Donal J. Reed

Properties of corticosterone-binding macromolecules from rat brain cytosol

Abstract A partial characterization has been made of [ 3 H]corticosterone-binding macromolecules from the cytosols of rat brain. The macromolecules are protein(s) and appear to have a molecular weight in excess of 200,000 because of migration characteristics on Sephadex and Sepharose 4B. Binding properties were measured which showed that the K dissoc is 2.7 × 10 −10 and 0.47 pmoles of corticosterone are bound/mg of protein, indicating a high affinity and limited capacity for [ 3 H]corticosterone. Competitive binding studies were performed which indicate that the protein(s) have a high degree of specificity for [ 3 H]corticosterone including stereospecificity. Based on its biological potency, dexamethasone did not compete with corticosterone for binding sites to the extent expected. Examination of rat brains with respect to their capacity to bind [ 3 H]corticosterone in vitro indicated that the greatest concentration of cytosol-binding molecules was localized in the hippocampus. Evidence was obtained that the corticosterone-binding protein in brain cytosol is not corticosteroid-binding globulin (CBG).

Active transport of sodium and potassium by the choroid plexus of the rat.

1. Choroid plexus from the lateral ventricle in the adult rat was found to contain approximately 54 m‐equiv Na+ and 89 m‐equiv K+ per kg wet tissue.

Corticosterone binding by rat brain cytosol.

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [3H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11‐dehydrocorticosterone, Cortisol and cortisone decreased the amount of [3H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [3H]corticosterone with macromolecules of the cytosol. Further evidence for protein‐steroid interaction was obtained by incubating [3H]corticosterone (B), [3H]cortisol (F), or 11‐[3H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.

Electrolyte and Acid-Base Parameters of Rat Cerebrospinal Fluid +

SummaryValues for various electrolytes and acid-base parameters of rat CSF were determined in adult animals anesthetized with pentobarbital or ether. In addition, the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO) between CSF and arterial and venous blood was measured in the same animals. It was found a) that CSF electrolyte and acid-base parameters are the same in ether-and pentobarbital-treated animals; b) that DMO distributions between CSF and blood are not determined solely by pH gradients; and c) that in rat CSF electrolyte concentrations (mEq/l) —Na=148.4; K=3.16, Cl=117.9 —and acid-base values —pH=7.38, H2CO3=1.30 mM/l; HCO3=24.5 mM/l —are very similar to those measured in other species.

Developmental studies of the compartmentalization of water and electrolytes in the choroid plexus of the neonatal rat brain.

Rats of various postnatal ages were utilized to study developmental changes in the distribution of Na, K and H2O between the various compartments of the lateral ventricular plexus (LVP). During the 3 weeks after birth, as the LVP grows from 0.5 to 0.8 mg, there is a significant increase in plexus K which is accompanied by a progressive decrease in Na and H2O. Also, during this postnatal period the decrease in [3h]inulin space in the plexus is proportional to the decrease in the Na space. Between 3 weeks and adulthood, the [3h]inulin and Na spaces are both augmented to a similar extent; moreover, during this same period of development there is a trebling of the residual [51cr]erythrocyte volume. Despite the substantial changes in the volume of the extracellular fluid and of the residual blood in the plexus with age, the calculated concentrations (mEquiv./kg H2O) of choroid cell Na (30-35) and K (145-155) are similar for all ages investigated. The derived data for cellular ionic concentration, together with the analysis of the ionic concentration gradients (cerebrospinal fluid/plasma H2O), suggest that the transport mechanism which translocates Na and K across the choroidal membrane is operative as early as 3-4 days postnatal. The important role of the choroid plexus in central nervous system homeostasis is discussed in relation to the developing brain.

The effect of furosemide on sodium-22 uptake into cerebrospinal fluid and brain

SummaryThe effect was studied of a saluretic agent on the uptake of sodium-22 into the CSF and cerebral cortex of nephrectomized rats. Furosemide, 1 mg/kg, was injected into the left lateral ventricle of the brain. Sodium-22 was injected intraperitioneally and the uptake of the isotope into CSF, cerebral cortex, skeletal muscle and plasma was measured at intervals from 0.25 hour to 24 hours. During the initial 4-hour period after injection of the sodium-22, the uptake of the isotope was reduced in the furosemide-treated animals. The maximum reduction in uptake occurred at the 0.25-hour time period. The CSF Na RSA of the treated animals was 37% less than that of the control animals. After about 8 hours there was no difference in sodium-22 uptake into the CSF between the control and treated animals.The uptake of sodium-22 into the cerebral cortex was decreased in the furosemide-treated animals. Since the brain extracellular space and the CSF appear to be in relatively free communication, the reduced brain radioactivity may be related to the decreased uptake of sodium-22 into the CSF.It is suggested that the decrease in sodium-22 uptake into CSF and brain in the furosemide-treated animals may be caused by inhibition of active sodium transport in the choroid plexus.

