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The effect of rubidium on the distribution and movement of potassium between blood, brain and cerebrospinal fluid in the rabbit
BRAIN RESEARCH
311
T H E E F F E C T OF R U B I D I U M ON T H E D I S T R I B U T I O N A N D M O V E M E N T OF POTASSIUM B E T W E E N BLOOD, BRAIN A N D C E R E B R O S P I N A L F L U I D IN T H E RABBIT
MICHAEL W. B. B R A D B U R Y
Division of Medicine, Cedars-Sinai Medical Center and Cedars-Sinai Medical Research Institute, Los Angeles, Calif. 90048 (U.S.A.) and Sherrington School of Physiology, St. Thomas's Hospital Medical School, London S.E.1 (Great Britain) (Accepted June 2nd, 1970)
INTRODUCTION
The relative distribution of potassium and rubidium between skeletal muscle tissue and blood plasma has been studied in the intact rat by Relman et al. s. In these animals in which about 40 ~ of the body potassium had been replaced by rubidium, rubidium was accumulated in the cell water relative to the extracellular fluid in preference to potassium. There is much other evidence that rubidium and potassium compete for membrane-transport sites in frog skeletal muscle 11,12 and in particular compete in the sodium-potassium pump of frog skeletal muscle z and of red blood cells lo. The potassium concentration in brain and cerebrospinal fluid (CSF) is kept very constant in the face of severe and chronic hypokalemia and hyperkalemia 4. The control of this stability depends in part on an influx of potassium into brain and CSF which increases little with the plasma concentration of potassium 4 and in part on an active mechanism inhibitable by ouabain which pumps potassium out of CSF into blood and is highly dependent on the CSF concentration of potassiumL It is not known whether this potassium pump exists solely at the choroid plexuses or is present at the blood-brain barrier as well. The latter hypothesis is likely since the potassium concentration in the interstitial fluid of the brain must be controlled as well as that in the CSF 4. In view of the known interactions between transport of potassium and rubidium, it seemed that a study of the effect of rubidium on potassium distribution, and movement in the system comprised by blood, brain and CSF might throw further light on the mechanisms involved in homeostasis of the concentration of potassium in CSF and in the interstitial fluid of brain. In particular, the results might indicate whether there is a potassium and rubidium pump sited at the blood-brain barrier which transfers these ions from the interstitial fluid of brain to blood in a similar manner to the mechanisms for potassium demonstrated as occurring between CSF and blood. Brain Research, 24 (1970) 311-321
312
M. W . B. B R A D B U R Y
The problem has been approached in two ways. Firstly, the distribution of potassium and rubidium ions between tissues and fluids has been studied in rabbits in which a proportion of the total body potassium has been replaced by rubidium. Secondly, the effect of rubidium ions on the influx of 42K into brain and CSF and on the effiux of 42K from CSF into brain and blood have been studied. MATERIALS A N D METHODS
Most of the experiments were carried out on adult female Dutch Belt rabbits, weighing 2.0-2.5 kg. The studies, involving movements of 4ZK, were made on New Zealand White rabbits of 2.5-3.0 kg (the latter breed was used in order that the results might be comparable to previous values of influx determined in this breed).
Replacement of body potassium with rubidium Rabbits were fed ad libitum on a special potassium free diet 4 for 3 weeks, the diet being begun on a Monday. Previous experience suggested that by this time the skeletal muscle potassium should fall to about 70 mequiv./kg wet weight. During the 4th week of the diet rubidium was given to replace the lost potassium. Initially, 10 sterile doses of 60 ml of glucose 5 % and 30 ml of rubidium.chloride 155 m M were given intraperitoneally through an indwelling cannula at intervals. This always caused temporary signs of peritoneal irritation and on occasions stopped the heart. Later, it was found more satisfactory to very slowly infuse RbC1 155 m M into an ear vein. 100 ml doses were given in this way at a rate of 0.25 ml/min on the Monday, Wednesday and Friday of the 4th week of the diet. Whatever the method, all rabbits received a total of 300 ml (46.5 mM) of RbC1 during the 4th week and no further injections after the Friday. Control animals received KC1 155 m M instead of RbC1. Rubidiumtreated rabbits were normal in appearance. Hyperactivity and hyperventilation were noted in some. Attempts to increase the degree of rubidium replacement further caused convulsions to occur.
Final studies on animals For steady-state studies, rabbits were killed on the Monday following the last injection of RbCI. This allowed at least 72 h for the final equilibration of the rubidium. Animals were anesthetized and decapitated. Two rabbits were kept for 6 days after the final rubidium injection, and 1 rabbit for 10 days, before sacrifice. These 3 animals received 5 mequiv./l RbC1 in their drinking water. The electrolytes were extracted from tissue samples with 0.5 N nitric acid. The procedure for collecting fluid samples, killing the rabbits and preparing the tissues have been described previously4. The electrical potential between ventricular CSF and jugular venous blood was measured in rubidium-treated Dutch Belt rabbits 4 on the Monday or Tuesday, following RbC1 infusion.
Brain Research, 24 (1970) 311-321
RB AND K IN BRAIN AND CSF
313
Transport of 42K into brain and CSF A constant concentration of 42K was maintained in the blood plasma of rubidium-replaced rabbits by intravenous infusion of this isotope in isotonic saline at a diminishing rate for 2 h 4. This was performed on the Tuesday following the rubidium replacement week. Since 4ZK movement into a given region of brain is determined by a single rate constant, a figure for influx of potassium in mequiv./kg/h can be simply calculated 4. Entry of 42K into CSF from blood follows more complex kinetics, which do not allow the calculation of influx from the activities and concentrations present at a single time. Hence the ratio of the activity in CSF to that in plasma has been recorded.
Ventriculo-cisternal perfusion of normal rabbits The clearance of 42K from CSF into blood, 'barrier clearance', and from CSF into brain was measured during ventriculo-cisternal perfusion of artificial CSF from both lateral ventricles to the cisterna magna of anesthetized normal rabbits for 2 h. The potassium concentration in the perfusion fluid was always 2.9 mequiv./1. Differing concentrations of rubidium between 1 and 15 mequiv./1 were obtained by adding increasing volumes of a similar perfusion fluid in which the NaCl had been completely replaced by RbCl. The details of the technique and of the treatment of the results have been fully describedL In brief, the total radioactivity lost from the perfusing CSF is compared with tbe total radioactivity gained by the central nervous system. The difference represents transfer into the blood stream. Clearances are obtained by dividing amounts of radioactivity transferred by the mean concentration of radioactivity in the perfusing CSF. The efflux of potassium into blood is the product of the barrier clearance of 42K and the mean concentration of potassium in the perfusing CSF.
Analytical methods Rubidium, potassium and sodium were analyzed in samples of plasma, CSF and nitric acid extracts of dried skeletal muscle and brain (1 ml 0.5 N HNOa/100 mg wet weight of tissue). All these fluids were diluted (1:10) in l0 mM cesium chloride0.02~ Acationox (Scientific Products). The emissions in an oxygen hydrogen flame were recorded in a Zeiss PMQ 11 spectro-flame photometer with double monochromator at 805 #m for rubidium, 757 #m for potassium and 589/tin for sodium. A slit width of 0.5 was used for rubidium and potassium and 0.01 for sodium. The rubidium and potassium settings were considerably removed from those giving optimum emission in order to reduce mutual interference between these two ions. Almost complete elimination of residual interference plus an almost two-fold potentiation of the reading was afforded by the presence of 10 mM cesium chlorideL Unknowns were 'bracketed' between two standards of approximately similar composition to the unknowns with respect to Rb, K and Na.
