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The Choroid Plexus as a Glucose Barrier
147
The Choroid Plexus as a Glucose Barrier Dcparttiierit of Ptiarniacology, University of Ker;tucky College of Medicitre, Lexirigton, Ketitrtcky ( U.S.A.)
The choroid plexus is composed of strongly vascularized connective tissue lined with an epithelial layer. It has long been considered a “barrier” between the blood and the cerebrospinal space. The luminal membrane of the epithelial lining shows a typical microvillus structure similar to that of the epithelium of the small intestine or kidney tubule (Millen and Woollam, 1962). As the latter organs are involved in the biological transport of sugars, by analogy a similar role could be presumed for the choroid plexus. The present paper summarizes the results of preliminary experiments aimed at clarifying the role of the choroid plexus in the maintenance of glucose concentration between the blood and cerebrospinal fluid (CSF). I t has been known for some time that CSF contains less glucose than blood does (Davson, 1956). The relationship is roughly 1 : 2. Moreover, a change in the glucose concentration of the blood plasma is followed, after some delay, by a corresponding change in the sugar concentration of the CSF,still maintaining the approximate 1 : 2 concentration ratio. It is clear that such an uneven equilibrium cannot be maintained by a passive barrier alone. Theoretically, three possibilities could be proposed for such a distribution : ( I ) Glucose could be filtered by the plexus and then metabolized in the CSF. (2) Glucose could be actively secreted from the blood into the cerebrospinal space by the choroid plexus. (3) Glucose could continually be filtered somewhere else and then actively reabsorbed by the choroid plexus. The first possibility would require a specific mechanism that would regulate the metabolic disappearance of glucose at a rate depending on the concentration of the sugar in the blood plasma. Although the existence of such metabolic regulatory re:eptors is theoretically not impossible, in actuality it has not been demonstrated. With the second possibility, it is somewhat difficult to envision a layer of epithelium that would secrete a solute from the blood into the CSF and maintain it at half its concentration level in the blood. There are glands, like the salivary glands, that produce secreta hypertonic to blood; however, this is done not by a simple transport across an epithelial layer, but by a process of filtration followed by selective reabsorption at a different site. The third possibility, namely that a plasma filtrate is continually produced and gluR
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T. Z . C S k K Y , B. M. R I G O R
coseis thenreabsorbed from it by the epithelium of the choroid plexus, would requirea low-capacity sugar pump in the latter. The function of such a pump would be limited by the concentration gradient against which it would have to transport. Thus it is quite possible that the limit of the gradient would be I : 2, which would result in the maintenance of the very uneven equilibrium between the blood and CSF as it was actually observed. In the following, evidence is presented that there exists a sugar pump in the choroid plexus, that this pump is of low capacity, and that it actively transports sugar from the CSF into the blood stream. Sugar-concentrating transport mechanism in the choroid plexus The following experimental procedure was performed (Csaky and Rigor, 1964). Choroid plexuses were removed from pentobarbital-anesthetized dogs and placed in a Krebs-Henseleit bicarbonate Ringer's solution containing a very small amount (7 pM)of 14C-labeled sugar. After gassing with 95 % 0 2 5 % COz, the vessels were shaken in a Dubnoff metabolic shaker for 1 h at 37" C. At the end of this period the plexuses were removed, gently blotted, and placed on a small piece of carefully preweighed filter paper. They were dried to obtain the total water content. The dry residue was combusted in a closed oxygen atmosphere, and the resulting W02was determined in a liquid-scintillation spectrometer using the scintillation liquid as described earlier (Kalberer and Rutschman, 1961). I n a number of experiments, radio-iodinated serum-albumin was also added to the Krebs-Ringer solution, and the distribution of radioiodine was determined in the choroid plexus. Assuming that this protein occupies only the extracellular space, the intracellular space of the choroid plexus was computed and the results calculated in such a fashion that the distribution ratio of radioactivity between the intracellular water of the choroid plexus and the incubating medium was
+
TABLE I R A T I O O F C O N C E N T R A T I O N O F R A D I O A C T I V I T Y I N T H E I N T R A C E L L U L A R W A T E R OVER T H E MEDIUM ( I / M ) I N T H E C H O R O I D P L E X U S F O L L O W I N G I N C U B A T I O N W I T H T H E L A B E L E D SUGARS
Sugar
Glucose Galactose 3-0-Methylglucose
I/
Number of experinrents
(mean 4 standard deviation)
26 35 25
21.34 & 3.01 19.70 t 3.36 0.70 +. 0.32
obtained. Table I shows that the radioactivity in experiments with glucose and galactose was concentrated more than twenty-fold in the intracellular space of the choroid plexus. However, it is interesting that 3-0-methylglucose (3-methylglucose) was not concentrated, although in other transport systems, such as the intestine, this sugarether behaves very much like glucose or galactose (Cshky, 1942, 1958). Glucose and galactose were metabolized by most tissues, whereas 3-methylglucose is not metabo-
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C H O R O I D P L E X U S AS G L U C O S E B A R R I E R
lized in animal ticsue (Csaky and Wilson, 1956). It was therefore of interest to examine whether or not the uneven distribution of the radioactivity between the intracellular water of the choroid plexus and the surrounding nutrient medium in the case of glucose and galactose was simply the result of transformation of the sugar to labeled metabolites, and not of true accumulation. For this reason choroid plexuses, having been incubated with radioactiveglucose or galactose, were extracted with hot distilled water. The protein-free filtrate was concentrated and chromatographed on filter paper, using either pyridine-water-butanol or acetic acid-water-butanol solvent mixtures (Tyszkiewicz, 1962). In the case of galactose it was found that about 70-75 % of the total radioactivity could be recovered in one spot, the migration of which was identical with that of the reference galactose. About 5 % of the radioactivity was found in another spot, the migration of which was identical with a reference galactose-I-phosphate. The rest of the radioactivity was distributed in various spots, the identity of which was not established. In the case of glucose, 60-70% of unchanged glucose was found in the extract. Also, glucose phosphate and unknown spots were located. These results indicate that even when making corrections for a possible slight metabolism there is a considerable concentration of true unchanged glucose and galactose in the choroid plexus. It is known that the intestinal sugar pump is inhibited by a number of factors, such as the lack of sodium in the medium; a low concentration of digitalis, 2,4-dinitrophenol, or phlorizin; or the lack of oxygen. In order to ascertain whether the choroid T A B L E 11 I N H I B I T I O N O F A C C U M U L A T I O N O F S U G A R S B Y L A C K O F S O D I U M , B Y I N H I B I T O R S A N D BY A N O X I A . F I G U R E S R E P R E S E N T T H E PER C E N T I N H I B I T I O N I N P A I R E D E X P E R I M E N T S
Glucose
Potassium Ringer
Lithium Ringer
Mannitol Ringer
Ouabain lO-4M
Phlorizin 10-3M
Dinitrophenol 10-3~
Anoxia
37 51 48 48
30 54 59 65 66 36
38 63 51 74 83
22 41 40 28 21
57 47 56 38 34
93 96 98 96
89 90 95 93
-
-
-
-
-
-
-
Average
46.0
51.7
61.8
30.4
46.4
96.3
91.7
Galactose
87 73 57 47 32
29 37 51 27 33
50 40 45 39
32 47 40 42
72 75 76 60 80 49
96 95 87 98
80 92 93 87
-
-
-
57
22
Average ~~
Rrfermces p . I54
-
__
-
-
53.6
35.4
43.5
40.3
68.7
94.0
88.0
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T. Z. C S A K Y , B. M. R I G O R
plexus sugar pump is similarly inhibited, the following experimental procedure was developed :two lateral ventricle choroid plexuses were removed from dog and compared in pairs. One was incubated as a control in a normal Krebs-Ringer solution; the other, in a modified solution or in the presence of the inhibitor. The results were then expressed as percentage of inhibition versus the control. Table 11 shows that the choroid plexus sugar pump is inhibited by the same factors as the intestinal sugar pump, however, to a different degree. Two facts deserve specific mention: the lack of absolute dependence ofthechoroid plexus sugar pump on sodium in the incubating medium and the very strong dependence on the presence of oxygen. The former is in contrast with what one finds in the intestine (Csaky, 1960). The possibility that sodium leaks out of the tissue into the medium is of little importance because the plexuses were incubated in a relatively large volume of the medium (the tissue water diluted at least 1 : 50). Moreover, it was found that all sodium-dependent transports are readily inhibited with relatively low concentration of digitalis (Csaky, 1963). On the one hand, the sugar pump of the choroid plexus is only partially depressed by the presence of the cardioglucoside. On the other hand, the strong dependence on oxygen and the complete inhibition of the pump by low concentration of 2,4-dinitrophenol suggest that the transport mechanism derives its energy mainly from a direct oxidative process. The capacity of the concentrating pump was examined by incubating the tissue with increasing amounts of the sugar. As can be seen in Table 111, the galactose-accumulating system became saturated between the concentrations of 0.5-1.1 mM. By analogy it can be assumed that the glucose pump is also a low capacity pump.
