Kinetics of 3-O-Methyl Glucose Transport in Red Blood Cells of [PDF]

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Kinetics of 3-O-Methyl Glucose Transport in Red Blood Cells of Newborn Pigs R O B E R T B. Z E I D L E R , P I N G LEE, and H. D. K I M From the Department of Physiology and Biophysics, West Virginia University Medical Center, Mogantown, West Virginia 26505 and the Department of Physiology, University of Arizona School of Medicine, Tucson, Arizona 85724

A B S T R A(2T The glucose-permeable fetal red cells in the pig are entirely replaced by glucose-impermeable adult red cells within a month after birth. This study investigates the kinetic parameters of the glucose transport mechanism in newborn pig red cells in comparison with immature adult red cells (reticulocytes) as well as the fully matured adult erythrocytes. Influx and efflux of the nonmetabolizable 3O-methyl glucose (3-O-M-G) in red cells of newborn pigs saturate at high substrate concentrations and exhibit typical Michaelis-Menten kinetics. Km values for efflux are 15.2 and 18.2 mM for 15 and 22°C, respectively. Q10 computed between 10 and 26 ° is 5.0. The energy of activation for the transport process is 34,000 cal mol -~. The effectiveness of hexoses in competing with 3-O-M-G in efflux is in the following order: D-glucose > D-mannose > D-fructose > D-galactose. Efflux of3-O-M-G does not increase with 3-O-M-G or D-ribose in the medium and is reduced by 2,4dinitroflurobenzene (DNFB), p-chloromercuriphenyl sulfonic acid (PCMBS), and phloridzin. The reticulocytes are shown to possess a carrier-mediated transport but with a considerably lower transport rate. As the reticulocytes mature into normal red cells, the carrier transport mechanism is lost. INTRODUCTION

T h e t r a n s f e r o f glucose across the plasma m e m b r a n e o f red blood cells obtained f r o m fetal animals is m u c h g r e a t e r than the t r a n s f e r which occurs in red blood cells obtained f r o m adult animals (Kozawa, 1914; Widdas, 1955). An outstanding e x a m p l e o f this p h e n o m e n o n occurs in pig r e d cells. Red cells f r o m n e w b o r n piglets are highly p e r m e a b l e to glucose, while the red cells f r o m adult pigs are nearly i m p e r m e a b l e to glucose. As a consequence, the adult pig red cell is unable to utilize glucose and the e n e r g y source used in vivo is not k n o w n (Kim a n d M c M a n u s , 1971 a, b). T h e p r e s e n t study was u n d e r t a k e n with two objectives in m i n d . T h e first was to characterize the glucose t r a n s f e r process in fetal pig red b l o o d cells. Since this process is lost in red cells f r o m the adult pig, it is o f interest to know if this t r a n s p o r t is any d i f f e r e n t f r o m the glucose t r a n s f e r process which occurs in the h u m a n red cells which are highly p e r m e a b l e to glucose at all stages o f h u m a n m a t u r a t i o n (Bowyer a n d Widdas, 1958; Karlish, 1972; Lacko et al., 1971; LeFevre, 1951; Levine a n d Stein, 1965; Widdas, 1953). Although Widdas (1955) indicated that a facilitated process m a y be present in fetal pig r e d blood cells, THE JOURNAL OF GENERAL PHYSIOLOGY ' VOLUME 6 7 ,

