Vincent A. Murphy

Saturable Transport of Manganese(II) Across the Rat Blood‐Brain Barrier

Unanesthetized adult male rats were infused intravenously with solutions containing 54Mn (II) and one of six concentrations of stable Mn(II). The infusion was timed to produce a near constant [Mn] in plasma for up to 20 min. Plasma was collected serially and on termination of the experiment, samples of CSF, eight brain regions, and choroid plexus (CP) were obtained. Influx of Mn (JMn) was calculated from uptake of 54Mn into tissues and CSF at two different times. Plasma [Mn] was varied 1,000‐fold (0.076–78 nmol/ml). Over this plasma concentration range, JMn increased 123 times into CP, 18–120 times into brain, and 706 times into CSF. CP and brain JMn values fit saturation kinetics with Km (nmol/ml) equal to 15 for CP and 0.7–2.1 for brain, and Vmix (10–2 nmol · g−1· s−1) of 27 for CP and 0.025–0.054 for brain. Brain JMn except at cerebral cortex had a nonsaturable component. CSF JMn varied linearly with plasma [Mn]. These findings suggest that Mn transport into brain and CP is saturable, but transport into CSF is nonsaturable.

Homeostasis of Brain and Cerebrospinal Fluid Calcium Concentrations During Chronic Hypo-and Hypercalcemia

Abstract: Three‐week‐old rats were made hypocalcemic or hypercalcemic by being fed diets low or high in Ca. Both total and ionized [Ca]s in the plasma decreased about 40% and remained depressed for 4 weeks in rats fed a low‐Ca diet. Plasma [Ca]s in rats fed a high‐Ca diet increased by 30% and remained elevated for 7 weeks. After 8 weeks on the diets, cerebrospinal fluid (CSF) [Ca] changed by <30% whereas brain [Ca] changed by <20% of the chronic changes in plasma ionized [Ca]. Assuming a brain extracellular volume of 20% and noting that brain extracellular volume equilibrates freely with CSF, the findings demonstrate only small perturbations in the Ca content of the brain cellular compartment during sustained hypo or hypercalcemia. Partial regulation of CSF and brain extracellular Ca suggests a role for the blood‐brain barrier in regulating CNS [Ca] during chronic changes in plasma [Ca].

Acidosis, acetazolamide, and amiloride: effects on 22Na transfer across the blood-brain and blood-CSF barriers.

Abstract Sprague‐Dawley rats were given treatments, known to decrease 22Na movement into choroid plexus and CSF, to investigate their effect on 22Na transfer across the cerebral capillaries. Acidic salts, acetazolamide, or amiloride was injected intraperitoneally into bilaterally nephrectomized rats, and the rate of 22Na uptake into parietal cortex, pons‐medulla, and CSF was determined at 12, 18, and 24 min. Severe acidosis (arterial pH 7.2), produced by HCl injection, decreased the rate of 22Na entry into both brain regions and CSF by 25%, whereas mild acidosis (pH 7.3) from NH.,C1 injection reduced brain entry by 18%, but CSF entry by only 10%. Like HCl acidosis, amiloride reduced transport into both brain and CSF by 22%. Penetration of 22Na into parietal cortex was unchanged by acetazolamide, but that into CSF was slowed 30%. Since uptake of 22Na into cortical regions is primarily movement of tracer across the cerebral capillaries when tracer uptake time is <30 min, the results indicate that both metabolic acidosis and amiloride decrease Na+ permeativity at the cerebral capillaries as well as at the choroid plexus. Acetazolamide, on the other hand, alters Na+ movement only across the choroidal epithelium.

Alteration of sodium transport by the choroid plexus with amiloride

Cerebrospinal fluid (CSF) production results from active transport of Na+ from blood to CSF, which is followed by H2O and anions. Amiloride reduces Na+ movement in epithelial tissues. To ascertain if amiloride alters transport of Na+ in the choroid plexus, the drug was administered either i.p. to male Sprague-Dawley rats that were bilaterally nephrectomized to determine in vivo effects, or added to artificial CSF to incubate the choroid plexus in vitro. Choroid cell [Na+] was reduced after amiloride treatment both in vivo and in vitro. In addition, the rate of 22Na uptake into the CSF and choroid plexus (CP) was decreased after amiloride. Alterations in choroid cell [Na+] and 22Na penetration into CSF and CP occurred at relatively high doses of drug (1 mumol/ml, in vitro and 100 micrograms/g in vivo), but lower doses were less effective (0.1 mumol/ml in vitro and 10 micrograms/g in vivo). It is concluded that the effects of amiloride on Na+ distribution and transport in the CP are due to inhibition of basolateral Na+-H+ exchange.

Increased transfer of45Ca into brain and cerebrospinal fluid from plasma during chronic hypocalcemia in rats

Recent studies have shown regulation of central nervous system [Ca] after chronic hypo- and hypercalcemia. To investigate the mechanism of this regulation, 3-week-old rats were fed diets for 8 weeks that contained low or normal levels of Ca. Plasma [Ca] was 40% less in rats fed the low Ca diet than in animals fed normal diet. Unidirectional transfer coefficients for Ca (KCa) and Cl (KCl) into cerebrospinal fluid (CSF) and brain were determined from the 10 min uptake of intravenously injected 45Ca and 36Cl in awake animals. KCa for CSF was 68% greater in low-Ca rats than in normal rats. Likewise, the values of KCa for brain regions with areas adjacent to the ventricles like the hippocampus and pons-medulla were 50% higher than in normal animals. On the other hand, KCas for parietal cortex, a brain region distant from the choroid plexus and not expected to be influenced by Ca entry into CSF, were similar between the groups. Comparison of the regional ratios of KCa/KCl revealed that a selective increase of Ca transport occurred into CSF and all brain regions except the parietal cortex in Ca-deficient rats. The results suggest that Ca homeostasis of CSF and brain [Ca] during chronic hypocalcemia is due to increased transfer of Ca from blood to brain, and that the regulation occurs via the CSF, possibly at the choroid plexus, but not via the cerebral capillaries.