T A B L E 111 I N H I B I T I O N OF T H E S U G A R C O N C E N T R A T I O N BY L A R G E A M O U N T OF S U G A R I N T H E M E D I U M . F I G U R E S R E P R E S E N T THE PER C E N T I N H I B I T I O N IN P A I R E D E X P E R I M E N T S ( C O N T R O L A N D E X P E R I M E N T A L P L E X U S FROM T H E SAME D O G )
Labeled sugars (7P M ) Glucose
Average
Average
Non-labeled sugars (30 m M ) Glucose
Galaciose 3-0-Meiliylglucose
99 96 98 98
55 75 49 77
54 53 69 49
97.8
61.2
-_
71 43 83 69
-
98 98 99 98
-
30 22 57 43
66.5
98.3
38.0
50
56.3
__
CHOROID PLEXUS AS GLUCOSE BARRIER
151
T A B L E 1V SATURATION O F THE GALACTOSE CONCENTRATING
Galactose in the Mediuni ltrert Radioactive Total (I'M)
(PM)
(ItM)
7
7 63 118 567 1127
0 56
7
560 1120
7
111
7 7
SYSTEM IN T H E C H O R O I D P L E X U S
Mean 11M
20.73 13.95 6.47 5.04 0.58
The possible active transport of 3-methylglucose in the choroid plexus merits further comment. As was seen, this sugar-ether is not concentrated in the plexus in vitro; however, the presence of a large amount of 3-methylglucose is capable of inhibiting the concentration of both glucose and galactose in the plexus (Table IV), as if the sugarether would compete with the other sugars for the transporting sites. Such behavior is not uncommon. In our laboratory we found that 3-methylglucose is most likely actively absorbed in the renal tubulues, yet in vitro it is not accumulated by the kidney slides*. Thus it is quite possible that, in spite of the lack of measurable accumulation, 3-methylglucose would still be actively transported across the epithelial layer of the plexus.
Direction of the sugar pump in dog choroid plexus in vivo The experiments described above demonstrate the presence of a concentrating pump in the choroid plexus but do not suggest anything about its direction. In other epithelial cells (kidney tubule or intestine), sugar is transported from the direction of the brush border towards the direction of the basal membrane. By analogy, then, the morphology of the choroid plexus epithelium suggests an active reabsorption of sugars from the CSF into the blood. To test this possibility, the following experiments were performed : dogs were anesthetized with sodium pentobarbital, and both lateral ventricles were tapped and cannulated, and an artificial CSF (Pappenheimer et al., 1961) was recirculated with a velocity of approximately 5 ml/min. The glucose content of the artificial CSF was less thanin the blood. From time to time, the glucose concentration in the perfusate was determined by the glgcostat technique (Teller, 1956). At the same time arterial blood samples were taken and the glucose concentration measured in the plasma by using the same method. Results of a set of typical experiments are reproduced in Fig. 1, which shows that there was a steady disappearapce of glucose from the cerebrospinal space into the blood. Because it probably proceeds up-hill, by definition the transport is active. This active transport is completely eliminated, however, if in the artificial CSF sodium was replaced by potassium or if digitalis or phlorizin was added to the medium.
* Unpublished Refcrenccs p . 154
observations.
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T. Z. C S A K Y , B. M. R I G O R
HR.
HR.
HR.
nn.
Fig. 1 . Perfusion of the lateral ventricles in the dog. Solid line (CSF): glucose concentration in the perfusate. Broken line: glucose concentration in the arterial blood plasma. First column : control; 2nd col.: Na+ was replaced by K+ in the perfusing medium; 3rd col.: Digitoxin (IO-4M) added to the perfusate; 4th col.: Phlorizin (lO-4M) added to the perfusate.
Sugar transport in the isolated choroid plexus of the horse
A more direct evidence regarding the direction of active sugar transport in the choroid plexus was obtained from experiments on the isolated horse choroid plexus in vitro. Healthy horses (obtained through the courtesy of the Department of Animal Pathology) were anesthetized with sodium pentobarbital, their skulls were opened, and the lateral plexus with some surrounding brain tissue was quickly removed, placed on ice, and carried rapidly to the laboratory, where the anterior and posterior choroidal arteries and the main choroidal vein were cannulated with small polyethylene tubings. The procedure between the removal of the plexus and the start of the experiment did not take longer than 10-20 min. The choroid plexus from the ventricle side was bathed in a Krebs-Ringer bicarbonated solution containing 50 mg% of galactose. An identical solution was infused slowly into the choroid artery. The outflow from the choroid vein was collected and analyzed for galactose by a method utilizing specific galactose oxidase*. The entire preparation was kept in a 37°C moist chamber, and oxygencarbon dioxide (95-5 %) was continuously bubbled through the bath. At the end ofthe experiment a dye was introduced into the artery. If the dye appeared in the bath, indicating a leak between the blood and the cerebral spinal space, the preparation was discarded. The galactose concentration of the venous outflow increased continuously during the experiment, as shown in Fig. 2. Such results would be possible only if the galactose was actually transported from the outside into the blood by active transport, or if the blood was losing water. In order to check the latter, in a few experiments
* Pamphlet 6-65
“Galactostat”, Worthington Biochem. Corp.