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kinetic d a t a are n o t available. S e c o n d l y , since fetal blood is p r i m a r i l y c o m p r i s e d o f cells o f y o u n g e r a g e , it is i m p o r t a n t to d e t e r m i n e if t h e d i f f e r e n c e in m e m b r a n e p e r m e a b i l i t y to glucose b e t w e e n fetal a n d a d u l t b l o o d is d u e to the age d i f f e r e n c e o f the cells. H e n c e , the glucose t r a n s p o r t in reticulocytes p r o d u c e d in a d u l t pig by p h e n y l h y d r a z i n e injection was also s t u d i e d . I n the p r e s e n t investigation a n o n m e t a b o l i z a b l e a n a l o g o f g l u c o s e , 3-O-methyl glucose (3-O-M-G) was e m p l o y e d to e x a m i n e the m e c h a n i s m o f glucose transfer. It was f o u n d t h a t the t r a n s f e r o f 3 - O - m e t h y l glucose in r e d cells o f n e w b o r n piglets is a c a r r i e r - m e d i a t e d t r a n s p o r t with e x t r e m e l y fast h a l f time f o r equilibr i u m a n d t h a t the reticulocytes in a d u l t pig still retain the c a r r i e r - m e d i a t e d t r a n s p o r t m e c h a n i s m b u t with a m u c h slower e q u i l i b r i u m time. T h i s carrierm e d i a t e d t r a n s p o r t is lost w h e n the reticulocytes m a t u r e to n o r m a l a d u l t r e d cells. M E T H O D S AND M A T E R I A L Fetal red cells were obtained from piglets born within 24 h when blood was taken. Blood was drawn by direct heart puncture into a syringe and coagulation was prevented by the use of l0 U of heparin for each milliliter of blood. The blood was centrifuged and the plasma and white bully coat were removed. The red cells were washed four times in icecold 0.9% NaCl by alternate resuspending and centrifuging. Reticulocytes were obtained by producing reticulocytosis in a 150-1b pig with injection of 1 g of phenylhydrazine per day for a week. The uptake of 3-O-methyl glucose in reticulocytes and adult pig cells was measured at 37°C. Since the transfer of glucose in fetal red cells is rapid at 37°C, the temperature at which influx and efflux are measured was lowered to 22°C. For efflux measurement a rapid sampling technique using a Millipore filter was employed (Mawe and Hempling, 1965). Red cells were loaded to a specific concentration of 14C-labeled 3-O-M-G. After centrifuging and removing supernatant fluid, an aliquot of 20-/zl packed cells was mixed into a 20-ml volume of a medium which contained 143 mM NaCI, 4.8 mM KCL, 9.35 mM Na HPO4, 1.65 mM Na H2PO4 at a pH of 7.4. Efflux is measured as the rate of appearance of labeled 3-O-M-G in the medium, by analysis of an accumulation compartment (Atkins, 1969). The modified equation which describes this accumulation of [14C]3O-M-G is In (1 - ( S t ~ S ® ) ) = - k t , where S t / S = is the ratio of activity in the medium at time t over the activity in the medium at time infinity and k is the rate constant and t is time. The rate constant is determined from the slope of the line when In (1 - ( S t ~ S ® ) ) vs. t is plotted. The flux of 3-O-M-G is calculated by multiplying the internal 3-O-M-G concentration by the rate constant. Intracellular concentrations of sugar were computed by determining the amount of 14C label initially present in the cells. Influx measurement was carried out by incubating 1 ml of washdd red cells in 10-ml media containing various 3-O-methyl glucose concentrations and electrolytes of essentially similar composition to the medium used for efflux measurement. [3-O-methyl-14C] glucose was added to serve as tracer and 3H inulin was used as an extracellular tag. At different times after incubation at 22°C a 0.7-ml aliquot sample was removed and squirted into 7 ml of ice-cold 0.9% NaCI solution with I mM HgCI2. The low temperature and HgCI~ served to stop further influx of 3-0methyl glucose and samples within 2-3 s may be taken. Quick sampling is necessary because of the fast transport o f 3-O-methyl glucose in fetal red ceils. The cold suspension was then quickly spun in a refrigerated centrifuge (Sorvall RC-2B, Dupont Instruments, Sorvall Operations, Newtown, Conn.) and the supernatant (SUP) solution was decanted. Red cells were then hemolyzed by adding 1 ml of distilled water and deproteinized by

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procedures using either perchloric acid extraction or Ba(OH)2 and Zn(SO4). The latter deproteinization procedure was accomplished by placing 0.2 cm 3 of a sample into 2.0 cm 3 of water. A volume of 0.2 cm 3 of 0.3 N Ba(OH)2 was added, and this suspension was heated in a water bath at 60°C for 5 min. After 5 min, 0.2 cm n of 5% ZnSO4 was added and the suspension vortexed. After centrifuging, aliquots of clear supernatant solution were used in liquid scintillation counting. For perchloric acid extraction, 0.2 cm 3 of a sample was placed into 2.0 cm 3 of 3.5% perchloric acid, vortexed, then centrifuged. To 1.5 cm 3 of the supernatant solution was added 0.1 cm 3 of 5.6 mM K2CO4 in order to neutralize the acid. Aliquots of the clear supernatant were used in counting the radioactivity. Since it has been shown that while the Ba(OH)2-Zn(SO)4 procedure results in removal of phosphorylated sugar, the PCA extraction does not (Thomas et al., 1970), red cells were extracted by both methods in order to determine whether 3-O-methyl glucose was phosphorylated. Table I shows the results of a 3-O-methyl glucose uptake experiment using the two different deproteinization procedures for extraction of tracer. T h e activities in cell by Ba(OH)2-Zn(SOh procedure were lower than those by PCA method. However, because of the scatter of the data, these differences are not considered significant. It is important TABLE I UPTAKE OF 14C-LABELED 3-O-METHYL GLUCOSE BY RED BLOOD CELLS OF A 1-DAY-OLD PIGLET Perchloricacid Hours incubation Activity* inside cells Activity* in medium Activity* in cell/ activity* in medium

