- Email: [email protected]
Development of chloride transport by the rat choroid plexus, in vitro
Brain Research, 624 (1993) 181-187 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
181
BRES 19272
Development of chloride transport by the rat choroid plexus, in vitro J.E. Preston, M. Dyas and C.E. Johanson Program in Neurosurgery, Department of Clinical Neurosciences, Brown University~Rhode Island Hospital, Providence, RI 02903 (USA) (Accepted 11 May 1993)
Key words: Chloride transport; Cerebrospinal fluid; Choroid plexus; DIDS; Bumetanide; Acetazolamide; DPC; Rat
The uptake and effiux of 36C1 in the lateral ventricle choroid plexus of 1-7-week-old rats were measured to determine if CI transport changed with age and if such transport responded to inhibitors of CSF secretion and ion transport. The steady-state (30 min) CI uptakes were 148+9.4 nmol'mg -x dry weight at 1 week and 139+7.0 nmoi.mg -~ dry weight at 7 weeks, (P > 0.05). The 36C1efflux was significantly slower in 1 and 2 week plexuses compared to more mature tissues (P<0.01) with k (rate coefficient)= 0.029+0.004 s -1, tl/2 =24.1:t:3.3 s at 1 week and k=0.041+0.003 s -1, tl/2=17.4+1.3 s at 7 weeks. 36C1 effiux at 1 week was unaffected by acetazolamide, bumetanide and DIDS (4,4 diisothiocyanato-stilbene-2,2 disulphonic acid), however, all these drugs substantially reduced efflux from 2, 3 and 7 week choroid plexuses. In contrast, the CI conductance blocker, DPC (diphenylamine carboxylate) at 10-aM reduced 360 efflux from both 1 and 7 week tissues by 43% and 39%, respectively. These findings suggest that some transport systems responsible for movement of C1 out of the epithelium are either absent or less functional in the immature rat choroid plexus and may account for the relatively low level of CSF secretion in younger animals. The unidirectional effiux of 36C1, J, was calculated for 1 week and adult rats, as a function of the choroid plexus volume to surface area ratio (V/A). Effiux was close to 550 nmol.cm-2.s-1 at both ages indicating that enhanced 36C1 efflux with age is mainly a function of increasing epithelial cell surface area.
INTRODUCTION
this time, d u e to p r o l i f e r a t i o n a n d e l o n g a t i o n o f the
microvilli 17. T h e c h o r o i d plexuses o f t h e lateral, t h i r d a n d f o u r t h ventricles, a r e t h e p r i m a r y s o u r c e o f c e r e b r o s p i n a l fluid ( C S F ) in t h e c e n t r a l n e r v o u s system, with s e c r e t i o n o f t h e fluid d e p e n d e n t on active t r a n s p o r t o f N a a n d CI 9. In s o m e m a m m a l s the system is fully d e v e l o p e d at b i r t h 7'21'22. H o w e v e r , t h e r a t is b o r n with a less m a t u r e brain; t h e b l o o d - C S F b a r r i e r has a g r e a t e r p e r m e a b i l ity at b i r t h t'12 a n d C S F p r o d u c t i o n is low 14. F o r t h e s e r e a s o n s t h e i m m a t u r e rat c h o r o i d plexus is a g o o d m o d e l for analyzing t h e d e v e l o p m e n t o f ion t r a n s p o r t m e c h a n i s m s which c o n t r i b u t e to t h e h o m e o s t a s i s o f C S F a n d b r a i n e x t r a c e l l u l a r fluid. T h e functions o f the c h o r o i d plexus a p p r o a c h m a t u rity d u r i n g t h e first 2 - 3 w e e k s a f t e r birth, in t e r m s o f Na, K - A T P a s e activity 23, C S F p r o d u c t i o n 14 a n d r e g u l a tion o f C S F a n d c h o r o i d e p i t h e l i u m ionic c o m p o s i t i o n a n d p H 1°'12'16. M o r p h o l o g i c a l l y , t h e C S F face o f t h e e p i t h e l i a l cells i n c r e a s e s g r e a t l y in surface a r e a d u r i n g
I n o r d e r to u n d e r s t a n d m o r e a b o u t the d e v e l o p m e n t o f ion t r a n s p o r t , which is i n t e g r a l to fluid p r o d u c t i o n , t h e efflux o f CI f r o m the in vitro rat c h o r o i d plexus was s t u d i e d at 1, 2, 3 a n d 7 w e e k s o f age, a n d the effect o f v a r i o u s inhibitors o f C S F s e c r e t i o n a n d a n i o n t r a n s p o r t i n v e s t i g a t e d a n d c o m p a r e d d u r i n g d e v e l o p m e n t . In vivo studies m a y b e c o m p l i c a t e d by inability to identify t h e site o f a c t i o n o f drugs, m e t a b o l i c d e g r a d a t i o n a n d p r o t e i n binding, a n d by effects on b l o o d flow to t h e tissue. F o r t h e s e reasons, i s o l a t e d l a t e r a l ventricle c h o r o i d plexuses w e r e u s e d to d e t e r m i n e CI u p t a k e a n d efflux in vitro.
MATERIALS AND METHODS Animals Sprague-Dawley rats, 6-8 weeks old, were obtained from Charles River Laboratories (Wilmington, MA). Pregnant animals were
Correspondence: J.E. Preston, Sherrington School of Physiology, UMDS, St. Thomas's Campus, Lambeth Palace Road, London, SE1 7EH, England, UK. Fax: (44) (71) 928 0729.
182 shipped 1 week before delivering. Pups were removed from mothers at 1, 2, and 3 weeks of age. The animals were anaesthetised with methoxyflurane (Metophane, Pitman-Moore, N J), exsanguinated and their circulation flushed with isotonic mannito123 to remove blood from the choroid plexus. Following decapitation, the lateral ventricle choroid plexuses were harvested; one for use as control, the other for experimentation. The plexuses were placed in separate incubation tubes, containing 1 ml artificial CSF (aCSF). All solutions of aCSF were warmed to 37°C in a water bath and gassed continuously with 95% 0 2 / 5 % C02.
Wet
weight
1.00.8"
, • 0.6
4, B
,#
.E
IO) BS
0.4
.0' weight
After a 5 min pre-incubation, the tissues were transferred to 0.5 ml aCSF containing 36C1 (5 p, C i / m l ) or 3H-mannitol (1 /xCi/ml) as an extracellular space marker. To allow sufficient isotope loading of the immature tissues, plexuses from 2 or 3 pups were pooled. After 20 min, 0.1 m M of the drug of interest was added to the loading solution and the drug vehicle added to the control aCSF. After an additional 10 min, the plexuses were removed, rinsed briefly (1 s) in aCSF and placed in an efflux bath consisting of a 35 m m tissue culture dish containing 2 ml warmed, gassed aCSF. The aCSF was stirred with a mini-magnetic bar on a submersible stirrer in a water bath at 37°C. Effiux bath samples of 200 tzl were taken every 20 s for 2 min and placed directly in liquid scintillation vials for determination of 36Cl efflux. The choroid plexus was removed at the time of the last bath sample, wiped on a glass slide to remove adhering aCSF, placed on a foil boat and weighed on a Cahn microbalance at 15 s intervals to determine the wet weight 12. The tissues were desiccated overnight for dry weight measurement, then placed in a vial with 25/zl H 2 0 for rehydration, and 75/xl of tissue solubilizer, Protosol. Scintillation fluid was added to both tissue and bath samples, which were analysed on a Beckman LS 5800 beta counter. The amount of isotope in the tissue at the beginning of the efflux procedure was determined from the sum of radioactivity released into the efflux bath, and that remaining in the tissue. As shown in Fig. 2, this total was taken as 100% labelling at time zero. The 36C] remaining in the tissue at each time point was then calculated by subtracting the efflux measured every 20 s. A logarithmic plot of 36C1 remaining in the tissue against time was linear and the slope of the line, determined by linear regression analysis, was taken as the efflux rate constant k ( s - l ) . The effiux tl/2 (s)was calculated as (In 2 ) / k . Efflux inhibition ( % ) w a s calculated as, 1--(kdrug/kcontml) X 100.
