306

Brain Research, 562 ( 199 t ) 306-31 [i © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993191/$03.50 I)O NIS 000689939124879(;

BRES 24879

Chloride efflux from

choroid plexus

Quentin R. Smith and Conrad E. Johanson Laboratory of Neurosciences, National Institute on Aging, NIH, Bethesda, MD 20892 (U.S.A.) and Program in Neurosurgerv, Department of Clinical Neurosciences, Brown University~Rhode Island Hospital, Providence, RI 02902 (U.S.A.) (Acccpted 9 July 1991)

Key words: Choroid plexus epithelium; Cerebrospinal fluid; 36C1 efflux coefficient; Acetazolamide; Disulfonic stilbene; Furosemide; lsethionate; Anion exchange

Chloride efflux was analyzed in adult rat lateral ventricle choroid plexus (LVCP) incubated in artificial CSF (aCSF) at 37 °C. Following steady-state loading of 36C1 in LVCP, the tracer release from plexus to aCSF was quantified by the effux coefficient (k, s-l), equal to In 2/t,. C1 efflux could be described by a 2-component model, with a tl for the 'fast' component matching well that for [3H]sucrose (extracetlular marker) and a slower, drug-inhibitable component of 36C1 release thought to reflect cellular washout. The cellular CI efflux was more than twice as fast at 37 °C than at t5 °C. There was progressively more rapid efflux (k) of 36C1 from cells as the aCSF was altered over a range of several pH values from 6.7 (k = 0.026 s -~) to 8.2 (0.070 s 1). CSF medium anion replacement (isethionate and HEPES for Cl and HCO 3, respectively) reduced the k for 36C1 by 57%. Acetazolamide (0.1 mM) and other CI transport inhibitors (disulfonic stilbenes a n d loop diuretic) reduced CI efflux by 35-55%. Acetazolamide inhibited CI release from LVCP into aCSF whether the latter contained Cl and HCO 3. or not. Overall, the findings suggest that CI extrusion from choroid plexus is by way of an anion exchanger and via channels.

The choroid plexus (CP) has a renal-like function to ensure homeostasis of the composition and volume of brain interstitial fluid and cerebrospinal fluid (CSF) 31. Secondary active transporters in the basolateral membrane of mammalian and amphibian CP move CI 'uphill' into the epithelium by C1-HCO 3 exchange 4'9"12 and by Na-C1 or Na-K-CI cotransport 2"13"23"3°. Such active inward transport of C1 into choroidal cells from the blood side results in this anion being the one at the highest concentration in both CP and C S F 29'3°. Thus the CSF [CI] of about 130 mmol/l in mammals is about 15 mmol/l greater than that in plasma 1~'~8. It has not been clear how CI, once accumulated above electrochemical equilibrium in mammalian CP epithelium 29"3°, is released from the cells. In this study, we characterized the nature of C1 extrusion from isolated CP. The rate of release of 36C1 from choroidal tissues was analyzed in artificial CSF (aCSF) containing various concentrations of anions, buffers or agents that interfere with C1 transport. Acetazolamide, as well as other inhibitors of CSF secretion, substantially reduced the ability of CP to release CI. Moreover, making aCSF acidotic suppressed CI flux out of the CP epithelium. We relate the findings to models of CSF secretion and acid-base regulation, and compare the choroidal CI efflux phenomena with those observed in other epithelia.

The following 5 sets of experiments were run to ascertain effects of temperature, ion substitution or pharmacologic agents on the ability of CP to transport C1 outward: (1) hypothermia of aCSF incubation medium, (2) replacement of C1 and HCO 3 in aCSF by isethionate and HEPES, (3) graded alterations in aCSF pH, (4) evaluation of the CSF secretory inhibitor, acetazolamide, in the presence or absence of CI and HCO 3, and (5) assessment of the transport-suppressing potential of diuretic-type drugs. Sprague-Dawley rats have been used extensively to develop the CSF transport model ~°, and so adult males of this species were used as the source of CP tissues. The plexuses, averaging 0.17 mg in dry weight, were removed after decapitation from lateral ventricles of 250350 g animals. In drug experiments, one CP was used to test for pharmacologic effects, whereas the contralateral tissue was utilized as control. In other experiments, each lateral ventricle plexus was used as an individual observation. Each CP was allowed to stabilize in aCSF for 25 min before exposing the tissue to drug or tracer. The aCSF was composed as previously described 29, except that [NaC1] was 122.6 mmol/l rather than 117.6 mmol/l. When C O 2 / H C O 3 was used as buffer, the aCSF was bubbled with 95% 02/5% CO 2. In experiments with HEPES as the buffering agent, the aCSF medium was

Correspondence: C.E. Johanson, Department of Clinical Neurosciences, Rhode Island Hospital, 593 Eddy Street, Providcncc, RI 02902, U.S.A.