2 183 186 1.0

6 200 174 1.1

Bariumhydroxidezincsulfate 2 146 186 0.8

6 176 174 1.0

* Activity = ["C]3-O-M-G cpm/ml. Red cells were incubated with 10.0 mM 3-O-M-G at 22.0°C for 6.0 h. Then they were deproteinized using perchloric acid or barium hydroxide and zinc sulfate. to note that the ratios of activities in and out of the cells were close to 1.0 indicating that the sugar was in equilibrium after 2 h of incubation. If there was any phosphorylation by the hexokinase reaction at all, the a m o u n t phosphorylated must be very small indeed. All samples were prepared for liquid scintillation counting by adding an aliquot to 11.5 cm 3 of Aquasol (a premixed scintillation cocktail sold by New England Nuclear, Boston, Mass.). Enough water was added to bring the total suspension to 15 cm a. T h e samples were counted in a model 222 Packard tri-carb liquid scintillation counter (Packard I n s t r u m e n t Co., Inc., Downers Grove. Ill.). All chemicals used in this study are commercially available. RESULTS

3-O-Methyl Glucose Uptake in Red Cells of Pigs T h e 3 - O - m e t h y l glucose (3-O-M-G) u p t a k e in r e d cells o f pigs shows drastic c h a n g e s as t h e pigs g r o w o l d e r . Fig. 1 shows the rate o f 3 - O - M - G u p t a k e i n r e d cells f r o m g r o w i n g pigs relative to t h e initial v a l u e o b t a i n e d at b i r t h . I n 30 days a f t e r b i r t h t h e glucose p e r m e a b i l i t y has a l r e a d y b e e n r e d u c e d to t h a t o f a d u l t level.

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T h e influx rate o f 3-O-methyl glucose in red cells f r o m 1-day-old piglets was very high. H a l f time o f equilibrium when external concentration is 1-5 m M is between 20-30 s. T h e initial 15-s flux rate (Fig. 2) is seen to be d e p e n d e n t on external 3-O-M-G concentrations, especially at lower substrate concentration;

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AFTER

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FIGURE 1. 3-O-Methyl glucose uptake in red cells from growing pigs. Uptake rates given are relative to the rate obtained in red cells from 1-day-old pigs. 2o ._= E

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FIGURE 2. 3-O-Methyl glucose uptake as a function of external 3-O-M-G concentrations. Measurements were carried out at 22°C. Insert shows the LineweaverBurk plot of the same data. the i n c r e m e n t in flux rate is smaller at higher external concentration, indicating a saturation type of t r a n s p o r t . T h e influx does not plateau completely at higher external concentration, instead it increases linearly with a m u c h smaller slope. This indicates that there is also a n o n s a t u r a b l e c o m p o n e n t (probably diffusion) in addition to a saturable system. A L i n e w e a v e r - B u r k plot (Fig. 2, right panel)

ZEIDLER ET AL,

Kineticsof 3-O-Methyl Glucose Transport

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yields a Km (sugar c o n c e n t r a t i o n at which o n e - h a l f o f the m a x i m a l t r a n s p o r t rate occurs) o f 23 m M a n d a V m a x ( m a x i m a l t r a n s p o r t rate) o f 27/.~mol/ml r e d b l o o d cells (RBC)/min.

Characteristics of 3-O-M-G Efflux T h e efflux o f 3-O-M-G is c h a r a c t e r i z e d by a t w o - c o m p a r t m e n t system. T h i s is seen in Fig. 3 A w h e n an intracellular c o n c e n t r a t i o n o f 18.8 m M 3-O-M-G is 1.0 0.8 0.6 ' 4

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FIGURE 3. Efflux of 14C-labeled 3-O-methyl glucose from red blood cells of l-dayold piglet. SJS®is the ratio of 14C activity in the efflux medium at time (t) and the activity at time infinity (o0). Panel A shows the efflux when the cells were loaded with 18.8 mM 3-O-M-G and panel B shows the efflux when the cells were loaded with 43.5 mM 3-O-M-G. Each efflux can be resolved into a two-compartment system represented by the dashed lines. The equation describing the two compartment efflux shown in panel A is Y = 0.58e -l'~t + 0.12e -°'°~t. The equation for the efflux seen in panel B is Y = 0.05e -°'9~t + 0.13e -°.°~t. p r e s e n t a n d in Fig. 3 B w h e n intracellular c o n c e n t r a t i o n is 43.5 m M 3-O-M-G. I n each case the flux is r e p r e s e n t e d by a large initial linear fast p o r t i o n followed by a smaller slow p o r t i o n . T h e initial fast p o r t i o n is linear a n d r e p r e s e n t s the majority o f the e f f l u x a n d the slope o f this line is u s e d as a m e a s u r e o f the rate c o n s t a n t o f 3-O-M-G efflux f o r c o m p u t a t i o n o f flux rate given in the followi n g sections.