Uptake After 15 min pre-incubation, each plexus was transferred to 0.5 ml aCSF, containing 36C1 ( 5 / ~ C i / m l ) and 3H-mannitol (2 /xCi/ml), for 12 s or 30 min, the times for half-maximal and steady-state 36Cl uptake by adult rat choroid plexuses 13. The tissues were then removed and rapidly rinsed, run over a glass slide to remove fluid, and weighed on a foil boat. Wet and dry weight measurements, and radioactivity of the tissues were determined as described for efflux experiments. CI transport inhibitors were added to both the pre-incubation and uptake solutions. The drug vehicle was added to the control solutions. Chloride uptake was determined from 36C1 labelling and corrected for extracellular isotope based on 3H-mannitol labelling. Solutions and chemicals The artificial CSF contained (in mM) 137.0 Na, 3.0 K, 1.4 Ca, 0.8 Mg, 125.0 CI, 18.0 H C O 3, 0.7 HPO4, 2.0 urea, 12.1 glucose, pH 7.4 and osmolality 280 m O s m o l ' k g -1 H 2 0 . Acetazolamide and DIDS (4,4 Diisothiocyanato-stilbene-2,2 disulphonic acid) were dissolved directly in aCSF. Bumetanide, a gift from Hoffmann-LaRoche, NJ, was dissolved in 95% ethanol as a stock solution at 10 -2 M, and DPC (diphenylamine carboxylate, Lancaster Synthesis Ltd NH) was dissolved in D M S O (dimethyl sulphoxide) as 1 0 - 2 M stock. The final amount of ethanol or D M S O in the loading solution was 1%. 36C1, 3H-mannitol and tissue solubilizer were obtained from New England Nuclear, Boston, MA. All other reagents and salts were from Sigma Chemical Company (St. Louis, MO).
0.2
B----'•" . . . . •" . . . . . . . . . . . . . . . . . . . . . .
0.0
0
•
l
I
I
I
i
i
i
1
2
3
4
5
6
7
Age in Weeks Fig. 1. The lateral ventricle choroid plexus weights from 1, 2, 3 and 7 week old rats. Tissues were allowed to air dry overnight, and dry weights were measured. The tissue water content was calculated as: 100 x (wet w e i g h t - dry weight)/wet weight, and was 82.2 + 0.8% at 1 week and 81.2-+0.4% at 7 weeks. Values are mean+-S.E, of 10 measurements. The S.E.'s of dry weight m e a s u r e m e n t s are contained within the points.
RESULTS
The wet weight of the lateral ventricle choroid plexus increases with age from 0.39 + 0.02 mg at 1 week to 0.92 + 0.04 mg at 7 weeks (Fig. 1). Plexuses from 2-3 pups were pooled during the effiux experiments to provide sufficient tissue for isotope loading, and hence effiux measurement. Uptake studies, however, were performed on individual choroid plexuses.
TABLE I
In vitro choroid plexus 36Cl uptake over 12 s (half maximal) and 30 min (steady-state) in 1 week old (infant) and 7-week-old (adult) rats All drug administrations were 10 -4 M. Values are the mean_+ S.E.; n in parenthesis.
12 s 36Cl uptake (nmol.mg - 1 dry weight)
Control Acetazolamide DIDS DPC DPC+DIDS
1 week
% Inhibition
7 weeks
70+- 4(16) 72-+12 (4) 72+- 6 (4) * 46_+ 3 (8) *48_+ 7 (4)
34.3 31.4
73+ 68-+ * 49+ * 47-+ * 49-+
% Inhibition 4(13) 9 (5) 3 (4) 6 (4) 9 (4)
32.9 35.6 32.9
30 min 36CI uptake (nmol. mg - 1 dry weight) 1 week Control Acetazolamide DPC
148_+ 9 (24) 148+_24 (5) 50+- 7 (7)
% lnhibition
65.8
• P < 0.01 compared with control.
7 weeks
% Inhibition
139+ 7 (14) 143+-13 (5) * 82+-13 (4)
41.1
183
O :6eel, 1Week 100 ~
•
Ill
,
,
0
20
40
, 60 80 Time (sec)
Cl, 7Weeks
,
,
100
120
Fig. 2. Cboroid plexuses from ]- and 7-week-old rats were incubated in aCSF containing 36C1and 3H-mannitol, then placed in isotope-free aCSF and samples taken every 20 s to determine 36C1 efflux and 3H-mannitol washout from the tissues. The rate coefficient, k, was calculated as described in Materials and Methods, and was 0.029 + 0.002 s -1 at 1 week ( R 2 = 0.997) and 0.040+0.003s -1 ( R 2 = 0.994) at 7 weeks. Values are m e a n + S.E., n = 5-6.
36C1 uptake The uptake of 36C1 (Table I), expressed as nmol. mg-~ dry weight, did not differ significantly at 1 week compared to 7 weeks, either during the 12 s uptake (half maximal) or 30 min uptake (steady-state) ~3. In the presence of acetazolamide uptake was unchanged, but was significantly decreased by the CI conductance blocker, diphenylamine carboxylate (DPC) at both ages and uptake times. However, the addition of DIDS resulted in different effects at the two ages, with no uptake inhibition at 1 week, but 32.9% inhibition at 7 weeks (Table I). A combination of DIDS and DPC (10 -4 M) reduced 12 s 36C1 uptake by 31% at 1 week and 33% at 7 weeks, but the inhibitions were not significantly different from those produced by DPC alone ( P > 0.05).
calculated as 24.1 + 3.3 s at 1 week and 17.4 + 1.3 s in adults. Flux was considered to be unidirectionally outward since the volume of the aCSF bath was over 1000 times that of the tissue, even at the end of the experiment. Measurements of 36C1 efflux were also taken for choroid plexuses from 2- and 3-week-old rats and the effect of inhibitors of CSF secretion and ion transport investigated. Efflux from 2 week plexuses was similar to that at 1 week with k = 0.031 + 0.003 s- 1 (n = 14), Fig. 3. The efflux coefficient at 3 weeks was significantly higher than that at the younger ages ( P < 0.01) with k = 0.043 + 0.004 s - t (n = 13), similar to that for adults. The carbonic anhydrase inhibitor, acetazolamide, reduced efflux from all age groups except at 1 week (Fig. 3). The fractional inhibition, shown in the inset of Fig. 3, was over 20% at 2, 3 and 7 weeks. The addition of bumetanide, a loop diuretic inhibiting Na, K, 2C1 co-transport, produced similar inhibitions, and efflux from all but the 1 week tissues was reduced (Fig. 4). However, inhibitions were greater than those produced by acetazolamide, notably in 2 week plexuses, where k was 34% less than control. 36C1 efflux was also reduced
4O
8 ,,,..
[] []
c:
Control Acetazolamide
0
|
!
2
3
0.05
Z
A
.
"7,
Z
/ /
~E 0.03
T
T
H 1
U.I
36El efflux The 3H-mannitol washout curve is shown in Fig. 2. Over 90% of the extracellular marker was washed out from the choroid plexus by the time of the first sampling at 20 s. This indicates that extracellular trapping of isotope is negligible over the last minute of the efflux experiment. 36C1 efflux from 1 and 7 week choroid plexuses did show differences (Fig. 2), unlike uptake measurements. Efflux was slower from the infant tissues with a coefficient, k of 0.029 + 0.004 s-1 (n = 23) compared to the adult k of 0.041 + 0.003 s -1 (n = 19). The t~/2 was
|
1
// // .f/ // //
H // ff
0.01 . 1
2
3
Age (weeks)
7
Fig. 3. The rate coefficients k, for 36C1 efflux from the rat choroid plexuses are shown for the ages 1, 2, 3 and 7 weeks. Since there are two lateral ventricle choroid plexuses available, control efflux is measured for one plexus (unshaded bars) and the effect of 10 -4 M acetazolamide measured with the other of the pair (shaded bars). Values are the m e a n + S.E. of 5 - 8 measurements. * P < 0.05, difference from control (paired Student t-test). Inset shows fractional inhibition (%) at each age (weeks).
184
A 4o
o~ [] []
°t
Control Bumetanide
o
0
0.05 ]
I
I
I
i
1
2
3
7
[] []
:5
Control DPC
--
0.05 m
T
T
I 0 o
o
II
"7
-r
x" 0.03 ZE
~- 0.03
UJ
U
U
0.01 1
3
2
0.01
7
1
Age (weeks) Fig. 4. 36C1 effiux from lateral ventricle choroid plexuses of 1 through 7 week old rats in the presence of l0 -4 M bumetamide (shaded bars). The drug vehicle, 95% ethanol (1% v/v) was added to the aCSF incubating the control tissues (unshaded bars). Values are mean + S.E. of 4-6 measurements. * P < 0.05, ** P < 0.01. Inset shows fractional inhibition at each age in weeks.
by the anion exchange inhibitor, DIDS, particularly at 3 and 7 weeks, but also with limited effect at 2 weeks (Fig. 5). While the DIDS-inhibitable component of
40
8 .'Q e-
[] II
Control DIDS
I
I
1
7
0.05
"7
O.O3 E
ILl
U t~
T
l'
0.01 1
2
3
I
7
Age (weeks) Fig. 5. Choroid plexus 36C1 efflux in the presence of 10 -4 M DIDS (shaded bars). Controls are unshaded. Values are mean+S.E, of 6-10 measurements. * P < 0.05, ** P < 0.01. Inset shows fractional inhibition at each age in weeks.
