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Mediated transport and metabolism of lactate in rat aorta
312
Biochimica et Biophysica Acta, 541 (1978)312--320
@)Elsevier/North-Holland Biomedical Press
BBA 28561 MEDIATED TRANSPORT AND METABOLISM OF LACTATE IN RAT AORTA
HOWARD KUTCHAI, LISA MANN GEDDIS and MARSHA S. MARTIN Department of Physiology, University of Virginia School of Medicine, Charlottesville, Va. 22901 (U.S.A.)
(Received November 14th, 1977) Summary 1. Under appropriate conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Oxidation of lactate to CO2 occurs under aerobic conditions. 2. L- and D-lactate are taken up into the cells when oxygen, glucose, or both oxygen and glucose are present in the incubation medium. Both L- and D-lactate are excluded from the cells when neither oxygen nor glucose is present. 3. D,L-Glyceraldehyde prevents the uptake of L-lactate. The effect is apparently n o t due to the inhibition of glucose metabolism by L-glyceraldehyde. 4. L-Lactate (20 mM) markedly inhibits the uptake of 5 mM D-lactate, but 20 mM D-lactate fails to inhibit the uptake of 5 mM L-lactate. 5. Raising the pH of the incubation medium markedly depresses the uptake of L-lactate. 6. The results provide evidence that L- and D-lactate enter the cells of rat aorta by a mediated transport system.
Introduction Until recently it has been assumed that lactic acid enters and leaves muscle cells by diffusing across their plasma membranes. Mainwood and WorsleyBrown [1] provided evidence that efflux of lactate from frog sartorius muscle does n o t occur by simple diffusion. They concluded that both undissociated lactic acid and lactate anion contributed to the efflux, in proportions that depended on the conditions of incubation. The mode of lactate transport in other types of muscle has n o t been considered. Lactic acid plays an especially prominent role in the metabolism of vascular smooth muscle [2]. Even in the presence of high oxygen tensions a large percentage of the glucose utilized is metabolized to lactate. In bovine mesenteric vein, Peterson and Paul [3] showed that about 30% of the ATP produced is
313 derived from aerobic glycolysis under a wide range of conditions. Under some circumstances lactate may serve as an important energy fuel for vascular smooth muscle. Coe et al. [4] f o u n d that lactate was more effective than glucose in restoring the contractility of substrate-depleted rabbit aortic strips. This suggests t h a t exogenous lactate enters the vascular smooth muscle cells and can be oxidized. L u n d h o l m and Mohme-Lundholm [5] found that lactate accumulates intracellularly to extremely high levels in bovine mesenteric vein u n d e r anaerobic conditions and can be released to the incubation medium at a rapid rate. The potential importance of the production and consumption of lactate in vascular smooth muscle stimulated our interest in the mechanisms by which lactate enters and leaves the cells of this tissue. This article reports our studies of the uptake of exogenous lactate by the cells of rat aorta. Methods
Preparation of tissue. Aortas were gently dissected from 200--300-g female Sprague-Dawley rats (Hilltop Farms) anesthetized with carbon dioxide. Each aorta was placed in a petri dish filled with Hanks' balanced salts solution (37°C, gassed with 95% 02/5% CO2). The segment of aorta between the aortic arch and the renal arteries was cannulated with polyethylene tubing and perfused with warmed, oxygenated Hanks' for the rest of the dissection. Fat and extraneous connective tissue were gently removed and th6 section of the aorta between the arch and the renal artery was either: (a) cut transversely into five sections {aortic rings) or (b) slit longitudinally along its length and then cut transversely into five sections {aortic strips). Uptake of L-lactate as a function of extracellular concentration. Aortic strips were incubated in a shaking water bath in glucose-free Hanks' solution containing various concentrations of L-lactate. The shaker was covered and gassed, with 95% 02/5% CO2. The incubation conditions were chosen to favor oxidation of exogenous lactate. At 30, 60, 90, 120 and 180 min of incubation, samples of the incubation medium were taken. Concentrations of L-lactate in medium samples were determined with the L-lactate dehydrogenase m e t h o d {Sigma Biochemical Corp.). The lactate uptake during a given time interval was c o m p u t e d from the a m o u n t of lactate that disappeared from the medium during that period and the results were expressed in nmoles of lactate taken up per min per mg wet tissue. Estimation of ~4C02 production from [~4C]lactate. Rat aortas were obtained as described above and were opened by a lengthwise cut and then cut transversely into two segments. Each artery segment was weighed and placed in a 13 × 100 mm test tube containing 0.5 ml Hanks' solution containing various amounts of L-[U-~4C]lactate. The tube was capped with a serum bottle stopper to the b o t t o m of which was attached a 0.5-ml polyethylene well (Kontes Glass Co.). The tubes were gassed with 95% 02/5% CO2 and incubated at 37°C in a shaking water bath. After 1, 2, 3 or 4 h of incubation, 0.4 ml hyamine hydroxide was added to the polyethylene well and 0.05 ml of concentrated H2SO4 was added to the incubation medium. Both additions were made via needles inserted through the serum bottle stopper. The tube was put back in
314 the incubator-shaker for 1 h to allow collection of the ~4CO2 by the hyam i ne h y d r o x id e. 14CO2 was quantified by counting an aliquot of the hyam i ne in toluene scintillation fluid. The lactate c o n c e n t r a t i o n in the incubation medium at the various time points was determined with the lactate dehydrogenase-based assay (Sigma Biochemical Corp.). The a m o u n t of COs p r o d u c e d from exogenous lactate was c o m p u t e d from the total dpm of ~4CO2 collected and the original d p m / a t o m carbon in the incubation medium.