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T h e net 3-O-M-G efflux is d e p e n d e n t on internal 3-O-M-G concentration. Fig. 4 shows the results o f m e a s u r e m e n t s o f the flux rate at 22 and 15°C. As the internal 3-O-M-G concentration increases, the flux rate increases and then reaches a plateau. This feature is consistent with Michaelis-Menten kinetics. By replotting the data in a Lineweaver-Burk plot (Fig. 4 top right corner), Vmax is f o u n d to be 58.8 p,mol/ml RBC/min and Km is 18.2 mM. These n u m b e r s are somewhat different from those f o u n d for influx, but it is doubtful that the 02

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FIGURr 4.

Effect of internal 3-O-methyl glucose on the efflux of 3-O-M-G from red blood cells of a 1-day-old piglet. Measurements were carried out at 22 and 15°C. The top right-hand corner shows the Lineweaver-Burk plot of data shown in main panel. difference is due to a significant asymmetry o f the transport system. Rather, it is more likely that this arises from the different p r o c e d u r e s o f m e a s u r i n g influx and efflux. It is i m p o r t a n t to note that both influx a n d efflux show saturation kinetics.

Apparent Energy of Activation T h e d e m o n s t r a t i o n o f saturable efflux o f 3-O-M-G in the previous section suggests that a carrier-mediated mechanism is involved. It is assumed here that

ZEmLER ET AL. Kinetics of 3-O-Methyl Glucose Transport

73

for the sugar molecule to cross the red cell m e m b r a n e , it m u s t react with the t r a n s p o r t carrier; t h e r e f o r e , an a p p a r e n t e n e r g y o f activation for the t r a n s p o r t process m a y be d e t e r m i n e d . T o e n s u r e that no secondary reactions are involved in this process, an A r r h e n i u s plot was d e t e r m i n e d between the t e m p e r a t u r e s o f 26 a n d 10°C (Fig. 5). Single linear results were f o u n d w h e n the log o f the rate constant was plotted against the reciprocal of the t e m p e r a t u r e indicating that no s e c o n d a r y reactions are i n t e r f e r i n g with the d e t e r m i n a t i o n o f the a p p a r e n t activation energy. T h e e n e r g y o f activation for the t r a n s p o r t process was determ i n e d for several 3-O-M-G concentrations at 15 and 22°C. T h e results are shown in T a b l e II. T h e a v e r a g e e n e r g y o f activation was 34,600 cal mo1-1. +0.4

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FIGURE 5. Effect of temperature on the efflux rate constants of 3-O-methyl glucose from red blood cells of a 1-day-old pig!et. The logarithm of the rate constant is plotted against the reciprocal of the temperature (°K).

Competitionfor Efflux I n o r d e r to e x a m i n e the specificity o f the t r a n s p o r t carrier, efflux o f 3-O-M-G was m e a s u r e d in the p r e s e n c e o f o t h e r structurally related sugars. T h e red cells were loaded with 40.0 m M 3-O-M-G a n d 40.0 mM of a n o t h e r s u g a r for 2 - 3 h to allow for equilibrium o f s u g a r to occur a n d the efflux of 3-O-M-G was m e a s u r e d . I f a particular substrate shares the s a m e carrier with 3-O-M-G, t h e n it will c o m p e t e with 3-O-M-G for t r a n s p o r t . A decrease in the efflux rate o f 3-O-M-G is e x p e c t e d , the extent o f the decrease d e p e n d s on the relative affinity o f the carrier for these two sugars. D-glucose, o-galactose, D-mannose a n d D-fructose were e m p l o y e d in this c o m p e t i t i o n study. T h e results are shown in Fig. 6. Galactose p r o d u c e s a 10% inhibition in 3-O-M-G efflux, fructose 14%, m a n n o s e 27%, a n d glucose 43%, Note that the 3-O-M-G efflux is d e c r e a s e d almost by one-

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h a l f by D-glucose; f u r t h e r m o r e , a d d i t i o n o f 40 m M o - g l u c o s e to 40 m M 3 - O - M - G results in a n e f f l u x r a t e c o n s t a n t n e a r l y e q u a l to the rate c o n s t a n t w h e n r e d cells were l o a d e d with 80 m M 3 - O - M - G ( T a b l e I I I ) . T h i s i n d i c a t e s t h a t t h e a f f i n i t y o f the c a r r i e r for 3 - O - M - G a n d D-glucose is n e a r l y the s a m e .