Brain Research, 24 (1970) 311-321
314
M . W . B . BRADBURY
o.,]
Rb Rb+K
Fig. 1. Mean ratio of Rb replacement, Rb/(Rb + K), in plasma, CSF, muscle and brain from 5 rubidium-treated rabbits. Limits are ± 2 S.E. TABLE I ELECTROLYTES AND WATER IN FLUIDS AND TISSUES FROM RABBITS IN WHICH DEPLETED K HAS BEEN REPLACED WITH Rb OR K Figures for animals on a diet of normal K content are given for comparison. Values are means ± S.E. Figures in parentheses are number of animals.
Rb replaced (5)
K replaced (3)
Normal (6)
Rb Plasma (mequiv./l H20) Muscle (mequiv./kg wet wt.) CSF (mequiv./1H~O) Brain (mequiv./kg wet wt.)
0.99 40.60 0.44 19.00
_[: ± ± ±
0.08 3.00 0.03 0.90
K Plasma (mequiv./1H20) Muscle (mequiv./kg wet wt.) CSF(mequiv./1HzO) Brain (mequiv./kg wet wt.)
2.23 60.00 2.02 69.80
± ± ± ±
0.16 2.60 0.09 1.50
I
I
I
2.84 102.00 2.83 91.60
± 0.47 ± 3.40 :[: 0.14 ± 0.80
3.96 102.20 2.89 87.90
± 0.10 ± 3.10 ± 0.06 4- 0.40
154.10 24.30 153.10 50.10
4- 6.10 4- 2.10 ± 5.90 ± 0.40
148.40 18.20 t45.60 51.50
± ± ± ±
Rb + K Plasma (mequiv./1HzO) Muscle (mequiv./kg wet wt.) CSF(mequiv./lH20) Brain (mequiv./kg wet wt.)
3.23 100.60 2.46 88.80
± 0.24 ± 1.70 ± 0.12 ± 2.20
150.30 22.80 157.80 48.10
± 12.70 ± 0.50 ± 1.60 ± 1.00
Na Plasma (mequiv./1 H20) Muscle (mequiv./kg wet wt.) CSF (mequiv./l H20) Brain (mequiv./kg wet wt.)
0.40 1.30 0.90 0.60
Water ( ~ wet weight) Muscle Brain
Brain Research, 24 (1970) 311-321
75.40 ± 78.80 ±
0.70 0.40
75.10 ± 0.20 78.90 ± 0.20
74.60 ± 0.30 79.50 ± 0.30
RB AND K IN BRAIN AND CSF
315
TABLE II RATIOS FOR RUBIDIUM AND POTASSIUM DISTRIBUTION IN RABBITS IN WHICH DEPLETED K
HAS BEEN
REPLACEDWITHRb OR K These ratios were calculated from fluid concentrations in mequiv./l and tissue concentrations in mequiv./kg wet weight and are means 4- S.E. Rb replaced
Muscle/plasma CSF/plasma Brain/plasma Brain/CSF
K replaced
Rb
K
Rb + K
K
42.20 4- 5.10 0.45 4- 0.03 19.40 4- 1.00 43.50 4- 2.00
27.20 ± 1.40 0.92 4- 0.05 31.60 4- 1.50 34.80 4- 1.70
31.80 ± 2.20 0.77 4- 0.05 27.90 4- 1.30 36.30 4- 1.80
37.70 4- 5.90 1.04 4- 0.15 33.80 4- 5.10 32.50 4- 1.90
RESULTS A considerable replacement of body potassium with rubidium has been achieved in our rabbits. I f the skeletal muscle values (Fig. 1 and Table I) are representative of the total body potassium, the mean replacement Rb/(Rb + K) must be about 40 %. N o t only has the lost potassium been replaced with rubidium, but this replacement would appear to be complete in that the (Rb + K) contents of skeletal muscle and of brain are very similar to the potassium contents of skeletal muscle and brain in the potassium-replaced and normal animals. Although the extracellular spaces of the tissues were not estimated in terms of chloride spaces or other parameters, the water and sodium contents of the tissues were sufficiently normal in the rubidium-replaced rabbits to indicate that there had been no great change in extracellular or cell volume. The p H of arterial blood from the rubidium-replaced rabbits was variable, being abnormally low in some animals (Table III). Pco2 measurements confirmed that this was a metabolic acidosis, perhaps due to a blocking of hydrogen ion secretion in the kidney by rubidium ions a. The ratios (muscle/plasma) of Table II confirm that rubidium is accumulated in the cells of skeletal muscle in preference to potassium at least at our plasma concentrations. However, when CSF and brain are compared to plasma, rubidium is selectively excluded from both relative to potassium, the ratios CSF/plasma and brain/ plasma for rubidium being about 0.5 and 0.6 of the corresponding values for potassium. That this was mainly due to causes other than a very slow entry of rubidium into brain and CSF was shown in 3 animals which were kept for longer periods after the cessation of the major rubidium load, their drinking water containing 5 mequiv./l RbC1. There appeared to be a slight increase in rubidium replacement in brain and CSF between the rabbits kept 6 days and those in the main group (Table II). There was no evidence of increased replacement beyond this time. Thus the mean ratios Rb/ (Rb -k K) for degree of rubidium replacement were 0.331 and 0.399 for plasma and muscles and 0.232 and 0.251 for CSF and brain at 6 days (2 rabbits). At 10 days the Brain Research, 24 (1970) 311-321
M . W . B . BRADBURY
316
TABLE III ELECTRICAL POTENTIAL BETWEEN VENTRICULAR C S F
AND J U G U L A R VENOUS BLOOD, IN Rb-TREATED
RABBITS
Experiment no.