7 Age (weeks) Fig 6. Choroid plexus 36C1 efflux in the presence of 10 -4 M DPC (shaded bars). The drug vehicle, 1% DMSO v/v, was added to the aCSF incubating the control tissues (unshaded bars). Values are the mean_+ S.E. for 4-5 measurements. ** P < 0.01. Inset shows fractional inhibition at 1 and 7 weeks.
effiux varies from 10% of control at 1 week to 37% at 7 weeks, the uninhibitable component had a similar k value in all age groups and averaged 0.026 s-1. Unlike the other inhibitors, DPC a C1 conductance blocker, significantly reduced 1 week k by over 40% to 0.018 ___0.005 s -1 with tl/2 of 38.7s (Fig. 6). Effiux from 7 week tissues was also reduced, from 0.041 + 0.003 s- ~ to 0.025 + 0.002 s- 1. The specificity of DPC reduction of CI movement needed clarification since at concentrations of 10 -3 M and above, there may be effects on the Na ÷, K +ATPase. To ascertain whether inactivation of the N a - K pump was occurring, the K ÷ content of the choroid plexus was measured by flame photometry after the tissue ions had been extracted overnight in 0.1 N nitric acid. After correction for extracellular space with 3Hmannitol, the K ÷ concentration was found to be 128 +_ 27.9 mmol. kg -1 cell H 2 0 in controls and 108 + 14.3 mmol .kg -1 H 2 0 in choroidal tissues treated for 20 min with 10-4M DPC (n = 3). While there was a slight reduction in K ÷ in the DPC-treated tissues, this was not statistically significant and would be unlikely to cause the large reduction of 36C1 effiux. A combination of drugs was then used to attempt additive inhibitions. However, as with uptake, DIDS and DPC ( 1 0 - 4 M ) together, failed to reduce 36C1 efflux any further than DPC alone in adult rats.
185 DISCUSSION This study has investigated the uptake and effiux of 36C1 at the rat lateral ventricle choroid plexus during development. Previous studies have shown this in vitro preparation to be viable over the time periods used 23'27. We now report differences, not only in baseline transport characteristics, but also in the response of the immature choroid plexus to pharmacological agents as the choroid plexus develops in size (Fig. 1) and capacity to secrete CSF. Chloride ions are accumulated in the choroidal epithelium above electrochemical equilibrium by an active mechanism 25'28. In the present studies the rate of uptake (12 s) was the same in 1 week and 7 week old rats (Table I). By comparison, K + uptake into 0.5-1 week rat choroid plexuses is less than that in 1.5-4 week tissues 22 so uptake processes for CI and K + seem to follow different developmental time courses. Unlike 36C1 uptake, the effiux was slower in the 1 week choroid plexus with k = 0.029 _+ 0.002 s- 1 (n = 23) compared to the adult k - 0.041 +_ 0.003 s-1 (n = 19) with corresponding t 1/2 values of 24 s and 17 s respectively. A combination of rapid uptake and slower effiux should result in the elevation of intracellular CI in the young choroid plexus as found by Smith, Woodbury and Johanson 29. The steady-state 36C1 uptake values in Table I are also consistent with relatively high cellular [C1] early in development. Again, such findings in infants probably reflect retention of choroid plexus [C1] due to an apical effiux that is slower than basolateral uptake. The lower k values for in vitro effiux in infants vs. adults are in line with previous in vivo findings that 36C1 movement from blood to CSF does not show a fast component in 1 week rats as in adults 29. The component missing in infants, but present in adults, probably represents net CI transport across the in vivo choroid plexus is. Measurement of in vitro 36C1 effiux from 2 week plexuses was also relatively slow, k = 0.031s-1, but at 3 weeks it was not significantly different from that of the adult. Of the inhibitors used to investigate C1 effiux, acetazolamide, bumetanide and DIDS were similar in that they reduced effiux from 2, 3 and 7 week choroid plexuses to varying degrees, but they did not significantly affect 1 week tissues. The carbonic anhydrase inhibitor, acetazolamide, decreases CSF secretion in vivo 3 and although it has been suggested that this is due to constriction of blood vessels in the choroid plexus 24 recent studies show that blood flow is actually increased 5. The inhibitory effects seem to be on ion transport 8, with decreased Na entry into CSF 4,3°, and we report here reduced CI effiux from the adult choroid
plexus after acetazolamide administration. Interestingly, acetazolamide does not reduce CI uptake (Table I) and so it may be affecting an apical anion channel 8. The loop diuretic bumetanide, which in adults reduces choroid plexus Na and C1 uptake in vitro 13 and C1 movement from blood to CSF in vivo 15, did reduce 36C1 efflux from adult choroid plexuses, but again there was no effect at 1 week. A bumetanide-sensitive Na, K, 2C1 cotransport system has been identified in the adult rat choroid plexus and it has a role in the uptake of these ions 2. Reduced uptake could indirectly affect C1 effiux by decreasing [CI] i and, therefore, effiux via anion channels. With the design of these efflux experiments, uptake may be inhibited during efflux measurement and lead to the reduced efflux rates observed. Bumetanide significantly inhibited 36C1 effiux at 2, 3 and 7 weeks indicating the presence of a cation-C1 cotransport system at these later stages of development. The disulphonic stilbene, DIDS, is an inhibitor of C1-HCO 3 exchange and it reduced 36C1 effiux with increasing effect from 1 week (10% inhibition) to 7 weeks (37% inhibition), leaving the uninhibited component of effiux close to k = 0.026 s-~ at all ages tested. DIDS also reduced 12 s uptake in adults but not in 1 week choroid plexuses. The functioning of this transporter appears to be in transition at 2 weeks since the % DIDS inhibition is only two-thirds that seen at 7 weeks. The anion exchanger has recently been located on the basolateral face of the choroid plexus 19 and it is one of the major mechanisms for C1 transport into the choroid plexus and thence into CSF 6. Each of the drugs analyzed did not significantly reduce 36CI efflux from 1 week tissues, although acetazolamide and bumetanide inhibitions at 2 weeks, and DIDS at 3 weeks, were similar to the adult tissues. Because these drugs are known to reduce CSF secretion or CSF [CI] in vivo, the implication is that slow CI efflux and CSF production at 1 week may be a result of limited functioning, or lack of these ion translocating mechanisms. In contrast with the other inhibitors used, the C1 conductance blocker DPC reduced 1 week effiux significantly with a 43% drop in k, similar to the 39% inhibition at 7 weeks, suggesting that a large component of 36C1 efflux is via this mechanism even in the immature choroid plexus. In addition, C1 uptake is also inhibited by DPC at both ages. It is improbable that DPC acts directly to prevent uptake via CI channels since C1 is above electrochemical equilibrium in the choroidal cell. It is more likely that CI efflux is blocked, leading to anion accumulation, thus slowing uptake. Indeed, in the rabbit parietal cell, DPC effects appear
186 to be localized to the CI secreting apical m e m b r a n e e°. If CI uptake is d e p e n d e n t on the rate of efflux, combining an uptake inhibitor with an efflux inhibitor may not result in additive inhibition of CI transport since the two systems are in series. In these studies C1 uptake was m e a s u r e d in the presence of both D I D S and DPC. D I D S blocks C1 uptake (Table I) via the basolateral C I - H C O 3 exchanger 1'), whilst D P C blocks CI efflux (Fig. 6) and this combination did not p r o d u c e additive uptake inhibition. It is likely that a large c o m p o n e n t of the observed 36C1 transport occurs at the apical m e m b r a n e because of its large surface area c o m p a r e d to the basolateral m e m b r a n e and close proximity to the incubating aCSF. An anion exchanger has been located at the basolateral face of the tissue 19 but this does not rule out the presence of pathways for chloride transport at the apical surface as has been seen at the brush-border m e m b r a n e of the bullfrog choroid plexus 26. A n anion exchanger has also been observed at the brush-border m e m b r a n e of the necturus m a c u l o s u s choroid plexus 33 and this may provide a pathway for 36C1 efflux from the loaded cell. In addition, a co-transport system has been described for KCI and H 2 0 at the apical face of the necturus choroid plexus 31. We have observed a C1 dep e n d e n t 86Rb efflux in both adult and 1-week-old rat choroid plexuses (unpublished results) which also points to the presence of KCI co-transport in this preparation. The coupling of CI and H 2 0 transport in the necturus plexus 3t'32 stresses the importance of C1 in secretion of fluid by the choroid plexus. This transport has been shown to be inhibited by the loop diuretic furosemide in vitro 3~, and in vivo causes the build up of C1 and K in the rat choroid plexus II, presumably by blocking ion m o v e m e n t across the apical m e m b r a n e into CSF. Based on these observations it is likely that the 36C1 efflux modulations observed at the rat choroid plexus are due to drug effects primarily at the apical, CSF secreting face of the choroid plexus. As the rat choroid plexus develops, not only does 36Cl efflux increase, but also the epithelial cell m e m brane surface area 17. Such an increase in surface area would allow insertion into the m e m b r a n e of more ion-transporting proteins and hence greater transport capabilities per unit volume of epithelium. T h e C1 flux as a function of the cell m e m b r a n e area can be calculated from; J = k~ V/A)[CI]~
where J is unidirectional flux ( m o l . cm - 2 " s - l ) , k is the efflux coefficient and V / A is the cell volume to surface area ratio. T h e m e a n values of k are 0.029 s - l
at 1 week, 0.041 s i at 7 weeks. [El] i was estimated from 36C1 30 rain uptake (Table I) and converted to m m o l . k g - l cell H 2 0 by subtracting the extracellular 3H-mannitol space i.e., extracellular volume, from the tissue wet weight. From the surface density ( A / V ) m e a s u r e m e n t s of Keep and Jones 17, values for V / A were extrapolated for 1 week rats, and values for 30 day rats used as approximations for adults. With these data, the 36C1 efflux, J, was 554 n m o l . cm 2. s-~ at 1 week and 549 n m o l - c m 2 . s - ~ for adults. The increase in m e m b r a n e surface area, particularly the apical m e m b r a n e , occurs over the same time course as the increase in 36C1 efflux and, therefore, J is similar in 1 and 7 week rats. We have shown that the choroid plexus of immature rats, in which the CSF secretory process is not yet complete, has less capacity to extrude C1 than the adult plexus. This may be due to incomplete m e m b r a n e transport development and is reflected in the ineffectiveness of the inhibitors used. This research was supported by NIH Grant NS 27601 and funds from the Rhode Island Hospital. We thank J.T. Parmelee for technical advice.