TABLE

II

ENERGY OF A C T I V A T I O N GLUCOSE-TRANSPORT C O M P O N E N T COMPLEX Internal 3-Omethyl glucose concentration

E n e r g y o f activation

cal mol -~

9.69 18.8 43.5 73.5

38,600 32,7O0 34,500 32,400 Average 34,600

The apparent activation energy (Ea) for the transport process was determined from the ratio of efflux rates of 3-O-M-G at 22 and 15°C using the Arrhenius equation: E~

4.56TIT2 T2 - T~

V~ log - Vj

I.CI i x

=,

r-

i

i,i

> 0.~

1

w

FI

FIcurt~ 6. The effect of hexoses on the efflux of 3-O-methyl glucose from red blood cells of a l-day-old piglet. Red cells were loaded with 40.0 mM 3-O-M-G and 40.0 mM of another sugar and efflux into a sugar-free medium was measured. The efflux rate constant of 3-O-M-G from cells loaded with only 40.0 mM 3-O-M-G is taken as control which is denoted as 1.0. Values plus range from four experiments are plotted and all values are significantly different from the control value (P < 0.05 as determined by the t test of E. Lord (1947),

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Inhibition by Drugs I f the transport o f 3-O-M-G d e p e n d s u p o n a particular configuration o f the transport c o m p o n e n t , it would be possible to attach specific groups on the c o m p o n e n t and disrupt the configuration thus altering the efflux o f 3-O-M-G. Newborn pig red cells were loaded with 40 mM 3-O-M-G and the 3-O-M-G was allowed to efflux into a m e d i u m which contained a specific drug. T h e concentrations were 1.0 raM, 2, 4 d i n i t r o f l u r o b e n z e n e (DNFB), 2,4,6-trintrobenzene sulfonic acid (TNBS) N-ethylmaleimide (NEM), u r e t h a n e , and phloridzin. A concentration o f 0.1 mM p - c h l o r o m e r c u r i p h e n y l sulfonic acid (PCMBS) was also employed. T h e results are shown in Fig. 7. T N B S , NEM, and u r e t h a n e have no effect, while DNFB produces 36% inhibition, PCMBS an 82% inhibition, and phloridzin an 85% inhibition. Efflux of 3-O-M-G into a Medium Containing 3-O-M-G or Ribose T h e transfer of 3-O-M-G implies that the m e m b r a n e transport c o m p o n e n t must have access to both sides o f the m e m b r a n e . Conceivably a n o t h e r sugar present in TABLE

III

EFFLUX RATE CONSTANTS OF 3-O-M-G AT VARIOUS CONCENTRATIONS WITH AND WITHOUT D-GLUCOSE Loaded sugar concentration

Rate c o n s t a n t

m/n i

40.0 mM 3-0M-G 80.0 mM 3-0M-G 40.0 mM 3-0M-G 40.0 mM o-Glucose

1.25 0.70 0.66

the efflux m e d i u m may attach to the t r a n s p o r t c o m p o n e n t . I f this happens, a change in efflux rate might be expected, d e p e n d i n g on whether the loaded carrier c o m p o n e n t is m o r e mobile than the free n o n l o a d e d carrier. Piglet red cells were loaded with a concentration 20 or 50 mM 3-O-M-G. Efflux rates o f 3-OM-G into a media which contains no sugar, 20, 40, 60, or 80 mM 3-O-M-G were measured. Media containing no sugar, 20, 40, or 80 mM ribose were also tested. Relative efflux rates were d e t e r m i n e d by dividing the experimentally determined rate constant f o u n d when no sugar was in the external m e d i u m , into the rate constant f o u n d when sugar was present in the external m e d i u m . T h e results shown in Fig. 8 indicate that neither 3-O-M-G n o r ribose in external m e d i u m affect the efflux o f 3-O-M-G.

Counter Transport Although newborn cells do not seem to show exchange flux, it was possible to d e m o n s t r a t e the existence o f c o u n t e r transport in these cells. W h e n glucose was

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a d d e d to the m e d i u m a f t e r x4C-labeled 3-O-M-G h a d r e a c h e d equilibrium, the 3O-M-G labels in m e d i u m increased, then fell back to its original level (Fig. 9). This observation is consistent with the c o n c e p t o f c a r r i e r - m e d i a t e d t r a n s p o r t existing in these cells.