Uptake of the 14C label from [14C]lactate under various conditions of incubation. Aortic strips or rings were preincubated at 37°C in Hanks' balanced salts solution f o r 30--45 min. T h e y were then incubated in Hanks' containing 5 or 10 mM L- or D-lactate (14C-labeled) and 5 mM sucrose (3H-labeled) under various conditions of incubation. After various periods of incubation, usually 15, 30, 60, 90 and 120 min, medium samples were taken and tissue pieces were removed f r o m the incubation flask. Aortic strips or rings were quickly rinsed in Hanks' solution on a fine stainless steel wire mesh held in a Millipore filter holder, which was attached to a vacuum flask. The aortic strips or rings were immediately weighed and the radioactivity was e x t r a c t e d by boiling in 1.5 ml of water f o r 30 min. The 3H and ~4C radioactivity in the medium samples and tissue extracts were de t e r m i ne d by double label liquid scintillation counting techniques (Beckman LS-230 counter) and the volumes of distribution (spaces) o f the two labels c o m p u t e d . The 3H space is taken as an estimate of the extracellular space o f the tissue. The different incubation conditions e m p l o y e d (in various combinations) were: the presence or absence of oxygen; the presence or absence of 11 mM glucose; the presence or absence of 30 pM 2,4-dinitrophenol; the presence or absence of 20 mM D,L-glyceraldehyde; the presence or absence of the opposite stereoisomer at 20 mM concent rat i on; and various pH values (7.1, 7.4 or 7.7) in the incubation medium. Sucrose is apparently a good marker for the extracellular space in arterial tissue. Jones and Swain [6] f o u n d t hat sucrose and 6°Co • EDTA distribute in the same space in arteries from several species and that inulin distributes in a considerably smaller volume. Using 6°Co • E D T A Jones [7] f o u n d the extracellular space of aorta from 300-g Sprague Dawley rats to be 0.425 ml/g wet tissue. The sucrose space values we obtained are in reasonable agreement with Jones' value. Our sucrose spaces for aortic rings were consistently less than those o f aortic strips, presumably due to less tissue damage in the f o r m e r preparation. We also f o u n d t hat the sucrose space varies considerably with the conditions of incubation. Estimation of total tissue water content. Total tissue water c o n t e n t was estim a t e d f r o m tissue weights before and after dessication and from the volume of distribution of ~4C-labelled urea. Aortic strips were incubated aerobically in Hanks' solution for 90 min. T h e y were then removed from the incubation medium, rinsed, b l ot t ed and immediately weighed on tared aluminum pans. The tissue pieces were t hen dried to cons t a nt weight in an oven at 95°C. From the wet and dry weights, the tissue water c o n t e n t was calculated to be 0.701 +_ 0.014 (S.E.) ml/g wet tissue. T o determine the volume of distribution of urea, aortic strips were incubated in Hanks' solution containing 3.33 mM urea (14Clabeled). Af ter 15, 30, 60, 120 and 180 min of incubation, samples of the incubation med iu m were taken and tissue samples were removed. The tissue pieces
315
were blotted on Whatman No. 50 filter paper, weighed and boiled in 1.5 ml of water to ex tr ac t the radioactivity. The space of 14C-labelled urea was at a steady-state value by 15 min of incubation and did n o t change over the rest of the incubation. The average urea space from 15 to 180 min of incubation was 0.709 +_0.015 (S.E.) ml/g wet tissue. Estimation o f the volume o f distribution o f unmetabolized [14C]lactate. In some of the experiments described above in which the uptake of [14C]lactate and [3H]sucrose were determined, the medium samples and tissue extracts were subjected to thin-layer c h r o m a t o g r a p h y on cellulose (Eastman Chromagram) with butanol/propionic acid/water, 63 : 31 : 5, as the developing solvent. The fraction of ~4C label co-migrating with a lactate standard was det erm i ned by liquid scintillation techniques. The dpm of 14C-lactate in the original medium sample or tissue e x t r a c t was then c o m p u t e d as the total ~4C dpm multiplied by the fraction present as unm e t a bol i z e d lactate. The volume of distribution of the u n m e t a b o l i z e d [~4C]lactate was c o m p u t e d from the dpm of unm et abol i zed lactate per mg wet tissue and the dpm of u n m e t a b o l i z e d lactate per tA incubation medium. Purity o f the D- and L-lactate utilized. The D- and L-lactates (~4C-labeled) used were obtained from Amersham-Searle and warranted by the supplier to be greater than 95% radiopure. This was c o n f i r m e d by thin-layer c h r o m a t o g r a p h y as described in the preceding paragraph. In addition we verified that the D - and L-lactates were pure in their stereoisomer c o n t e n t in the following m anner [8]. Approx. 0.5 t~mol of 14C-labeled D- or L-lactate was incubated with L-lactate dehydrogenase, NAD ÷, glutamic pyruvic transaminase and glutamate for 45 min at 37°C. The reaction m i xt ur e was deproteinized and subjected to thinlayer c h r o m a t o g r a p h y as described above. Greater than 98% of the L-lactate was co n v er ted to pyruvate and alanine, but essentially none of the D-lactate was converted to these products. Thus, within the limits of sensitivity of this m e t h o d , we find t hat the L-[14C]lactate is pure and is u n c o n t a m i n a t e d with D-[~4C]lactate. The D-[~4C]lactate is n o t c o n t a m i n a t e d with L-[~4C]lactate in measurable amounts. Results and Discussion
Dependence of L-lactate uptake rate on the concentration o f L-lactate in the incubation medium. The rate of lactate upt ake during the first 30 min of incubation was high. This is a t t r i but ed to the rapid distribution of lactate in the extracellular space of the tissue. During the remainder of the incubation the rate of lactate u p t a k e was lower and relatively const ant for at least 120 min more. Lactate u p ta ke was c o m p u t e d as the mean of the upt ake rate values for four 30-min incubation periods after the initial 30-min period. The rate of lactate uptake increased with increasing lactate c o n c e n t r a t i o n in the incubation medium up t o approx. 5 mM lactate. Increasing the medium lactate level above 5 mM did n o t cause a f ur t her increase in the rate of lactate uptake. We f o u n d that the m a x i m u m rate of lactate upt ake u n d e r these conditions was a b o u t 0.05 n m o l / m i n per mg wet wt., and the lactate c o n c e n t r a t i o n in the med iu m for half-maximal upt ake rate was approx. 2.5 mM. In similar experiments with aortic rings incubated in 10 mM L-lactate, the rate of lactate
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F i g . 1. T h e r a t e o f p r o d u c t i o n o f 1 4 C O 2 b y s t r i p s o f r a t a o r t a f r o m L - [ U - 1 4 C ] l a c t a t e w e t t i s s u e is p l o t t e d vs. t h e c o n c e n t r a t i o n o f L - l a c t a t e i n t h e i n c u b a t i o n m e d i u m .
in nmol/min
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F i g . 2. A e r o b i c i n c u b a t i o n o f a o r t i c r i n g s i n t h e p r e s e n c e a n d a b s e n c e o f g l u c o s e . T h e a v e r a g e s p a c e s i n ml/g wet tissue of 14C (from L-[14C]lactate) a n d 3 H ( f r o m [3 H ] s u c r o s e ) a r e p l o t t e d v e r s u s t i m e . T h e horizontal bar indicates the total tissue water space. The vertical bars represent standard errors.