CHOROID PLEXUS AS GLUCOSE BARRIER
153
Fig. 2. Horse choroid plexus perfused in vifro. Broken line: galactose concentration in the arteria input. Solid line: galactose concentration in the venous outflow. First column: perfusion with KrebsRinger: 2nd col.: Na+ replaced by K+ in the perfusing Krebs-Ringer: 3rd col.: perfused with KrebsRinger, gassed with N2.
radioactive inulin was added to the fluid which was pumped into the artery. The inulin concentration did not change in the venous outflow, indicating that there was no appreciable loss of water from the blood in the preparation. Consequently, one must conclude that in this preparation galactose was actually transported from the outside into the bloodstream against a concentration gradient by active transport. It is also noteworthy that this active transport was eliminated by the absence of sodium or by anoxia. CONCLUSIONS
Evidence is presented of an accumulating low-capacity sugar pump in the choroid plexus that transports glucose or galactose from the ventricle into the bloodstream. The experimental results would be in accord with the following working hypothesis: the CSF is essentially a blood filtrate in which glucose is originally present in the same concentration as in the plasma. The choroid plexus possesses an actively transporting pump system which continuously reabsorbs glucose from the cerebral spinal fluid into the blood. Because the pump is of low capactiy, the net transport will be determined by the gradient against which the transport has to take place. As a result, the glucose content in the CSF is always below that in the plasma but is in rough accord with the concentration fluctuations in the latter. As it happens, the pump is regulated so that a ratio of roughly 1 : 2 is maintained. Rrfercnces p. 154
154
T. Z. C S A K Y , B. M. R l G O R ACKNOWLEDGEMENT
This work was supported in part by research grants from the U.S.P.H.S. and the American Heart Association. REFERENCES CSAKY,T. Z. (1942) Hoppe-Seyler’s Z . Physiol. Chem., 277, 47. CSAKY, T. Z. (1958) Intern. Abstr. Biol. Sci. Suppl., 6 , 79. CSAKY, T. Z. (1963) Biochim. Biophys. Acta, 74, 160. CSAKY,T. Z. AND RIGOR,B. M. (1964) Li/e Sci., 3, 931. CSAKY, T. Z. AND THALE, M. (1960) J . Physiol. (Lond.), 151, 59. CSAKY,T. Z. AND WILSON, J. E. (1956) Biochim. Biophys. Acta, 22, 185. DAVSON, H. (1956) Physiology of the Ocular ana Cerebrospinal Fluids. Boston, Little Brown. KALBERER, F. AND RUTSCHMAN, J . (1961) Helv. Chim. Acta, 44, 1956. MILLEN, J. W. AND WOOLLAM, D. H. M. (1962) The Anatomy of the Cerebrospinal F h i d Londcn, Oxford University Press. J. R., HEISEY, S . R. AND JORDAN, E. F. (1961) Amer. J . Physiol., 200, 1. PAPPENHEIMER, TELLER, J. D. (1956) Abstract of Papers, 130th Meeting, Amer. Chem. SOC.,69c. TYSZKIEWICZ, E. (1962) Anal. Biochem., 3, 164. D I S C U S S I ON D. B. TOWER: I think this is very interesting work of Dr. Csaky’s, and I would like to ask several related questions. It has been known for a good many years in clinical practice that when the blood glucose level rises above a certain point, and if my memory serves me correctly it is about 300 mg%, then the cerebrospinal fluid glucose level no longer rises. One has the impression in looking at these figures that the pump is saturated in the opposite direction. In other words, the glucose is pumped into the central nervous system, and if one increases the glucose level on the side from which the .glucose is being pumped, the pump can be saturated. This obviously does not agree with your data; so I am wondering if you have an explanation for this clinical observation. The second point relates t o some experiments that Dr. Fishman did in the dog. Unfortunately I don’t think he published the data, but he told me that he studied the effects of phlorhizin, both given into the blood and injected into the cerebrospinal fluid. I think he used up to 1 0 - 3 M concentrations, and got no effect on the entry of glucose into the central nervous system. I just wonder how you would interpret this observation of Dr. Fishman’s on the studies in vivo in the dog. Finally, I wonder what is the role of glucose metabolism in the central nervous system in your system; since glucose is the major substrate being used by the brain and a t a very substantial rate (about 65% of the glucose consumed by the body per unit time is consumed by the central nervous system). I find it a little difficult to visualize how one may conceive of an outward pump for glucose together with this type of metabolic system.
T. Z. CSAKY:For the first question, regarding the effect of phlorizin, I can provide only a partial answer. In our experiments we obtained an inhibition with this glucoside both in vitro and in vivo, but only a partial one. So it is possible that the sugar carriers are different in different anatomical entities. Concerning sugar transport into the brain at very high sugar concentration: we did not study such a phenomenon directly. However, one can speculate about this problem by analogy. E.g., it is known that if the metabolic pattern of glucose is changed, its transport rate also changes in some tissues. Thus, semi-starvation leads t o an increase in the rate of glucose absorption from the intestine. Perhaps it would be conceiveable that the high glucose containing blood “overfeeds” the brain cells and thereby decreases the rate of transport. With regard to your third point about the slices: I do not think that the brain cells are fed through the choroid plexus, but that glucose enters the brain from the capillaries via the mediation of the glia. In connection with this point I may mention that in our laboratory we recently found an active sugar pump in the isolated glial cells grown in tissue culture.
C H O R O I D P L E X U S A S GLUCOSE B A R R I E R
1 S5
D. P. RALL:In which direction? T. Z. CSAKY:Working essentially with a cell suspension we cannot at present answer this question. We could only demonstrate an energy dependent accumulation of the sugar in the cell. But it is not inconceivable that the glia pumps glucose from the blood into the brain. Of course, in brain slices the glia c:lls are also included. I think the choroid plexus is different. Its epithelium morphologically and perhaps also functionally seems to be like that of the kidney tubule or intestinal mucosa.
D . B. TOWER:Could I just make a comment then? I a m glad that we put this in about the glia, because it would make it much easier t o understand the total picture. I think it would also make it easier to understand Fishman's results, because if I remember his experimental set-up, he was following the influx of glucose into the central nervous system. If you will allow this for the sake of argument, that the glia more or less overpower the pump in the choroid plexus, then you would not see the plexus effect in an overall result. I think this may make it easier t o put the whole picture together.
D. M. WOODBURY: HOWd o you know that it is pumped into the glia? T. Z. CSAKY:We incubated cultured glia cells with radioactive sugars and found an accumulation wiiich was inhibited by cooling, anoxia, metabolic poisons and by digitalis.
D. P. RALL:What about neurons? T. Z. CSAKY: We have not cultured neuron cells. D. P. RALL:Just one question: You perfused dogs ventricles at a rate of 15 ml per minute? T. Z. CSAKY:Yes.
D. P. RALL:And you perfused them with a sodium-free potassium Ringer? T. Z. CSAKY:Yes. D. P. RALL:Are those dogs alive?
T. Z. CSAKY: Yes, they are. D. P. RALL:Seriously, I am sure they are, because you are doing it from one ventricle t o the other, but I think you must destroy the tissue adjacent to your perfusions. And I would assume that the fact that the glucose is increasing there is because you have tissue breakdown. Definitely. We d o not claim that the choroid plexus alone has contributed t o the glucose T. Z. CSAKY: ontflux. We claim only that in these experiments the choroid plexus does not function the same way as in normal Ringer. D. P. RALL:You think the choroid plexus is still alive? T. Z. CSAKY: The animal is, judged from blood pressure. D. P. RALL:Can you d o this from ventricle to cisterna? That is the critical thing.
T. Z. CSAKY:We were not interested in examining the effect of potassium as such, only in demonstrating, that in vivo, as irr vitro, the choroid plexus does not pump sugar if sodium ions are lacking from the perfusate. As far as the active sugar transport is concerned the plexus is dead. This is my only point.
