COMPARTMENTATION OF THE INULIN SPACE IN MOUSE BRAIN SLICES

Abstract

(1) Mouse cerebrum slices swell in tris-buffered Krebs-Ringer medium. Swelling is rapid at first, then slows to a more or less constant rate. Even after 3 hr incubation, water content/g of tissue dry wt. shows no sign of an asymptotic limit. Swelling is the same at 37 degrees and at 0 degree. (2) Tissue water measured by incubation with tritiated water is equal to total tissue water measured by drying slices. Equilibration between tritiated water and tissue water is complete within 2 min. (3) Tissue liquid can be divided into three phenomenologically distinguishable compartments: first inulin space, which is the compartment permeable to inulin at both 0 degree and 37 degrees; second inulin space, which is the compartment permeable to inulin at 37 degrees but not at 0 degree; and 37 degrees non-inulin space, which is the compartment impermeable to inulin at both 0 degree and 37 degrees. The evidence for this is: (a) Penetration of inulin into tissue is greater at 37 degrees than at 0 degree. After the first 20 min the rate of penetration at 0 degree is approximately equal to the rate of penetration at 37 degrees, and only slightly less than the rate of increase of total tissue water. Therefore the smaller inulin space observed at 0 degree cannot be due to slower entry of inulin. (b) The inulin content of slices incubated in inulin-containing medium at 37 degrees and cooled to 0 degree in the same medium is the same as the inulin content of tissue incubated at 37 degrees without subsequent cooling. In contrast, the inulin content of tissues preincubated in inulin-free medium at 37 degrees and then incubated in inulin-containing medium at 0 degree is the same as the inulin content of tissues incubated in inulin-containing medium at 0 degree without preincubation at 37 degrees. Therefore the smaller inulin space at 0 degree than at 37 degrees can be due neither to a reversible temperature-dependent change in the size of one single inulin space nor to an irreversible, greater swelling of a single inulin space at the higher temperature, but is due to some portion of the 37 degrees inulin space becoming impermeable to inulin at 0 degree. (c) Some inulin is retained by tissue incubated with inulin at 37 degrees, then transferred to inulin-free medium at 0 degree; the amount of retained inulin is equal to the difference between inulin content of tissue incubated with inulin at 37 degrees and tissue incubated with inulin at 0 degree This confirms 3b above and in addition shows that inulin which has entered the second inulin space at 37 degrees is trapped there when this space becomes impermeable to inulin at 0 degree. (4) The penetration of the amino acids, L-lysine and D-glutamate at 0 degree is equal to the penetration of inulin at 37 degrees. This confirms the real existence of the 37 degrees inulin space at 0 degree, and shows that the barrier at 0 degree between the first and second inulin spaces does not exist for these substances. (5) The amino acids L-leucine and glycine penetrate total tissue water at 0 degree. L-leucine is actively transported at this temperature. (6) The amino acids alpha-aminoisobutyric acid, L-leucine, and L-lysine at 2 mM have no effect at 37 degrees on either the inulin space or the non-inulin space. (7) The inulin space is insensitive at 37 degrees to physiologically significant changes in the medium. In contrast, the non-inulin space is quite sensitive to these changes. Addition of D-glutamate greatly increases the non-inulin space; addition of ouabain or cyanide, or omission of glucose, increases the non-inulin space slightly; and replacement of Na+ ion by choline+ ion greatly decreases this space. These changes are independent and roughly additive.