Fluid secretion by the frog choroid plexus

Exp. Eye Res. (1977) Suppl., 149-155 Fluid Secretion by the Frog Choroid Plexus ERNEST M. WRIGHT, G(~NTHERWIEDNER AND GERttARD RUMRICtI

Physiology Department, University of California Medical Center, Los Angeles, California 90024, U.S.A. and Max Planck-Institut fi~r Biophysik, Frankfurt/Main, West Germany Experimental work on the mechanism of CSF secretion by the in vitro choroid plexus of the bullfrog is presented and discussed in this paper. The results show that this tissue secretes a hypertonic fluid containing sodium, chloride and bicarbonate ions and that this is brought about by a sodium pump located in the apical membrane of the choroidal epithelium. An analysis of several possible osmotic mechanisms of fluid secretion across the choroid plexus is made.

1. Introduction The frog provides a useful and convenient model for the study of the physiology of the cerebrospinal fluid (CSF) : the ultrastructure of the meninges, the composition of the CSF and the regulation of the CSF composition are essentially similar to those in higher animals despite the fact that the frog brain is relatively simple (see Wright, 1975). A major advantage of using amphibians is that they provide a source of viable tissues that tolerate a wide range of in vitro experimental conditions. In this brief paper we will review progress that has been made towards the elucidation of the mechanism of CSF secretion of the frog choroid plexus. We will show that the tissue secretes a hypertonic fluid containing sodium, chloride and bicarbonate ions, and that this is brought about by a sodium pump located in the apical membrane of the epithelium.

2. Methods All experiments were carried out on the plexus isolated from the fourth ventricle of the Bullfrog: this plexus forms the roof of the open ventricle, and a single layer of epithelial cells covers the ventricular surface of the plexus. The cuboidal cells are joined together at the apical surface by the so-called tight junctions, and the epithelium rests on a thin connective tissue richly endowed with blood vessels. The isolated plexus was mounted between lucite flux chambers that enabled the measurement of (i) radioactive tracer fluxes across the epithelium and across the apical cell membrane (Wright, 1972, 1974 a, b), (ii) the electrical properties of the tissue (Wright, 1972), (iii) the rate and direction of water flow, and (iv) the composition of the freshly secreted CSF. Water flows across the epithelium were detected by two methods: the first was to measure the rate of change of concentration of an impermeable marker ([14C]dextran, tool. wt. 17 000) contained in a known volume of fluid on the ventricular side of the tissue; and the second was to record the changes in volume of the fluid on each side of the epithelium using the DIMEQ TE 200 transducer equipment (Wayne Kerr, Bognor Regis, England). This instrument was calibrated to measure the change in volunfe of fluid in the compartments on each side of the epithelium by recording the changes in capacitance (distance) between capacitive probes and the surfaces of the fluid. Using this procedure it was possible to measure volume changes (flows) as low as 1 nl (see Wiedner, 1976). The composition of the freshly secreted CSF was determined by covering the ventricular surface of the plexus with oil and collecting the nascent fluid from the tissue/oil interface at regular intervals of time. Microanalysis of 0-1-0-5 nl samples of the fluid was carried out to obtain the Na, K and C1 concentrations and the osmolarity of the fluids. Chloride was 149

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E. M. W R I G H T ,

G. W I E D N E R

A N D G. R U M R I C H

estimated by electrometric titration, Na and K by flame photometry, and the osmolarity by depression of freezing point. Experiments were carried out in Ringer's solutions buffered to pH 7.2 with either phosphate or bicarbonate buffers. Each solution was equilibrated with appropriate gas mixtures containing oxygen, and all experiments were carried out at 23°C. 3. Results and Discussion

Ion transport The unidirectional fluxes of ions across the isolated choroidal epithelium incubated in bicarbonate Ringer's solutions are summarized in Table I. In the absence of elecffrochemical potential gradients across the tissue there was net secretion of Na and C1 from the blood into the ventricular solution, and there was a small net absorption of K from the CSF. The deficit between the net cation and anion movements is TABLE I

Ion fluxes across the choroid plexus Flux /~equiv./cm2/hr

Sodium Chloride Potassium Glycodiazine

Jsv

Jvs

Jnet

2.9 ± 0 . 2 (22) 2.7 :]:0.1 (36) 0.094-0.01 (6) 0.80±0.11 (7)