307 gassed with 100% 0 2 . Drugs were o b t a i n e d from Sigma Chemical Co., except for furosemide which was bought from Hoechst-Roussel Pharmaceuticals. 36C1 (as NaC1, 12.7 mCi/g) and [3H]sucrose (11.4 Ci/mmol) were supplied by ICN and New England Nuclear Co., respectively. Loading of tracer into tissue was accomplished by incubating CP for 5 min in a vial containing aCSF and 36C1 or [3H]sucrose (5 /~Ci/ml). The 'hot' tissue was then transferred, sequentially, into a series of vials containing a C S F identical in composition (most experiments) to preincubation m e d i u m , but lacking radioactivity. The initial sample time interval was 5 s, beginning with the transfer of tracer-loaded tissue to the first vial of tracerfree aCSF; thereafter, succeeding time periods for sampiing were at 5-s or, later, at 10-s intervals (Fig. 1). The half-time (t½) for 36C1 release from each CP was calculated from a semi-logarithmic plot of points describing the serial a p p e a r a n c e , at precisely-timed intervals, of dpms in successive samples of a C S F (Fig. 1). Linear regression analysis revealed two c o m p o n e n t s of 36C1 release, a 'fast' and 'slow' with t~ values of about 2.5 and 12 s, respectively. In other experiments [3H]sucrose, a m a r k e r that distributes extracellularly, displayed a single c o m p o n e n t of release with a t½ of 2 - 4 s, and a k of 0.25 _+ 0.04 (n = 3). Therefore we conclude that the rapid release of 36C1 was from the extracellular c o m p a r t m e n t , and that the slower c o m p o n e n t (modifiable by drugs or ion substitution in aCSF) is attributable to release of tracer CI from p a r e n c h y m a l cells. T e m p e r a t u r e reduction m a r k e d l y decreased C1 efflux. Lowering the a C S F t e m p e r a t u r e from 37 °C control to 15 °C caused a 51% decrease in k. Thus in 4 hypothermic incubations the mean k was only 0.033 + 0.002 s -1. Corresponding values for the t~s of washout were 10.4 _+ 0.44 s at 37 °C and 21.5 -+ 1.4 s at 15 °C. Such slowing of C1 efflux would elevate cellular [C1] if CP uptake of CI were not p r o p o r t i o n a l l y reduced. In this regard, Smith and Johanson 27 r e p o r t e d that cell [C1] did increase by 23 mmol/kg H 2 0 when CP was incubated at 15 °C. Extrusion of cellular C1 by the CP C1-HCO 3 exchanger was h a m p e r e d when extracellular (aCSF) H C O 3 and C1 were r e m o v e d briefly in the efflux solution. Thus, the k for C1 release was decreased by 23%, 34% or 58% as [HCO3] o, [Clio or [ H C O 3 + C1]o in a C S F was taken as close to zero as possible by r e p l a c e m e n t with organic anions with much lower affinity for the anion exchanger (Table I). This series of ion substitutions provides kinetic evidence for the considerable ability of C1 and H C O 3 outside the cell to counter-exchange for C1 inside the epithelium. Previous steady-state experiments provided indirect evidence for anion exchange in rat CP 12, which has since been localized by antibody staining to basolateral m e m b r a n e 15.

