The effect of Pronase on choroid plexus transport

Brain Research, 134 (1977) 573-576 © Elsevier/North-Holland Biomedical Press

573

The effect of Pronase on choroid plexus transport

REYNOLD SPECTOR Department of Neuroscience, Children's Hospital Medical Center and the Department of Medicine, Peter Bent Brigham Hospital and Harvard Medical School, Boston, Mass. 02115 (U.S.A.)

(Accepted June 9th, 1977)

The choroid plexus is the anatomical locus of the blood--cerebrospinal fluid (CSF) barrier a. The choroid plexus epithelial cells, joined by tight junctions, impede the transfer of small, water-soluble substances between the permeable choroid plexus' capillaries and the CSF ~. However, the choroid plexus epithelial cells, like the kidney tubule cells, contain a group of separate, saturable transport systems that carry certain substances from CSF to blood (e.g. iodide and leucine) 3,6 and from blood to CSF (e.g., vitamins and (scyllitol9,12. The ability of the isolated choroid plexus to concentrate these water-soluble substances in vitro by a process distinct from binding is generally taken as an indication of the ability of the choroid plexus to transport these substances in vivo 1. However, the direction of the transport - - i.e. from CSF to blood or vice versa - - cannot be ascertained by in vitro studies n. The nature and location of these transport processes in the choroid plexus epithelial cells is unclear. However, carriers have been postulated to exist on the cell surfacesLU. In some cases, these putative carriers appear to transport molecules into the choroid plexus epithelial cells against large concentration gradients 11. The transported molecule then appears to leave the epithelial cells on the other side down a concentration gradient H. The purposes of the present experiments were to provide evidence (1) that the putative carriers associated with the choroid plexus are protein, at least in part, and (2) that these carriers are located on the choroid plexus cell surfaces. If the carriers are protein and protrude sufficiently from the plasma membrane into the extracellular space, then proteolytic enzymes might be able to inactivate sensitive carrier proteins. Hormone receptors on the plasma membrane can be altered by proteolytic enzymes 2. Therefore, using proteolytic enzymes as a probe, isolated choroid plexuses were incubated in artificial CSF containing a proteolytic enzyme (Pronase) at a concentration that did not lyse the cells. The ability of the Pronase-treated isolated choroid plexuses to concentrate several substances was then tested. Substances were chosen that have previously been shown to be concentrated by the isolated choroid plexus in vitro and to be transported from blood to CSF or vice versa in vivo ~,3,5,6,9,12. Also, the concentration of the choroid plexus folic acid binding protein (which has been implicated in