Potential (mY)
,4rterial p H
1
+ 10.9
2 3 4
+ 5.4 + 6.5 + 3.0
7.25 7.43 7.32 7.46
Plasma K Plasma Rb (mequiv./l H20) : (mequiv./l HeO)
0.50 0.71 0.70 0.72
1.63
2.56 1.52 1.78
TABLE IV 42K INFLUXINTODIFFERENTREGIONSOF BRAINFROMRUBIDIUM-TREATEDRABBITS The 4~K ratio is that of concentration of activity in CSF H20 to concentration of activity in plasma HuO. aSK influx in absence of rubidium was obtained from Bradbury and Kleeman4. Limits are S.E. Experiment
1
2
3
4
Plasma(K, mequiv./lH~O) CSF (K, mequiv./l H20) Plasma (Rb, mequiv./l HzO) CSF (Rb, mequiv./1H20)
2.28 2.25 0.50 0.23
1.73 2.21 0.45 0.40
2.20 2.37 0.73 0.41
3.17 2.63 0.95 0.43
42K ratio at 2 h CSF/Plasma
0.18
0.20
0.22
0.21
42K influx (mequiv./kg/h) Cerebral cortex White centrum ovale Hippocampus Residium Midbrain Cerebellum Pons and medulla
1.68 1.20 1.39 1.64 0.89 1.55 1.52
0.99 0.59 0.55 0.80 0.51 0.96 0.94
1.35 0.94 1.11 1.18 0.60 1.35 1.15
1.68 L.35 1.27 1.45 0.84 1.63 1.43
0.79 ± 0.74 ± 0.84 ± 0.91 i 0:74± 0.83 ! 0.75 ±
0.07* 0.10" 0.14" 0.12" 0.09" 0.07* 0.08*
* Mean ratio of 42K influx in Rb-treated rabbits to 4gK influx at the same plasma K conc. in non-Rb-treated rabbits. ratios were 0.317 and 0.356 for p l a s m a and muscle and 0.200 and 0.247 f o r C S F and brain (1 rabbit). T h e average positive potential o f 6.5 mV, recorded between C S F and blood, was rather higher (Table II) t h a n that in the n o r m a l and h y p o k a l e m i c animals studied previously, 4.3 and 4.6 m V respectively 4. It was confirmed in 2 r u b i d i u m - t r e a t e d animals that the potential varied in the usual fashion with arterial p H ( A p D/0.1 p H unit =- 2.5 mV); the arterial p H in these animals was altered by artificial respiration with gas mixtures c o n t a i n i n g different p r o p o r t i o n s o f CO2. Thus, if the potentials are corrected to an arterial p H o f 7.40, they are all within n o r m a l limits. Brain Research, 24 (1970) 311-321
RB AND K 1N BRAIN AND CSF
317
-•8C I=
•
p
4c
tl.I
d Rb + K in PERFUSION FLUIC mEq/I
Fig. 2. The relation between clearance of 42K from CSF into brain and the combined Rb and K concentration in the inflow±rig fluid during ventriculo-cisternal perfusion. Fluid was infused into each lateral ventricle at 30 pl/min and always contained 2.9 mequiv./l of K. The interrupted line represents the relation for a varying K concentration in the inflowing fluid in the absence of Rb from Bradbury and Stulcova4. The 42K influxes into different regions of the brain, measured over 2 h are recorded in Table IV. There is probably some dependence of the influx on plasma potassium, as in non-rubidium-treated animals 4. I f the 4ZK influx is expressed as a ratio to the normal influx at the relevant value of the plasma potassium, it is apparent that the presence of rubidium reduced 42K influx into brain, although influx from blood into CSF cannot be calculated for the reasons given above. The 42K ratio, concentration of activity in CSF H~O : concentration of activity in plasma HeO, at a mean of 0.20 was considerably less than the mean 4~K ratio for potassiumdepleted rabbits 4. The value for these was 0.306 ± 0.013, the concentration of potassium in blood plasma being similar at 2.29 ± 0.14 mequiv./1 Hence movement of potassium into CSF as well as into brain must have been suppressed by the presence of rubidium. At low concentrations of rubidium in the perfusing CSF the clearance of 42K from CSF into brain was similar to that occurring in the absence of rubidium (Fig. 2). At the higher concentrations of rubidium, 10 and 15 mequiv./1, the clearances of a2K into brain were less, 43.6 + 3.6 and 44.4 ± 2.6/zl/min, than those at 0, 1, 2 and 5 mequiv./l, namely 57.7 ± 4.45, 54.8 ± 1.4, 57.7 ± 7.0 and 53.4 ± 6.0/zl/min. The probability of the differences between the clearances at 0 and 15 mequiv./1 being due to chance is between 0.05 and 0.02. Pooling of the results at low and high concentrations would yield a smaller P value. A similar suppression of clearance was caused by 15 mequiv./1 of potassium in the perfusing CSF 5. The barrier clearance of 42K was markedly increased by increasing concentrations of rubidium (Fig. 3). The increase in relation to concentration difference was greatest between 2 and 5 mequiv./1 of rubidium in the perfusing CSF, but an increment still occurred between 10 and 15 mequiv./1. There was no suggestion of a decrement in 42K clearance such as took place between 10 and 15 mequiv./l potassium in the perfusing CSF 5. Brain Research, 24 (1970) 311-321
318
M.W.B.
BRADBURY
4C
::L
,,,30 "5 hi U
hi .J U rr lU I C n,
O Rb in PERFUSION FLUID mEq,/I
Fig. 3. The relation between the clearance of 42K from CSF into blood (barrier clearance) and the Rb concentration in the inflowing fluid during ventriculo-cisternal perfusion. Fluid was infused into each lateral ventricle at 30/~l/min and always contained 2.9 mequiv./l of K. Limits are the standard deviations of the means of 3 or 4 experiments. 3.0 UJ E F-" Z
2.0
_1 Lt. LL ,W
Z
_D
0
I
I
I
2
TIME OF PEP,FUSION,
HOURS.
Fig. 4. The K concentration in the effluent during ventriculo-cisternal perfusion of fluids containing Rb 1 m M (open circles) or Rb 10 m M (closed circles). Fluid was infused into each lateral ventricle at 30/~l/min and always contained 2.9 mequiv./l of K (interrupted line, analyzed value). Limits are the standard deviations of the mean of 3 experiments.
In association with the increased barrier clearance of 4gK from the perfusing CSF in the presence of rubidium, there was a significant drop in the absolute concentration of potassium in the effluent at the cisterna magna (Fig. 4). The fall was greatest in the experiments involving perfusion fluids containing 5 mequiv./l and 10 mequiv./1. In these experiments, the potassium concentration in the effluent dropped from 2.9 mequiv./l to a minimum of 2.45 mequiv./l and 2.49 mequiv./1 respectively. The greatest fall was during the hour following the first 20 min collection. Since the changes in potassium concentration during perfusion are limited, Brain Research, 24 (1970) 311-321
RB AND K IN BRAIN AND CSF
319
42K effiux from CSF into blood is necessarily related to the rubidium concentration in the perfusing CSF in a similar way to that in which the barrier clearance o f 42K is (Fig. 3). The relation is sigmoid and qualitatively similar to that between 42K efflux and the potassium concentration in CSF. DISCUSSION
Our results fully confirm the observation of Relman et al. s in rats that rubidium is accumulated in the cells of skeletal muscle in preference to potassium. As they pointed out this means that both cations cannot be passively distributed in conformity with the membrane potential. If this were so for potassium, then rubidium ions must be actively transported into the cell or absorbed within it. Recent studies, involving measurement of the membrane potential during the recovery of sodium loaded frog skeletal muscle in potassium-containing and rubidium-containing solutions and the effect on the process of digitalis alkaloids, prove that rubidium ions must be actively transported in association with sodium pumping 2. Membrane permeability of skeletal muscle fibers to rubidium is less than their permeability to potassium, as determined either by isotopic exchange or by the effect of changing concentrations on the membrane potentiall,ll, 12. This factor could by itself cause a relative accumulation of rubidium, if both ions are actively transported into the cell at not too dissimilar rates; another factor might be a greater affinity of the N a - K pump for rubidium than for potassium ions as originally suggested by Relman et al. s. It is considered that CSF is more nearly representative of the true interstitial fluid of brain than is plasma 7. This being so it is of interest that with respect to degree of rubidium replacement brain appears to bear a similar relation to CSF to that which muscle bears to plasma, i.e., rubidium is accumulated relatively more in the tissue than in the fluid. The mechanisms also could be similar. The relative accumulation of Rb in brain with respect to CSF is not as great as that in muscle with respect to plasma. Kernan observed greater accumulation of rubidium in red muscle than in white muscle from the rat 6. He attributed this to a greater passive permeability of the cells of the latter tissue to potassium ions. A similar explanation might apply to brain cells. In addition, there is a relative lack of rubidium in CSF and brain, when compared with blood and plasma. This exists over periods as long as 10 days after a near steady state has been established. It is likely that active transport out of CSF and brain into blood occurs, a hypothesis supported by the potential findings. The presence of a potassium pump, linked to sodium influx, inhibitable by ouabain and directed from CSF to blood has already been demonstrated 5. This active transport mechanism is activated by rubidium. Most likely it is also capable of transporting rubidium in the same direction as potassium. This appears to be a general property of the N a - K pump. Less relative influx of rubidium or greater pumping of this ion would then easily explain its deficiency in CSF. That brain is comparatively as deficient in rubidium as CSF suggests that such a pump capable of transporting both potassium and rubidium ions is present at the blood-brain barrier as well as between CSF and blood. Brain Research, 24 (1970) 311-321
320
M.W.B.