There was progressively slower efflux of Cl from CP upon altering the extracellular p H over a wide range from alkaline to acidic values (Fig. 2). This p h e n o m e non has also been observed in erythrocytes 3'21, and has been attributed to a mechanism titratable with extracellular hydrogen ions into less functional forms for CI transport 5. In our series, no [HCO3] or C O 2 was a d d e d to aCSF while extracellular p H was varied over a range of 1.4 p H units across several experiments. M o r e o v e r , CP p H i is not well buffered in vitro, and so choroid cell p H tends to follow the p H of the aCSF 12. Thus the slower release of C1 by CP in the acidosis experiments (aCSF p H = 6.7 and 7.1) may also be due in part to

LVCP 36CI EFFLUX I0000600

t ~ ( Fost): 2.5 Sec 3000-

t/

(Slow) • 1212 Sec

< i,~ [,,

I000-

600" [--

300-

I0060-

30-

Fost Component

I0

Slow Component

,'o

3'o

;o TIME

,;o

! 130

(Sec)

Fig. 1. Kinetics of release of 36C1 from choroid plexus (CP) into artificial CSF (aCSF) medium of control ionic composition. Tissue was excised from an adult rat, then preincubated for 25 min at 37 °C in aCSF at pH 7.4. Five min before ending preincubation, 36C1 was added to the aCSF in order to load the tissue with tracer. The 36Cl-laden tissue was subsequently transferred sequentially into a series of vials containing aCSF identical in composition to preincubation medium, but lacking radioactivity. The efflux curve resolved into a 'fast' component (extracellular) and a 'slow' component (parenchymal cellular). The equation, c(t) = A1 x exp(-kl x t + A2 x exp(-k x t), was fit to the data (t,c) using weighted non-linear least squares. The 'fast' component was obtained by a curve-stripping procedure 3°. The baseline data depicted for this plexus was one set in a series of 6 tissues which yielded the following average values _+ S.E.M.: 'fast' component of CI efflux, k = 0.25 -+ 0.04 s -~ (t½ of 2.8 s), and 'slow' component of C1 efflux, k = 0.055 _+ 0.004 s -l (t~ of 12.6 s). t is the half time of the disappearance of tracer from CP.

308

reduced cytoplasmic pH which can suppress C1 transport via anion exchange ~7"2~'26. Acetazolamide (0.1 mM) decreased CI release from CP by 40%, with or without CO 2, H C O 3 and CI in aCSF (Fig. 3). Normally functioning CI-HCO 3 exchange in CP needs levels of CO 2, H C O 3 and C1 in aCSF similar to those in vivo. Full inhibition by acetazolamide of 36C1 efflux, even when CO 2, HCO3 and CI were negligible in aCSF, indicates that this drug effect was mainly on CI

channels rather than anion exchange. This is consistent with the finding by Seifter and Aronson :5 that acetazolamide does not directly interfere with C1-HCO:~ cxchange, and with the view by several investigators that acetazolamide blocks anion channels (summarized in ref. 9). A CI channel has been electrophysiologically demonstrated in the apical membrane of bullfrog CP 24. The existence of CI channels in rat CP has been inferred by the finding that diphenylamine carboxylate (DPC), a CI channel blocker, decreased CI efflux from rat CP by nearly 40% 22. Other transport inhibitors, like furosemide (loop diuretic), and DIDS and SITS (disulfonic stilbenes), decrease CSF formation 4 and so they would be expected to alter CSF uptake of CI, the main anion in CSF secretion 14. All 3 agents substantially reduced CI release from our in vitro preparation, as 1 mM furosemide, DIDS and SITS decreased k for CI by 51%, 56% and 38%, respectively, to values of 0.030 ± 0.003, 0.027 ± 0.004 and 0.038 ± 0.003 s -a. In erythrocytes, the anionic form of furosemide interacts with the Cl transport mechanism at a site separate from both the transport site and the halide-reactive modifier site 3. I11 proximal tubule, furosemide and the stilbene agents (DIDS and SITS) interfere with Cl transport via anion exchange 1. Thus the marked effects of these inhibitors provide further, albeit deductive, pharmacological evidence for CI-HCO 3 exchange in rat CP. CSF-formation inhibitors and extracellular metabolic acidosis decrease the penetration of Cl and Na from blood across CP into C S F 4"l~'l~'16'~s'2~ Such altered fluxes in vivo could occur either paracellutarly (between

CI

CI

TABLE I

Effects o f a C S F anion substitution on chloride efflux f r o m the choroid plexus Values are means -+ S.E.M. Radioisotope efflux measurements were made at 37 °C. Tissues were preincubated in aCSF of normal composition, and then transferred to aCSF depleted in H C O 3 and/or Cl. The [Clio and [HCO3] o refer to concentrations of these anions in artificial CSE [Clio and [HCOs] o were reduced to ~ 0 mM in aCSF by replacement of the respective anions with isethionate and HEPES. CSF p H was held near 7.40 in all incubations, k is the rate coefficient for Cl efflux (see text for calculation), zl k refers to the average % change from the mean control value. The t~ is the half-time for the CI release from the choroid plexus. *P < 0.05, anion substitution vs control, by Dunnett's test.