Binding of natural and synthetic glucocorticoids in rat brain

Abstract The protein(s) from rat brain cytosol which bind corticosterone are selective, stereospecific and have a high affinity ( K dissoc . = 3.8 × 10 −9 M) and low capacity ( n = 4.7 × 10 −13 mol/mg protein) for [1,2- 3 H]-corticosterone. These or similar molecules also bind [1,2,4- 3 H]-dexamethasone but have a lower capacity ( n = 2.4 × 10 −13 mol/mg protein), lower affinity ( K dissoc = 2.5 × 10 −9 M) and were less selective for this synthetic glucocorticoid than for corticosterone. The amount of [1,2- 3 H]-corticosterone bound by brain receptor proteins has a diurnal pattern which is the mirror image of the circulating levels of endogenous corticosterone in blood plasma. This cyclic variation in binding was abolished by adrenalectomy. Thus, as the plasma concentration of endogenous corticosterone increases, the number of specific receptor sites in the cytosol of whole brains filled by endogenous corticosterone also is increased quantitatively. Following the bilateral removal of the adrenal glands, the amount of [1,2- 3 H]-corticosterone bound/ mg protein to brain cytosol protein rapidly increases during the first 24 h after adrenalectomy and approaches values seen at 3 days. During the same period the plasma corticosterone concentration is rapidly decreasing. By 30 days after adrenalectomy the amount of [1,2- 3 H]-corticosterone bound per mg of protein has increased over those values measured at 1 day after removal of the adrenals. At 60 and 120 days post-adrenalectomy, the amount of [1,2- 3 H]-corticosterone bound is near control values (3 day) but decreases below this value by 180 days after removal of the adrenals. In newborn rats (up to 50 g), the amount of [1,2- 3 H]-corticosterone bound/mg receptor protein increases with increasing weight. However, from 195 g to 450 g the amount of [1,2- 3 H]-corticosterone bound/mg protein decreases with increasing weight. The increase in binding of corticosterone to brain protein of new born rats has been correlated with other maturational events.

BINDING OF NATURAL AND SYNTHETIC GLUCOCORTICOIDS IN RAT BRAIN

The protein(s) from rat brain cytosol which bind corticosterone are selective, stereospecific and have a high affinity (Kdissoc = 3·8 × 10–9 M) and low capacity (n = 4·7 × 10–13 mol/mg protein) for [1,2–3H]-corticosterone. These or similar molecules also bind [1,2,4-3H]-dexamethasone but have a lower capacity (n = 2·4 × 10–13 mol/mg protein), lower affinity (Kdissoc = 2·5 × 10–9 M) and were less selective for this synthetic glucocorticoid than for corticosterone. The amount of [1,2-3H]-corticosterone bound by brain receptor proteins has a diurnal pattern which is the mirror image of the circulating levels of endogenous corticosterone in blood plasma. This cyclic variation in binding was abolished by adrenalectomy. Thus, as the plasma concentration of endogenous corticosterone increases, the number of specific receptor sites in the cytosol of whole brains filled by endogenous corticosterone also is increased quantitatively. Following the bilateral removal of the adrenal glands, the amount of [1,2-3H]-corticosterone bound/mg protein to brain cytosol protein rapidly increases during the first 24 h after adrenalectomy and approaches values seen at 3 days. During the same period the plasma corticosterone concentration is rapidly decreasing. By 30 days after adrenalectomy the amount of [1,2-3H]-corticosterone bound per mg of protein has increased over those values measured at 1 day after removal of the adrenals. At 60 and 120 dayspost-adrenalectomy, the amount of [1,2-3H]-corticosterone bound is near control values (3 day) but decreases below this value by 180 days after removal of the adrenals. In newborn rats (up to 50 g), the amount of [1,2-3H]-corticosterone bound/mg receptor protein increases with increasing weight. However, from 195 g to 450 g the amount of [1,2-3H]-corticosterone bound/mg protein decreases with increasing weight. The increase in binding of corticosterone to brain protein of new born rats has been correlated with other maturational events.

Comparison of bincling of [3H]corticosterone and [3H]dexamethasone by rat brain cytosol

Publisher Summary This chapter compares the binding of [ 3 H] corticosterone and [ 3 H]dexametha by rat brain cytosol. A protein “receptor” for corticosterone (B k ) is demonstrated in cytoplasm and nuclei of rat brain. Because dexamethasone (D x ) has important central nervous system effects, it is important to determine whether there are specific receptor molecules for D x and whether these are the same as the receptors for B k. Adult male adrenalectomized (adrex) or non-adrenalectomized (intact) rats were perfused ventriculocisternally with [1,2,4- 3 H] D x. Data obtained from competitive binding experiments in vitro appear to indicate a difference in the binding characteristics of “receptor sites” for [ 3 H]D X and [ 3 H]B k . The cytosol from hippocampus bound more [ 3 H] D X and [ 3 H] B k than comparable fractions from other brain region. In addition, the chapter also deals with the influence of hypothalamic extract on the uptake of steroid hormones by the anterior pituitary gland in rats.