BRADBURY
The concentration of potassium in the CSF of rubidium-replaced rabbits was lower than that ever recorded in either normal or severely potassium-depleted rabbits. This depression of CSF potassium concentration by rubidium can be related to the effect of rubidium on the movements of 42K. Since influx of potassium into CSF and brain is reduced and active efflux from CSF is stimulated, a lower steady-state concentration of potassium in CSF in the presence of rubidium is the reasonable result. An enhancement of sodium pumping 2 and oxygen consumption 3 in relation to rubidium acting on the N a - K pump occurs in other tissues. Furthermore rubidium at certain concentration will actually increase active influx of potassium ions into the human red blood celP o. A similar enhancement of potassium transport between CSF and blood has been demonstrated here and provides further proof of the existence of a similar pump between CSF and blood. The fact that rubidium in the perfusing CSF causes a reduction in the actual concentration of potassium in the outflowing CSF indicates that the rubidium is actually increasing the net movement of potassium ions out of the CSF and not just enhancing a bidirectional flux. That the change in outflow concentration is not as great as the change in the barrier clearance of 42K might suggest could be due to a net leakage of potassium ions from brain cells. The findings further characterize the nature of potassium movements between blood on one hand and brain and CSF on the other hand. A summary may be made of the known properties of potassium influx into the brain-CSF system and effiux from it into blood. Influx of potassium into both brain and CSF is little dependent on the plasma potassium concentration4; it is energy dependent at least during hypokalemia4; it shows some interaction with rubidium. Efflux from CSF into blood is largely dependent on an N a - K pump inhibitable by ouabainS; it is energy dependent, at least during hyperkalemia 4; it is highly dependent on both potassium 5 and rubidium concentrations in CSF and shows a sigmoid relation with the concentrations of both these ions. In general, the results confirm the concept of a close relation between the ionic composition of CSF and that of the interstitial fluid of brain. Thus in respect to rubidium accumulation, brain is to CSF as muscle is to plasma. The simplest explanation of this finding is that the CSF is reflecting the ionic composition of the interstitial fluid of brain. As has been discussed, the results taken together with previous findings indicate the presence of a mechanism capable of actively transporting rubidium and potassium ions from CSF and/or interstitial fluid of brain to blood. The exact site or sites of this mechanism is not proven, but it is probably present at the choroid epithelia or the blood-brain barrier. The hypothesis that it occurs at both these interphases is most completely compatible with the results. SUMMARY
(1) About 4 0 ~ of skeletal muscle potassium was replaced with rubidium in rabbits by feeding a potassium-free diet and injecting rubidium chloride. Brain Research, 24 (1970) 311-321
RB AND K IN BRAIN AND C S F
321
(2) T h e ratio o f r u b i d i u m replacement, R b / ( R b + K), was 30 ~ greater in muscle t h a n in p l a s m a . (3) R u b i d i u m was relatively lacking in b r a i n a n d C S F , the ratio o f r u b i d i u m r e p l a c e m e n t for b r a i n being 52 ~ o f t h a t for muscle a n d the r a t i o f o r C S F being 58 ~o o f t h a t for muscle. The ratio for b r a i n was 12 ~ higher t h a n t h a t for C S F . (4) R u b i d i u m h a d no direct effect on the electrical p o t e n t i a l between C S F a n d blood. (5) P o t a s s i u m influx m e a s u r e d with 4ZK into b r a i n a n d C S F was decreased by r u b i d i u m r e p l a c e m e n t by a b o u t 20 ~ . (6) Ventriculo-cisternal perfusion o f artificial C S F c o n t a i n i n g a n o r m a l 2.9 mequiv./l o f p o t a s s i u m a n d between 1-15 mequiv./1 o f r u b i d i u m m a r k e d l y enhanced p o t a s s i u m efflux into b l o o d . The relation o f p o t a s s i u m flux to r u b i d i u m c o n c e n t r a t i o n was sigmoid. (7) P r o b a b l y p o t a s s i u m a n d r u b i d i u m are t r a n s p o r t e d into b r a i n a n d C S F across the same sites at specific rates. B o t h ions m a y be actively m o v e d out o f C S F a n d b r a i n into b l o o d by a s o d i u m - p o t a s s i u m p u m p sited at the c h o r o i d e p i t h e l i u m a n d / o r the b l o o d - b r a i n barrier. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t N B 05905 a n d in p a r t b y a g r a n t f r o m the M e d i c a l Research Council. I n v a l u a b l e assistance was rendered by Miss Helen B a g d o y a n , Miss Alice Berberian a n d Miss J a n e t Wilson. REFERENCES 1 ADRIAN, R. H., The rubidium and potassium permeability of frog muscle membrane, J. Physiol. (Lond.), 175 (1964) 134-159. 2 ADRIAN,R. H., AND SLAYMAN,C. L., Membrane potential and conductance during transport of sodium, potassium and rubidium in frog muscle, J. Physiol. (Lond.), 184 (1966) 970-1014. 3 BAKER, P. F., AND CONNELLY , C. M., Some properties of the external activation site of the sodium pump in crab nerve, J. Physiol. (Lond.), 185 (1966) 270-297. 4 BRADBURY,M. W. B., AND KLEEMAN)C. R., Stability of the potassium content of cerebrospinal fluid and brain, Amer. J. Physiol., 213 (1967) 519-528. 5 BRADBURY,M. W. B., AND STULCOVA)B., Efiqluxmechanism contributing to the stability of the potassium concentration in cerebrospinal fluid, J. Physiol. (Lond.}, 208 (1970) 415-430. 6 KERNAN, R. D., Accumulation of caesium and rubidium in vivo by red and white muscles of the rat, J. Physiol. (Lond.), 204 (1969) 195-205. 7 PAPPENHEIMER,J. R., The ionic composition of cerebral extracellular fluid and its relation to control of breathing, Harvey Lect., 61 (1967) 71-94. 8 RELMAN,A. S., LAMBIE,A. T., BURROWS,B. A., AND ROY, A. M., Cation accumulation by muscle tissue: the displacement of potassium by rubidium and cesium in the living animal, J. clin. Invest., 36 (1957) 1249-1256. 9 RELMAN,A. S., ROY, A. M., AND SCHWARTZ)W. B., The acidifying effect of rubidium in normal and potassium deficient alkalotic rats, J. clin. Invest., 34 (1955) 538-544. 10 SACHS, J. R., AND WELT, L. C., The concentration dependence of active potassium transport in the human red blood cell, J. clin. Invest., 46 (1967) 65-76. 11 SJODIN, R. A., Rubidium and cesium fluxes in muscle as related to the membrane potential, J. gen. Physiol., 42 (1959) 983-1003. 12 SJODIN, R. A., Some cation interactions in muscle, J. gen. Physiol., 44 (1961) 929-962. Brain Research, 24 (1970) 311-321
311
T H E E F F E C T OF R U B I D I U M ON T H E D I S T R I B U T I O N A N D M O V E M E N T OF POTASSIUM B E T W E E N BLOOD, BRAIN A N D C E R E B R O S P I N A L F L U I D IN T H E RABBIT
MICHAEL W. B. B R A D B U R Y
Division of Medicine, Cedars-Sinai Medical Center and Cedars-Sinai Medical Research Institute, Los Angeles, Calif. 90048 (U.S.A.) and Sherrington School of Physiology, St. Thomas's Hospital Medical School, London S.E.1 (Great Britain) (Accepted June 2nd, 1970)
INTRODUCTION