3-O-Methyl Glucose Transport in Reticulocytes and Adult Pig Red Cells Fig. 10 shows the 3-O-methyl glucose influx in reticulocytes a n d m a t u r e red cells f r o m adult pig. Because o f the slow u p t a k e rate o f 3-O-methyl glucose in reticulocytes a n d m a t u r e adult red cells, m e a s u r e m e n t s were carried out at 38°C instead o f 22°C which influx m e a s u r e m e n t o f 3-O-M-G in n e w b o r n cells were p e r f o r m e d . I n f l u x o f 3-O-methyl glucose in reticulocytes is c o n c e n t r a t i o n de-

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Z "~

0

FIGURE 7. Effect of drugs on the efflux of 3-O-methyl glucose from red cells of a 1-day-old piglet. Efflux rates in medium-containing drugs are computed relative to the control rate obtained in medium with no drug. Values plus range from four experiments are plotted. Asterisk denotes values which are significantly different from the control values (P < 0.01 as determined by the t test of E. Lord. Red cells were loaded with 40.0 mM 3-O-M-G and placed into a medium containing 1.0 mM DNFB, TNBS, NEM, urethane, phloridzin or 0.1 mM PCMBS. p e n d e n t at low substrate concentrations but tends to saturate at h i g h e r substrate concentrations. This indicates that reticulocytes retain a c a r r i e r - m e d i a t e d transp o r t m e c h a n i s m a l t h o u g h the t r a n s p o r t rate is m u c h slower t h a n that f o u n d in n e w b o r n cells. W h e n the reticulocytes lose their reticulum a n d m a t u r e to n o r m a l adult cells, the c a r r i e r - m e d i a t e d t r a n s p o r t m e c h a n i s m is lost. In contrast to reticulocytes, the influx o f 3-O-M-G in the adult red cells is a concentrationd e p e n d e n t first-order t r a n s p o r t process. D I S C U S S I O N

T h e results p r e s e n t e d above clearly show that a c a r r i e r - m e d i a t e d m e c h a n i s m is involved in the 3-O-methyl glucose t r a n s p o r t in r e d cells o f n e w b o r n pigs. I n this r e g a r d , this investigation c o n f i r m s a n d e x t e n d s the initial observation by Widdas (1955) who postulated the carrier m e c h a n i s m for fetal pig r e d cells.

ZEIDLER

ET AL.

Kinetics of 3-O-Methyl Glucose Transport

77

A l t h o u g h the overall t r a n s p o r t characteristics in piglet red cells are not unlike those f o u n d in h u m a n red cells (Widdas, 1953; L e F e v r e and Davies, 1951; L e F e v r e a n d McGinness, 1960) a n d rabbit red cells (Regen, 1964), there are several distinct differences which are worthy to note. T h e t r a n s p o r t rate in piglet red cells is m u c h slower t h a n that f o u n d for h u m a n red cells. At 22°C the maximal t r a n s p o r t rate (Vm) in piglet red cells is a b o u t o n e - t h i r d that for adult h u m a n red cells (Mawe a n d H e m p l i n g , 1965) but Km value (the substrate A I.O

0,5 • o

20raM 5OmM

x

,-,

o

tJJ

EXTERNAL

_>

3-O-M-G

(raM)

B

tAJ n.. I.O

v

0.5

2'0 EXTERNAL

6'0 RIBOSE (rnM)

FIGURE 8. Efflux of 3-O-methyl glucose from red blood cells of a 1-day-old piglet into a medium containing 3-O-M-G or ribose. Panel A shows results obtained from two animals when red cells were loaded with 20.0 or 50.0 mM 3-O-M-G and effluxes into an external media containing no sugar, 20, 40, 60, or 80 mM 3-O-M-G were determined. Panel B shows the results from two animals when these red cells were placed in a medium which contained no sugar, 20, 40, or 80 mM ribose and efflux rates were determined. concentration at which the flux rate is o n e - h a l f maximal) is similar to those f o u n d for net fluxes in h u m a n red cells (Karlish et al., 1972). It seems that the t r a n s p o r t m e c h a n i s m for glucose in those two cell types shows similar affinities for glucose but since the m a x i m u m flux rate is smaller in piglet red cells, the latter m a y have fewer t r a n s p o r t sites (or carriers) or the t u r n o v e r rate (mobility o f carrier) is slower. T h e very high activation energy, 34,600 cal mo1-1 a n d the high Ql0 value (5 as c o m p a r e d to 2 - 3 f o u n d for h u m a n red cells), m a y suggest that the rate-limiting steps in the t r a n s p o r t are probably (a) at the site o f reaction w h e r e s u g a r binds with the carrier, and (b) the translocation o f the sugar-carrier