uptake was 0.068 + 0.015 (S.E.) n m o l / m i n per mg wet tissue. We are n o t aware of other determinations of the rate of uptake of L-lactate by vascular tissues. It is n o t e w o r t h y that our maximal rates of lactate uptake are comparable to the rates of glucose utilization that have been reported for arterial tissue and fall within the ranges of glucose utilization values reported for swine aorta [9], rabbit aorta [9] and rat aorta [10]. The concentration of lactate in the arterial blood of Wistar rats anesthetized with N20 is 2.84 mmol/1 of blood under control conditions [11]. The blood lactate level increased about 4.5-fold when the arterial blood pressure was dropped to 25--35 mmHg by bleeding [12] and arterial lactate increased 3-fold due to a 3-min period of asphyxia [11]. The control level of blood lactate is close to the concentration that, in our experiments, results in half-maximal lactate uptake by rat aortic strips. The blood levels reached in certain types of physiological stress are apparently sufficient to saturate lactate uptake by the aorta. Production of 14C02 from L-[14C]lactate. As shown in Fig. 1, a major fraction of the 14C carbons of the 14C-labeled L-lactate taken up by the rat aortic strips can be collected as 14CO2. This supports the contention that under these experimental conditions L-lactate is readily oxidized by rat aorta. The high rate of lactate uptake by the tissue and its apparent ease of oxidation are consistent with the idea that, under some circumstances, oxidation of exogenous L-lactate may be an important energy source for arterial tissue. Dependence of L-lactate uptake on the presence of glucose and oxygen in
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Fig. 3. A n a e r o b i c i n c u b a t i o n of a o r t i c rings in t h e p r e s e n c e o f 11 m M glucose ( p a n e l A ) a n d in t h e a b s e n c e of glucose ( p a n e l B). T h e spaces of 14C ( f r o m [ 1 4 C ] l a c t a t e ) a n d 3 H ( f r o m [ 3 I - I ] s u c r o s e ) i n m l ] g w e t tissue are p l o t t e d vs. t i m e . T h e v e r t i c a l bars n e a r t h e d a t a p o i n t s r e p r e s e n t t h e s t a n d a r d errors. T h e h o r i z o n t a l lines i n d i c a t e t h e t o t a l tissue w a t e r space.
the incubation medium. As shown in Fig. 2, under aerobic conditions L-lactate uptake did n o t depend on the presence of glucose in the incubation medium. Both with and w i t h o u t glucose, the ~4C label from the lactate distributed in a space that exceeded the total tissue water. Thin layer chromatography of tissue extracts showed that a substantial fraction of the 14C in the tissue was present as compounds that did not co-migrate with lactate and thus are presumably metabolic products of L-lactate. Under anaerobic conditions, as shown in Fig. 3, L-lactate was readily taken up in the presence of glucose in the incubation medium. However, when glucose was omitted from the medium, the 14C label from lactate was confined to a volume of distribution that did n o t exceed the sucrose space of the tissue. This strongly suggests that under these conditions L-lactate failed to enter the cells of the aorta. Thin-layer chromatography of tissue extracts showed t h a t in the presence of glucose metabolites were present in the tissue, while in the absence of glucose at least 95% of the tissue 14C co-chromatographed with lactate. It thus appears that oxidative metabolism is n o t required for the uptake of L-lactate provided that glucose is present. This interpretation is consistent with the finding that 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, had no significant effect on the rate of L-lactate uptake under aerobic conditions (Fig. 4). Effect of D,L-glyceraldehyde on L-lactate uptake. As shown in Fig. 5, the presence of D,L-glyceraldehyde markedly inhibited the uptake of ~4C from L-[~4C]lactate u n d e r anaerobic conditions in the presence of glucose. Under these conditions the 14C label was apparently confined to the extracellular space of the tissue when D,L-glyceraldehyde was present. This suggests that D,L-glyceraldehyde acted to inhibit transport of lactate into the cells. We originally believed that the effect of glyceraldehyde might be due to inhibition of glucose metabolism since L-glyceraldehyde is an inhibitor of hexokinase. However, we f o u n d that glyceraldehyde had the same inhibitory effect under aerobic conditions in the absence of glucose, which argues against this inter-
318
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Fig. 4. A e r o b i c i n c u b a t i o n o f aortic strips in t h e p r e s e n c e o f 3 0 # M 2 , 4 - d i n i t r o p h e n o l and in the a b s e n c e o f the inhibitor. T h e s p a c e s o f 1 4 C ( f r o m [ 1 4 C ] l a c t a t e ) and 3 H ( f r o m [ 3 H ] s u c r o s e ) in m l / g w e t tissue are the averages o f results f r o m t w o e x p e r i m e n t s . T h e h o r i z o n t a l bar i n d i c a t e s t h e t o t a l tissue w a t e r space. Fig. 5. E f f e c t o f D , L - g l y c e r a l d e h y d e . A o r t i c rings w e r e i n c u b a t e d a n a e r o b i c a l l y w i t h 1 1 m M g l u c o s e in the p r e s e n c e o f 2 0 m M D , L - g l y c e r a l d e h y d e and in its a b s e n c e . S p a c e s o f 1 4 C ( f r o m [ 1 4 C ] l a c t a t e ) and 3 H ( f r o m [3 H ] s u c r o s e ) in m l / g w e t tissue are p l o t t e d vs. t i m e . 3 H s p a c e s in the a b s e n c e o f g l y c e r a l d e h y d e have b e e n o m i t t e d for clarity; t h e y did n o t d i f f e r s i g n i f i c a n t l y f r o m the 3 H ( s u c r o s e ) s p a c e s s h o w n . T h e h o r i z o n t a l bar d e n o t e s t h e t o t a l tissue w a t e r space.
pretation. It is possible that D- or L-glyceraldehyde more directly inhibits lactate entry. Uptake of D-lactate. Under aerobic incubation conditions with no glucose in the incubation medium, 14C label from D-[14C]lactate distributed in a volume that significantly e x c e e d e d t h e total tissue water. Thin-layer chromatography of tissue extracts showed/that a significant fraction of the 14C did n o t co-migrate with D-lactate, indicating the presence of metabolic products of D-lactate. As was the case for L-lactate, in the absence of both o x y g e n and glucose in the incubation medium ~4C from D-lactate was confined to a space equal to the sucrose space. This suggests that under these conditions D-lactate did n o t enter the cells of the tissue. Effect of the presence of the opposite stereoisomer on lactate uptake. As s h o w n in Fig. 6A, the presence of 20 mM L-lactate markedly inhibited the uptake of 5 mM D-[14C]lactate. On the other hand, the presence of D-lactate at 20 mM had no detectable effect on the rate of uptake of L-[14C]lactate (Fig. 6B). The space of unmetab01ized D-[14C]lactate was determined in the presence and absence of 20 m M L-lactate and was f o u n d to be essentially constant from 30 to 120 min of incubation. The average space of unmetabolized D-[~4C]lactate in the absence of L-lactate was 0.502 + 0.023 (S.E.) ml/g wet tissue and in the presence of L-lactate was 0 . 4 6 9 + 0 . 0 0 8 (S.E.) ml/g wet tissue. If the effect of L-lactate were to inhibit the metabolism of D-[14C]lactate, the
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Fig. 6. E f f e c t o f o p p o s i t e s t e r e o i s o m e r o n l a c t a t e u p t a k e . A o r t i c rings w e r e i n c u b a t e d in t h e p r e s e n c e of glucose a n d o x y g e n w i t h 5 r a m [ 1 4 C ] l a c t a t e in t h e p r e s e n c e a n d a b s e n c e o f t h e o p p o s i t e s t e r e o i s o m e r a t 20 raM. Panel A; t h e u p t a k e of 14C f r o m D - [ 1 4 C ] l a c t a t e w i t h ( . . . . . . ) and without ( ) 20 raM L - l a c t a t e . S u c r o s e s p a c e s are s h o w n b y t h e l o w e r p o i n t s . P a n e l B: t h e u p t a k e o f 14C f r o m L - [ 1 4 C ] l a e t a t e in t h e p r e s e n c e ( e ) a n d a b s e n c e (o) o f 20 m M D - l a c t a t e . S p a c e in r a l / g w e t tissue is p l o t t e d vs. t i m e . T h e v e r t i c a l b a r s r e p r e s e n t s t a n d a r d errors.