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T. 2. C S A K Y , B. M. R I G O R
D. P. RALL:Is the level of potassium as high as the sodium level? T. Z. CSAKY:It was a complete Ringer solution, potassium replacing sodium iso-osmotically. Other similar epithelial cells, e.g., in the kidney or in the gut can be exposed reversibly to such solutions.
R. CUTLER: But the brain is different, I hope! T. Z. CSAKY:Not necessarily. R. CUTLER: It has a zero-membrane potential.
T. Z. CSAKY:We are discussing now the epithelium of the choroid plexus and not necessarily the "brain". In other epithelial tissues zero potential can be readily obtained, reversibly, by changing the ionic environment. J. FOLCH-PI: You have not mentioned exchange between the cerebrospinal fluid and the brain parenchyma. The brain parenchyma has about 1/5 of the concentration of glucose that you find in the blood, and the cerebrospinal fluid seems to be in between the two. So, if you assume as a logical consequence that a steady state will be reached, you would have an ultrafiltrate of plasma entering the cerebrospinal fluid, and from there your ultrafiltrate glucose would be remove4 by the sink. An injection into the brain parenchyma produces a sink into which glucose can be definitely forced. Can you gather such evidence by studying the point of the possible exchange by whatever mechanism it is, between CSF and the whole brain parenchyma?
T. Z. CSAKY:Not in such experiments as I have just described. Here we demonstrated only the presence of the sugar pump in the choroid plexus and its direction. We cannot at present assess the quantitative contribution of the choroid plexus sugar pump to the steady state level of glucose in the brain. J. FOLCH-PI: Now a general question to anybody: Does anybody know if there is any evidence concerning a possible exchange of glucose between the cerebrospinal fluid and brain parenchyma in general?
R. CUTLER: There is the evidence of Tschergi, I believe it was, who has perfused the CSF with glucose, which did not maintain the living brain.
J. FOLCH-PI: No, that is different. The amount of glucose you need to maintain a living brain is phenomenal. You would have to inject syrup which you could not possibly provide for the brain from the cerebrospinal fluid. K. A. C. ELLIOTT: What is the relation of the accumulation of the sugar by the choroid plexus t o the transport of sugar from the cerebrospinal fluid to the blood? The accumulation within the choroid plexus does not constitute sufficient evidence of transport through the choroid because glucose must be taken up from the one side and passed on t o the other side. I went along with the idea that substituting potassium for sodium is the same as leaving out sodium. Are you satisfied that the effect of substitution of potassium for sodium is indeed due to lack of sodium and not to the presence of the extra potassium? T. 2.CSAKY:Let me answer the two points separately. About the substitution: in vitro, as we had shown, the pump was inhibited if the sodium was replaced, regardless whether with potassium, mannitol or lithium. We have not used choline, but we could have and probably would have obtained the same results. Tris and magnesium have been used by us and by others as sodium substitute and we could probably find fifty other cations in the Merck index which could be used for the same purpose. The point which our experiment stresses is, that the sugar pump in the choroid plexus, like sugar pumps in other tissues, requires sodium. Your other question refers to the relationship between intracellular accumulation and transcellular transport in the epithelial cell. The general concept is that cross-cellular up-hill transport is preceeded by an intracellular accumulation. The sugar pump is most likely localized in the brush
C H O R O I D PLEXUS AS GLUCOSE BARRIER
157
border and is responsible forcreatinga concentration difference between the two sidesofthemembrane. The basal membrane is probably rather freely permeable for the substrate. Consequently the actual degree of intracellular accumulation depends on the ratio between the pumping and the leakage on the o t h x side. I f the leak is great the intracellular accumulation may besosmall that it may escape our analytical skill. Thus, I fully agree with you that a clear-cut demonstration of intracellular accumulation is not an absolute requirement for a transcellular up-hill transport; but it is quite different the other way around: if one can demonstrate a true intracellular accumulation of the free substrate it is a proof of the pump. In our experiments this was the case with glucose and galactose so that we can be certain that these sugars are actively transported. The failure in demonstrating the accumulation of 3-methylglucose does not necessarily disprove active transport. 1 hope this answers your question.
K. A. C. ELLIOTT:I agree immediately that there is a pump involved in the accumulation, but I would tend to use the word “transport” in the case of movement through a cell, as transport could
include two membranes: the entrance and theexit.
T. Z. CSAKY:The word “transport” means only translocation, active or non-active alike. In case of active transport energy is invested and the free energy of the transported solute increases. For such transport the expression “pump” is an excellent choice.
K. A. C . ELLIOTT: But I am wondering how does the sugar get out again. T. Z. CSAKY: I t leaks out passively.
K. A. C . ELLIOTT: But if it is all in the same cell, how can it be pumping in actively, and leaking out at the same time? T. Z. CSAKY: Such a combination is very common occurrence in nature. In the red blood cell there is a steady state equilibrium of sodium and potassium caused by the pump-leak ratio. If the pump is inhibited by digitalis, the leak can be beautifully demonstrated.
K. A. C. ELLIOTT: In the same place? T. Z. CSAKY: Probably not, but indeed in the same membrane.
D. M. WOODBURY: Certainly, in the choroid plexus, if it pumps potassium out, you can measure the ratio, whether it is lower in the CSF than in the plasma. It concentrates in the choroid plexus so that this pump is on on: side, and it just leaks out passively on the other side. The same situation can apply here. T. Z. CSAKY:This would fit admirably with the postulate that there must be a sodium pump in the choroid plexus. I t has been amply demonstrated in other tissues that a functioning sodium pump is a prerequisite for the functioning of the sugar pump.
D. M. WOODBURY: Yes, and the sodium is pumped of course in the other direction. D. P. RALL:I am a bad mathematician, but as I calculate it I mM glucose is about 20 mg%. If that is what it saturates, then it seems to me this system has essentially no biological usefulness, because there is generally around 50 or 60 mgo4 glucose in the spinal fluid. And if it was saturated back down at 20 mg‘:”! T. Z. CSAKY: Your arithmetic is almost correct (actually 18 nigO/;) but 1 am afraid your conclusion regarding the possible biological significance of the choroid plexus sugar pump is too hasty. First, you should remember that I showed that the in v i m saturation of the galactose (not glucose) pump occurs at about 1 mM. Furthermore, there is a difference between in virro and in vivo. In the former case both brush border and basal membrane of the epithelial cells are exposed to the same solution while iri vivo the polarity of the cell is maintained. From similar experiments conducted in other tis-
158
T. 2. C S A K Y , B. M. R I G O R
sues we know that the steady state accumulations in the two preparations are only roughly comparable and, if the polarity of the cell is maintained, the tissue-to-medium values are somewhat higher. Thus, we should only make an intelligent guess that the sugar pump is of low capacity but whether the in vivo limit in case ofglucose is 1,5, or 10 mM cannot be said at this moment. Davson 1967, (Physiology of the Cerebrospinal Fluid, Little, Brown and ComW. W. TOURTELLOTTE: pany, Boston, Table XX, p. 188) quoting other investigators’ data stated that the concentration of glucose was higher in the ventricles than in the cisterna magna, and least concentrated in the lumbar sac. Have you conducted experiments which were designed to explain the concentration gradient of glucose in the cerebrospinal fluid axis? T. Z. CSIIKY:As you may recall Pappenheimer suggested that various choroid plexuses may have different transport properties not unlike the epithelial cells in the different areas of the kidney tubule. We compared sugar pumps in plexuses obtained from different regions but could not detect any gross differences in vitro. We have not yet reached the point where we perfuse the individual ventricles and measure glucose fluxes. Such experiments may yield the information you are asking for.