Acknowledgements.
REFERENCES 1 Amtorp, O. and Sorensen, S.C., The ontogenetic development of concentration differences for protein and ions between plasma and cerebrospinal fluid in rabbits and rats, J. Physiol., 243 (1974) 387-400. 2 Bairamian, D., Johanson, C.E., Parmelee, J.T. and Epstein, M., Potassium cotransport with sodium and chloride in the choroid plexus, J. Neurochem., 56 (1991) 1623-1629. 3 Davson, H. and Pollay, M., The turnover of 24Na in the cerebrospinal fluid and its bearing on the blood brain barrier, J. Physiol., 167 (1963) 247-255. 4 Davson, H. and Segal, M.B., The effect of some inhibitors and accelerators of sodium transport on the turnover of 22Na in the cerebrospinal fluid and the brain, J. Physiol., 209 (1970) 131-153. 5 Faraci, F.M., Mayhan, W.G. and Heistad, D.D., Vascular effects of acetazolamide on the choroid plexus, J. Pharmacol. Exp. Ther., 254 (1990) 23-27. 6 Frankel, H. and Kazemi, H., Regulation of CSF compositionblocking chloride-bicarbonate exchange, J. App. Physiol., 55 (1983) 177-182. 7 Hodson, W.A., Fenner, A., Brumley, G., Chernick, V. and Avery, M.E., Cerebrospinal fluid and blood acid-base relationships in fetal and neonatal lambs and pregnant ewes, Resp. Physiol., 4 (1968) 322-332. 8 Johanson, C.E., Differential effects of acetazolamide, benzolamide and systemic acidosis on hydrogen and bicarbonate gradients across the apical and basolateral membranes of the choroid plexus, J. Pharmacol. Exp. Ther., 231 (1984) 502-511. 9 Johanson, C.E., Potential for pharmacological manipulation of the blood-cerebrospinal fluid barrier. In E. Neuwelt (Ed.), Implications of the Blood-Brain Barrier and its Manipulation. Basic Science Aspects, 11"ol. 1, Plenum Press, New York, 1989, pp.
223-260. 10 Johanson, C.E., Allen, J. and Withrow, C.D., Regulation of pH and HCO3 in brain and CSF of the developing mammalian central nervous system, Dev. Brain Res., 38 (1988) 255-264. 11 Johanson, C.E., Murphy, V.A. and Dyas, M., Ethacrynic acid and
187 furosemide alter CI, K and Na distribution between blood, choroid plexus, CSF and brain, Neurochem. Res., 17 (1992) 1079-1085. 12 Johanson, C.E., Reed, D.J. and Woodbury, D.M., Developmental studies of the compartmentalization of water and electrolytes in the choroid plexus of neonatal rat brain, Brain Res., 116 (1976) 35-48. 13 Johanson, C.E., Sweeney, S.M., Parmelee, J.T. and Epstein, M.H., Cotransport of sodium and chloride by the adult mammalian choroid plexus, Am. J. Physiol., 258 (1990) C211-C216. 14 Johanson, C.E. and Woodbury, D.M., Changes in CSF flow and extracellular space in the developing rat. In A. Vernadakis and N. Weiner (Eds.), Drugs and the Developing Brain, Plenum Press, New York, 1974, pp. 281-287. 15 Johnson, D.C., Singer, S., Hoop, B. and Kazemi, H., Chloride flux from blood to CSF: inhibition by furosemide and bumetanide, J. App. Physiol., 63 (1987) 1591-1600 16 Jones, H.C. and Keep, R.F., The control of potassium concentration in the cerebrospinal fluid and brain interstitial fluid of developing rats, J. Physiol., 383 (1987) 441-453. 17 Keep, R.F. and Jones, H.C., A morphometric study on the development of the lateral ventricle choroid plexus, choroid plexus capillaries and ventricular ependyma in the rat, Dev. Brain Res., 56 (1990) 47-53. 18 Levin, V. and Patlak, C.S., A compartmental analysis of 24Na kinetics in rat cerebrum, sciatic nerve and cerebrospinal fluid, J. Physiol., 224 (1972) 559-581. 19 Lindsey, A,E., Schneider, K., Simmons, D.M., Baron, R., Lee, B.S. and Kopito, R.R., Functional expression and subcellular localization of an anion exchanger cloned from choroid plexus, Proc. Nat. Acad. Sci. USA, 87 (1990) 5278-5282. 20 Malinowska, D.H., CI- channel blockers inhibit acid secretion in rabbit parietal cells, Am. J. PhysioL, 259 (1990) G536-G543. 21 Mitchell, W., Kim, C.S., O'Tuama, L.A., Pritchard, J.B. and Pick, J.R., Choroid plexus, brain and kidney Na, K+-ATPase: comparative activities in fetal, newborn and young adult rabbits, Neurosci. Lett., 31 (1982) 37-40. 22 Nattie, E.E. and Edwards, W.H., CSF acid-base regulation and ventilation during acute hypercapnia in the new born dog, J. Appl. PhysioL, 50 (1981) 566-574.
23 Parmelee, J.T. and Johanson, C.E., Development of potassium transport capability by choroid plexus of infant rats, Am. J. Physiol., 256 (1989) R786-R791. 24 Politoff, A. and Macri, F., Pharmacologic differences between isolated, perfused arteries of the choroid plexus and of brain parenchyma, Int. J. Neuropharm., 5 (1966) 155-162 25 Saito, Y, and Wright, E.M., Regulation of bicarbonate transport across the brush border membrane of the bull-frog choroid plexus, J. Physiol., 350 (1984) 327-342. 26 Saito, Y. and Wright, E.M., Regulation of intracellular chloride in bullfrog choroid plexus, Brain Res., 417 (1987) 267-272. 27 Smith, Q.R. and Johanson, C.E., Active transport of chloride by lateral ventricle choroid plexus of the rat, Am. J. Physiol., 249 (1985) F470-F477. 28 Smith, Q.R., Woodbury, D.M. and Johanson, C.E., Uptake of CI-36 and Na-22 by the choroid plexus-cerebrospinal fluid system: evidence for active chloride transport by the choroidal epithelium, J. Neurochem., 37 (1981) 107-116. 29 Smith, Q.R., Woodbury, D.M. and Johanson, C.E., Kinetic analysis of [36C1]-, [22Na]- and [3H]mannitol uptake into the in vivo choroid plexus-cerebrospinal fluid system: ontogeny of the blood-brain and blood-CSF barriers, DeL'. Brain Res., 3 (1982) 181-198. 30 Vogh, B.P. and Doyle, A.S., The effect of carbonic anhydrase inhibitors and other drugs on sodium entry to cerebrospinal fluid, J. Pharmacol. Exp. Ther., 217 (1981) 51-56. 31 Zeuthen, T., Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus Maculosus, J. Physiol., 444 (1991) 133-151. 32 Zeuthen, T., Secondary active transport of water across ventricular cell membrane of choroid plexus of Necturus Maculosus, J. Physiol., 444 (1991) 153-173. 33 Zeuthen, T., Christensen, O., Baerentsen, J.H. and la Cour, M., The mechanism of electrodiffusive K ÷ transport in leaky epithelia and some of its consequences for anion transport, Pfliigers Arch., 408 (1987) 260-266.