space of unmetabolized D-[14C]lactate should have been larger in the presence of L-lactate than in its absence. We observed the reverse, which supports the interpretation that L-lactate inhibits the transport of D-lactate into the cells. Effect of the pH of the incubation medium on uptake of L-lactate. As shown in Fig. 7, increasing the pH of the incubation medium caused a marked
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Fig. 7. E f f e c t of t h e p H of t h e i n c u b a t i o n m e d i u m . A o r t i c rings w e r e i n c u b a t e d a e r o b i c a l l y w i t h 11 raM glucose in i n c u b a t i o n m e d i a w i t h p H 7.1, 7.4 o r 7.7. T h e s p a c e o f 14C ( f r o m [ 1 4 C ] l a c t a t e ) a n d 3 H ( f r o r a [ 3 H ] s u c r o s e ) in r a l / g w e t tissue are p l o t t e d vs. t i m e . T h e v e r t i c a l bars n e a r t h e d a t a p o i n t s r e p r e s e n t stand a r d errors. T h e d a t a f o r p H 7.7 are t h e m e a n s o f t w o e x p e r i m e n t s w i t h n e a r l y i d e n t i c a l results. T h e u p p e r c u r v e s ( w i t h t h e p H values n o t e d a l o n g s i d e ) are 14C s p a c e s a n d t h e l o w e r c u r v e s are t h e 3 H spaces. T h e v e r t i c a l bars r e p r e s e n t s t a n d a r d e r r o r s .
320
decrease in the rate of uptake of the 14C from L-[14C]lactate. At pH 7.1 only 0.1% of the total lactate in the incubation medium is in the form of the undissociated acid. Increasing the pH by 0.3 units will halve the concentration of undissociated acid, but will change the concentration of lactate anion negligibly. The data are consistent with the interpretation that a substantial fraction of the lactate that enters the cell does so as the undissociated acid. An alternative interpretation is that the data reflect primarily the pH dependence of the activity of a membrane lactate anion carrier. Mainwood and Worsley-Brown [1] concluded that in frog sartorius both the anion and acid forms may contribute to lactate efflux from the cells. Interpretations of the results. We have shown that under the appropriate incubation conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Some of our results are inconsistent with the idea that lactate entered the cells by simply diffusing across the plasma membrane. These are: (1) that L- and D-lactate failed to enter the cells when both glucose and oxygen were absent from the incubation medium, (2) that D,L-glyceraldehyde prevented the entry of L-lactate into the cells, and (3) that L-lactate was able to markedly depress the rate of transport of D-lactate by the tissue. These results are consistent with the interpretation that L- and D-lactate enter the cells of rat aorta by a mediated transport system. The inhibition )f the uptake of D-lactate by a 4-fold excess of L-lactate, together with the failure of a 4-fold excess D-lactate to inhibit uptake of L-lactate, is consistent with the interpretation that the mediated transport system has a higher affinity for L- then for D-lactate. These results provide support for the idea that lactate enters the cells of rat aorta by a mediated transport system. Our studies have done little to define the nature of this transport system, however. The form of lactate that is transported, the structural specificity of the transport system, and the nature of the apparent relationship of the transport system to cellular metabolism remain to be determined. Acknowledgements This research was supported by grants HL 15716 and HL 17967 and by Research Career Development Award HL 00014 to H.K. from the National Institutes of Health. It is a pleasure to acknowledge the participation of Charles Epstein in the early stages of this study. References 1 M a i n w o o d , G.W. a n d W o r s l e y - B r o w n , P. ( 1 9 7 5 ) J. P h y s i o l . 2 5 0 , 1 - - 2 2 2 L e h n i n g e r , A . L . ( 1 9 5 9 ) in T h e A r t e r i a l Wall ( L a n s i n g , V . A . , e d . ) , p p . 2 2 0 - - 2 4 6 , Williams a n d Wilkins, Baltimore 3 P c t e r s o n , J.W. a n d P a u l , R . J . ( 1 9 7 4 ) B i o c h e m . B i o p h y s . A c t a 3 5 7 , 1 6 7 - - 1 7 6 4 C o e , J., D e t a r , R. a n d B o h r , D . F . ( 1 9 6 8 ) A m . J. P h y s i o l . 2 1 4 , 2 4 5 - - 2 5 0 5 L u n d h o l m , L. a n d M o h m e - L u n d h o l m , E. ( 1 9 6 0 ) A c t a P h a r m a c o l . T o x i c o l . 1 6 , 3 7 4 - - 3 8 8 6 J o n e s , A.W. a n d S w a i n , M.L. ( 1 9 7 2 ) A m . J. P h y s i o l . 2 2 3 , 1 1 1 0 - - 1 1 1 8 7 J o n e s , A.W. ( 1 9 7 3 ) Circ. Res. 3 3 , 5 6 3 - - 5 7 2 8 Lowry, O.H. and Passonneau, J.V. (1972) A Flexible System of Enzymatic Analysis, pp. 194--199 A c a d e m i c Press, N e w Y o r k 9 M o r r i s o n , E.S., S c o t t , R . F . , K r o m s , M. a n d F r i c k , J. ( 1 9 7 2 ) A t h e r o s c l e r o s i s 16, 1 7 5 - - 1 8 4 1 0 D a l y , M.M. ( 1 9 7 6 ) A m . J. P h y s i o l . 2 3 0 , 3 0 - - 3 3 11 K a a s i k , A . E . , Nilsson, L. a n d SiesjS, B . K . ( 1 9 7 0 ) A c t a P h y s i o l . S c a n d . 7 8 , 4 3 3 - - 4 4 7 12 K a a s i k , A . E . , N i l s s o n , L. a n d SiesjS, B.K. ( 1 9 7 0 ) A c t a P h y s i o l . S c a n d . 7 8 , 4 4 8 - - 4 5 8
Biochimica et Biophysica Acta, 541 (1978)312--320
@)Elsevier/North-Holland Biomedical Press
BBA 28561 MEDIATED TRANSPORT AND METABOLISM OF LACTATE IN RAT AORTA
HOWARD KUTCHAI, LISA MANN GEDDIS and MARSHA S. MARTIN Department of Physiology, University of Virginia School of Medicine, Charlottesville, Va. 22901 (U.S.A.)