Prevention of nerve edema and increased blood-nerve barrier permeability-surface area product in galactosemic rats by aldose reductase or thromboxane synthetase inhibitors.

Nerve water content and the permeability–surface area product (PA) to [3H]- or [14C]sucrose at the blood-nerve barrier were determined in unanesthetized control rats fed a normal diet and in rats fed galactose with or without an aldose reductase inhibitor (Statil or AL 1576) or a thromboxane synthetase inhibitor (CGS 12970). Nerve water content was determined by taking the difference between dry and wet weights of whole tibial nerves. PA was determined by an intravenous bolus injection of radiotracer with multiple–time-point graphic and quantitative autoradiographic methods. The mean nerve water content in galactosemic rats was 15% higher than in control rats after 7–11 mo on the diet. Statil and AL 1576 prevented nerve edema, but CGS 12970 was only partially effective in preventing an increase in nerve water content in galactose-fed rats. In galactosemic rats, the mean PA to sucrose at the blood-nerve barrier, calculated from nerve dry weight, was twofold higher than in control rats. Treatment with Statil, AL 1576, or CGS 12970 prevented increased PA. Our results suggest that nerve edema and increased blood-nerve barrier PA are secondary to polyol production and can be prevented by inhibiting aldose reductase.

Uptake and concentrations of calcium in rat choroid plexus during chronic hypo- and hypercalcemia.

The choroid plexus has been implicated in the regulation of cerebrospinal fluid (CSF) [Ca], but little information is available concerning Ca transport by this epithelium. We determined the transfer coefficients for 45Ca uptake into choroid plexus from blood, as well as tissue [Ca], in weanling Fischer-344 rats fed low, normal, or high Ca diets for 8 weeks. Plasma [Ca] decreased by 45% with low Ca diet and increased by 25% with high Ca diet. Choroid plexus 45Ca uptake varied inversely with plasma [Ca]. This relation was due largely to changes in extracellular Ca binding rather than to entry from blood, as the transfer coefficient was independent of plasma [Ca]. The extracellular Ca distribution in choroid plexus, the intercept of a plot of tissue 45Ca distribution against time, was reciprocally related to plasma [Ca]. Changes in total cell [Ca] during hypercalcemia were equivalent to those in plasma, and in hypocalcemia were 70% of those in plasma. These findings indicate that regulation of CSF [Ca] does not involve saturable transport of Ca into the choroid epithelium from blood, and that the apical membrane of the choroid epithelium is involved in homeostasis of CSF [Ca].

Method for determination of sodium, potassium, calcium, magnesium, chloride, and phosphate in the rat choroid plexus by flame atomic absorption and visible spectroscopy.

A rapid simple technique for the measurement of Na+, K+, Mg2+, Ca2+, PO4(3-), and Cl- was developed to analyze ion contents in the choroid plexus of the rat. The technique involves digestion in piperidine, precipitation of proteins with HClO4, and analysis of Na+, K+, Ca2+, and Mg2+ by atomic absorption spectroscopy and Cl- and PO4(3-) by visible spectroscopy. The coefficient of variation for the measurement of eight replicates was 1-3% for all ions. Analysis of choroid plexuses from eight rats yielded coefficients of variation of about 6% and the values for Na+, K+, and Cl- compared favorably to previous works. The analytical procedure described in this paper allows the determination of six major physiologic ions in rat choroid plexus (4 mg wet wt).

Regulation of brain and cerebrospinal fluid calcium by brain barrier membranes following vitamin D-related chronic hypo- and hypercalcemia in rats

Abstract: Male Fischer‐344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36C1 transfer coefficients (KCa and KC1) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. ATCa values at CSF, parietal cortex, and pons‐medulla were negatively correlated with plasma ionized [Ca], whereas KC1 values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo‐ or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25‐dihydroxyvitamin D3.

Calcium deficiency enhances cadmium accumulation in the central nervous system

Weanling male rats were administered 1 of 4 diets for 40 days: control (CONT), low Ca (LOCA), control plus Cd (CONT + Cd) or low Ca plus Cd (LOCA + Cd). After 40 days, Cd was analyzed in 7 brain regions, spinal cord, serum, liver, kidney, muscle and femur by atomic absorption spectrophotometry with Zeeman background correction. No significant difference in Cd between CONT and LOCA was found except in femur, where it was increased. In CONT + Cd rats, peripheral tissues showed an increase in Cd of 30-71 fold above CONT rats. Brain regions exhibited a more modest 7-10 fold change, and serum Cd was 8.5 times above control. LOCA + Cd rats showed a 25-fold increase of Cd above LOCA in serum, 25-100 fold in peripheral tissues, and a 14-20 times in brain. These findings show that brain Cd is increased during Ca deficiency, but that central nervous system Cd changes less than peripheral tissue Cd. This increase in brain Cd could alter brain function.