The relative distribution of potassium and rubidium between skeletal muscle tissue and blood plasma has been studied in the intact rat by Relman et al. s. In these animals in which about 40 ~ of the body potassium had been replaced by rubidium, rubidium was accumulated in the cell water relative to the extracellular fluid in preference to potassium. There is much other evidence that rubidium and potassium compete for membrane-transport sites in frog skeletal muscle 11,12 and in particular compete in the sodium-potassium pump of frog skeletal muscle z and of red blood cells lo. The potassium concentration in brain and cerebrospinal fluid (CSF) is kept very constant in the face of severe and chronic hypokalemia and hyperkalemia 4. The control of this stability depends in part on an influx of potassium into brain and CSF which increases little with the plasma concentration of potassium 4 and in part on an active mechanism inhibitable by ouabain which pumps potassium out of CSF into blood and is highly dependent on the CSF concentration of potassiumL It is not known whether this potassium pump exists solely at the choroid plexuses or is present at the blood-brain barrier as well. The latter hypothesis is likely since the potassium concentration in the interstitial fluid of the brain must be controlled as well as that in the CSF 4. In view of the known interactions between transport of potassium and rubidium, it seemed that a study of the effect of rubidium on potassium distribution, and movement in the system comprised by blood, brain and CSF might throw further light on the mechanisms involved in homeostasis of the concentration of potassium in CSF and in the interstitial fluid of brain. In particular, the results might indicate whether there is a potassium and rubidium pump sited at the blood-brain barrier which transfers these ions from the interstitial fluid of brain to blood in a similar manner to the mechanisms for potassium demonstrated as occurring between CSF and blood. Brain Research, 24 (1970) 311-321
312
M. W . B. B R A D B U R Y
The problem has been approached in two ways. Firstly, the distribution of potassium and rubidium ions between tissues and fluids has been studied in rabbits in which a proportion of the total body potassium has been replaced by rubidium. Secondly, the effect of rubidium ions on the influx of 42K into brain and CSF and on the effiux of 42K from CSF into brain and blood have been studied. MATERIALS A N D METHODS
Most of the experiments were carried out on adult female Dutch Belt rabbits, weighing 2.0-2.5 kg. The studies, involving movements of 4ZK, were made on New Zealand White rabbits of 2.5-3.0 kg (the latter breed was used in order that the results might be comparable to previous values of influx determined in this breed).
Replacement of body potassium with rubidium Rabbits were fed ad libitum on a special potassium free diet 4 for 3 weeks, the diet being begun on a Monday. Previous experience suggested that by this time the skeletal muscle potassium should fall to about 70 mequiv./kg wet weight. During the 4th week of the diet rubidium was given to replace the lost potassium. Initially, 10 sterile doses of 60 ml of glucose 5 % and 30 ml of rubidium.chloride 155 m M were given intraperitoneally through an indwelling cannula at intervals. This always caused temporary signs of peritoneal irritation and on occasions stopped the heart. Later, it was found more satisfactory to very slowly infuse RbC1 155 m M into an ear vein. 100 ml doses were given in this way at a rate of 0.25 ml/min on the Monday, Wednesday and Friday of the 4th week of the diet. Whatever the method, all rabbits received a total of 300 ml (46.5 mM) of RbC1 during the 4th week and no further injections after the Friday. Control animals received KC1 155 m M instead of RbC1. Rubidiumtreated rabbits were normal in appearance. Hyperactivity and hyperventilation were noted in some. Attempts to increase the degree of rubidium replacement further caused convulsions to occur.
Final studies on animals For steady-state studies, rabbits were killed on the Monday following the last injection of RbCI. This allowed at least 72 h for the final equilibration of the rubidium. Animals were anesthetized and decapitated. Two rabbits were kept for 6 days after the final rubidium injection, and 1 rabbit for 10 days, before sacrifice. These 3 animals received 5 mequiv./l RbC1 in their drinking water. The electrolytes were extracted from tissue samples with 0.5 N nitric acid. The procedure for collecting fluid samples, killing the rabbits and preparing the tissues have been described previously4. The electrical potential between ventricular CSF and jugular venous blood was measured in rubidium-treated Dutch Belt rabbits 4 on the Monday or Tuesday, following RbC1 infusion.
Brain Research, 24 (1970) 311-321
RB AND K IN BRAIN AND CSF
313
Transport of 42K into brain and CSF A constant concentration of 42K was maintained in the blood plasma of rubidium-replaced rabbits by intravenous infusion of this isotope in isotonic saline at a diminishing rate for 2 h 4. This was performed on the Tuesday following the rubidium replacement week. Since 4ZK movement into a given region of brain is determined by a single rate constant, a figure for influx of potassium in mequiv./kg/h can be simply calculated 4. Entry of 42K into CSF from blood follows more complex kinetics, which do not allow the calculation of influx from the activities and concentrations present at a single time. Hence the ratio of the activity in CSF to that in plasma has been recorded.
Ventriculo-cisternal perfusion of normal rabbits The clearance of 42K from CSF into blood, 'barrier clearance', and from CSF into brain was measured during ventriculo-cisternal perfusion of artificial CSF from both lateral ventricles to the cisterna magna of anesthetized normal rabbits for 2 h. The potassium concentration in the perfusion fluid was always 2.9 mequiv./1. Differing concentrations of rubidium between 1 and 15 mequiv./1 were obtained by adding increasing volumes of a similar perfusion fluid in which the NaCl had been completely replaced by RbCl. The details of the technique and of the treatment of the results have been fully describedL In brief, the total radioactivity lost from the perfusing CSF is compared with tbe total radioactivity gained by the central nervous system. The difference represents transfer into the blood stream. Clearances are obtained by dividing amounts of radioactivity transferred by the mean concentration of radioactivity in the perfusing CSF. The efflux of potassium into blood is the product of the barrier clearance of 42K and the mean concentration of potassium in the perfusing CSF.
Analytical methods Rubidium, potassium and sodium were analyzed in samples of plasma, CSF and nitric acid extracts of dried skeletal muscle and brain (1 ml 0.5 N HNOa/100 mg wet weight of tissue). All these fluids were diluted (1:10) in l0 mM cesium chloride0.02~ Acationox (Scientific Products). The emissions in an oxygen hydrogen flame were recorded in a Zeiss PMQ 11 spectro-flame photometer with double monochromator at 805 #m for rubidium, 757 #m for potassium and 589/tin for sodium. A slit width of 0.5 was used for rubidium and potassium and 0.01 for sodium. The rubidium and potassium settings were considerably removed from those giving optimum emission in order to reduce mutual interference between these two ions. Almost complete elimination of residual interference plus an almost two-fold potentiation of the reading was afforded by the presence of 10 mM cesium chlorideL Unknowns were 'bracketed' between two standards of approximately similar composition to the unknowns with respect to Rb, K and Na.