(Received November 14th, 1977) Summary 1. Under appropriate conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Oxidation of lactate to CO2 occurs under aerobic conditions. 2. L- and D-lactate are taken up into the cells when oxygen, glucose, or both oxygen and glucose are present in the incubation medium. Both L- and D-lactate are excluded from the cells when neither oxygen nor glucose is present. 3. D,L-Glyceraldehyde prevents the uptake of L-lactate. The effect is apparently n o t due to the inhibition of glucose metabolism by L-glyceraldehyde. 4. L-Lactate (20 mM) markedly inhibits the uptake of 5 mM D-lactate, but 20 mM D-lactate fails to inhibit the uptake of 5 mM L-lactate. 5. Raising the pH of the incubation medium markedly depresses the uptake of L-lactate. 6. The results provide evidence that L- and D-lactate enter the cells of rat aorta by a mediated transport system.
Introduction Until recently it has been assumed that lactic acid enters and leaves muscle cells by diffusing across their plasma membranes. Mainwood and WorsleyBrown [1] provided evidence that efflux of lactate from frog sartorius muscle does n o t occur by simple diffusion. They concluded that both undissociated lactic acid and lactate anion contributed to the efflux, in proportions that depended on the conditions of incubation. The mode of lactate transport in other types of muscle has n o t been considered. Lactic acid plays an especially prominent role in the metabolism of vascular smooth muscle [2]. Even in the presence of high oxygen tensions a large percentage of the glucose utilized is metabolized to lactate. In bovine mesenteric vein, Peterson and Paul [3] showed that about 30% of the ATP produced is
313 derived from aerobic glycolysis under a wide range of conditions. Under some circumstances lactate may serve as an important energy fuel for vascular smooth muscle. Coe et al. [4] f o u n d that lactate was more effective than glucose in restoring the contractility of substrate-depleted rabbit aortic strips. This suggests t h a t exogenous lactate enters the vascular smooth muscle cells and can be oxidized. L u n d h o l m and Mohme-Lundholm [5] found that lactate accumulates intracellularly to extremely high levels in bovine mesenteric vein u n d e r anaerobic conditions and can be released to the incubation medium at a rapid rate. The potential importance of the production and consumption of lactate in vascular smooth muscle stimulated our interest in the mechanisms by which lactate enters and leaves the cells of this tissue. This article reports our studies of the uptake of exogenous lactate by the cells of rat aorta. Methods
Preparation of tissue. Aortas were gently dissected from 200--300-g female Sprague-Dawley rats (Hilltop Farms) anesthetized with carbon dioxide. Each aorta was placed in a petri dish filled with Hanks' balanced salts solution (37°C, gassed with 95% 02/5% CO2). The segment of aorta between the aortic arch and the renal arteries was cannulated with polyethylene tubing and perfused with warmed, oxygenated Hanks' for the rest of the dissection. Fat and extraneous connective tissue were gently removed and th6 section of the aorta between the arch and the renal artery was either: (a) cut transversely into five sections {aortic rings) or (b) slit longitudinally along its length and then cut transversely into five sections {aortic strips). Uptake of L-lactate as a function of extracellular concentration. Aortic strips were incubated in a shaking water bath in glucose-free Hanks' solution containing various concentrations of L-lactate. The shaker was covered and gassed, with 95% 02/5% CO2. The incubation conditions were chosen to favor oxidation of exogenous lactate. At 30, 60, 90, 120 and 180 min of incubation, samples of the incubation medium were taken. Concentrations of L-lactate in medium samples were determined with the L-lactate dehydrogenase m e t h o d {Sigma Biochemical Corp.). The lactate uptake during a given time interval was c o m p u t e d from the a m o u n t of lactate that disappeared from the medium during that period and the results were expressed in nmoles of lactate taken up per min per mg wet tissue. Estimation of ~4C02 production from [~4C]lactate. Rat aortas were obtained as described above and were opened by a lengthwise cut and then cut transversely into two segments. Each artery segment was weighed and placed in a 13 × 100 mm test tube containing 0.5 ml Hanks' solution containing various amounts of L-[U-~4C]lactate. The tube was capped with a serum bottle stopper to the b o t t o m of which was attached a 0.5-ml polyethylene well (Kontes Glass Co.). The tubes were gassed with 95% 02/5% CO2 and incubated at 37°C in a shaking water bath. After 1, 2, 3 or 4 h of incubation, 0.4 ml hyamine hydroxide was added to the polyethylene well and 0.05 ml of concentrated H2SO4 was added to the incubation medium. Both additions were made via needles inserted through the serum bottle stopper. The tube was put back in
314 the incubator-shaker for 1 h to allow collection of the ~4CO2 by the hyam i ne h y d r o x id e. 14CO2 was quantified by counting an aliquot of the hyam i ne in toluene scintillation fluid. The lactate c o n c e n t r a t i o n in the incubation medium at the various time points was determined with the lactate dehydrogenase-based assay (Sigma Biochemical Corp.). The a m o u n t of COs p r o d u c e d from exogenous lactate was c o m p u t e d from the total dpm of ~4CO2 collected and the original d p m / a t o m carbon in the incubation medium.
