoglycerate NMR resonances change position with red cell volume as shown in Fig. 1, in which the resonances are identified according to Moon and Richards [ 1]. The separation we have observed between the two resonances in dog red cells is 32 Hz in reasonable agreement with the 28 Hz found in human red cells by Henderson et al. [2]. The left hand portion of the figure shows the results when sucrose is added to the isosmolar medium. As the cells shrink the resonances move downfield from a phosphoric acid external standard. The right hand portion of the figure shows a similar change in resonance position when the cells are shrunken by the addition of choline chloride or swollen by the removal of NaC1 from the medium. Results for cell swelling with NaC1 were essentially similar to those with choline chloride as are the sucrose ones w~henconverted :to relative cell volume. Thus, the change in resonance positions is independent of the nature of the solute causing the change of cell volume. Heustis and Raftery [5] have shown that binding of 2,3-diphosphoglycerate to hemoglobin produces a change in resonance position. Thus, it was first necessary to see whether the results in Fig. 1 could be attributed to an interaction of 2,3-diphosphoglycerate with hemoglobin. Since 2,3-diphosphoglycerate binding to the oxygenated and carbon m o n o x y g e n a t e d forms of hemoglobin is minimal, studies were made of the effect of oxygen and carbon monoxide. When additional oxygen was passed through the red cell suspension prior to measurement, the resonance of phosphate-3 was shifted upfield by 1.6 Hz and the resonance of phosphate-2 was shifted upfield by 0.8 Hz. When the red cell was treated with carbon monoxide prior to measurement, the resonances shifted upfield by 0.2 and 0.4 Hz respectively for phosphates-2 and -3 in normal cells and 0.3 and 0.2 Hz for cells shrunken by the addition of 200 mM sucrose. Since these shifts are small compared with those shown
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Fig.1. Shift in ~IP-NMR p e a k s o f 2 , 3 - d i p h o s p h o g l y c e r a t e ( D P G ) w i t h d o g red cell v o l u m e . T h e l e f t h a n d s e c t i o n s h o w s t h e e f f e c t o f a d d i n g s u c r o s e t o a m e d i u m i s o s m o l a r w i t h d o g b l o o d (in o n e e x p e r i m e n t , t y p i c a l o f s e v e n ) and the right hand s e c t i o n s h o w s t h e r e s o n a n c e shifts as a f u n c t i o n o f relative cell v o l u m e . T h e cells w e r e s u s p e n d e d in b u f f e r a n d e i t h e r NaCI w a s r e m o v e d t o sv~ell t h e cells, or c h o l i n e c h l o r i d e w a s a d d e d t o shrink t h e cells.
in Fig. 1, it is co n c l uded t hat t he effect we observe does n o t reflect t he interaction with hemoglobin. F u r t h e r m o r e , Heustis and R a f t e r y [5] have shown t h a t one resonance shifts almost twice as m u c h as the o t h e r when 2,3-diphosphoglycerate is b o u n d to hemoglobin. T o the contrary, changes in cell volume cause b o t h resonances to shift by a b o u t the same a m o u n t , as shown in the difference curve at t he b o t t o m o f the sucrose data in Fig. 1. Moon and Richards [ 1 ] have shown t h a t a pH shift from pH 6.5 t o 7.5 causes a shift o f a b o u t 5--10 Hz in the difference b e t w e e n the 2-phosphate and th e 3-phosphate resonances in carbon m o n o x i d e - t r e a t e d rabbit red cell hemolysates. Our experiments were carried o u t on whole red cells f r o m a d if f er en t species, and we have t h e r e f o r e measured the effect of pH directly. For this purpose the buffer pH was shifted by a b o u t one unit, f r o m pH 7.7 to 6.8. D u h m [6] has shown t hat under normal conditions t he cellular pH in h u m a n red cells tracks t he extracellular pH t o within 0.1--0.2 pH units. Since th e D o n n a n equilibrium for C1- in the dog is the same as in man, an external pH change o f 1.1 units should p r o d u c e an intracellular change of a b o u t 1 unit. This pH change caused t he 2-phosphate resonance t o shift by 0.5 Hz for normal dog red cells and 0.1 Hz f or cells shrunken with 200 mM sucrose. For the 3-phosphate resonance the shifts were 0.1 and 0.3 Hz. Thus, the chemical shifts in Fig. 1 m a y n o t be at t r i b ut ed to changes in red cell pH. When th e dog red cell shrinks or swells, t he c o n c e n t r a t i o n of each of the i m p e r m e a n t solutes also changes; some or all of these c o n c e n t r a t i o n changes could be responsible for the chemical shift. T h e cell water volume is
508 markedly different at the two extremes in Fig. 1. Addition of 200 mM sucrose causes the red cell volume to decrease to 77% of control (400 mM, 67%) and the cell water volume to decrease to 67% of control (400 mM, 53%}. A control experiment was carried out to study the effect of ionic strength by examining how KC1 concentration affects the-2,3-diphosphoglycerate 31P-NMR spectrum in free solution. 