The Choroid Plexus as a Glucose Barrier Dcparttiierit of Ptiarniacology, University of Ker;tucky College of Medicitre, Lexirigton, Ketitrtcky ( U.S.A.)
The choroid plexus is composed of strongly vascularized connective tissue lined with an epithelial layer. It has long been considered a “barrier” between the blood and the cerebrospinal space. The luminal membrane of the epithelial lining shows a typical microvillus structure similar to that of the epithelium of the small intestine or kidney tubule (Millen and Woollam, 1962). As the latter organs are involved in the biological transport of sugars, by analogy a similar role could be presumed for the choroid plexus. The present paper summarizes the results of preliminary experiments aimed at clarifying the role of the choroid plexus in the maintenance of glucose concentration between the blood and cerebrospinal fluid (CSF). I t has been known for some time that CSF contains less glucose than blood does (Davson, 1956). The relationship is roughly 1 : 2. Moreover, a change in the glucose concentration of the blood plasma is followed, after some delay, by a corresponding change in the sugar concentration of the CSF,still maintaining the approximate 1 : 2 concentration ratio. It is clear that such an uneven equilibrium cannot be maintained by a passive barrier alone. Theoretically, three possibilities could be proposed for such a distribution : ( I ) Glucose could be filtered by the plexus and then metabolized in the CSF. (2) Glucose could be actively secreted from the blood into the cerebrospinal space by the choroid plexus. (3) Glucose could continually be filtered somewhere else and then actively reabsorbed by the choroid plexus. The first possibility would require a specific mechanism that would regulate the metabolic disappearance of glucose at a rate depending on the concentration of the sugar in the blood plasma. Although the existence of such metabolic regulatory re:eptors is theoretically not impossible, in actuality it has not been demonstrated. With the second possibility, it is somewhat difficult to envision a layer of epithelium that would secrete a solute from the blood into the CSF and maintain it at half its concentration level in the blood. There are glands, like the salivary glands, that produce secreta hypertonic to blood; however, this is done not by a simple transport across an epithelial layer, but by a process of filtration followed by selective reabsorption at a different site. The third possibility, namely that a plasma filtrate is continually produced and gluR
148
T. Z . C S k K Y , B. M. R I G O R
coseis thenreabsorbed from it by the epithelium of the choroid plexus, would requirea low-capacity sugar pump in the latter. The function of such a pump would be limited by the concentration gradient against which it would have to transport. Thus it is quite possible that the limit of the gradient would be I : 2, which would result in the maintenance of the very uneven equilibrium between the blood and CSF as it was actually observed. In the following, evidence is presented that there exists a sugar pump in the choroid plexus, that this pump is of low capacity, and that it actively transports sugar from the CSF into the blood stream. Sugar-concentrating transport mechanism in the choroid plexus The following experimental procedure was performed (Csaky and Rigor, 1964). Choroid plexuses were removed from pentobarbital-anesthetized dogs and placed in a Krebs-Henseleit bicarbonate Ringer's solution containing a very small amount (7 pM)of 14C-labeled sugar. After gassing with 95 % 0 2 5 % COz, the vessels were shaken in a Dubnoff metabolic shaker for 1 h at 37" C. At the end of this period the plexuses were removed, gently blotted, and placed on a small piece of carefully preweighed filter paper. They were dried to obtain the total water content. The dry residue was combusted in a closed oxygen atmosphere, and the resulting W02was determined in a liquid-scintillation spectrometer using the scintillation liquid as described earlier (Kalberer and Rutschman, 1961). I n a number of experiments, radio-iodinated serum-albumin was also added to the Krebs-Ringer solution, and the distribution of radioiodine was determined in the choroid plexus. Assuming that this protein occupies only the extracellular space, the intracellular space of the choroid plexus was computed and the results calculated in such a fashion that the distribution ratio of radioactivity between the intracellular water of the choroid plexus and the incubating medium was
+
TABLE I R A T I O O F C O N C E N T R A T I O N O F R A D I O A C T I V I T Y I N T H E I N T R A C E L L U L A R W A T E R OVER T H E MEDIUM ( I / M ) I N T H E C H O R O I D P L E X U S F O L L O W I N G I N C U B A T I O N W I T H T H E L A B E L E D SUGARS
Sugar
Glucose Galactose 3-0-Methylglucose
I/
Number of experinrents
(mean 4 standard deviation)
26 35 25
21.34 & 3.01 19.70 t 3.36 0.70 +. 0.32
obtained. Table I shows that the radioactivity in experiments with glucose and galactose was concentrated more than twenty-fold in the intracellular space of the choroid plexus. However, it is interesting that 3-0-methylglucose (3-methylglucose) was not concentrated, although in other transport systems, such as the intestine, this sugarether behaves very much like glucose or galactose (Cshky, 1942, 1958). Glucose and galactose were metabolized by most tissues, whereas 3-methylglucose is not metabo-
I49
C H O R O I D P L E X U S AS G L U C O S E B A R R I E R
lized in animal ticsue (Csaky and Wilson, 1956). It was therefore of interest to examine whether or not the uneven distribution of the radioactivity between the intracellular water of the choroid plexus and the surrounding nutrient medium in the case of glucose and galactose was simply the result of transformation of the sugar to labeled metabolites, and not of true accumulation. For this reason choroid plexuses, having been incubated with radioactiveglucose or galactose, were extracted with hot distilled water. The protein-free filtrate was concentrated and chromatographed on filter paper, using either pyridine-water-butanol or acetic acid-water-butanol solvent mixtures (Tyszkiewicz, 1962). In the case of galactose it was found that about 70-75 % of the total radioactivity could be recovered in one spot, the migration of which was identical with that of the reference galactose. About 5 % of the radioactivity was found in another spot, the migration of which was identical with a reference galactose-I-phosphate. The rest of the radioactivity was distributed in various spots, the identity of which was not established. In the case of glucose, 60-70% of unchanged glucose was found in the extract. Also, glucose phosphate and unknown spots were located. These results indicate that even when making corrections for a possible slight metabolism there is a considerable concentration of true unchanged glucose and galactose in the choroid plexus. It is known that the intestinal sugar pump is inhibited by a number of factors, such as the lack of sodium in the medium; a low concentration of digitalis, 2,4-dinitrophenol, or phlorizin; or the lack of oxygen. In order to ascertain whether the choroid T A B L E 11 I N H I B I T I O N O F A C C U M U L A T I O N O F S U G A R S B Y L A C K O F S O D I U M , B Y I N H I B I T O R S A N D BY A N O X I A . F I G U R E S R E P R E S E N T T H E PER C E N T I N H I B I T I O N I N P A I R E D E X P E R I M E N T S
Glucose
Potassium Ringer
Lithium Ringer
Mannitol Ringer
Ouabain lO-4M
Phlorizin 10-3M
Dinitrophenol 10-3~
Anoxia
37 51 48 48
30 54 59 65 66 36
38 63 51 74 83
22 41 40 28 21
57 47 56 38 34
93 96 98 96
89 90 95 93
-
-
-
-
-
-
-
Average
46.0
51.7
61.8
30.4
46.4
96.3
91.7
Galactose
87 73 57 47 32
29 37 51 27 33
50 40 45 39
32 47 40 42
72 75 76 60 80 49
96 95 87 98
80 92 93 87
-
-
-
57
22
Average ~~
Rrfermces p . I54
-
__
-
-
53.6
35.4
43.5
40.3
68.7
94.0
88.0
150
T. Z. C S A K Y , B. M. R I G O R
plexus sugar pump is similarly inhibited, the following experimental procedure was developed :two lateral ventricle choroid plexuses were removed from dog and compared in pairs. One was incubated as a control in a normal Krebs-Ringer solution; the other, in a modified solution or in the presence of the inhibitor. The results were then expressed as percentage of inhibition versus the control. Table 11 shows that the choroid plexus sugar pump is inhibited by the same factors as the intestinal sugar pump, however, to a different degree. Two facts deserve specific mention: the lack of absolute dependence ofthechoroid plexus sugar pump on sodium in the incubating medium and the very strong dependence on the presence of oxygen. The former is in contrast with what one finds in the intestine (Csaky, 1960). The possibility that sodium leaks out of the tissue into the medium is of little importance because the plexuses were incubated in a relatively large volume of the medium (the tissue water diluted at least 1 : 50). Moreover, it was found that all sodium-dependent transports are readily inhibited with relatively low concentration of digitalis (Csaky, 1963). On the one hand, the sugar pump of the choroid plexus is only partially depressed by the presence of the cardioglucoside. On the other hand, the strong dependence on oxygen and the complete inhibition of the pump by low concentration of 2,4-dinitrophenol suggest that the transport mechanism derives its energy mainly from a direct oxidative process. The capacity of the concentrating pump was examined by incubating the tissue with increasing amounts of the sugar. As can be seen in Table 111, the galactose-accumulating system became saturated between the concentrations of 0.5-1.1 mM. By analogy it can be assumed that the glucose pump is also a low capacity pump.