574 the transfer of folates between blood and CSF) was measured in Pronase-treated choroid plexuses l°. Choriod plexuses, weighing about 5-6 mg each, were isolated rapidly from the brains of 1.5-2.0 kg New Zealand White rabbits 11,~2. Each isolated choroid plexus was preincubated for 45 rain in 3 ml artificial CSF containing 3 mg glucose and 3 mg bovine serum albumin (BSA) or Pronase (Type VI protease from Streptomyces griseus; Sigma Chemical Co., St. Louis, Mo.)at 37 °C under 95% Oz and 50/o CO2 in a metabolic shakerg, T M . At the end of the preincubation, the choroid plexuses were washed for 10 sec in 10 ml artificial CSF and then used for various experiments. Tissue-to-medium ratios (T/M) were obtained for [aH]folic acid (156Ci/mmole), [3H]scyllitol (5 mCi/mmole), Na 12~l (carrier-free), [11C]L-leucine (5 mCi/mmole), and [3H]2-deoxyglucose (10 Ci/mmole) by incubating the pre-incubated choroid plexuses in 3 ml artificial CSF containing the isotope and 3 mg glucose for 30 rain at 37 °C under 95 % 02 and 5 ~o CO2 in a shaker 3,5,6,9,12. At the end ofthe incubation, the choroid plexuses were wiped on a glass slide and weighed, and the T/M were obtained by dividing the disintegration per minute (dpm) per ml of tissue intracellular water by the dpm/ml in the mediumg, 12. Iodide, folic acid, and scyllitol are not metabolized by the choroid plexus during a 30-min incubation 3,9,~2. The per cent of [3H]deoxyglucose phosphorylated in the choroid plexus at the end of the incubation was determined by separating the [3H]deoxyglucose from the [3H]deoxyglucose phosphate on microcolumns 4. Unlike glucose, 2-deoxyglucose cannot be metabolized beyond the initial phosphorylation step 4. The [3H]folic acid complexed to the folic acid binding protein in the choroid plexus at the end of the incubation was measured by solubilizing the [aH]folic acid-folic acid binding protein complex in Triton X-100 and separating the complexed [3H]folic acid from the unbound [3H]folic acid on 13-cm Sephadex G-25 columns TM. At the end of the incubation experiments, the cellular integrity of the choroid plexus was measured by light microscopy of hematoxylin and eosin stained 6-#m sections, trypan blue exclusion and the total lactic acid dehydrogenase content (LDH) of the tissues determined colorimetrically8. Since LDH is an omnipresent cytoplasmic enzyme and readily leaks out of damaged tissue, LDH tissue concentrations have been widely used as a measure of cellular integrity7. Also, the ability of isolated choroid plexuses to retain [3H]scyllitol in the presence of Pronase was tested. Choroid plexuses were preincubated in 10 # M [3H]scyllitol for 60 rain as above and achieved T/M of 21.8. At the end of the preincubation in the [3H]scyllitol, each choroid plexus was washed for 10 sec and then incubated at 37 °C under 95~,, O2:5 °/,, CO2 in 3 ml artificial CSF containing 1 mg/ml glucose and 1 mg/ml Pronase or BSA. After 45 rain, the tissue weight and ZH content of the choroid plexus and medium were determinedg, 12. The choroid plexus T/M for iodide, scyllitol, and folic acid were markedly decreased by a 45-rain preincubation in Pronase (A, Table I). The T/M for 2-deoxyglucose and leucine were less affected (A, Table I). The decrease in [ZH]deoxyglucose TIM was due to a significant decrease in phosphorylation of [ZH]deoxyglucose (A, Table I). By light microscopy and trypan blue exclusion, the Pronase-treated choroid

575 TABLE I Effect o f Pronase treatment on T/M, folate binding protein, L D H content, and scyllitol retention Control and experimental choroid plexuses were preincubated for 45 rain in either BSA or Pronase respectively before the 30 min T/M ratios shown in (A) were obtained. In (B) are shown the pmoles of [aH]folic acid bound to the folic acid binding protein per g of choroid plexus in both control and experimental groups. In (C) are shown the LDH content in arbitrary absorbance units per g8. Rabbit serum had 36 units per ml. In (D) are shown the percentages of [3H]scyllitol retained by choroid plexuses that had been preincubated in 10/~M [3H]scyllitol and then incubated in BSA or Pronase for 45 rain. In parentheses are shown the number of choroid plexuses used. Substance (concentration)

Control group

Experimental group

[ZH]folic acid (4 n M ) Na 125I (5 pCi/ml) 13H]scyllitol (31 # M ) [14C]leucine (8/~M) [ZH]deoxyglucose (0.2 # M ) ( ~ phosphorylated)

(A) 82.0 45.4 13.3 3.47 3.7l (71

24.4 1.8 2.0 1.32 2.10 (40

T I M ratios ~ 5.0 (8) I-_-3.8 (6) ± 0.6 (9) ± 0.57 (3) ± 0.52 (3) ~z 4)

~ 4.3 ~z 0.2 ± 0.3 ~ 0.08 :L 0.20 i 5)

(9) (6) (9) (3) (3)

Per cent control

31" 4* 15' 38** 57** 56*

(B) [3H]folic acid bound** * 107.6 ± 1.8 (3) 41.9 ~: 9.8 (3)

39*

(C) L D H content 2978 ~ 131 (6)

96

2859 ± 423 (6)

( D) [3H]scyllitol retention 20 ~ 1 (6) 21 ± 2

(6)

105

*,** Values designated by * and ** have P < 0.01 and P < 0.05 respectively by Student's t-test, 2-tailed. *** The pmoles of unbound [SHlfolic acid per gram of choroid plexus were 44.0 ~ 8.8 (3) and 26.8 ± 7.0 (3) in the control and experimental groups respectively (P > 0.05).