1.9 :~0.2 (17) 2.1 ±0-1 (36) 0.12~0.01 (5) 0-50=[0.03 (6)

+1.0 +0.6 --0.03 +0.3

All experiments, except those with glycodiazine, were carried out in bicarbonate Ringer's solution and gassed with 5 % C02/95% 02. [aH]glycodiazine fluxes were measured in a Ringer's solution in which the 25 mM-NaHCOa was replaced with 25 mM-Na glycodiazine. This solution was buffered at p H 7.2 with a 2.5 mM-Na phosphate buffer a n d it was equilibrated with 100% 02. Glycodiazine (pK 5.9) mimics the effect of bicarbonate in a n u m b e r of tissue including the choroid plexus (see text). Unidirectional fluxes across the epithelium were obtained from radioactive tracer fluxes, the errors are quoted as standard errors of the mean, and the n u m b e r of experiments are given in parentheses. Jsv = t h e unidirectional flux from the serosal (vascular) side to the ventricular (CSF) side of t h e epithelium and Jvs = the unidirectional flux in the opposite direction. A positive n e t flux means secretion into t h e CSF, and a negative net flux m e a n s net absorption from the CSF into the serosal compartment. The composition of the saline in both the ventricular and serosal compartments was identical throughout the experiments, and the spontaneous electrical potential across the epithelium was less t h a n 1 mV, i.e. fluxes were obtained in the absence of appreciable electrochemical potential gradients across the plexus. These results were taken from Wright (1972) and unpublished data. Glycodiazine (Bayer) and [aH]glycodiazine were generous gifts from Professor K. Ullrich.

probably accounted for by the secretion of bicarbonate ions into the CSF. Evidence to support this conclusion is : (i) bicarbonate is the only additional ion present in the Ringer's solution to any significant amount; (ii) the net secretion of Na is reduced 50% by omission of bicarbonate from the incubation media (Wright, 1972); (iii) when 25 mM-Na glycodiazine was used to replace the 25 mM-NatICO a in normal Ringer's solution there was a net secretion of glycodiazine into the CSF (Table I). Glycodiazine is a buffer (pK 5.9) that is able to mimic the effects of bicarbonate ions in the renal tubule (Ullrieh, Radtke and Rumrich, 1971), pancreas (Sehultz, 1971) and choroid plexus (unpublished observations). The primary event in these net ion movements

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151

across the plexus appears to be the active secretion of sodium across the apical cell membrane by a Na/K exchange pump. This view comes from experiments that show: (i) Ouabain, a potent inhibitor of Na/K ATPases, inhibits the transport of Na, K, C1 and glycodiazine (Wright, 1972 and unpublished observations). Ouabain is only effective in the ventricular solution (Wright, 1972), and the glycoside is specifically bound to the apical cell membrane (Quinton, Wright and Tormey, 1973); (ii) Na/K ATPases are localized in the apical membranes (unpublished observations on plasma membranes from the frog choroid plexus) ; (iii) K is actively accmnulated within the epithelimn by an ouabain sensitive, sodium dependent pump in the apical cell membrane (Wright, 1976). Active K accumulation, like active sodium secretion, is stimulated by the presence of bicarbonate or glycodiazine in the Ringer's solutions; (iv) Chloride is passively transported across the plexus (Wright, 1972). These flux experiments show that a mixture of NaC1 and NaHCO a is secreted across the frog ehoroid plexus from the blood into the cerebrospinal fluid, and that this is due to the presence of the Na/K exchange pump in the apical membrane of the epithelium.