2,3-Diphosphoglycerate was dissolved in a buffer of the following composition: 5 mM NaC1/25 mM Tris/20% 2H2 0 plus KC1 which varied from 100 to 300 mM. This corresponds to a cell volume excursion from 124 to 72% of normal. The pH was adjusted to 7.4 for each sample. The change in the KC1 concentration from 100 to 250 mM shifted the 3-phosphate resonance by 1.1 Hz; raising the KCI concentration to 300 mM caused an additional shift of 1.1 Hz. Since these shifts are much smaller than the 8--10 Hz shifts in Fig. 1 over the same volume range, it is unlikely that the cell-shrinking effect may be attributed to changes in cellular ionic strength. This conclusion was extended in experiments in which the cell membranes in a 2 ml sample of packed red cells were disrupted with 0.5% Triton X-100. Normal buffer was then added in steps of 0.5 ml up to a total of 2.5 ml. This produced small shifts* in the resonances in the direction opposite to that observed when whole cells swell. Between the first and last addition, the 2-phosphate resonance shifted by 2 Hz and the 3-phosphate resonance by 1.5 Hz. It was also possible that changes in diamagnetic susceptibility could contribute significantly to the effect we have observed. To test this, cells were incubated for 3 h with 25 mM phosphate {plus NaC1) to increase intracellular phosphate. After washing to remove extracellular phosphate, the cells were shrunken with 200, 300 and 400 mosM sucrose. The phosphate resonance shifted only slightly with cell volume; the total excursion between control and 400 mosM sucrose amounted* to 2 Hz. Since this is small compared with the shifts in Fig. 1, the resonance shift produced by cell swelling may not be attributed to changes in diamagnetic susceptibility. The effect of Mg2÷ on the 2,3-diphosphoglycerate spectrum was also measured in free solution. Since both Mg2÷ and 2,3-diphosphoglycerate permeate the red cell membrane slowly, if at all, changes in cell volume should not affect the concentration ratio of these two constituents. For this reason the Mg 2÷ concentration was altered by + 20% for the control experiment. The control solution consisted of: 5 mM MgC12/90 mM NaC1/ 10 mM KCI/5 mM 2,3-diphosphoglycerate/100 mM Tris, pH 7.4/20% ~H20. Reducing the Mg2+ concentration to 4 mM shifted the 3-phosphate resonance upfield by 0.2 Hz and increasing it to 6 mM shifted it 0.2 Hz in the same direction. Hence it seems unlikely that the effect of cell shrinking on the spectrum may be attributed to a change in the Mg2+/2,3-diphosphoglycerate concentration ratio in the intracellular free water volume. The concentration of all the cell solutes varies in a similar manner and it is not possible to make direct measurements such as those above for each solute. Instead, we have altered the physical state of the red cell membrane *These spectra were measured on a JEOL, FX 60 NMR spectrometer (operating at 24.15 MHz for ~P) and the observed shifts have been multiplied by 100/60 to make them comparable with the measurements on the Varian XL-100-15 machine.
509 +5C CHLORPROMAZINE
normol
shrunken
~_ +2C z
~_ +1(
O0 -05
~6 x w
i
g 21
I
i0-6
2x10-6
I
6xlO-6
I
2xlO-5
I
6xlO-5
I
2xlO 4
I
6xlO-4
CHLORPROMAZINE (M)
Fig. 2. Shift in 31P-NMR r e s o n a n c e o f t h e 2 - p h o s p h a t e o f 2 , 3 - d i p h o s p h o g l y c e r a t e w i t h increasing a m o u n t s o f c h l o r p r o m a z i n e in o n e e x p e r i m e n t t y p i c a l o f t w o . T h e b o t t o m c u r v e s h o w s m e m b r a n e a r e a r e d r a w n f r o m S e e m a n et al. [ 7 ] .
b y an o th er mechanism, Seeman and his colleagues have studied the effects o f a wide variety of anesthetics on red cell properties. In the course of these studies t h e y have shown [7] t hat the dissolution of anesthetics in the red cell m e m b r a n e f r e q u e n t l y casuses an increase in m e m b r a n e area. In t he case o f c h l o r p r o m a z i n e the h u m a n red cell m e m b r a n e area passes t h r o u g h a m a x i m u m at a b o u t 6% expansion, as shown in the b o t t o m section o f Fig. 2. The t o p p o r t i o n of Fig. 2 shows t ha t t he addition of chl orprom azi ne also caused t h e 2-phosphate resonance to shift t h r o u g h a m axi m um , analogous t o t h e ef f ect on m e m b r a n e area. Since t he area data were obtained on h u m a n red cells and th e NMR data on dog red cells, t he difference in m a x i m a is p ro bab ly n o t significant, particularly because the red cell m e m b r a n e lipids in the t w o species differ appreciably [ 8 ] . Fig. 2 shows t hat t he 2-phosphate resonance in the shrunken red cells also shifts t h r o u g h a similar m axi m um , even f u r t h e r displaced f r o m t he m a x i m u m in m e m b r a n e area. A similar b u t smaller shift was observed in the 3-phosphate resonance. To make sure t hat the shifts in Fig. 2 were of a general nature rather t h a n specific to chlorpromazine, similar experiments were carried o u t with a n o t h er anesthetic, 1-pentanol, whose effect on red cell area had also been measured b y Seeman et al. T he results o f these experiments, shown in Fig. 3, are similar to those with c h l o r p r o m a z i n e for normal cells t h o u g h t here are differences in th e behavior of shrunken cells. Th e measurements o f cell area were made on spherical h u m a n red cell ghosts so t h a t t h e cell volume also increases with t he area. In the case of the biconcave disc which t he nor m a l red cell approximates, Jacobs [9] has
510
1-PENTANOL o shrunken
x °---°-
/0~
=
f3~
~F37
uJ 136 o_
r3~ i x ! O -2
/ I
L
1
2 x l O -2
4xlO 2
6xlO 2
~___
8xlO z
1 -PENT,~NOL(M)
Fig,3. Shift in 31P-NMR resonance of t h e 2 - p h o s p h a t e o f 2 , 3 - d i p h o s p h o g l y c e r a t e w i t h increasing a m o u n t s o f 1 - p e n t a n o l in o n e e x p e r i m e n t ~ typical of two. The b o t t o m curve s h o w s m e m b r a n e area r e d r a w n f r o m S e e m a n et al. [7].