T A B L E 111 I N H I B I T I O N OF T H E S U G A R C O N C E N T R A T I O N BY L A R G E A M O U N T OF S U G A R I N T H E M E D I U M . F I G U R E S R E P R E S E N T THE PER C E N T I N H I B I T I O N IN P A I R E D E X P E R I M E N T S ( C O N T R O L A N D E X P E R I M E N T A L P L E X U S FROM T H E SAME D O G )
Labeled sugars (7P M ) Glucose
Average
Average
Non-labeled sugars (30 m M ) Glucose
Galaciose 3-0-Meiliylglucose
99 96 98 98
55 75 49 77
54 53 69 49
97.8
61.2
-_
71 43 83 69
-
98 98 99 98
-
30 22 57 43
66.5
98.3
38.0
50
56.3
__
CHOROID PLEXUS AS GLUCOSE BARRIER
151
T A B L E 1V SATURATION O F THE GALACTOSE CONCENTRATING
Galactose in the Mediuni ltrert Radioactive Total (I'M)
(PM)
(ItM)
7
7 63 118 567 1127
0 56
7
560 1120
7
111
7 7
SYSTEM IN T H E C H O R O I D P L E X U S
Mean 11M
20.73 13.95 6.47 5.04 0.58
The possible active transport of 3-methylglucose in the choroid plexus merits further comment. As was seen, this sugar-ether is not concentrated in the plexus in vitro; however, the presence of a large amount of 3-methylglucose is capable of inhibiting the concentration of both glucose and galactose in the plexus (Table IV), as if the sugarether would compete with the other sugars for the transporting sites. Such behavior is not uncommon. In our laboratory we found that 3-methylglucose is most likely actively absorbed in the renal tubulues, yet in vitro it is not accumulated by the kidney slides*. Thus it is quite possible that, in spite of the lack of measurable accumulation, 3-methylglucose would still be actively transported across the epithelial layer of the plexus.
Direction of the sugar pump in dog choroid plexus in vivo The experiments described above demonstrate the presence of a concentrating pump in the choroid plexus but do not suggest anything about its direction. In other epithelial cells (kidney tubule or intestine), sugar is transported from the direction of the brush border towards the direction of the basal membrane. By analogy, then, the morphology of the choroid plexus epithelium suggests an active reabsorption of sugars from the CSF into the blood. To test this possibility, the following experiments were performed : dogs were anesthetized with sodium pentobarbital, and both lateral ventricles were tapped and cannulated, and an artificial CSF (Pappenheimer et al., 1961) was recirculated with a velocity of approximately 5 ml/min. The glucose content of the artificial CSF was less thanin the blood. From time to time, the glucose concentration in the perfusate was determined by the glgcostat technique (Teller, 1956). At the same time arterial blood samples were taken and the glucose concentration measured in the plasma by using the same method. Results of a set of typical experiments are reproduced in Fig. 1, which shows that there was a steady disappearapce of glucose from the cerebrospinal space into the blood. Because it probably proceeds up-hill, by definition the transport is active. This active transport is completely eliminated, however, if in the artificial CSF sodium was replaced by potassium or if digitalis or phlorizin was added to the medium.
* Unpublished Refcrenccs p . 154
observations.
152
T. Z. C S A K Y , B. M. R I G O R
HR.
HR.
HR.
nn.
Fig. 1 . Perfusion of the lateral ventricles in the dog. Solid line (CSF): glucose concentration in the perfusate. Broken line: glucose concentration in the arterial blood plasma. First column : control; 2nd col.: Na+ was replaced by K+ in the perfusing medium; 3rd col.: Digitoxin (IO-4M) added to the perfusate; 4th col.: Phlorizin (lO-4M) added to the perfusate.
Sugar transport in the isolated choroid plexus of the horse
A more direct evidence regarding the direction of active sugar transport in the choroid plexus was obtained from experiments on the isolated horse choroid plexus in vitro. Healthy horses (obtained through the courtesy of the Department of Animal Pathology) were anesthetized with sodium pentobarbital, their skulls were opened, and the lateral plexus with some surrounding brain tissue was quickly removed, placed on ice, and carried rapidly to the laboratory, where the anterior and posterior choroidal arteries and the main choroidal vein were cannulated with small polyethylene tubings. The procedure between the removal of the plexus and the start of the experiment did not take longer than 10-20 min. The choroid plexus from the ventricle side was bathed in a Krebs-Ringer bicarbonated solution containing 50 mg% of galactose. An identical solution was infused slowly into the choroid artery. The outflow from the choroid vein was collected and analyzed for galactose by a method utilizing specific galactose oxidase*. The entire preparation was kept in a 37°C moist chamber, and oxygencarbon dioxide (95-5 %) was continuously bubbled through the bath. At the end ofthe experiment a dye was introduced into the artery. If the dye appeared in the bath, indicating a leak between the blood and the cerebral spinal space, the preparation was discarded. The galactose concentration of the venous outflow increased continuously during the experiment, as shown in Fig. 2. Such results would be possible only if the galactose was actually transported from the outside into the blood by active transport, or if the blood was losing water. In order to check the latter, in a few experiments
* Pamphlet 6-65
“Galactostat”, Worthington Biochem. Corp.