Water transport The frog choroid plexus secreted CSF in vitro at a rate of 8-10 t~l/cm2/hr in bicarbonate Ringer's solution at 23°C (to be published). This corresponds to 0.07/~l/min/mg (plexus wet weight), and this is comparable to estimates of CSF secretion in the dogfish. In mammals, where experiments are carried out at 37°C, estimates of CSF secretion vary between 0.2 and 0.6 ttl/min/mg (see Cserr, 1971). Addition of ouabain (5 x 10-4 M) to the ventricular compartment in six bullfrog experiments reduced the spontaneous volume flow by 7.6±2-5/~l/cm~/hr. Addition of sucrose to the fluid bathing one side of the epithelium produced osmotic volume flows across the tissue. The steady-state hydraulic conductivity (L~) in ten experiments was 2.2±0.1 × 10-a cm/sec, which is similar in magnitude to that reported for the rabbit choroid plexus by Welch, Sadler and Gold (1966). The L~ in the frog plexus was independent of the rate and direction of flow with osmotic gradients up to 100 mosmol, but with higher gradients a lower L~ was observed when the flow was in the direction of blood to CSF, e.g. with 300 raM-sucrose in the ventricular fluid the L~ was 1.6=~0.2 × 10-3 cm/sec. This decrease in the hydraulic resistance of the tissue is probably due to the collapse of the lateral intercellular spaces (see Wright, Smulders and Tormey, 1972). The diffusional water permeability of the frog ehoroid plexus (P~) was found to be 0-07 ~=0-01(30) × 10-a cm/sec (Wright and Pietras, 1974). The ratio of the osmotic to the diffusional water permeabilities (32) is very close to that obtained for the frog gall bladder (22) and frog urinary bladder (29). The reason for these high ratios of the water permeabilities is not quite clear, but it is perhaps related to the high values of the absolute water permeabilities in these tissues. This view stems from two observations in the toad bladder: in the absence of ADH, where the absolute water permeabilities are an order of magnitude less than the three frog tissues, the ratio is close to 3; and secondly, in the presence of ADH, where the permeabilities are comparable to those in the frog tissues, the ratio increases to 23 (see House, 1974).

The composition of the transported fluid The freshly secreted CSF was collected from the surface of the isolated choroid plexus by micropipette in experiments similar to those carried out in vivo by Ames,

152

E. M. W R I G H T ,

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A N D G. R U M R I C t t

Sakanoue and Endo (1964). The results of one experiment where the nascent CSF was collected at intervals during a 5-hr experiment are shown in Fig. 1. The CSF was hypertonic (254 vs. 205 mosmol in saline), and the higher osmolarity was largely accounted for by the high sodium concentration in the fluid (125 vs. 110 mEq/1 in the saline bathing the vascular side of the tissue). As the chloride concentration in the secreted fluid was not significantly different from that in saline, it is concluded that bicarbonate ions account for the anion deficit. This is consistent with the observations on the ion fluxes across the tissue that a mixture of NaC1 and NaHCO s is secreted across the tissue (see Table I and p. 150). Sodium chloride 120

ine

,]

80

40 >

g E

0

Potassium

Osmolarity [osmol]

200 "G

~ 100 O E 1

Z 5 4 Time (hr) FIG. 1. The composition of freshly secreted CSF. In this experiment the frog choroid plexus was m o u n t e d in a lucite chamber t h a t enabled the serosal (vascular) side of the epithelium to be bathed with Ringer's solution, and the vcntricular surface of the epithelium to be covered with mineral oil. The freshly secreted CSF, which formed under the mineral oil, was collected at 1 hr intervals a n d was analyzed for Na, K, Cl and osmolarity. The first collection was discarded to avoid possible contamination with saline not removed from the surface of the plexus at the start of the experiment. The figure shows the composition of the CSF collected over the subsequent 4 hr, and the composition of t h e saline (shown as the continuous horizontal lines on each graph). In the upper graph the sodium concentration is indicated by the hatched bars while the chloride concentration is indicated by the solid bars. Leaks of saline across the tissue were ruled out by adding [14C]dextran (mol. wt. 17 000) to the saline bathing the serosal side of the:plexus, and monitoring the appearance of the isotope in the freshly secreted CSF.

These observations on the composition of the freshly secreted CSF are similar to those reported by others for comparable experiments with intact mammalian preparations (for reviews see Cserr, 1971 and Rapoport, 1976). However, in the frog both the sodium and osmotic gradients between the freshly secreted fluid and plasma are substantially higher than those in mammals; 15 mEq./l and 49 mosmol in the frog vs. 9 mEq/1 and 6-9 mosmol in mammals. It should also be noted that the sodium concentration of bulk CSF in both amphibians and mammals is significantly higher than in plasma.