181
BRES 19272
Development of chloride transport by the rat choroid plexus, in vitro J.E. Preston, M. Dyas and C.E. Johanson Program in Neurosurgery, Department of Clinical Neurosciences, Brown University~Rhode Island Hospital, Providence, RI 02903 (USA) (Accepted 11 May 1993)
Key words: Chloride transport; Cerebrospinal fluid; Choroid plexus; DIDS; Bumetanide; Acetazolamide; DPC; Rat
The uptake and effiux of 36C1 in the lateral ventricle choroid plexus of 1-7-week-old rats were measured to determine if CI transport changed with age and if such transport responded to inhibitors of CSF secretion and ion transport. The steady-state (30 min) CI uptakes were 148+9.4 nmol'mg -x dry weight at 1 week and 139+7.0 nmoi.mg -~ dry weight at 7 weeks, (P > 0.05). The 36C1efflux was significantly slower in 1 and 2 week plexuses compared to more mature tissues (P<0.01) with k (rate coefficient)= 0.029+0.004 s -1, tl/2 =24.1:t:3.3 s at 1 week and k=0.041+0.003 s -1, tl/2=17.4+1.3 s at 7 weeks. 36C1 effiux at 1 week was unaffected by acetazolamide, bumetanide and DIDS (4,4 diisothiocyanato-stilbene-2,2 disulphonic acid), however, all these drugs substantially reduced efflux from 2, 3 and 7 week choroid plexuses. In contrast, the CI conductance blocker, DPC (diphenylamine carboxylate) at 10-aM reduced 360 efflux from both 1 and 7 week tissues by 43% and 39%, respectively. These findings suggest that some transport systems responsible for movement of C1 out of the epithelium are either absent or less functional in the immature rat choroid plexus and may account for the relatively low level of CSF secretion in younger animals. The unidirectional effiux of 36C1, J, was calculated for 1 week and adult rats, as a function of the choroid plexus volume to surface area ratio (V/A). Effiux was close to 550 nmol.cm-2.s-1 at both ages indicating that enhanced 36C1 efflux with age is mainly a function of increasing epithelial cell surface area.
INTRODUCTION
this time, d u e to p r o l i f e r a t i o n a n d e l o n g a t i o n o f the
microvilli 17. T h e c h o r o i d plexuses o f t h e lateral, t h i r d a n d f o u r t h ventricles, a r e t h e p r i m a r y s o u r c e o f c e r e b r o s p i n a l fluid ( C S F ) in t h e c e n t r a l n e r v o u s system, with s e c r e t i o n o f t h e fluid d e p e n d e n t on active t r a n s p o r t o f N a a n d CI 9. In s o m e m a m m a l s the system is fully d e v e l o p e d at b i r t h 7'21'22. H o w e v e r , t h e r a t is b o r n with a less m a t u r e brain; t h e b l o o d - C S F b a r r i e r has a g r e a t e r p e r m e a b i l ity at b i r t h t'12 a n d C S F p r o d u c t i o n is low 14. F o r t h e s e r e a s o n s t h e i m m a t u r e rat c h o r o i d plexus is a g o o d m o d e l for analyzing t h e d e v e l o p m e n t o f ion t r a n s p o r t m e c h a n i s m s which c o n t r i b u t e to t h e h o m e o s t a s i s o f C S F a n d b r a i n e x t r a c e l l u l a r fluid. T h e functions o f the c h o r o i d plexus a p p r o a c h m a t u rity d u r i n g t h e first 2 - 3 w e e k s a f t e r birth, in t e r m s o f Na, K - A T P a s e activity 23, C S F p r o d u c t i o n 14 a n d r e g u l a tion o f C S F a n d c h o r o i d e p i t h e l i u m ionic c o m p o s i t i o n a n d p H 1°'12'16. M o r p h o l o g i c a l l y , t h e C S F face o f t h e e p i t h e l i a l cells i n c r e a s e s g r e a t l y in surface a r e a d u r i n g
I n o r d e r to u n d e r s t a n d m o r e a b o u t the d e v e l o p m e n t o f ion t r a n s p o r t , which is i n t e g r a l to fluid p r o d u c t i o n , t h e efflux o f CI f r o m the in vitro rat c h o r o i d plexus was s t u d i e d at 1, 2, 3 a n d 7 w e e k s o f age, a n d the effect o f v a r i o u s inhibitors o f C S F s e c r e t i o n a n d a n i o n t r a n s p o r t i n v e s t i g a t e d a n d c o m p a r e d d u r i n g d e v e l o p m e n t . In vivo studies m a y b e c o m p l i c a t e d by inability to identify t h e site o f a c t i o n o f drugs, m e t a b o l i c d e g r a d a t i o n a n d p r o t e i n binding, a n d by effects on b l o o d flow to t h e tissue. F o r t h e s e reasons, i s o l a t e d l a t e r a l ventricle c h o r o i d plexuses w e r e u s e d to d e t e r m i n e CI u p t a k e a n d efflux in vitro.
MATERIALS AND METHODS Animals Sprague-Dawley rats, 6-8 weeks old, were obtained from Charles River Laboratories (Wilmington, MA). Pregnant animals were
Correspondence: J.E. Preston, Sherrington School of Physiology, UMDS, St. Thomas's Campus, Lambeth Palace Road, London, SE1 7EH, England, UK. Fax: (44) (71) 928 0729.
182 shipped 1 week before delivering. Pups were removed from mothers at 1, 2, and 3 weeks of age. The animals were anaesthetised with methoxyflurane (Metophane, Pitman-Moore, N J), exsanguinated and their circulation flushed with isotonic mannito123 to remove blood from the choroid plexus. Following decapitation, the lateral ventricle choroid plexuses were harvested; one for use as control, the other for experimentation. The plexuses were placed in separate incubation tubes, containing 1 ml artificial CSF (aCSF). All solutions of aCSF were warmed to 37°C in a water bath and gassed continuously with 95% 0 2 / 5 % C02.
Wet
weight
1.00.8"
, • 0.6
4, B
,#
.E
IO) BS
0.4
.0' weight
After a 5 min pre-incubation, the tissues were transferred to 0.5 ml aCSF containing 36C1 (5 p, C i / m l ) or 3H-mannitol (1 /xCi/ml) as an extracellular space marker. To allow sufficient isotope loading of the immature tissues, plexuses from 2 or 3 pups were pooled. After 20 min, 0.1 m M of the drug of interest was added to the loading solution and the drug vehicle added to the control aCSF. After an additional 10 min, the plexuses were removed, rinsed briefly (1 s) in aCSF and placed in an efflux bath consisting of a 35 m m tissue culture dish containing 2 ml warmed, gassed aCSF. The aCSF was stirred with a mini-magnetic bar on a submersible stirrer in a water bath at 37°C. Effiux bath samples of 200 tzl were taken every 20 s for 2 min and placed directly in liquid scintillation vials for determination of 36Cl efflux. The choroid plexus was removed at the time of the last bath sample, wiped on a glass slide to remove adhering aCSF, placed on a foil boat and weighed on a Cahn microbalance at 15 s intervals to determine the wet weight 12. The tissues were desiccated overnight for dry weight measurement, then placed in a vial with 25/zl H 2 0 for rehydration, and 75/xl of tissue solubilizer, Protosol. Scintillation fluid was added to both tissue and bath samples, which were analysed on a Beckman LS 5800 beta counter. The amount of isotope in the tissue at the beginning of the efflux procedure was determined from the sum of radioactivity released into the efflux bath, and that remaining in the tissue. As shown in Fig. 2, this total was taken as 100% labelling at time zero. The 36C] remaining in the tissue at each time point was then calculated by subtracting the efflux measured every 20 s. A logarithmic plot of 36C1 remaining in the tissue against time was linear and the slope of the line, determined by linear regression analysis, was taken as the efflux rate constant k ( s - l ) . The effiux tl/2 (s)was calculated as (In 2 ) / k . Efflux inhibition ( % ) w a s calculated as, 1--(kdrug/kcontml) X 100.