Brain Research, 24 (1970) 311-321
314
M . W . B . BRADBURY
o.,]
Rb Rb+K
Fig. 1. Mean ratio of Rb replacement, Rb/(Rb + K), in plasma, CSF, muscle and brain from 5 rubidium-treated rabbits. Limits are ± 2 S.E. TABLE I ELECTROLYTES AND WATER IN FLUIDS AND TISSUES FROM RABBITS IN WHICH DEPLETED K HAS BEEN REPLACED WITH Rb OR K Figures for animals on a diet of normal K content are given for comparison. Values are means ± S.E. Figures in parentheses are number of animals.
Rb replaced (5)
K replaced (3)
Normal (6)
Rb Plasma (mequiv./l H20) Muscle (mequiv./kg wet wt.) CSF (mequiv./1H~O) Brain (mequiv./kg wet wt.)
0.99 40.60 0.44 19.00
_[: ± ± ±
0.08 3.00 0.03 0.90
K Plasma (mequiv./1H20) Muscle (mequiv./kg wet wt.) CSF(mequiv./1HzO) Brain (mequiv./kg wet wt.)
2.23 60.00 2.02 69.80
± ± ± ±
0.16 2.60 0.09 1.50
I
I
I
2.84 102.00 2.83 91.60
± 0.47 ± 3.40 :[: 0.14 ± 0.80
3.96 102.20 2.89 87.90
± 0.10 ± 3.10 ± 0.06 4- 0.40
154.10 24.30 153.10 50.10
4- 6.10 4- 2.10 ± 5.90 ± 0.40
148.40 18.20 t45.60 51.50
± ± ± ±
Rb + K Plasma (mequiv./1HzO) Muscle (mequiv./kg wet wt.) CSF(mequiv./lH20) Brain (mequiv./kg wet wt.)
3.23 100.60 2.46 88.80
± 0.24 ± 1.70 ± 0.12 ± 2.20
150.30 22.80 157.80 48.10
± 12.70 ± 0.50 ± 1.60 ± 1.00
Na Plasma (mequiv./1 H20) Muscle (mequiv./kg wet wt.) CSF (mequiv./l H20) Brain (mequiv./kg wet wt.)
0.40 1.30 0.90 0.60
Water ( ~ wet weight) Muscle Brain
Brain Research, 24 (1970) 311-321
75.40 ± 78.80 ±
0.70 0.40
75.10 ± 0.20 78.90 ± 0.20
74.60 ± 0.30 79.50 ± 0.30
RB AND K IN BRAIN AND CSF
315
TABLE II RATIOS FOR RUBIDIUM AND POTASSIUM DISTRIBUTION IN RABBITS IN WHICH DEPLETED K
HAS BEEN
REPLACEDWITHRb OR K These ratios were calculated from fluid concentrations in mequiv./l and tissue concentrations in mequiv./kg wet weight and are means 4- S.E. Rb replaced
Muscle/plasma CSF/plasma Brain/plasma Brain/CSF
K replaced
Rb
K
Rb + K
K
42.20 4- 5.10 0.45 4- 0.03 19.40 4- 1.00 43.50 4- 2.00
27.20 ± 1.40 0.92 4- 0.05 31.60 4- 1.50 34.80 4- 1.70
31.80 ± 2.20 0.77 4- 0.05 27.90 4- 1.30 36.30 4- 1.80
37.70 4- 5.90 1.04 4- 0.15 33.80 4- 5.10 32.50 4- 1.90
RESULTS A considerable replacement of body potassium with rubidium has been achieved in our rabbits. I f the skeletal muscle values (Fig. 1 and Table I) are representative of the total body potassium, the mean replacement Rb/(Rb + K) must be about 40 %. N o t only has the lost potassium been replaced with rubidium, but this replacement would appear to be complete in that the (Rb + K) contents of skeletal muscle and of brain are very similar to the potassium contents of skeletal muscle and brain in the potassium-replaced and normal animals. Although the extracellular spaces of the tissues were not estimated in terms of chloride spaces or other parameters, the water and sodium contents of the tissues were sufficiently normal in the rubidium-replaced rabbits to indicate that there had been no great change in extracellular or cell volume. The p H of arterial blood from the rubidium-replaced rabbits was variable, being abnormally low in some animals (Table III). Pco2 measurements confirmed that this was a metabolic acidosis, perhaps due to a blocking of hydrogen ion secretion in the kidney by rubidium ions a. The ratios (muscle/plasma) of Table II confirm that rubidium is accumulated in the cells of skeletal muscle in preference to potassium at least at our plasma concentrations. However, when CSF and brain are compared to plasma, rubidium is selectively excluded from both relative to potassium, the ratios CSF/plasma and brain/ plasma for rubidium being about 0.5 and 0.6 of the corresponding values for potassium. That this was mainly due to causes other than a very slow entry of rubidium into brain and CSF was shown in 3 animals which were kept for longer periods after the cessation of the major rubidium load, their drinking water containing 5 mequiv./l RbC1. There appeared to be a slight increase in rubidium replacement in brain and CSF between the rabbits kept 6 days and those in the main group (Table II). There was no evidence of increased replacement beyond this time. Thus the mean ratios Rb/ (Rb -k K) for degree of rubidium replacement were 0.331 and 0.399 for plasma and muscles and 0.232 and 0.251 for CSF and brain at 6 days (2 rabbits). At 10 days the Brain Research, 24 (1970) 311-321
M . W . B . BRADBURY
316
TABLE III ELECTRICAL POTENTIAL BETWEEN VENTRICULAR C S F
AND J U G U L A R VENOUS BLOOD, IN Rb-TREATED
RABBITS
Experiment no.