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As indicated in Fig. 1 the potassium concentration in the secreted fluid is higher than in saline, 6.6 vs. 2 mEq/1. The reason for the discrepancy between these results and those shown in Table I is not yet clear, but it should be noted that in this series of experiments exposure of the plexus to ouabain increase the K concentration in the fluid to greater than 20 mEq/1 (cf. Ames, Higashi and Nesbett, 1965). Ouabain also lowered the Na concentration and osmolarity of the fluid towards that in the saline bathing the vascular surface of the plexus. Additional experiments were performed where the composition of the nascent CSF was measured as a function of the osmolarity of the bathing solution. The osmolarity was varied between ]00 and 300 mosmol/1 by changing the NaCl concentration. The osmolarity of the secreted fluid varied linearly with the osmolarity of the bathing fluid, and the secreted fluid remained hypertonic by 30 50 mosmol/1 over the entire range of the osmolarity of the bathing fluid. T]~e mechanisms of CSF secretion

Our experiments suggest an osmotic mechanism for CSF secretion in the frog. First, sodimn is actively secreted by a pump located in the apical membrane of the epithelium. Chloride and bicarbonate ions accompany sodimn movement into the CSF. Second, the nascent CSF is hypertonic to the saline bathing the vascular side of the epithelium, and the secreted fluidremains hypertonic over a threefold range in the osmolarity of the bathing fluid. Finally, a specific inhibitor of sodium/potassium ATPase, ouabain, blocks sodium secretion and fluid formation with a concomitant reduction in the osmolarity of the fluid formed on the surface of the plexus. The simplest mechanism that could account for net water flow across the plexus into the CSF is osmotic flow in response to an osmotic gradient between the bulk CSF and plasma generated by active sodium secretion. Although the osmolarity of amphibian CSF is unknown, the sodium concentration of bulk CSF is higher than plasma (see Wright, 1975), and in mammals the bulk CSF is hypertonic by about 6 mosmol (see Rapoport, 1976). The rate of osmotic flow across a membrane (J~,) is given by the relation: J~ = (~L~RTAC where a is the reflection coefficient, RTAC the osmotic gradient, and L~ the hydraulic conductivity of the membrane. Assuming a sigma of 1.0 and a L, of 1.6 × 10-6 cm/ sec/atm, an osmotic gradient of 50 mosmol is required to account for the rate of CSF secretion in the frog. This mechanism, however, will not explain the in vitro results which show that CSF secretion occurs in the absence of bulk phase osmotic gradients. A more promising approach is that active solute transport across the plexus generates local osmotic gradients, i.e. solutes are pumped into a diffusion restricted compartment adjacent to the epithelium and the local osmotic gradient draws water across the plexus. The steady-state osmolarity of the fluid in the local osmotic compartment (C) is equal to the osmolarity of the final transported fluid, and is related to the rate of active solute transport (M0) and the osmotic permeability of the epithelimn (L~). A precise relationship between C, M 0 and L~ is given by Diamond (1964) as Oo + C =~

0~'o

M0 -4-RTL----~

where 00 is the ba+:hing solution osmolarity. It is assumed that the reflection coefficient of the actively transported solute is 1, and that solvent drag is the main

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E. M. WRIGHT, G. WIEDNER AND G. RUMRICH

mechanism for solute movement from the local osmotic compartment into the bulk CSF. If solute transfer occurs by diffusion rather than solvent drag, the osmolarity of the secreted fluid will be higher than that predicted. Using the experimental data gathered for CSF secretion by the frog choroid plexus, i.e. 0 0 = 205 moslnol, M 0 = 2 x 10-3 mosmol/cm2/hr, and L~ = 5.9 x 10-a cm/hr/atm, the predicted osmolarity of the secreted fluid is 275 nlosmol. The observed value was 254 mosmol. The agreement is close particularly in light of the fact that the observed steady-state osmotic permeability may be seriously underestimated (see Wright et al., 1972). In the event that L~ is underestimated by an order of magnitude the predicted minimum value for the osmolarity of the secreted fluid becomes 215 mosmol. The anatomical location of the local osmotic compartment in the choroid plexus may be the interspaces between the microvilli of the apical cell membrane. Accordingly, active sodium transport across the apical membrane into the spaces between the nficrovilli raises the tonicity of the fluid and this osmotically draws water across the plexus. An alternative view is that the local osmotic compartment is the unstirred layer of CSF adjacent to the plexus. The volume ftow across the plexus (J~) in this case would be related to the rate of solute transport (Mo), the osmotic water permeability (L,) and the thickness of the unstirred layer (8). Dainty and House (1966) derived the equation :