Uptake After 15 min pre-incubation, each plexus was transferred to 0.5 ml aCSF, containing 36C1 ( 5 / ~ C i / m l ) and 3H-mannitol (2 /xCi/ml), for 12 s or 30 min, the times for half-maximal and steady-state 36Cl uptake by adult rat choroid plexuses 13. The tissues were then removed and rapidly rinsed, run over a glass slide to remove fluid, and weighed on a foil boat. Wet and dry weight measurements, and radioactivity of the tissues were determined as described for efflux experiments. CI transport inhibitors were added to both the pre-incubation and uptake solutions. The drug vehicle was added to the control solutions. Chloride uptake was determined from 36C1 labelling and corrected for extracellular isotope based on 3H-mannitol labelling. Solutions and chemicals The artificial CSF contained (in mM) 137.0 Na, 3.0 K, 1.4 Ca, 0.8 Mg, 125.0 CI, 18.0 H C O 3, 0.7 HPO4, 2.0 urea, 12.1 glucose, pH 7.4 and osmolality 280 m O s m o l ' k g -1 H 2 0 . Acetazolamide and DIDS (4,4 Diisothiocyanato-stilbene-2,2 disulphonic acid) were dissolved directly in aCSF. Bumetanide, a gift from Hoffmann-LaRoche, NJ, was dissolved in 95% ethanol as a stock solution at 10 -2 M, and DPC (diphenylamine carboxylate, Lancaster Synthesis Ltd NH) was dissolved in D M S O (dimethyl sulphoxide) as 1 0 - 2 M stock. The final amount of ethanol or D M S O in the loading solution was 1%. 36C1, 3H-mannitol and tissue solubilizer were obtained from New England Nuclear, Boston, MA. All other reagents and salts were from Sigma Chemical Company (St. Louis, MO).
0.2
B----'•" . . . . •" . . . . . . . . . . . . . . . . . . . . . .
0.0
0
•
l
I
I
I
i
i
i
1
2
3
4
5
6
7
Age in Weeks Fig. 1. The lateral ventricle choroid plexus weights from 1, 2, 3 and 7 week old rats. Tissues were allowed to air dry overnight, and dry weights were measured. The tissue water content was calculated as: 100 x (wet w e i g h t - dry weight)/wet weight, and was 82.2 + 0.8% at 1 week and 81.2-+0.4% at 7 weeks. Values are mean+-S.E, of 10 measurements. The S.E.'s of dry weight m e a s u r e m e n t s are contained within the points.
RESULTS
The wet weight of the lateral ventricle choroid plexus increases with age from 0.39 + 0.02 mg at 1 week to 0.92 + 0.04 mg at 7 weeks (Fig. 1). Plexuses from 2-3 pups were pooled during the effiux experiments to provide sufficient tissue for isotope loading, and hence effiux measurement. Uptake studies, however, were performed on individual choroid plexuses.
TABLE I
In vitro choroid plexus 36Cl uptake over 12 s (half maximal) and 30 min (steady-state) in 1 week old (infant) and 7-week-old (adult) rats All drug administrations were 10 -4 M. Values are the mean_+ S.E.; n in parenthesis.
12 s 36Cl uptake (nmol.mg - 1 dry weight)
Control Acetazolamide DIDS DPC DPC+DIDS
1 week
% Inhibition
7 weeks
70+- 4(16) 72-+12 (4) 72+- 6 (4) * 46_+ 3 (8) *48_+ 7 (4)
34.3 31.4
73+ 68-+ * 49+ * 47-+ * 49-+
% Inhibition 4(13) 9 (5) 3 (4) 6 (4) 9 (4)
32.9 35.6 32.9
30 min 36CI uptake (nmol. mg - 1 dry weight) 1 week Control Acetazolamide DPC
148_+ 9 (24) 148+_24 (5) 50+- 7 (7)
% lnhibition
65.8
• P < 0.01 compared with control.
7 weeks
% Inhibition
139+ 7 (14) 143+-13 (5) * 82+-13 (4)
41.1
183
O :6eel, 1Week 100 ~
•
Ill
,
,
0
20
40
, 60 80 Time (sec)
Cl, 7Weeks
,
,
100
120
Fig. 2. Cboroid plexuses from ]- and 7-week-old rats were incubated in aCSF containing 36C1and 3H-mannitol, then placed in isotope-free aCSF and samples taken every 20 s to determine 36C1 efflux and 3H-mannitol washout from the tissues. The rate coefficient, k, was calculated as described in Materials and Methods, and was 0.029 + 0.002 s -1 at 1 week ( R 2 = 0.997) and 0.040+0.003s -1 ( R 2 = 0.994) at 7 weeks. Values are m e a n + S.E., n = 5-6.
36C1 uptake The uptake of 36C1 (Table I), expressed as nmol. mg-~ dry weight, did not differ significantly at 1 week compared to 7 weeks, either during the 12 s uptake (half maximal) or 30 min uptake (steady-state) ~3. In the presence of acetazolamide uptake was unchanged, but was significantly decreased by the CI conductance blocker, diphenylamine carboxylate (DPC) at both ages and uptake times. However, the addition of DIDS resulted in different effects at the two ages, with no uptake inhibition at 1 week, but 32.9% inhibition at 7 weeks (Table I). A combination of DIDS and DPC (10 -4 M) reduced 12 s 36C1 uptake by 31% at 1 week and 33% at 7 weeks, but the inhibitions were not significantly different from those produced by DPC alone ( P > 0.05).
calculated as 24.1 + 3.3 s at 1 week and 17.4 + 1.3 s in adults. Flux was considered to be unidirectionally outward since the volume of the aCSF bath was over 1000 times that of the tissue, even at the end of the experiment. Measurements of 36C1 efflux were also taken for choroid plexuses from 2- and 3-week-old rats and the effect of inhibitors of CSF secretion and ion transport investigated. Efflux from 2 week plexuses was similar to that at 1 week with k = 0.031 + 0.003 s- 1 (n = 14), Fig. 3. The efflux coefficient at 3 weeks was significantly higher than that at the younger ages ( P < 0.01) with k = 0.043 + 0.004 s - t (n = 13), similar to that for adults. The carbonic anhydrase inhibitor, acetazolamide, reduced efflux from all age groups except at 1 week (Fig. 3). The fractional inhibition, shown in the inset of Fig. 3, was over 20% at 2, 3 and 7 weeks. The addition of bumetanide, a loop diuretic inhibiting Na, K, 2C1 co-transport, produced similar inhibitions, and efflux from all but the 1 week tissues was reduced (Fig. 4). However, inhibitions were greater than those produced by acetazolamide, notably in 2 week plexuses, where k was 34% less than control. 36C1 efflux was also reduced
4O
8 ,,,..
[] []
c:
Control Acetazolamide
0
|
!
2
3
0.05
Z
A
.
"7,
Z
/ /
~E 0.03
T
T
H 1
U.I
36El efflux The 3H-mannitol washout curve is shown in Fig. 2. Over 90% of the extracellular marker was washed out from the choroid plexus by the time of the first sampling at 20 s. This indicates that extracellular trapping of isotope is negligible over the last minute of the efflux experiment. 36C1 efflux from 1 and 7 week choroid plexuses did show differences (Fig. 2), unlike uptake measurements. Efflux was slower from the infant tissues with a coefficient, k of 0.029 + 0.004 s-1 (n = 23) compared to the adult k of 0.041 + 0.003 s -1 (n = 19). The t~/2 was
|
1
// // .f/ // //
H // ff
0.01 . 1
2
3
Age (weeks)
7
Fig. 3. The rate coefficients k, for 36C1 efflux from the rat choroid plexuses are shown for the ages 1, 2, 3 and 7 weeks. Since there are two lateral ventricle choroid plexuses available, control efflux is measured for one plexus (unshaded bars) and the effect of 10 -4 M acetazolamide measured with the other of the pair (shaded bars). Values are the m e a n + S.E. of 5 - 8 measurements. * P < 0.05, difference from control (paired Student t-test). Inset shows fractional inhibition (%) at each age (weeks).
184
A 4o
o~ [] []
°t
Control Bumetanide
o
0
0.05 ]
I
I
I
i
1
2
3
7
[] []
:5
Control DPC
--
0.05 m
T
T
I 0 o
o
II
"7
-r
x" 0.03 ZE
~- 0.03
UJ
U
U
0.01 1
3
2
0.01
7
1
Age (weeks) Fig. 4. 36C1 effiux from lateral ventricle choroid plexuses of 1 through 7 week old rats in the presence of l0 -4 M bumetamide (shaded bars). The drug vehicle, 95% ethanol (1% v/v) was added to the aCSF incubating the control tissues (unshaded bars). Values are mean + S.E. of 4-6 measurements. * P < 0.05, ** P < 0.01. Inset shows fractional inhibition at each age in weeks.
by the anion exchange inhibitor, DIDS, particularly at 3 and 7 weeks, but also with limited effect at 2 weeks (Fig. 5). While the DIDS-inhibitable component of
40
8 .'Q e-
[] II
Control DIDS
I
I
1
7
0.05
"7
O.O3 E
ILl
U t~
T
l'
0.01 1
2
3
I
7
Age (weeks) Fig. 5. Choroid plexus 36C1 efflux in the presence of 10 -4 M DIDS (shaded bars). Controls are unshaded. Values are mean+S.E, of 6-10 measurements. * P < 0.05, ** P < 0.01. Inset shows fractional inhibition at each age in weeks.