[C!] o ~ [ H C O f l o --~ k (s -l) %A k t~ ~,) %A t n=

efflux

Control 130 m M 18 m M

130 m M 0 mM

0 mM 18 m M

0 mM 0 mM

0.0563 ±0.0023 12.3 +_0.6 12

0.0435 ±0.0027 -23% 15.9" ±0.9 +29% 7

0.0373 _+0.0022 -34% 18.6" ± l.l +51% 8

0.0235 ±0.(XH7 -58% 29.4* .~2.2 + 139c~ 7

(k, sec "1)

efflux

(k, sec "1)

0.08 A

Iflln

~

0.08

0.04 0.02 0.08

6.8

7.1-

7.4

7.7

8.1

art. CSF pH Fig. 2. Effect of alterations in the pH of aCSF medium (pHo) on C1 effiux from isolated CP. Each bar is the mean for 4-7 tissues. The unfilled bar represents the control. The rate coefficient, k, describing efflux was calculated as In 2 + t. CO 2 and H C O 3 were not added to aCSF, so H E P E S (10 raM) was used as buffer. Limits are S.E.M. Standard error for k was estimated as [In 2 + t] (S.E. t~ mean t,).

DRUG

~

BUFFER ==:>

NONE HCO 3

ACTZ HCO 3

ACTZ HEPES

Fig. 3. Effect of acetazolamide, with or without H C O 3 and CI in aCSF, on the C1 washout from CP CSF pH and [acetazolamide] were 7.4 and 0.l mM, respectively. Bars are means ± S.E.M. for 6-8 CPs. Unfilled bar is the control. *P < 0.05. acetazolamidc (ACTZ) vs control (no drug). From left to right, the corresponding t, values were 12.2 + 0.6. 20.3 ' 1.2, and 21.5 ~ 0.9 s.

309

epithelial cells) or transcellularly ~°'1~'~8. We p r o v i d e ad-

port inhibitors (e.g. D I D S , SITS or a c e t a z o l a m i d e ) are

ditional e v i d e n c e h e r e for effects on the transcellular

c h a l l e n g e d with m e t a b o l i c alkalosis v o r r e s p i r a t o r y aci-

r o u t e , b e c a u s e w h e n isolated C P was e x p o s e d to diuretic

dosis 8'9'2°. O t h e r investigators h a v e i m p l i e d a role for

agents or d i m i n i s h e d e x t r a c e l l u l a r p H , the cellular re-

the c h o r o i d a l t r a n s p o r t of CI in C S F acid-base b a l a n c e 2°.

lease of C1 was substantially slowed. T h e r e f o r e o u r ob-

T h e direct analysis of C P in the p r e s e n t study points to

servations are consistent with t h o s e on intact animals

C1 t r a n s p o r t m e c h a n i s m s possibly u n d e r l y i n g such C S F

(subjected

which C S F t o o k up CI and Na m o r e slowly f r o m b l o o d ~ '

p H regulation. In s u m m a r y , C1 is r e l e a s e d f r o m rat C P via an a n i o n

ls.2~. O v e r a l l , t h e r e is s t r o n g c o r r e l a t i v e and direct evi-

e x c h a n g e r , and by a m e c h a n i s m sensitive to acetazola-

d e n c e which indicates that t r e a t m e n t s suppressing C S F

m i d e but not r e q u i r i n g C1 and H C O 3 in the m e d i u m .

f o r m a t i o n do so by altering C1 t r a n s p o r t t h r o u g h C P ep-

T h e latter p a t h w a y is likely a C1 c h a n n e l , and it is cur-

ithelium. C S F acid-base b a l a n c e is m o d u l a t e d by CI and H C O 3

rently u n d e r i n v e s t i g a t i o n in d e v e l o p i n g and m a t u r e animals 22.