CHOROID PLEXUS AS GLUCOSE BARRIER
153
Fig. 2. Horse choroid plexus perfused in vifro. Broken line: galactose concentration in the arteria input. Solid line: galactose concentration in the venous outflow. First column: perfusion with KrebsRinger: 2nd col.: Na+ replaced by K+ in the perfusing Krebs-Ringer: 3rd col.: perfused with KrebsRinger, gassed with N2.
radioactive inulin was added to the fluid which was pumped into the artery. The inulin concentration did not change in the venous outflow, indicating that there was no appreciable loss of water from the blood in the preparation. Consequently, one must conclude that in this preparation galactose was actually transported from the outside into the bloodstream against a concentration gradient by active transport. It is also noteworthy that this active transport was eliminated by the absence of sodium or by anoxia. CONCLUSIONS
Evidence is presented of an accumulating low-capacity sugar pump in the choroid plexus that transports glucose or galactose from the ventricle into the bloodstream. The experimental results would be in accord with the following working hypothesis: the CSF is essentially a blood filtrate in which glucose is originally present in the same concentration as in the plasma. The choroid plexus possesses an actively transporting pump system which continuously reabsorbs glucose from the cerebral spinal fluid into the blood. Because the pump is of low capactiy, the net transport will be determined by the gradient against which the transport has to take place. As a result, the glucose content in the CSF is always below that in the plasma but is in rough accord with the concentration fluctuations in the latter. As it happens, the pump is regulated so that a ratio of roughly 1 : 2 is maintained. Rrfercnces p. 154
154
T. Z. C S A K Y , B. M. R l G O R ACKNOWLEDGEMENT
This work was supported in part by research grants from the U.S.P.H.S. and the American Heart Association. REFERENCES CSAKY,T. Z. (1942) Hoppe-Seyler’s Z . Physiol. Chem., 277, 47. CSAKY, T. Z. (1958) Intern. Abstr. Biol. Sci. Suppl., 6 , 79. CSAKY, T. Z. (1963) Biochim. Biophys. Acta, 74, 160. CSAKY,T. Z. AND RIGOR,B. M. (1964) Li/e Sci., 3, 931. CSAKY, T. Z. AND THALE, M. (1960) J . Physiol. (Lond.), 151, 59. CSAKY,T. Z. AND WILSON, J. E. (1956) Biochim. Biophys. Acta, 22, 185. DAVSON, H. (1956) Physiology of the Ocular ana Cerebrospinal Fluids. Boston, Little Brown. KALBERER, F. AND RUTSCHMAN, J . (1961) Helv. Chim. Acta, 44, 1956. MILLEN, J. W. AND WOOLLAM, D. H. M. (1962) The Anatomy of the Cerebrospinal F h i d Londcn, Oxford University Press. J. R., HEISEY, S . R. AND JORDAN, E. F. (1961) Amer. J . Physiol., 200, 1. PAPPENHEIMER, TELLER, J. D. (1956) Abstract of Papers, 130th Meeting, Amer. Chem. SOC.,69c. TYSZKIEWICZ, E. (1962) Anal. Biochem., 3, 164. D I S C U S S I ON D. B. TOWER: I think this is very interesting work of Dr. Csaky’s, and I would like to ask several related questions. It has been known for a good many years in clinical practice that when the blood glucose level rises above a certain point, and if my memory serves me correctly it is about 300 mg%, then the cerebrospinal fluid glucose level no longer rises. One has the impression in looking at these figures that the pump is saturated in the opposite direction. In other words, the glucose is pumped into the central nervous system, and if one increases the glucose level on the side from which the .glucose is being pumped, the pump can be saturated. This obviously does not agree with your data; so I am wondering if you have an explanation for this clinical observation. The second point relates t o some experiments that Dr. Fishman did in the dog. Unfortunately I don’t think he published the data, but he told me that he studied the effects of phlorhizin, both given into the blood and injected into the cerebrospinal fluid. I think he used up to 1 0 - 3 M concentrations, and got no effect on the entry of glucose into the central nervous system. I just wonder how you would interpret this observation of Dr. Fishman’s on the studies in vivo in the dog. Finally, I wonder what is the role of glucose metabolism in the central nervous system in your system; since glucose is the major substrate being used by the brain and a t a very substantial rate (about 65% of the glucose consumed by the body per unit time is consumed by the central nervous system). I find it a little difficult to visualize how one may conceive of an outward pump for glucose together with this type of metabolic system.
T. Z. CSAKY:For the first question, regarding the effect of phlorizin, I can provide only a partial answer. In our experiments we obtained an inhibition with this glucoside both in vitro and in vivo, but only a partial one. So it is possible that the sugar carriers are different in different anatomical entities. Concerning sugar transport into the brain at very high sugar concentration: we did not study such a phenomenon directly. However, one can speculate about this problem by analogy. E.g., it is known that if the metabolic pattern of glucose is changed, its transport rate also changes in some tissues. Thus, semi-starvation leads t o an increase in the rate of glucose absorption from the intestine. Perhaps it would be conceiveable that the high glucose containing blood “overfeeds” the brain cells and thereby decreases the rate of transport. With regard to your third point about the slices: I do not think that the brain cells are fed through the choroid plexus, but that glucose enters the brain from the capillaries via the mediation of the glia. In connection with this point I may mention that in our laboratory we recently found an active sugar pump in the isolated glial cells grown in tissue culture.
C H O R O I D P L E X U S A S GLUCOSE B A R R I E R
1 S5
D. P. RALL:In which direction? T. Z. CSAKY:Working essentially with a cell suspension we cannot at present answer this question. We could only demonstrate an energy dependent accumulation of the sugar in the cell. But it is not inconceivable that the glia pumps glucose from the blood into the brain. Of course, in brain slices the glia c:lls are also included. I think the choroid plexus is different. Its epithelium morphologically and perhaps also functionally seems to be like that of the kidney tubule or intestinal mucosa.
D . B. TOWER:Could I just make a comment then? I a m glad that we put this in about the glia, because it would make it much easier t o understand the total picture. I think it would also make it easier to understand Fishman's results, because if I remember his experimental set-up, he was following the influx of glucose into the central nervous system. If you will allow this for the sake of argument, that the glia more or less overpower the pump in the choroid plexus, then you would not see the plexus effect in an overall result. I think this may make it easier t o put the whole picture together.
D. M. WOODBURY: HOWd o you know that it is pumped into the glia? T. Z. CSAKY:We incubated cultured glia cells with radioactive sugars and found an accumulation wiiich was inhibited by cooling, anoxia, metabolic poisons and by digitalis.
D. P. RALL:What about neurons? T. Z. CSAKY: We have not cultured neuron cells. D. P. RALL:Just one question: You perfused dogs ventricles at a rate of 15 ml per minute? T. Z. CSAKY:Yes.
D. P. RALL:And you perfused them with a sodium-free potassium Ringer? T. Z. CSAKY:Yes. D. P. RALL:Are those dogs alive?
T. Z. CSAKY: Yes, they are. D. P. RALL:Seriously, I am sure they are, because you are doing it from one ventricle t o the other, but I think you must destroy the tissue adjacent to your perfusions. And I would assume that the fact that the glucose is increasing there is because you have tissue breakdown. Definitely. We d o not claim that the choroid plexus alone has contributed t o the glucose T. Z. CSAKY: ontflux. We claim only that in these experiments the choroid plexus does not function the same way as in normal Ringer. D. P. RALL:You think the choroid plexus is still alive? T. Z. CSAKY: The animal is, judged from blood pressure. D. P. RALL:Can you d o this from ventricle to cisterna? That is the critical thing.
T. Z. CSAKY:We were not interested in examining the effect of potassium as such, only in demonstrating, that in vivo, as irr vitro, the choroid plexus does not pump sugar if sodium ions are lacking from the perfusate. As far as the active sugar transport is concerned the plexus is dead. This is my only point.