7 Age (weeks) Fig 6. Choroid plexus 36C1 efflux in the presence of 10 -4 M DPC (shaded bars). The drug vehicle, 1% DMSO v/v, was added to the aCSF incubating the control tissues (unshaded bars). Values are the mean_+ S.E. for 4-5 measurements. ** P < 0.01. Inset shows fractional inhibition at 1 and 7 weeks.
effiux varies from 10% of control at 1 week to 37% at 7 weeks, the uninhibitable component had a similar k value in all age groups and averaged 0.026 s-1. Unlike the other inhibitors, DPC a C1 conductance blocker, significantly reduced 1 week k by over 40% to 0.018 ___0.005 s -1 with tl/2 of 38.7s (Fig. 6). Effiux from 7 week tissues was also reduced, from 0.041 + 0.003 s- ~ to 0.025 + 0.002 s- 1. The specificity of DPC reduction of CI movement needed clarification since at concentrations of 10 -3 M and above, there may be effects on the Na ÷, K +ATPase. To ascertain whether inactivation of the N a - K pump was occurring, the K ÷ content of the choroid plexus was measured by flame photometry after the tissue ions had been extracted overnight in 0.1 N nitric acid. After correction for extracellular space with 3Hmannitol, the K ÷ concentration was found to be 128 +_ 27.9 mmol. kg -1 cell H 2 0 in controls and 108 + 14.3 mmol .kg -1 H 2 0 in choroidal tissues treated for 20 min with 10-4M DPC (n = 3). While there was a slight reduction in K ÷ in the DPC-treated tissues, this was not statistically significant and would be unlikely to cause the large reduction of 36C1 effiux. A combination of drugs was then used to attempt additive inhibitions. However, as with uptake, DIDS and DPC ( 1 0 - 4 M ) together, failed to reduce 36C1 efflux any further than DPC alone in adult rats.
185 DISCUSSION This study has investigated the uptake and effiux of 36C1 at the rat lateral ventricle choroid plexus during development. Previous studies have shown this in vitro preparation to be viable over the time periods used 23'27. We now report differences, not only in baseline transport characteristics, but also in the response of the immature choroid plexus to pharmacological agents as the choroid plexus develops in size (Fig. 1) and capacity to secrete CSF. Chloride ions are accumulated in the choroidal epithelium above electrochemical equilibrium by an active mechanism 25'28. In the present studies the rate of uptake (12 s) was the same in 1 week and 7 week old rats (Table I). By comparison, K + uptake into 0.5-1 week rat choroid plexuses is less than that in 1.5-4 week tissues 22 so uptake processes for CI and K + seem to follow different developmental time courses. Unlike 36C1 uptake, the effiux was slower in the 1 week choroid plexus with k = 0.029 _+ 0.002 s- 1 (n = 23) compared to the adult k - 0.041 +_ 0.003 s-1 (n = 19) with corresponding t 1/2 values of 24 s and 17 s respectively. A combination of rapid uptake and slower effiux should result in the elevation of intracellular CI in the young choroid plexus as found by Smith, Woodbury and Johanson 29. The steady-state 36C1 uptake values in Table I are also consistent with relatively high cellular [C1] early in development. Again, such findings in infants probably reflect retention of choroid plexus [C1] due to an apical effiux that is slower than basolateral uptake. The lower k values for in vitro effiux in infants vs. adults are in line with previous in vivo findings that 36C1 movement from blood to CSF does not show a fast component in 1 week rats as in adults 29. The component missing in infants, but present in adults, probably represents net CI transport across the in vivo choroid plexus is. Measurement of in vitro 36C1 effiux from 2 week plexuses was also relatively slow, k = 0.031s-1, but at 3 weeks it was not significantly different from that of the adult. Of the inhibitors used to investigate C1 effiux, acetazolamide, bumetanide and DIDS were similar in that they reduced effiux from 2, 3 and 7 week choroid plexuses to varying degrees, but they did not significantly affect 1 week tissues. The carbonic anhydrase inhibitor, acetazolamide, decreases CSF secretion in vivo 3 and although it has been suggested that this is due to constriction of blood vessels in the choroid plexus 24 recent studies show that blood flow is actually increased 5. The inhibitory effects seem to be on ion transport 8, with decreased Na entry into CSF 4,3°, and we report here reduced CI effiux from the adult choroid
plexus after acetazolamide administration. Interestingly, acetazolamide does not reduce CI uptake (Table I) and so it may be affecting an apical anion channel 8. The loop diuretic bumetanide, which in adults reduces choroid plexus Na and C1 uptake in vitro 13 and C1 movement from blood to CSF in vivo 15, did reduce 36C1 efflux from adult choroid plexuses, but again there was no effect at 1 week. A bumetanide-sensitive Na, K, 2C1 cotransport system has been identified in the adult rat choroid plexus and it has a role in the uptake of these ions 2. Reduced uptake could indirectly affect C1 effiux by decreasing [CI] i and, therefore, effiux via anion channels. With the design of these efflux experiments, uptake may be inhibited during efflux measurement and lead to the reduced efflux rates observed. Bumetanide significantly inhibited 36C1 effiux at 2, 3 and 7 weeks indicating the presence of a cation-C1 cotransport system at these later stages of development. The disulphonic stilbene, DIDS, is an inhibitor of C1-HCO 3 exchange and it reduced 36C1 effiux with increasing effect from 1 week (10% inhibition) to 7 weeks (37% inhibition), leaving the uninhibited component of effiux close to k = 0.026 s-~ at all ages tested. DIDS also reduced 12 s uptake in adults but not in 1 week choroid plexuses. The functioning of this transporter appears to be in transition at 2 weeks since the % DIDS inhibition is only two-thirds that seen at 7 weeks. The anion exchanger has recently been located on the basolateral face of the choroid plexus 19 and it is one of the major mechanisms for C1 transport into the choroid plexus and thence into CSF 6. Each of the drugs analyzed did not significantly reduce 36CI efflux from 1 week tissues, although acetazolamide and bumetanide inhibitions at 2 weeks, and DIDS at 3 weeks, were similar to the adult tissues. Because these drugs are known to reduce CSF secretion or CSF [CI] in vivo, the implication is that slow CI efflux and CSF production at 1 week may be a result of limited functioning, or lack of these ion translocating mechanisms. In contrast with the other inhibitors used, the C1 conductance blocker DPC reduced 1 week effiux significantly with a 43% drop in k, similar to the 39% inhibition at 7 weeks, suggesting that a large component of 36C1 efflux is via this mechanism even in the immature choroid plexus. In addition, C1 uptake is also inhibited by DPC at both ages. It is improbable that DPC acts directly to prevent uptake via CI channels since C1 is above electrochemical equilibrium in the choroidal cell. It is more likely that CI efflux is blocked, leading to anion accumulation, thus slowing uptake. Indeed, in the rabbit parietal cell, DPC effects appear
186 to be localized to the CI secreting apical m e m b r a n e e°. If CI uptake is d e p e n d e n t on the rate of efflux, combining an uptake inhibitor with an efflux inhibitor may not result in additive inhibition of CI transport since the two systems are in series. In these studies C1 uptake was m e a s u r e d in the presence of both D I D S and DPC. D I D S blocks C1 uptake (Table I) via the basolateral C I - H C O 3 exchanger 1'), whilst D P C blocks CI efflux (Fig. 6) and this combination did not p r o d u c e additive uptake inhibition. It is likely that a large c o m p o n e n t of the observed 36C1 transport occurs at the apical m e m b r a n e because of its large surface area c o m p a r e d to the basolateral m e m b r a n e and close proximity to the incubating aCSF. An anion exchanger has been located at the basolateral face of the tissue 19 but this does not rule out the presence of pathways for chloride transport at the apical surface as has been seen at the brush-border m e m b r a n e of the bullfrog choroid plexus 26. A n anion exchanger has also been observed at the brush-border m e m b r a n e of the necturus m a c u l o s u s choroid plexus 33 and this may provide a pathway for 36C1 efflux from the loaded cell. In addition, a co-transport system has been described for KCI and H 2 0 at the apical face of the necturus choroid plexus 31. We have observed a C1 dep e n d e n t 86Rb efflux in both adult and 1-week-old rat choroid plexuses (unpublished results) which also points to the presence of KCI co-transport in this preparation. The coupling of CI and H 2 0 transport in the necturus plexus 3t'32 stresses the importance of C1 in secretion of fluid by the choroid plexus. This transport has been shown to be inhibited by the loop diuretic furosemide in vitro 3~, and in vivo causes the build up of C1 and K in the rat choroid plexus II, presumably by blocking ion m o v e m e n t across the apical m e m b r a n e into CSF. Based on these observations it is likely that the 36C1 efflux modulations observed at the rat choroid plexus are due to drug effects primarily at the apical, CSF secreting face of the choroid plexus. As the rat choroid plexus develops, not only does 36Cl efflux increase, but also the epithelial cell m e m brane surface area 17. Such an increase in surface area would allow insertion into the m e m b r a n e of more ion-transporting proteins and hence greater transport capabilities per unit volume of epithelium. T h e C1 flux as a function of the cell m e m b r a n e area can be calculated from; J = k~ V/A)[CI]~
where J is unidirectional flux ( m o l . cm - 2 " s - l ) , k is the efflux coefficient and V / A is the cell volume to surface area ratio. T h e m e a n values of k are 0.029 s - l
at 1 week, 0.041 s i at 7 weeks. [El] i was estimated from 36C1 30 rain uptake (Table I) and converted to m m o l . k g - l cell H 2 0 by subtracting the extracellular 3H-mannitol space i.e., extracellular volume, from the tissue wet weight. From the surface density ( A / V ) m e a s u r e m e n t s of Keep and Jones 17, values for V / A were extrapolated for 1 week rats, and values for 30 day rats used as approximations for adults. With these data, the 36C1 efflux, J, was 554 n m o l . cm 2. s-~ at 1 week and 549 n m o l - c m 2 . s - ~ for adults. The increase in m e m b r a n e surface area, particularly the apical m e m b r a n e , occurs over the same time course as the increase in 36C1 efflux and, therefore, J is similar in 1 and 7 week rats. We have shown that the choroid plexus of immature rats, in which the CSF secretory process is not yet complete, has less capacity to extrude C1 than the adult plexus. This may be due to incomplete m e m b r a n e transport development and is reflected in the ineffectiveness of the inhibitors used. This research was supported by NIH Grant NS 27601 and funds from the Rhode Island Hospital. We thank J.T. Parmelee for technical advice.