to a c e t a z o l a m i d e

or systemic

acidosis)

in

t r a n s p o r t b e t w e e n C P and C S F 6"~9. T h e sensitivity of in vitro C P CI t r a n s p o r t (efflux) to changes in a C S F p H , i.e. a b o u t 2-fold c h a n g e f r o m p H 7.1 to 7.7, suggests a tion is d i s r u p t e d w h e n animals p r e t r e a t e d with C1 trans-

This study was supported by funds from NIA and from NIH Grant NS 27601. We thank J. Preston for critical reading of the manuscript.

1 Aronson, P.S., The renal proximal tubule: a model for diversity of anion exchangers and stilbene-sensitive anion transporters, Annu. Rev. Physiol., 51 (1989) 419-441. 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 Brazy, P.C. and Gunn, R.B., Furosemide inhibition of chloride transport in human red blood cells, J. Gen. Physiol., 68 (1976) 583-599. 4 Deng, Q.S. and Johanson, C.E., Stilbenes inhibit exchange of chloride between blood, choroid plexus and cerebrospinal fluid, Brain Research, 501 (1989) 183-187. 5 Gunn, R.B., Dalmark, M., Tosteson, D.C. and Wieth, J.O., Characteristics of chloride transport in human red blood cells, J. Gen. Physiol., 61 11973) 185-206. 6 Husted, R.E and Reed, D.J., Regulation of cerebrospinal fluid bicarbonate by the cat choroid plexus, J. Physiol., 267 (1977) 411-428. 7 Javaheri, S. and Weyne, J., Effect of the anion blocker 'SITS' on cerebrospinal fluid [HCO 3 ] in acute acid-base perturbations. In M. Schalfke, H. Koepchen, and W. See (Eds.), Central Neurone Environment and the Control Systems o f Breathing and Circulation, Springer, Berlin, 1983, pp. 22-28. 8 Javaheri, S. and Weyne, J., Effects of 'DIDS', an anion transport blocker, on CSF [HCO~ ] in respiratory acidosis, Resp. Physiol., 57 (1984) 365-376. 9 Johanson, C.E., Differential effects of acetazolamidc, benzolamide and systemic acidosis on hydrogen and bicarbonate gradients across the apical and basolateral membranes of the chorold plexus, J. Pharmacol. Exp. Ther, 23l (1984) 502-511. 10 Johanson, C.E., Potential for pharmacologic manipulation of the blood-cerebrospinal fluid barrier. In E.A. Neuwelt (Ed.), lmplic~ttions of" the Blood-Brain Barrier and Its Manipulation, Vol. I, Ch. 9, Basic Science Aspects, Plenum Press, 1989, pp. 223-261t. 11 Johanson, C.E. and Murphy, V.A., Acetazolamide and insulin alter choroid plexus epithelial cell [Na+], pH, and volume, Am. J. Physiol., 258 (1990) F1538-FI546. 12 Johanson, C.E., Parandoosh, Z. and Smith, Q.R., CI-HCO~ exchange in choroid plexus: analysis by the DMO method for cell pH, Am. J. Physiol., 249 (1985) F478-F484. 13 Johanson, C.E., Swecncy, 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.

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role in C S F p H h o m e o s t a s i s . C S F p H or H C O 3 regula-

310 coefficients to sodium, potassium and chloride, J. Neurochem., 46 (1986) 1732-1742. 29 Smith, Q.R., Woodbury, D.M. and Johanson, C.E., Uptake of 36CI and 22Na by the choroid plexus-eerebrospinal fluid system: evidence for active chloride transport by the choroidal epithelium, J. Neurochem., 37 (1981) 107-116. 30 Smith, Q.R., Woodbury, D.M. and Johanson, C.E., Kinetic

analysis of CI-36, Na-22 and 1-1-3 mannitol uptakc into the m vivo choroid plexus-cerebrospinal fluid system: ontogcny of the blood-brain and blood-CSF barricrs, Dev. Braitl Res.. 3 (1982) 181-198. 31 Spector, R. and Johanson, C.I-'L, l'he mammalian choroid plexus, Sci. Am., 261 (1989) 68- 74,