Potential (mY)
,4rterial p H
1
+ 10.9
2 3 4
+ 5.4 + 6.5 + 3.0
7.25 7.43 7.32 7.46
Plasma K Plasma Rb (mequiv./l H20) : (mequiv./l HeO)
0.50 0.71 0.70 0.72
1.63
2.56 1.52 1.78
TABLE IV 42K INFLUXINTODIFFERENTREGIONSOF BRAINFROMRUBIDIUM-TREATEDRABBITS The 4~K ratio is that of concentration of activity in CSF H20 to concentration of activity in plasma HuO. aSK influx in absence of rubidium was obtained from Bradbury and Kleeman4. Limits are S.E. Experiment
1
2
3
4
Plasma(K, mequiv./lH~O) CSF (K, mequiv./l H20) Plasma (Rb, mequiv./l HzO) CSF (Rb, mequiv./1H20)
2.28 2.25 0.50 0.23
1.73 2.21 0.45 0.40
2.20 2.37 0.73 0.41
3.17 2.63 0.95 0.43
42K ratio at 2 h CSF/Plasma
0.18
0.20
0.22
0.21
42K influx (mequiv./kg/h) Cerebral cortex White centrum ovale Hippocampus Residium Midbrain Cerebellum Pons and medulla
1.68 1.20 1.39 1.64 0.89 1.55 1.52
0.99 0.59 0.55 0.80 0.51 0.96 0.94
1.35 0.94 1.11 1.18 0.60 1.35 1.15
1.68 L.35 1.27 1.45 0.84 1.63 1.43
0.79 ± 0.74 ± 0.84 ± 0.91 i 0:74± 0.83 ! 0.75 ±
0.07* 0.10" 0.14" 0.12" 0.09" 0.07* 0.08*
* Mean ratio of 42K influx in Rb-treated rabbits to 4gK influx at the same plasma K conc. in non-Rb-treated rabbits. ratios were 0.317 and 0.356 for p l a s m a and muscle and 0.200 and 0.247 f o r C S F and brain (1 rabbit). T h e average positive potential o f 6.5 mV, recorded between C S F and blood, was rather higher (Table II) t h a n that in the n o r m a l and h y p o k a l e m i c animals studied previously, 4.3 and 4.6 m V respectively 4. It was confirmed in 2 r u b i d i u m - t r e a t e d animals that the potential varied in the usual fashion with arterial p H ( A p D/0.1 p H unit =- 2.5 mV); the arterial p H in these animals was altered by artificial respiration with gas mixtures c o n t a i n i n g different p r o p o r t i o n s o f CO2. Thus, if the potentials are corrected to an arterial p H o f 7.40, they are all within n o r m a l limits. Brain Research, 24 (1970) 311-321
RB AND K 1N BRAIN AND CSF
317
-•8C I=
•
p
4c
tl.I
d Rb + K in PERFUSION FLUIC mEq/I
Fig. 2. The relation between clearance of 42K from CSF into brain and the combined Rb and K concentration in the inflow±rig fluid during ventriculo-cisternal perfusion. Fluid was infused into each lateral ventricle at 30 pl/min and always contained 2.9 mequiv./l of K. The interrupted line represents the relation for a varying K concentration in the inflowing fluid in the absence of Rb from Bradbury and Stulcova4. The 42K influxes into different regions of the brain, measured over 2 h are recorded in Table IV. There is probably some dependence of the influx on plasma potassium, as in non-rubidium-treated animals 4. I f the 4ZK influx is expressed as a ratio to the normal influx at the relevant value of the plasma potassium, it is apparent that the presence of rubidium reduced 42K influx into brain, although influx from blood into CSF cannot be calculated for the reasons given above. The 42K ratio, concentration of activity in CSF H~O : concentration of activity in plasma HeO, at a mean of 0.20 was considerably less than the mean 4~K ratio for potassiumdepleted rabbits 4. The value for these was 0.306 ± 0.013, the concentration of potassium in blood plasma being similar at 2.29 ± 0.14 mequiv./1 Hence movement of potassium into CSF as well as into brain must have been suppressed by the presence of rubidium. At low concentrations of rubidium in the perfusing CSF the clearance of 42K from CSF into brain was similar to that occurring in the absence of rubidium (Fig. 2). At the higher concentrations of rubidium, 10 and 15 mequiv./1, the clearances of a2K into brain were less, 43.6 + 3.6 and 44.4 ± 2.6/zl/min, than those at 0, 1, 2 and 5 mequiv./l, namely 57.7 ± 4.45, 54.8 ± 1.4, 57.7 ± 7.0 and 53.4 ± 6.0/zl/min. The probability of the differences between the clearances at 0 and 15 mequiv./1 being due to chance is between 0.05 and 0.02. Pooling of the results at low and high concentrations would yield a smaller P value. A similar suppression of clearance was caused by 15 mequiv./1 of potassium in the perfusing CSF 5. The barrier clearance of 42K was markedly increased by increasing concentrations of rubidium (Fig. 3). The increase in relation to concentration difference was greatest between 2 and 5 mequiv./1 of rubidium in the perfusing CSF, but an increment still occurred between 10 and 15 mequiv./1. There was no suggestion of a decrement in 42K clearance such as took place between 10 and 15 mequiv./l potassium in the perfusing CSF 5. Brain Research, 24 (1970) 311-321
318
M.W.B.
BRADBURY
4C
::L
,,,30 "5 hi U
hi .J U rr lU I C n,
O Rb in PERFUSION FLUID mEq,/I
Fig. 3. The relation between the clearance of 42K from CSF into blood (barrier clearance) and the Rb concentration in the inflowing fluid during ventriculo-cisternal perfusion. Fluid was infused into each lateral ventricle at 30/~l/min and always contained 2.9 mequiv./l of K. Limits are the standard deviations of the means of 3 or 4 experiments. 3.0 UJ E F-" Z
2.0
_1 Lt. LL ,W
Z
_D
0
I
I
I
2
TIME OF PEP,FUSION,
HOURS.
Fig. 4. The K concentration in the effluent during ventriculo-cisternal perfusion of fluids containing Rb 1 m M (open circles) or Rb 10 m M (closed circles). Fluid was infused into each lateral ventricle at 30/~l/min and always contained 2.9 mequiv./l of K (interrupted line, analyzed value). Limits are the standard deviations of the mean of 3 experiments.
In association with the increased barrier clearance of 4gK from the perfusing CSF in the presence of rubidium, there was a significant drop in the absolute concentration of potassium in the effluent at the cisterna magna (Fig. 4). The fall was greatest in the experiments involving perfusion fluids containing 5 mequiv./l and 10 mequiv./1. In these experiments, the potassium concentration in the effluent dropped from 2.9 mequiv./l to a minimum of 2.45 mequiv./l and 2.49 mequiv./1 respectively. The greatest fall was during the hour following the first 20 min collection. Since the changes in potassium concentration during perfusion are limited, Brain Research, 24 (1970) 311-321
RB AND K IN BRAIN AND CSF
319
42K effiux from CSF into blood is necessarily related to the rubidium concentration in the perfusing CSF in a similar way to that in which the barrier clearance o f 42K is (Fig. 3). The relation is sigmoid and qualitatively similar to that between 42K efflux and the potassium concentration in CSF. DISCUSSION
Our results fully confirm the observation of Relman et al. s in rats that rubidium is accumulated in the cells of skeletal muscle in preference to potassium. As they pointed out this means that both cations cannot be passively distributed in conformity with the membrane potential. If this were so for potassium, then rubidium ions must be actively transported into the cell or absorbed within it. Recent studies, involving measurement of the membrane potential during the recovery of sodium loaded frog skeletal muscle in potassium-containing and rubidium-containing solutions and the effect on the process of digitalis alkaloids, prove that rubidium ions must be actively transported in association with sodium pumping 2. Membrane permeability of skeletal muscle fibers to rubidium is less than their permeability to potassium, as determined either by isotopic exchange or by the effect of changing concentrations on the membrane potentiall,ll, 12. This factor could by itself cause a relative accumulation of rubidium, if both ions are actively transported into the cell at not too dissimilar rates; another factor might be a greater affinity of the N a - K pump for rubidium than for potassium ions as originally suggested by Relman et al. s. It is considered that CSF is more nearly representative of the true interstitial fluid of brain than is plasma 7. This being so it is of interest that with respect to degree of rubidium replacement brain appears to bear a similar relation to CSF to that which muscle bears to plasma, i.e., rubidium is accumulated relatively more in the tissue than in the fluid. The mechanisms also could be similar. The relative accumulation of Rb in brain with respect to CSF is not as great as that in muscle with respect to plasma. Kernan observed greater accumulation of rubidium in red muscle than in white muscle from the rat 6. He attributed this to a greater passive permeability of the cells of the latter tissue to potassium ions. A similar explanation might apply to brain cells. In addition, there is a relative lack of rubidium in CSF and brain, when compared with blood and plasma. This exists over periods as long as 10 days after a near steady state has been established. It is likely that active transport out of CSF and brain into blood occurs, a hypothesis supported by the potential findings. The presence of a potassium pump, linked to sodium influx, inhibitable by ouabain and directed from CSF to blood has already been demonstrated 5. This active transport mechanism is activated by rubidium. Most likely it is also capable of transporting rubidium in the same direction as potassium. This appears to be a general property of the N a - K pump. Less relative influx of rubidium or greater pumping of this ion would then easily explain its deficiency in CSF. That brain is comparatively as deficient in rubidium as CSF suggests that such a pump capable of transporting both potassium and rubidium ions is present at the blood-brain barrier as well as between CSF and blood. Brain Research, 24 (1970) 311-321
320
M.W.B.