shown that red cell area is independent of cell volume in the range between the biconcave disc and the sphere and Sha'afi et al. [10] have argued t h a t this also applies when cells are shrunken. Thus we should not expect that the changes in membrane area produced in the biconcave disc by chlorpromazine and 1-pentanol would necessarily be accompanied by any significant change in volume in the range around the normal volume. Furthermore, Weed and Chailley [11] have pointed out that chlorpromazine causes cups to form in human red cell membranes, whereas alcohols such as ethanol and butanol cause spikes to form. With low drug concentrations, the surface modifications of either kind do not produce a change in volume. Hence the changes in resonance positions in Figs. 2 and 3 appear to be due to physical changes in the cell membrane rather than being mediated through changes in cell volume. The results with the anesthetics, taken together with those in which red cell volume was changed by osmotic pressure, lead us to conclude that there is an interaction between 2,3-diphosphoglycerate and a binding site that is dependent upon the physical condition of the dog red cell membrane. This finding is of particular relevance to cation transport since in this species similar alterations in osmotic pressure cause profound changes in Na ÷ transport which appear to be closely related to phosphoglycerate metabolism. This p r o j e c t has b e e n s u p p o r t e d in part by the I n s t i t u t e of General Medical Sciences, National I n s t i t u t e s o f Health, U.S.A., t h r o u g h grant 5 RO1 G M 1 5 6 9 2 - 0 8 and also in part by United States Public Health Service grant AM 07300. We a c k n o w l e d g e w i t h t h a n k s the use o f the Varian XL-100-15 NMR i n s t r u m e n t ( p r o v i d e d by the National Science F o u n d a t i o n u n d e r grant GP-32317) at t h e D e p a r t m e n t o f Chemistry. O n e o f us ( E . T . F . ) t h a n k s the Muscular D y s t r o p h y A s s o c i a t i o n s o f A m e r i c a for a fellowship. We s h o u l d like
511
t o e x p r e s s o u r t h a n k s t o Dr. J. H e r z f e l d f o r s t i m u l a t i n g d i s c u s s i o n , t o M i c h a e l R. T o o n a n d M u r r a y Welt f o r t h e i r t e c h n i c a l a s s i s t a n c e a n d t o Drs. E. K i r k a n d P e t e r A. M a r o k o f o r g e n e r o u s l y m a k i n g s u p p l i e s o f d o g b l o o d available.
References 1 2 3 4 5 6 7 8 9 10 11
Moon, R.B. and Richards, J.H. (1973) J. Biol. Chem. 248, 7276--7278 Henderson, T.O., Costello, A.J.R. and Omachi, A. (1974) Proc. Natl. Acad, Sci. U.S. 71, 2 4 8 7 - 2 4 9 0 Elford, B.C. and Solomon, A.K. (1974) Biochim. Biophys. Acta 373, 253--264 Parker, J.C. and Hoffman, J.F. (1964) Fed. Proc. 24, 589 Heustis, W.H. and Raftery, M.A. (1972) Biochem. Biophys. Res. Commun. 49, 428--433 Duhm, J. (1972) Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (It~rth, M. and Astrup, P., eds.) pp. 583--594, Munksgaard, Copenhagen and Academic Press, New York Seeman, P., Kwant, W.O. and Sauks, T. (1969) Biochim. Biophys. Acta 183, 499--511 Rouser, G., Nelson, G.J., Fleisher, S. and Simon, G. (1968) Biological Membranes, Physical Fact and Function (Chapman, D., ed.), pp. 5--69, Academic Press, London Jacohs, M.H. (1932) Biol. Bull 62, 178--194 Sha'afi, R.I., Rich, G.T., Sidel, V.W., Bossert, W. and Solomon, A.K. (1967) J. Gen. Physiol. 50, 1377--1399 Weed, R.I. and Chailley, B. (1973) in Red Cell Shape, Physiology, Pathology, Ultrastructure (Bessis, M., Weed, R.I. and LeBlond, P.F., eds.), pp. 55--68, Springer Verlag, New York