plexuses appeared identical to the controls. Further evidence that the depression in the T/M was not due to tissue damage and leakage of intracellular contents was the comparable LDH content of the control and Pronase-treated goups (C, Table I). Finally, the ability of choroid plexus to retain [3H]scyllitol (mol. wt., 180) was not depressed by incubation of the tissue in Pronase (D, Table I). The weight of the control and experimental choroid plexuses (A, Table I) were 5.73 ± 0.51 mg (S.E.M. ; n = 29) and 5.29 Jz 0.43 (n == 30), respectively (P ~ 0.05). The possibility that the Pronase treatment secondarily decreased the T/M (A, Table I) (as by decreasing energy production) was clearly not the only factor, at least in the case of the depression of the [ZH]folic acid T/M. The amount of the folate binding protein able to complex [3H]folic acid was also significantly decreased (B, Table 1)10. Furthermore, in vitro, interference with energy production (as by incubating isolated choroid plexuses in l m M dinitrophenol) does not decrease the ability of the choroid plexus to concentrate [3H]folic acid 9,12. However, these studies do not exclude the possibility that preincubation of the choroid plexus in Pronase decreased the T/M (A, Table I) by altering membrane structure non-specifically, rather than specifically inactivating the carrier proteins. Also, these studies do not exclude the possi-

576 bility t h a t p r e i n c u b a t i o n o f the c h o r o i d plexus in Pronase decreased 2-cleoxygiucose p h o s p h o r y l a t i o n rather than t r a n s p o r t (A, Table 1). In s u m m a r y , these experiments p r o v i d e s u p p o r t for the n o t i o n that the c h o r o i d plexus t r a n s p o r t m e c h a n i s m s (carriers) for folate a n d p r o b a b l y iodide, scyllitol, leucine and glucose consist, at least in part, o f proteins which exist on the cell surfaces a n d are accessible to extracellular Pronase. The a u t h o r wishes to t h a n k Peter Levy for o u t s t a n d i n g technical assistance and Drs. N o r m a n U r e t s k y and A. V. L o r e n z o for their counsel and support. This w o r k was s u p p o r t e d by N . I . H . G r a n t I R 0 1 - N S 12274 and by a G r a n t from the N a t i o n a l F o u n d a t i o n - M a r c h o f Dimes.

I Cserr, H., Physiology of the choroid plexus, Physiol. Rev., 51 (1971) 273-31 I. 2 Cuatrecasas, P., Perturbation of the insulin receptor of isolated fat cells with proteolytic enzymes, J. biol. Chem., 246 (1971) 6522-6531. 3 Davson, H., Psychology of the Cerebrospinal Fluid, Little, Brown, Boston, 1967, pp. 1-100. 4 Diamond, I. and Fishman, R.A., High affinity transport and phosphorylation of 2-deoxy-Dglucose in synaptosomes, J. Neurochem., 20 (1973) 1533-1542. 5 Lorenzo, A. V., The uptake and metabolism of D-[U-14Clglucose by the choroid plexus, Brain Research, 112 (1976) 435-441. 6 Lorenzo, A. V. and Snodgrass, S. R., Leucine transport from the ventricles and the cranial subarachnoid space in the cat, J. Neurochem., 19 (1972) 1287-1298. 7 Riddall, D. R., Leach, M.J. and Davison, A. N., Neurotransmitter uptake into slices of rat cerebral cortex in vitro: effect of slice size, J. Neurochem., 27 (1976) 835-839. 8 Sigma Technical Bulletin No. 500, The Colorimetric Determination of Lactic Dehydrogenase. 9 Spector, R., The specificity and sulfhydryl sensitivity of the inositol transport system of the central nervous system, J. Neurochem., 27 (1976) 229-236. 10 Spector, R., Identification of a folate binding macromolecule in rabbit choroid plexus, J. bioL Chem., 252 (1977) 3364-3370. I 1 Spector, R. and Lorenzo, A. V., Ascorbic acid homeostasis in the central nervous system, Amer. J. PhysioL, 225 (1973) 757-763. 12 Spector, R. and Lorenzo, A. V., Folate transport by the choroid plexus in vitro, Science, 187 (1975) 540-542.