BRADBURY
The concentration of potassium in the CSF of rubidium-replaced rabbits was lower than that ever recorded in either normal or severely potassium-depleted rabbits. This depression of CSF potassium concentration by rubidium can be related to the effect of rubidium on the movements of 42K. Since influx of potassium into CSF and brain is reduced and active efflux from CSF is stimulated, a lower steady-state concentration of potassium in CSF in the presence of rubidium is the reasonable result. An enhancement of sodium pumping 2 and oxygen consumption 3 in relation to rubidium acting on the N a - K pump occurs in other tissues. Furthermore rubidium at certain concentration will actually increase active influx of potassium ions into the human red blood celP o. A similar enhancement of potassium transport between CSF and blood has been demonstrated here and provides further proof of the existence of a similar pump between CSF and blood. The fact that rubidium in the perfusing CSF causes a reduction in the actual concentration of potassium in the outflowing CSF indicates that the rubidium is actually increasing the net movement of potassium ions out of the CSF and not just enhancing a bidirectional flux. That the change in outflow concentration is not as great as the change in the barrier clearance of 42K might suggest could be due to a net leakage of potassium ions from brain cells. The findings further characterize the nature of potassium movements between blood on one hand and brain and CSF on the other hand. A summary may be made of the known properties of potassium influx into the brain-CSF system and effiux from it into blood. Influx of potassium into both brain and CSF is little dependent on the plasma potassium concentration4; it is energy dependent at least during hypokalemia4; it shows some interaction with rubidium. Efflux from CSF into blood is largely dependent on an N a - K pump inhibitable by ouabainS; it is energy dependent, at least during hyperkalemia 4; it is highly dependent on both potassium 5 and rubidium concentrations in CSF and shows a sigmoid relation with the concentrations of both these ions. In general, the results confirm the concept of a close relation between the ionic composition of CSF and that of the interstitial fluid of brain. Thus in respect to rubidium accumulation, brain is to CSF as muscle is to plasma. The simplest explanation of this finding is that the CSF is reflecting the ionic composition of the interstitial fluid of brain. As has been discussed, the results taken together with previous findings indicate the presence of a mechanism capable of actively transporting rubidium and potassium ions from CSF and/or interstitial fluid of brain to blood. The exact site or sites of this mechanism is not proven, but it is probably present at the choroid epithelia or the blood-brain barrier. The hypothesis that it occurs at both these interphases is most completely compatible with the results. SUMMARY
(1) About 4 0 ~ of skeletal muscle potassium was replaced with rubidium in rabbits by feeding a potassium-free diet and injecting rubidium chloride. Brain Research, 24 (1970) 311-321
RB AND K IN BRAIN AND C S F
321
(2) T h e ratio o f r u b i d i u m replacement, R b / ( R b + K), was 30 ~ greater in muscle t h a n in p l a s m a . (3) R u b i d i u m was relatively lacking in b r a i n a n d C S F , the ratio o f r u b i d i u m r e p l a c e m e n t for b r a i n being 52 ~ o f t h a t for muscle a n d the r a t i o f o r C S F being 58 ~o o f t h a t for muscle. The ratio for b r a i n was 12 ~ higher t h a n t h a t for C S F . (4) R u b i d i u m h a d no direct effect on the electrical p o t e n t i a l between C S F a n d blood. (5) P o t a s s i u m influx m e a s u r e d with 4ZK into b r a i n a n d C S F was decreased by r u b i d i u m r e p l a c e m e n t by a b o u t 20 ~ . (6) Ventriculo-cisternal perfusion o f artificial C S F c o n t a i n i n g a n o r m a l 2.9 mequiv./l o f p o t a s s i u m a n d between 1-15 mequiv./1 o f r u b i d i u m m a r k e d l y enhanced p o t a s s i u m efflux into b l o o d . The relation o f p o t a s s i u m flux to r u b i d i u m c o n c e n t r a t i o n was sigmoid. (7) P r o b a b l y p o t a s s i u m a n d r u b i d i u m are t r a n s p o r t e d into b r a i n a n d C S F across the same sites at specific rates. B o t h ions m a y be actively m o v e d out o f C S F a n d b r a i n into b l o o d by a s o d i u m - p o t a s s i u m p u m p sited at the c h o r o i d e p i t h e l i u m a n d / o r the b l o o d - b r a i n barrier. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t N B 05905 a n d in p a r t b y a g r a n t f r o m the M e d i c a l Research Council. I n v a l u a b l e assistance was rendered by Miss Helen B a g d o y a n , Miss Alice Berberian a n d Miss J a n e t Wilson. REFERENCES 1 ADRIAN, R. H., The rubidium and potassium permeability of frog muscle membrane, J. Physiol. (Lond.), 175 (1964) 134-159. 2 ADRIAN,R. H., AND SLAYMAN,C. L., Membrane potential and conductance during transport of sodium, potassium and rubidium in frog muscle, J. Physiol. (Lond.), 184 (1966) 970-1014. 3 BAKER, P. F., AND CONNELLY , C. M., Some properties of the external activation site of the sodium pump in crab nerve, J. Physiol. (Lond.), 185 (1966) 270-297. 4 BRADBURY,M. W. B., AND KLEEMAN)C. R., Stability of the potassium content of cerebrospinal fluid and brain, Amer. J. Physiol., 213 (1967) 519-528. 5 BRADBURY,M. W. B., AND STULCOVA)B., Efiqluxmechanism contributing to the stability of the potassium concentration in cerebrospinal fluid, J. Physiol. (Lond.}, 208 (1970) 415-430. 6 KERNAN, R. D., Accumulation of caesium and rubidium in vivo by red and white muscles of the rat, J. Physiol. (Lond.), 204 (1969) 195-205. 7 PAPPENHEIMER,J. R., The ionic composition of cerebral extracellular fluid and its relation to control of breathing, Harvey Lect., 61 (1967) 71-94. 8 RELMAN,A. S., LAMBIE,A. T., BURROWS,B. A., AND ROY, A. M., Cation accumulation by muscle tissue: the displacement of potassium by rubidium and cesium in the living animal, J. clin. Invest., 36 (1957) 1249-1256. 9 RELMAN,A. S., ROY, A. M., AND SCHWARTZ)W. B., The acidifying effect of rubidium in normal and potassium deficient alkalotic rats, J. clin. Invest., 34 (1955) 538-544. 10 SACHS, J. R., AND WELT, L. C., The concentration dependence of active potassium transport in the human red blood cell, J. clin. Invest., 46 (1967) 65-76. 11 SJODIN, R. A., Rubidium and cesium fluxes in muscle as related to the membrane potential, J. gen. Physiol., 42 (1959) 983-1003. 12 SJODIN, R. A., Some cation interactions in muscle, J. gen. Physiol., 44 (1961) 929-962. Brain Research, 24 (1970) 311-321