Uptake of the 14C label from [14C]lactate under various conditions of incubation. Aortic strips or rings were preincubated at 37°C in Hanks' balanced salts solution f o r 30--45 min. T h e y were then incubated in Hanks' containing 5 or 10 mM L- or D-lactate (14C-labeled) and 5 mM sucrose (3H-labeled) under various conditions of incubation. After various periods of incubation, usually 15, 30, 60, 90 and 120 min, medium samples were taken and tissue pieces were removed f r o m the incubation flask. Aortic strips or rings were quickly rinsed in Hanks' solution on a fine stainless steel wire mesh held in a Millipore filter holder, which was attached to a vacuum flask. The aortic strips or rings were immediately weighed and the radioactivity was e x t r a c t e d by boiling in 1.5 ml of water f o r 30 min. The 3H and ~4C radioactivity in the medium samples and tissue extracts were de t e r m i ne d by double label liquid scintillation counting techniques (Beckman LS-230 counter) and the volumes of distribution (spaces) o f the two labels c o m p u t e d . The 3H space is taken as an estimate of the extracellular space o f the tissue. The different incubation conditions e m p l o y e d (in various combinations) were: the presence or absence of oxygen; the presence or absence of 11 mM glucose; the presence or absence of 30 pM 2,4-dinitrophenol; the presence or absence of 20 mM D,L-glyceraldehyde; the presence or absence of the opposite stereoisomer at 20 mM concent rat i on; and various pH values (7.1, 7.4 or 7.7) in the incubation medium. Sucrose is apparently a good marker for the extracellular space in arterial tissue. Jones and Swain [6] f o u n d t hat sucrose and 6°Co • EDTA distribute in the same space in arteries from several species and that inulin distributes in a considerably smaller volume. Using 6°Co • E D T A Jones [7] f o u n d the extracellular space of aorta from 300-g Sprague Dawley rats to be 0.425 ml/g wet tissue. The sucrose space values we obtained are in reasonable agreement with Jones' value. Our sucrose spaces for aortic rings were consistently less than those o f aortic strips, presumably due to less tissue damage in the f o r m e r preparation. We also f o u n d t hat the sucrose space varies considerably with the conditions of incubation. Estimation of total tissue water content. Total tissue water c o n t e n t was estim a t e d f r o m tissue weights before and after dessication and from the volume of distribution of ~4C-labelled urea. Aortic strips were incubated aerobically in Hanks' solution for 90 min. T h e y were then removed from the incubation medium, rinsed, b l ot t ed and immediately weighed on tared aluminum pans. The tissue pieces were t hen dried to cons t a nt weight in an oven at 95°C. From the wet and dry weights, the tissue water c o n t e n t was calculated to be 0.701 +_ 0.014 (S.E.) ml/g wet tissue. T o determine the volume of distribution of urea, aortic strips were incubated in Hanks' solution containing 3.33 mM urea (14Clabeled). Af ter 15, 30, 60, 120 and 180 min of incubation, samples of the incubation med iu m were taken and tissue samples were removed. The tissue pieces
315
were blotted on Whatman No. 50 filter paper, weighed and boiled in 1.5 ml of water to ex tr ac t the radioactivity. The space of 14C-labelled urea was at a steady-state value by 15 min of incubation and did n o t change over the rest of the incubation. The average urea space from 15 to 180 min of incubation was 0.709 +_0.015 (S.E.) ml/g wet tissue. Estimation o f the volume o f distribution o f unmetabolized [14C]lactate. In some of the experiments described above in which the uptake of [14C]lactate and [3H]sucrose were determined, the medium samples and tissue extracts were subjected to thin-layer c h r o m a t o g r a p h y on cellulose (Eastman Chromagram) with butanol/propionic acid/water, 63 : 31 : 5, as the developing solvent. The fraction of ~4C label co-migrating with a lactate standard was det erm i ned by liquid scintillation techniques. The dpm of 14C-lactate in the original medium sample or tissue e x t r a c t was then c o m p u t e d as the total ~4C dpm multiplied by the fraction present as unm e t a bol i z e d lactate. The volume of distribution of the u n m e t a b o l i z e d [~4C]lactate was c o m p u t e d from the dpm of unm et abol i zed lactate per mg wet tissue and the dpm of u n m e t a b o l i z e d lactate per tA incubation medium. Purity o f the D- and L-lactate utilized. The D- and L-lactates (~4C-labeled) used were obtained from Amersham-Searle and warranted by the supplier to be greater than 95% radiopure. This was c o n f i r m e d by thin-layer c h r o m a t o g r a p h y as described in the preceding paragraph. In addition we verified that the D - and L-lactates were pure in their stereoisomer c o n t e n t in the following m anner [8]. Approx. 0.5 t~mol of 14C-labeled D- or L-lactate was incubated with L-lactate dehydrogenase, NAD ÷, glutamic pyruvic transaminase and glutamate for 45 min at 37°C. The reaction m i xt ur e was deproteinized and subjected to thinlayer c h r o m a t o g r a p h y as described above. Greater than 98% of the L-lactate was co n v er ted to pyruvate and alanine, but essentially none of the D-lactate was converted to these products. Thus, within the limits of sensitivity of this m e t h o d , we find t hat the L-[14C]lactate is pure and is u n c o n t a m i n a t e d with D-[~4C]lactate. The D-[~4C]lactate is n o t c o n t a m i n a t e d with L-[~4C]lactate in measurable amounts. Results and Discussion
Dependence of L-lactate uptake rate on the concentration o f L-lactate in the incubation medium. The rate of lactate upt ake during the first 30 min of incubation was high. This is a t t r i but ed to the rapid distribution of lactate in the extracellular space of the tissue. During the remainder of the incubation the rate of lactate u p t a k e was lower and relatively const ant for at least 120 min more. Lactate u p ta ke was c o m p u t e d as the mean of the upt ake rate values for four 30-min incubation periods after the initial 30-min period. The rate of lactate uptake increased with increasing lactate c o n c e n t r a t i o n in the incubation medium up t o approx. 5 mM lactate. Increasing the medium lactate level above 5 mM did n o t cause a f ur t her increase in the rate of lactate uptake. We f o u n d that the m a x i m u m rate of lactate upt ake u n d e r these conditions was a b o u t 0.05 n m o l / m i n per mg wet wt., and the lactate c o n c e n t r a t i o n in the med iu m for half-maximal upt ake rate was approx. 2.5 mM. In similar experiments with aortic rings incubated in 10 mM L-lactate, the rate of lactate
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F i g . 1. T h e r a t e o f p r o d u c t i o n o f 1 4 C O 2 b y s t r i p s o f r a t a o r t a f r o m L - [ U - 1 4 C ] l a c t a t e w e t t i s s u e is p l o t t e d vs. t h e c o n c e n t r a t i o n o f L - l a c t a t e i n t h e i n c u b a t i o n m e d i u m .
in nmol/min
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F i g . 2. A e r o b i c i n c u b a t i o n o f a o r t i c r i n g s i n t h e p r e s e n c e a n d a b s e n c e o f g l u c o s e . T h e a v e r a g e s p a c e s i n ml/g wet tissue of 14C (from L-[14C]lactate) a n d 3 H ( f r o m [3 H ] s u c r o s e ) a r e p l o t t e d v e r s u s t i m e . T h e horizontal bar indicates the total tissue water space. The vertical bars represent standard errors.