I56
T. 2. C S A K Y , B. M. R I G O R
D. P. RALL:Is the level of potassium as high as the sodium level? T. Z. CSAKY:It was a complete Ringer solution, potassium replacing sodium iso-osmotically. Other similar epithelial cells, e.g., in the kidney or in the gut can be exposed reversibly to such solutions.
R. CUTLER: But the brain is different, I hope! T. Z. CSAKY:Not necessarily. R. CUTLER: It has a zero-membrane potential.
T. Z. CSAKY:We are discussing now the epithelium of the choroid plexus and not necessarily the "brain". In other epithelial tissues zero potential can be readily obtained, reversibly, by changing the ionic environment. J. FOLCH-PI: You have not mentioned exchange between the cerebrospinal fluid and the brain parenchyma. The brain parenchyma has about 1/5 of the concentration of glucose that you find in the blood, and the cerebrospinal fluid seems to be in between the two. So, if you assume as a logical consequence that a steady state will be reached, you would have an ultrafiltrate of plasma entering the cerebrospinal fluid, and from there your ultrafiltrate glucose would be remove4 by the sink. An injection into the brain parenchyma produces a sink into which glucose can be definitely forced. Can you gather such evidence by studying the point of the possible exchange by whatever mechanism it is, between CSF and the whole brain parenchyma?
T. Z. CSAKY:Not in such experiments as I have just described. Here we demonstrated only the presence of the sugar pump in the choroid plexus and its direction. We cannot at present assess the quantitative contribution of the choroid plexus sugar pump to the steady state level of glucose in the brain. J. FOLCH-PI: Now a general question to anybody: Does anybody know if there is any evidence concerning a possible exchange of glucose between the cerebrospinal fluid and brain parenchyma in general?
R. CUTLER: There is the evidence of Tschergi, I believe it was, who has perfused the CSF with glucose, which did not maintain the living brain.
J. FOLCH-PI: No, that is different. The amount of glucose you need to maintain a living brain is phenomenal. You would have to inject syrup which you could not possibly provide for the brain from the cerebrospinal fluid. K. A. C. ELLIOTT: What is the relation of the accumulation of the sugar by the choroid plexus t o the transport of sugar from the cerebrospinal fluid to the blood? The accumulation within the choroid plexus does not constitute sufficient evidence of transport through the choroid because glucose must be taken up from the one side and passed on t o the other side. I went along with the idea that substituting potassium for sodium is the same as leaving out sodium. Are you satisfied that the effect of substitution of potassium for sodium is indeed due to lack of sodium and not to the presence of the extra potassium? T. 2.CSAKY:Let me answer the two points separately. About the substitution: in vitro, as we had shown, the pump was inhibited if the sodium was replaced, regardless whether with potassium, mannitol or lithium. We have not used choline, but we could have and probably would have obtained the same results. Tris and magnesium have been used by us and by others as sodium substitute and we could probably find fifty other cations in the Merck index which could be used for the same purpose. The point which our experiment stresses is, that the sugar pump in the choroid plexus, like sugar pumps in other tissues, requires sodium. Your other question refers to the relationship between intracellular accumulation and transcellular transport in the epithelial cell. The general concept is that cross-cellular up-hill transport is preceeded by an intracellular accumulation. The sugar pump is most likely localized in the brush
C H O R O I D PLEXUS AS GLUCOSE BARRIER
157
border and is responsible forcreatinga concentration difference between the two sidesofthemembrane. The basal membrane is probably rather freely permeable for the substrate. Consequently the actual degree of intracellular accumulation depends on the ratio between the pumping and the leakage on the o t h x side. I f the leak is great the intracellular accumulation may besosmall that it may escape our analytical skill. Thus, I fully agree with you that a clear-cut demonstration of intracellular accumulation is not an absolute requirement for a transcellular up-hill transport; but it is quite different the other way around: if one can demonstrate a true intracellular accumulation of the free substrate it is a proof of the pump. In our experiments this was the case with glucose and galactose so that we can be certain that these sugars are actively transported. The failure in demonstrating the accumulation of 3-methylglucose does not necessarily disprove active transport. 1 hope this answers your question.
K. A. C. ELLIOTT:I agree immediately that there is a pump involved in the accumulation, but I would tend to use the word “transport” in the case of movement through a cell, as transport could
include two membranes: the entrance and theexit.
T. Z. CSAKY:The word “transport” means only translocation, active or non-active alike. In case of active transport energy is invested and the free energy of the transported solute increases. For such transport the expression “pump” is an excellent choice.
K. A. C . ELLIOTT: But I am wondering how does the sugar get out again. T. Z. CSAKY: I t leaks out passively.
K. A. C . ELLIOTT: But if it is all in the same cell, how can it be pumping in actively, and leaking out at the same time? T. Z. CSAKY: Such a combination is very common occurrence in nature. In the red blood cell there is a steady state equilibrium of sodium and potassium caused by the pump-leak ratio. If the pump is inhibited by digitalis, the leak can be beautifully demonstrated.
K. A. C. ELLIOTT: In the same place? T. Z. CSAKY: Probably not, but indeed in the same membrane.
D. M. WOODBURY: Certainly, in the choroid plexus, if it pumps potassium out, you can measure the ratio, whether it is lower in the CSF than in the plasma. It concentrates in the choroid plexus so that this pump is on on: side, and it just leaks out passively on the other side. The same situation can apply here. T. Z. CSAKY:This would fit admirably with the postulate that there must be a sodium pump in the choroid plexus. I t has been amply demonstrated in other tissues that a functioning sodium pump is a prerequisite for the functioning of the sugar pump.
D. M. WOODBURY: Yes, and the sodium is pumped of course in the other direction. D. P. RALL:I am a bad mathematician, but as I calculate it I mM glucose is about 20 mg%. If that is what it saturates, then it seems to me this system has essentially no biological usefulness, because there is generally around 50 or 60 mgo4 glucose in the spinal fluid. And if it was saturated back down at 20 mg‘:”! T. Z. CSAKY: Your arithmetic is almost correct (actually 18 nigO/;) but 1 am afraid your conclusion regarding the possible biological significance of the choroid plexus sugar pump is too hasty. First, you should remember that I showed that the in v i m saturation of the galactose (not glucose) pump occurs at about 1 mM. Furthermore, there is a difference between in virro and in vivo. In the former case both brush border and basal membrane of the epithelial cells are exposed to the same solution while iri vivo the polarity of the cell is maintained. From similar experiments conducted in other tis-
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sues we know that the steady state accumulations in the two preparations are only roughly comparable and, if the polarity of the cell is maintained, the tissue-to-medium values are somewhat higher. Thus, we should only make an intelligent guess that the sugar pump is of low capacity but whether the in vivo limit in case ofglucose is 1,5, or 10 mM cannot be said at this moment. Davson 1967, (Physiology of the Cerebrospinal Fluid, Little, Brown and ComW. W. TOURTELLOTTE: pany, Boston, Table XX, p. 188) quoting other investigators’ data stated that the concentration of glucose was higher in the ventricles than in the cisterna magna, and least concentrated in the lumbar sac. Have you conducted experiments which were designed to explain the concentration gradient of glucose in the cerebrospinal fluid axis? T. Z. CSIIKY:As you may recall Pappenheimer suggested that various choroid plexuses may have different transport properties not unlike the epithelial cells in the different areas of the kidney tubule. We compared sugar pumps in plexuses obtained from different regions but could not detect any gross differences in vitro. We have not yet reached the point where we perfuse the individual ventricles and measure glucose fluxes. Such experiments may yield the information you are asking for.