78

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PHYSIOLOGY

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67 • 1976

complex f r o m one side o f the m e m b r a n e to the other. T h e similarity Km values f o u n d for piglet and h u m a n red cells suggests that the m a g n i t u d e o f the e n e r g y barrier involved in substrate-carrier combination is approximately the same. H e n c e , the difference in rate o f t r a n s p o r t is most likely to be due to the translocation o f the sugar-carrier complex. It is possible that this translocation in piglet red cells is m o r e sensitive to t e m p e r a t u r e change than that in h u m a n red cells. 2.6 2.4 2.2 2,0 1.8 u') ~ 1.6 ir I,-

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1.4 1,2 1.0 ~

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-

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T

0.8

40mM Glucose Added to Medium

Loading

0f[I4c]3-O-M-G

0.6

-,6o

-~'o

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~ TIME

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) MINUTES

FIGURE 9. Countel transport of 3-O-methyl glucose. Red cells from a 1-day-old piglet were incubated at 22°C with 0.1 and 40 mM a4C-labeled 3-O-M-G for 2 h to allow 3-O-M-G to come into equilibrium. Cold glucose was then added to the medium to give a final concentration of 40 raM. I4C counts in the medium (SUP) were monitored. The ordinate gives the ratios of I4C counts in one ml medium relative to the counts in 1-ml cell water. A n o t h e r i m p o r t a n t difference between n e w b o r n pig red cells a n d h u m a n red cells is that in adult h u m a n red cells, the rate o f efflux o f a sugar into a m e d i u m which contains sugar is two to three times faster than the rate o f efflux o f a sugar into a sugar-free m e d i u m (Lacko and Berger, 1963; Levine a n d Stein, 1965; Mawe and H e m p l i n g , 1965). I n n e w b o r n pig red cells, no e n h a n c e m e n t o f fluxes is shown with sugar present in the external m e d i u m . This is similar to results f o u n d in adult rabbit red cells. (Regen, 1964) and bovine red cells (Hoos et al., 1972). T h e r f o r e , it seems reasonable to conclude that the loaded carrier moves no faster than the u n l o a d e d carrier in n e w b o r n pig red cells.

ZEIDLERET AL. Kinetics of 3-O-Methyl Glucose Transport

79

It is o f interest to note t h a n w h e n the pig m a t u r e s , s o m e h o w its red cells no longer possess this p a r t o f the t r a n s p o r t m a c h i n e r y . It is unlikely that the d i f f e r e n c e f o u n d between n e w b o r n red cells a n d adult r e d cells is d u e to differences in cell ages. T h e reticulocytes still retain a c a r r i e r - m e d i a t e d t r a n s p o r t f o r 3-O-methyl glucose which is lost w h e n the cells m a t u r e . T h e m a g n i t u d e o f this t r a n s p o r t , however, is at least two or t h r e e o r d e r s o f m a g n i t u d e lower than that f o u n d in n e w b o r n cells. T h e r e f o r e , t h e r e seem to be basic differences between fetal cells a n d adult cells. W h e t h e r this is d u e to a loss o f this c o m p o n e n t in the r e d cells p r o d u c e d a f t e r birth or to a masking effect owing to the presence o f o t h e r materials is not k n o w n at this time. As a consequence o f this loss o f the m e m b r a n e permeability to glucose, pig red cells b e c o m e incapable o f utilizing glucose for the p r o d u c t i o n o f necessary free e n e r g y to maintain their cellular integrity (Kim et al., 1973; Kim a n d McManus a, b, 1971). Despite this metabolic a n o m a l y , the postnatal r i g h t - h a n d shift o f c

0.3

temp = 38°C

/

_

m

~ E q

0.2

o.