Acknowledgements.
REFERENCES 1 Amtorp, O. and Sorensen, S.C., The ontogenetic development of concentration differences for protein and ions between plasma and cerebrospinal fluid in rabbits and rats, J. Physiol., 243 (1974) 387-400. 2 Bairamian, D., Johanson, C.E., Parmelee, J.T. and Epstein, M., Potassium cotransport with sodium and chloride in the choroid plexus, J. Neurochem., 56 (1991) 1623-1629. 3 Davson, H. and Pollay, M., The turnover of 24Na in the cerebrospinal fluid and its bearing on the blood brain barrier, J. Physiol., 167 (1963) 247-255. 4 Davson, H. and Segal, M.B., The effect of some inhibitors and accelerators of sodium transport on the turnover of 22Na in the cerebrospinal fluid and the brain, J. Physiol., 209 (1970) 131-153. 5 Faraci, F.M., Mayhan, W.G. and Heistad, D.D., Vascular effects of acetazolamide on the choroid plexus, J. Pharmacol. Exp. Ther., 254 (1990) 23-27. 6 Frankel, H. and Kazemi, H., Regulation of CSF compositionblocking chloride-bicarbonate exchange, J. App. Physiol., 55 (1983) 177-182. 7 Hodson, W.A., Fenner, A., Brumley, G., Chernick, V. and Avery, M.E., Cerebrospinal fluid and blood acid-base relationships in fetal and neonatal lambs and pregnant ewes, Resp. Physiol., 4 (1968) 322-332. 8 Johanson, C.E., Differential effects of acetazolamide, benzolamide and systemic acidosis on hydrogen and bicarbonate gradients across the apical and basolateral membranes of the choroid plexus, J. Pharmacol. Exp. Ther., 231 (1984) 502-511. 9 Johanson, C.E., Potential for pharmacological manipulation of the blood-cerebrospinal fluid barrier. In E. Neuwelt (Ed.), Implications of the Blood-Brain Barrier and its Manipulation. Basic Science Aspects, 11"ol. 1, Plenum Press, New York, 1989, pp.
223-260. 10 Johanson, C.E., Allen, J. and Withrow, C.D., Regulation of pH and HCO3 in brain and CSF of the developing mammalian central nervous system, Dev. Brain Res., 38 (1988) 255-264. 11 Johanson, C.E., Murphy, V.A. and Dyas, M., Ethacrynic acid and
187 furosemide alter CI, K and Na distribution between blood, choroid plexus, CSF and brain, Neurochem. Res., 17 (1992) 1079-1085. 12 Johanson, C.E., Reed, D.J. and Woodbury, D.M., Developmental studies of the compartmentalization of water and electrolytes in the choroid plexus of neonatal rat brain, Brain Res., 116 (1976) 35-48. 13 Johanson, C.E., Sweeney, S.M., Parmelee, J.T. and Epstein, M.H., Cotransport of sodium and chloride by the adult mammalian choroid plexus, Am. J. Physiol., 258 (1990) C211-C216. 14 Johanson, C.E. and Woodbury, D.M., Changes in CSF flow and extracellular space in the developing rat. In A. Vernadakis and N. Weiner (Eds.), Drugs and the Developing Brain, Plenum Press, New York, 1974, pp. 281-287. 15 Johnson, D.C., Singer, S., Hoop, B. and Kazemi, H., Chloride flux from blood to CSF: inhibition by furosemide and bumetanide, J. App. Physiol., 63 (1987) 1591-1600 16 Jones, H.C. and Keep, R.F., The control of potassium concentration in the cerebrospinal fluid and brain interstitial fluid of developing rats, J. Physiol., 383 (1987) 441-453. 17 Keep, R.F. and Jones, H.C., A morphometric study on the development of the lateral ventricle choroid plexus, choroid plexus capillaries and ventricular ependyma in the rat, Dev. Brain Res., 56 (1990) 47-53. 18 Levin, V. and Patlak, C.S., A compartmental analysis of 24Na kinetics in rat cerebrum, sciatic nerve and cerebrospinal fluid, J. Physiol., 224 (1972) 559-581. 19 Lindsey, A,E., Schneider, K., Simmons, D.M., Baron, R., Lee, B.S. and Kopito, R.R., Functional expression and subcellular localization of an anion exchanger cloned from choroid plexus, Proc. Nat. Acad. Sci. USA, 87 (1990) 5278-5282. 20 Malinowska, D.H., CI- channel blockers inhibit acid secretion in rabbit parietal cells, Am. J. PhysioL, 259 (1990) G536-G543. 21 Mitchell, W., Kim, C.S., O'Tuama, L.A., Pritchard, J.B. and Pick, J.R., Choroid plexus, brain and kidney Na, K+-ATPase: comparative activities in fetal, newborn and young adult rabbits, Neurosci. Lett., 31 (1982) 37-40. 22 Nattie, E.E. and Edwards, W.H., CSF acid-base regulation and ventilation during acute hypercapnia in the new born dog, J. Appl. PhysioL, 50 (1981) 566-574.
23 Parmelee, J.T. and Johanson, C.E., Development of potassium transport capability by choroid plexus of infant rats, Am. J. Physiol., 256 (1989) R786-R791. 24 Politoff, A. and Macri, F., Pharmacologic differences between isolated, perfused arteries of the choroid plexus and of brain parenchyma, Int. J. Neuropharm., 5 (1966) 155-162 25 Saito, Y, and Wright, E.M., Regulation of bicarbonate transport across the brush border membrane of the bull-frog choroid plexus, J. Physiol., 350 (1984) 327-342. 26 Saito, Y. and Wright, E.M., Regulation of intracellular chloride in bullfrog choroid plexus, Brain Res., 417 (1987) 267-272. 27 Smith, Q.R. and Johanson, C.E., Active transport of chloride by lateral ventricle choroid plexus of the rat, Am. J. Physiol., 249 (1985) F470-F477. 28 Smith, Q.R., Woodbury, D.M. and Johanson, C.E., Uptake of CI-36 and Na-22 by the choroid plexus-cerebrospinal fluid system: evidence for active chloride transport by the choroidal epithelium, J. Neurochem., 37 (1981) 107-116. 29 Smith, Q.R., Woodbury, D.M. and Johanson, C.E., Kinetic analysis of [36C1]-, [22Na]- and [3H]mannitol uptake into the in vivo choroid plexus-cerebrospinal fluid system: ontogeny of the blood-brain and blood-CSF barriers, DeL'. Brain Res., 3 (1982) 181-198. 30 Vogh, B.P. and Doyle, A.S., The effect of carbonic anhydrase inhibitors and other drugs on sodium entry to cerebrospinal fluid, J. Pharmacol. Exp. Ther., 217 (1981) 51-56. 31 Zeuthen, T., Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus Maculosus, J. Physiol., 444 (1991) 133-151. 32 Zeuthen, T., Secondary active transport of water across ventricular cell membrane of choroid plexus of Necturus Maculosus, J. Physiol., 444 (1991) 153-173. 33 Zeuthen, T., Christensen, O., Baerentsen, J.H. and la Cour, M., The mechanism of electrodiffusive K ÷ transport in leaky epithelia and some of its consequences for anion transport, Pfliigers Arch., 408 (1987) 260-266.