uptake was 0.068 + 0.015 (S.E.) n m o l / m i n per mg wet tissue. We are n o t aware of other determinations of the rate of uptake of L-lactate by vascular tissues. It is n o t e w o r t h y that our maximal rates of lactate uptake are comparable to the rates of glucose utilization that have been reported for arterial tissue and fall within the ranges of glucose utilization values reported for swine aorta [9], rabbit aorta [9] and rat aorta [10]. The concentration of lactate in the arterial blood of Wistar rats anesthetized with N20 is 2.84 mmol/1 of blood under control conditions [11]. The blood lactate level increased about 4.5-fold when the arterial blood pressure was dropped to 25--35 mmHg by bleeding [12] and arterial lactate increased 3-fold due to a 3-min period of asphyxia [11]. The control level of blood lactate is close to the concentration that, in our experiments, results in half-maximal lactate uptake by rat aortic strips. The blood levels reached in certain types of physiological stress are apparently sufficient to saturate lactate uptake by the aorta. Production of 14C02 from L-[14C]lactate. As shown in Fig. 1, a major fraction of the 14C carbons of the 14C-labeled L-lactate taken up by the rat aortic strips can be collected as 14CO2. This supports the contention that under these experimental conditions L-lactate is readily oxidized by rat aorta. The high rate of lactate uptake by the tissue and its apparent ease of oxidation are consistent with the idea that, under some circumstances, oxidation of exogenous L-lactate may be an important energy source for arterial tissue. Dependence of L-lactate uptake on the presence of glucose and oxygen in
317 1--
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Fig. 3. A n a e r o b i c i n c u b a t i o n of a o r t i c rings in t h e p r e s e n c e o f 11 m M glucose ( p a n e l A ) a n d in t h e a b s e n c e of glucose ( p a n e l B). T h e spaces of 14C ( f r o m [ 1 4 C ] l a c t a t e ) a n d 3 H ( f r o m [ 3 I - I ] s u c r o s e ) i n m l ] g w e t tissue are p l o t t e d vs. t i m e . T h e v e r t i c a l bars n e a r t h e d a t a p o i n t s r e p r e s e n t t h e s t a n d a r d errors. T h e h o r i z o n t a l lines i n d i c a t e t h e t o t a l tissue w a t e r space.
the incubation medium. As shown in Fig. 2, under aerobic conditions L-lactate uptake did n o t depend on the presence of glucose in the incubation medium. Both with and w i t h o u t glucose, the ~4C label from the lactate distributed in a space that exceeded the total tissue water. Thin layer chromatography of tissue extracts showed that a substantial fraction of the 14C in the tissue was present as compounds that did not co-migrate with lactate and thus are presumably metabolic products of L-lactate. Under anaerobic conditions, as shown in Fig. 3, L-lactate was readily taken up in the presence of glucose in the incubation medium. However, when glucose was omitted from the medium, the 14C label from lactate was confined to a volume of distribution that did n o t exceed the sucrose space of the tissue. This strongly suggests that under these conditions L-lactate failed to enter the cells of the aorta. Thin-layer chromatography of tissue extracts showed t h a t in the presence of glucose metabolites were present in the tissue, while in the absence of glucose at least 95% of the tissue 14C co-chromatographed with lactate. It thus appears that oxidative metabolism is n o t required for the uptake of L-lactate provided that glucose is present. This interpretation is consistent with the finding that 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, had no significant effect on the rate of L-lactate uptake under aerobic conditions (Fig. 4). Effect of D,L-glyceraldehyde on L-lactate uptake. As shown in Fig. 5, the presence of D,L-glyceraldehyde markedly inhibited the uptake of ~4C from L-[~4C]lactate u n d e r anaerobic conditions in the presence of glucose. Under these conditions the 14C label was apparently confined to the extracellular space of the tissue when D,L-glyceraldehyde was present. This suggests that D,L-glyceraldehyde acted to inhibit transport of lactate into the cells. We originally believed that the effect of glyceraldehyde might be due to inhibition of glucose metabolism since L-glyceraldehyde is an inhibitor of hexokinase. However, we f o u n d that glyceraldehyde had the same inhibitory effect under aerobic conditions in the absence of glucose, which argues against this inter-
318
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Fig. 4. A e r o b i c i n c u b a t i o n o f aortic strips in t h e p r e s e n c e o f 3 0 # M 2 , 4 - d i n i t r o p h e n o l and in the a b s e n c e o f the inhibitor. T h e s p a c e s o f 1 4 C ( f r o m [ 1 4 C ] l a c t a t e ) and 3 H ( f r o m [ 3 H ] s u c r o s e ) in m l / g w e t tissue are the averages o f results f r o m t w o e x p e r i m e n t s . T h e h o r i z o n t a l bar i n d i c a t e s t h e t o t a l tissue w a t e r space. Fig. 5. E f f e c t o f D , L - g l y c e r a l d e h y d e . A o r t i c rings w e r e i n c u b a t e d a n a e r o b i c a l l y w i t h 1 1 m M g l u c o s e in the p r e s e n c e o f 2 0 m M D , L - g l y c e r a l d e h y d e and in its a b s e n c e . S p a c e s o f 1 4 C ( f r o m [ 1 4 C ] l a c t a t e ) and 3 H ( f r o m [3 H ] s u c r o s e ) in m l / g w e t tissue are p l o t t e d vs. t i m e . 3 H s p a c e s in the a b s e n c e o f g l y c e r a l d e h y d e have b e e n o m i t t e d for clarity; t h e y did n o t d i f f e r s i g n i f i c a n t l y f r o m the 3 H ( s u c r o s e ) s p a c e s s h o w n . T h e h o r i z o n t a l bar d e n o t e s t h e t o t a l tissue w a t e r space.
pretation. It is possible that D- or L-glyceraldehyde more directly inhibits lactate entry. Uptake of D-lactate. Under aerobic incubation conditions with no glucose in the incubation medium, 14C label from D-[14C]lactate distributed in a volume that significantly e x c e e d e d t h e total tissue water. Thin-layer chromatography of tissue extracts showed/that a significant fraction of the 14C did n o t co-migrate with D-lactate, indicating the presence of metabolic products of D-lactate. As was the case for L-lactate, in the absence of both o x y g e n and glucose in the incubation medium ~4C from D-lactate was confined to a space equal to the sucrose space. This suggests that under these conditions D-lactate did n o t enter the cells of the tissue. Effect of the presence of the opposite stereoisomer on lactate uptake. As s h o w n in Fig. 6A, the presence of 20 mM L-lactate markedly inhibited the uptake of 5 mM D-[14C]lactate. On the other hand, the presence of D-lactate at 20 mM had no detectable effect on the rate of uptake of L-[14C]lactate (Fig. 6B). The space of unmetab01ized D-[14C]lactate was determined in the presence and absence of 20 m M L-lactate and was f o u n d to be essentially constant from 30 to 120 min of incubation. The average space of unmetabolized D-[~4C]lactate in the absence of L-lactate was 0.502 + 0.023 (S.E.) ml/g wet tissue and in the presence of L-lactate was 0 . 4 6 9 + 0 . 0 0 8 (S.E.) ml/g wet tissue. If the effect of L-lactate were to inhibit the metabolism of D-[14C]lactate, the
319
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Fig. 6. E f f e c t o f o p p o s i t e s t e r e o i s o m e r o n l a c t a t e u p t a k e . A o r t i c rings w e r e i n c u b a t e d in t h e p r e s e n c e of glucose a n d o x y g e n w i t h 5 r a m [ 1 4 C ] l a c t a t e in t h e p r e s e n c e a n d a b s e n c e o f t h e o p p o s i t e s t e r e o i s o m e r a t 20 raM. Panel A; t h e u p t a k e of 14C f r o m D - [ 1 4 C ] l a c t a t e w i t h ( . . . . . . ) and without ( ) 20 raM L - l a c t a t e . S u c r o s e s p a c e s are s h o w n b y t h e l o w e r p o i n t s . P a n e l B: t h e u p t a k e o f 14C f r o m L - [ 1 4 C ] l a e t a t e in t h e p r e s e n c e ( e ) a n d a b s e n c e (o) o f 20 m M D - l a c t a t e . S p a c e in r a l / g w e t tissue is p l o t t e d vs. t i m e . T h e v e r t i c a l b a r s r e p r e s e n t s t a n d a r d errors.