0.1

d

~

Erythrocytes

i

?

i 50

0.0 0

i I00

i 150

/ 2O0

EXT 3-O-M-G CONCENTRATION(mM)

FIGURE 10. 3-O-Methyl glucose (3-O-M-G) uptake in reticulocytes and in mature red cells from adult pig. Measurements were carried out at 38°C. o x y g e n h e m o g l o b i n dissociation curve o f t e n f o u n d in n e w b o r n m a m m a l s still takes place in the pig mainly d u e to a rapid rise of red cell 2,3-DPG content (Kim and D u h m , 1974). Clearly, the transition f r o m glycolytic fetal red cells to nonglycolytic adult red cells has little b e a r i n g u p o n the red cells' p r i m a r y role of delivering oxygen to the tissue. T h e intriguing p r o b l e m o f how nonglycolytic m a m m a l i a n red cells can survive in circulation awaits f u r t h e r studies. This study was supported by N1H grant HL-13237 to P. Lee and NIH grant AM-17723 and S. Arizona Heart Grant to H. D. Kim. Part of this work appears in a dissertation submitted to West Virginia University by R. B. Zeidler in partial fulfillment of the requirements for the Degree of Doctor of Philosophy. Receivedfor publication 10 September1974. REFERENCES

ATKINS,G. L. 1969. Multicompartment Models for Biological Systems. Methuen and Co., Ltd., London.

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BOWYER, F., and W. F. WIDDAS. 1958. T h e action of inhibitors on the facilitated transfer

system in erythrocytes.J. Physiol. (Lond.). 141:219. H o o s , R. T., H. L. TARPLEY, and D. M. REGEN. 1972. Sugar transport in beef erythrocytes. Biochim. Biophys. Acta. 206:1974. KARLISH, S., W . LIEB, D. RAM, and W. STEIN. 1972. Kinetic parameters of glucose efflux from h u m a n red blood cells u n d e r zero-trans conditions. Biochim. Biophys. Acta. 255:126. KIM, HYUN DJU, and JOCHEN DUHM. 1974. Postnatal decrease in the oxygen affinity of pig blood induced by red cell 2,3-DPG. Am. J. Physiol. 220:(4):1001. KIM, HYUN DJU, and T. J. McMANuS. 1971 a. Studies on the energy metabolism of pig red cells. 1. T h e limiting role o f membrane permeability in glycolysis. Biochim. Biophys. Acta. 230:1. KIM, H YUN DJU, and T . J . M C M A N u S , 1971 b. Studies on the energy metabolism of pig red cells. 2. Lactate formation from free ribose and deoxyribose with maintenance o f ATP. Biochim. Biophys. Acta. 230:12. KIM, H. D., T. J. McMANuS, and G. R. BARTLETT. 1973. Transitory changes in the metabolism of pig red cells d u r i n g neonatal development. In Erythrocytes, T h r o m b o cytes, Leukocytes: Recent Advances in Membrane and Metabolic Research. E. Gerlach, K. Moser, E. Deutch, and W. Williams, editors. Thieme, Stuttgart, W. Germany. 146148. KOZAWA, S. 1914. Artdifferezen in der Durchl~issigkeit d e r roten Bluk6rperchen. Biochem. Z. 60:232. LACKO, L., and M. BERGER. 1963. Kinetics comparison of exchange transport of sugars with non-exchange transport in h u m a n erythrocytes.J. Biol. Chem. 238:3478. LACKO, L. B., B. WITTKE, and P. GECK. 1971. T h e pH d e p e n d e n c e of exchange transport of glucose in h u m a n erythrocytes. J. Cell Physiol. 80:73. LEFEVRE, P. G., and R. DAVIES. 1951. Active transport into the h u m a n erythrocyte. Evidence from comparative kinetics and competition among monosaccharides.J. Gen. Physiol. 34:515. LEFEvRE, P. G., and G. McGINNESS. 1960. T r a c e r uptake vs. net uptake of glucose through h u m a n red cell surfaces.J. Gen. Physiol. 44:87. LEVlNE, M. OXENDER, and W. D. STEIN. 1965. T h e substrate-facilitated transport of the glucose carrier across the h u m a n erythrocyte membrane. Biochim. Biophys. Acta. 109:151. LORD, E. 1947. Biometrika. 34:56. MAWE, R. L., and H. G. HEMPLING. 1965. T h e exchange of 14C-glucose across the m e m b r a n e of the h u m a n erythrocyte.J. Cell. Comp. Physiol. 66:95. REGEN, D. M. 1964. Studies of the glucose-transport system in the rabbit erythrocyte. Biochim. Biophys. Acta. 79:151. THOMAS, J. A . , M. MAWHINNEY, and G. WENGER. 1970. Enzymatic measurement of prostrate gland fructose.J. Reprod. Fertil. 22:21. WIDDAS, W. F. 1953. Kinetics of glucose transfer across the h u m a n erythrocyte membrane. J. Physiol. (Lond. ). 120:23. WIDDAS, W. F. 1955. Hexose permeability o f fetal erythrocytes. J. Physiol. (Lond.). 127:318.