J~-

MoL~RT8 198+CfiL~RT

where D~ is the free solution diffusion coefficient of the actively transported solute, and C~ is the concentration of the solute in the bulk solutions. Taking the ventricular unstirred layer as 300 t~ (Wright and Prather, 1970) and the experimental data summarized in this paper, the predicted CSF flow rate is 0.2 ~l/cme/hr. To account for the observed rate of flow, 10/xl/cm2/hr, the true osmotic water permeability needs to be about 50-fold higher than the steady-state value reported here. These tests of osmotic mechanisms of fluid secretion across the choroid plexus provide one illustration of the more general problem of fluid transport across epithelial membranes. The validity of all models proposed rests upon the value chosen for the osmotic permeability of the epithelium. In the absence of reliable estimates of the true osmotic permeability of the choroid plexus it is not possible to distinguish one osmotic mechanism from another. ACKNOWLEDGMENTS This work was supported in part by grants from the USPHS (NS-09666), Merk Sharp & Dohme, and the Max-Planck-Gesellshaft. REFERENCES Ames, A., III, Higashi, K. and Nesbett, F. B. (1965). Effects of Pco2, acetazolamide and ouabain on volume and composition of choroid plexus fluid. J. Physiol. (Lond.) 181~516-24. Ames, A., III, Sakanoue, M. and Endo, S. (1964). Na+, K+, Ca~+, Mg2+ and C1- concentrations in choroid plexus fluid and cisternal fluid compared with plasma ultrafiltrate. J. Neurophysiol. 27, 672-81. Cserr, H. F. (1971). Physiology of the choroid plexus. Physiol. Ray. 51, 273-307. Dainty, J. and House, C. R. (1966). Unstirred layers in frog skin. J. Physiol. (Lond.) 182, 66-78. Diamond, J. M. (1964). The mechanism of isotonic water transport. J. Gen. Physiol. 48, 15-42.

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House, C. R. (1974). Water Transport in Cells and Tissues. Edward Arnold Ltd, London. Quinton, P. M., Wright, E. M. and Tormey, J. McD. (1973). Localization of sodium pumps in the choroid plexus epithelimn. J. Cell Biol. 58, 724-30. Rapoport, S. I. (1974). Blood Brain Barrier in Physiology and Medicine. Raven Press, New York. Schultz, I. (1971). Influence of bicarbonate-CO2- and glycodiazine buffer on the secretion of the isolated cat's pancreas. Pfliigers Arch. 329, 283-306. Ullrich, K. J., Radtke, H. W. and Rumrich, G. (1971). The role of bicarbonate and other buffers on isotonic fluid absorption in the proximal convolution of the rat kidney. Pillagers Arch. 330, 149-61. Welch, K., Sadler, K. and Gold, G. (1966). Volume flow across choroidal ependyma of the rabbit. Am. J. Physiol. 210, 232-6. Wiedner, G. (1976). Method to detect volume flows in the nanoliter range. Rev. Sc. Inst. 47, 775-6. Wright, E. M. (1972). Mechanisms of ion transport across the choroid plexus. J. Physiol. (Lond.) 226, 545-71. Wright, E. M. (1974). Active transport of iodide and other anions across the choroid plexus. J. Physiol. (Lond.) 240, 535-66. Wright, E. M. (1975). Solute transport across the frog choroid plexus. In Fluid Environment of the Brain. Pp. 139-56. Academic Press, Inc., New York. Wright, E. M. (1974). Active potassium transport by the choroid plexus. The Physiologist 19, 416. Wright, E. M. and Pietras, R. J. (1974). Routes of nonelectrolyte permeation across epithelial membranes. J. Membr. Biol. 17, 293-312. Wright, E. M. and Prather, J. W. (1970). The permeability of the frog choroid plexus to nonelectrolytes. J. Membr. Biol. 2, 127-49. Wright, E. M., Smulders, A. P. and Tormey, J. McD. (1972). The role of the lateral intercellular spaces and solute polarization effects in the passive flow of water across the rabbit gallbladder. J. Membr. Biol. 7, 198-219.