space of unmetabolized D-[14C]lactate should have been larger in the presence of L-lactate than in its absence. We observed the reverse, which supports the interpretation that L-lactate inhibits the transport of D-lactate into the cells. Effect of the pH of the incubation medium on uptake of L-lactate. As shown in Fig. 7, increasing the pH of the incubation medium caused a marked
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Fig. 7. E f f e c t of t h e p H of t h e i n c u b a t i o n m e d i u m . A o r t i c rings w e r e i n c u b a t e d a e r o b i c a l l y w i t h 11 raM glucose in i n c u b a t i o n m e d i a w i t h p H 7.1, 7.4 o r 7.7. T h e s p a c e o f 14C ( f r o m [ 1 4 C ] l a c t a t e ) a n d 3 H ( f r o r a [ 3 H ] s u c r o s e ) in r a l / g w e t tissue are p l o t t e d vs. t i m e . T h e v e r t i c a l bars n e a r t h e d a t a p o i n t s r e p r e s e n t stand a r d errors. T h e d a t a f o r p H 7.7 are t h e m e a n s o f t w o e x p e r i m e n t s w i t h n e a r l y i d e n t i c a l results. T h e u p p e r c u r v e s ( w i t h t h e p H values n o t e d a l o n g s i d e ) are 14C s p a c e s a n d t h e l o w e r c u r v e s are t h e 3 H spaces. T h e v e r t i c a l bars r e p r e s e n t s t a n d a r d e r r o r s .
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decrease in the rate of uptake of the 14C from L-[14C]lactate. At pH 7.1 only 0.1% of the total lactate in the incubation medium is in the form of the undissociated acid. Increasing the pH by 0.3 units will halve the concentration of undissociated acid, but will change the concentration of lactate anion negligibly. The data are consistent with the interpretation that a substantial fraction of the lactate that enters the cell does so as the undissociated acid. An alternative interpretation is that the data reflect primarily the pH dependence of the activity of a membrane lactate anion carrier. Mainwood and Worsley-Brown [1] concluded that in frog sartorius both the anion and acid forms may contribute to lactate efflux from the cells. Interpretations of the results. We have shown that under the appropriate incubation conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Some of our results are inconsistent with the idea that lactate entered the cells by simply diffusing across the plasma membrane. These are: (1) that L- and D-lactate failed to enter the cells when both glucose and oxygen were absent from the incubation medium, (2) that D,L-glyceraldehyde prevented the entry of L-lactate into the cells, and (3) that L-lactate was able to markedly depress the rate of transport of D-lactate by the tissue. These results are consistent with the interpretation that L- and D-lactate enter the cells of rat aorta by a mediated transport system. The inhibition )f the uptake of D-lactate by a 4-fold excess of L-lactate, together with the failure of a 4-fold excess D-lactate to inhibit uptake of L-lactate, is consistent with the interpretation that the mediated transport system has a higher affinity for L- then for D-lactate. These results provide support for the idea that lactate enters the cells of rat aorta by a mediated transport system. Our studies have done little to define the nature of this transport system, however. The form of lactate that is transported, the structural specificity of the transport system, and the nature of the apparent relationship of the transport system to cellular metabolism remain to be determined. Acknowledgements This research was supported by grants HL 15716 and HL 17967 and by Research Career Development Award HL 00014 to H.K. from the National Institutes of Health. It is a pleasure to acknowledge the participation of Charles Epstein in the early stages of this study. References 1 M a i n w o o d , G.W. a n d W o r s l e y - B r o w n , P. ( 1 9 7 5 ) J. P h y s i o l . 2 5 0 , 1 - - 2 2 2 L e h n i n g e r , A . L . ( 1 9 5 9 ) in T h e A r t e r i a l Wall ( L a n s i n g , V . A . , e d . ) , p p . 2 2 0 - - 2 4 6 , Williams a n d Wilkins, Baltimore 3 P c t e r s o n , J.W. a n d P a u l , R . J . ( 1 9 7 4 ) B i o c h e m . B i o p h y s . A c t a 3 5 7 , 1 6 7 - - 1 7 6 4 C o e , J., D e t a r , R. a n d B o h r , D . F . ( 1 9 6 8 ) A m . J. P h y s i o l . 2 1 4 , 2 4 5 - - 2 5 0 5 L u n d h o l m , L. a n d M o h m e - L u n d h o l m , E. ( 1 9 6 0 ) A c t a P h a r m a c o l . T o x i c o l . 1 6 , 3 7 4 - - 3 8 8 6 J o n e s , A.W. a n d S w a i n , M.L. ( 1 9 7 2 ) A m . J. P h y s i o l . 2 2 3 , 1 1 1 0 - - 1 1 1 8 7 J o n e s , A.W. ( 1 9 7 3 ) Circ. Res. 3 3 , 5 6 3 - - 5 7 2 8 Lowry, O.H. and Passonneau, J.V. (1972) A Flexible System of Enzymatic Analysis, pp. 194--199 A c a d e m i c Press, N e w Y o r k 9 M o r r i s o n , E.S., S c o t t , R . F . , K r o m s , M. a n d F r i c k , J. ( 1 9 7 2 ) A t h e r o s c l e r o s i s 16, 1 7 5 - - 1 8 4 1 0 D a l y , M.M. ( 1 9 7 6 ) A m . J. P h y s i o l . 2 3 0 , 3 0 - - 3 3 11 K a a s i k , A . E . , Nilsson, L. a n d SiesjS, B . K . ( 1 9 7 0 ) A c t a P h y s i o l . S c a n d . 7 8 , 4 3 3 - - 4 4 7 12 K a a s i k , A . E . , N i l s s o n , L. a n d SiesjS, B.K. ( 1 9 7 0 ) A c t a P h y s i o l . S c a n d . 7 8 , 4 4 8 - - 4 5 8