Marcel Debray

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood–brain barrier. As P‐glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P‐glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4u2003µg/mL puromycin for the first 2u2003days of culture or 3u2003µg/mL puromycin for the first 3u2003days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin‐treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75u2003×u200310−6u2003cm/s) and a high electrical resistance (up to 500u2003Ωu2003×u2003cm2). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood–brain barrier for cellular and molecular biology studies as well as pharmacological investigations.

Effect of dietary α-linolenic acid deficiency on habituation

Abstract Three weeks before mating, two groups of SWISS OF 1 mice were fed a diet that was similar but contained either peanut oil poor in alpha-linolenic acid [18: 3 (n-3)] (n − 3 deficient = deficient mice = (n-3)−) or peanut + rapeseed oil rich in alpha-linolenic acid (n − 3 nondeficient = controls = (n − 3)+). Pups, fed the same diet as their dams, aged 45 to 62 days were used for brain lipid analysis and for behavioral experiments, aimed at determining whether there is a relation between the dietary intake of alpha-linolenate and a simple form of learning: habituation. The behavior of mice was compared using four models: exploration recorded in a photocell actimeter, activity in an open-field, duration of immobility in the forced swimming test and number of escape attempts from a small closed space. Habituation was measured by testing the mice in the same situation after some time had elapsed since the first test. Exploration in the photocell actimeter was significantly reduced between day 1 and 4 in nondeficient mice, but, not in deficient mice. The number of square crossings in the open-field was significantly reduced on the second test neither in the control nor in the deficient mice. In the forced swimming test, the habituation (increase in duration of immobility) was significantly greater (255%) in nondeficient than in deficient mice (163%) In the escape attempt experiment, the habituation showed a trend to be greater in controls than in deficient mice (p = 0.061) and was significantly greater in females than in males (p = 0.028) These results suggest that a simple form of learning, habituation, occurs more slowly in mice fed a diet deficient in alpha-linolenic acid.

Morphine pharmacokinetics and pain assessment after intracerebroventricular administration in patients with terminal cancer

Morphine pharmacokinetics and pain relief were evaluated after intracerebroventricular administration of morphine (0.4 ±0.11 mg) in seven patients with cancer suffering from intractable pain. Ventricular cerebrospinal fluid (CSF), lumbar CSF, and plasma morphine concentrations were analyzed by a specific morphine radioimmunoassay. A two‐compartment model was sufficient to describe the kinetics of morphine in ventricular CSF. Morphine diffuses to the lumbar level, and the mean maximum concentration was 192 ± 105 ng/ml at 4.5 ±1.3 hours. Ventricular and lumbar CSF morphine kinetics showed a similar decline during the elimination phase, with terminal half‐lives of 3.8 ± 0.6 hours and 4.2 ± 1.6 hours, respectively. Pain relief was evaluated by a visual analog scale: the test showed a rapid onset of analgesia (<10 minutes). Analgesic effectiveness reached a maximum between 6 and 10 hours. The relationship between pharmacologie effect and morphine concentrations in ventricular CSF resulted in an anticlockwise hysteresis curve. The presence of morphine in lumbar CSF suggested an additive spinal action of morphine, which probably plays a role in the duration of analgesia.

Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

Isolation impairs place preference conditioning to morphine but not aversive learning in mice

Abstract Morphine (8–100u2005mg/kg IP) induces place preference conditioning in mice. The effect of two different periods of isolation (15 and 30 days) was examined. Mice isolated for 15 days but not 30 days exhibited place preference conditioning to morphine (8u2005mg/kg). After 30 days of isolation morphine could not induce place preference conditioning with the following doses (8,u200516,u200564,u2005100u2005mg/kg). Social regrouping of male mice previously isolated for 30 days with naive female mice for 15 or 30 days resulted in a reappearance of the conditioned place preference to morphine (16u2005mg/kg). The specificity of this associative deficit was examined by testing learning in isolated compared to non-isolated mice in two distinct settings: escape learning in the Morris water maze and passive avoidance acquisition and retention. On the Morris water maze isolated mice did not differ from non-isolated mice regarding place learning, the probe trial or extinction. Isolated mice were unimpaired in passive avoidance acquisition and retention. It was concluded that the deficits in place preference conditioning were not the result of a global learning impairment in isolated mice.

Effect of dietary α-linolenic acid on functional characteristic of Na+/K+-ATPase isoenzymes in whole brain membranes of weaned rats

The influence of dietary fatty acids on Na+ sensitivity and ouabain affinity of Na+/K(+)-ATPase isoenzymes of whole brain membranes were studied in weaned rats fed for two generations with diets either devoid of alpha-linolenic acid (sunflower oil diet) or rich in alpha-linolenic acid (soya oil diet). The (n--3) deficiency induced by the sunflower oil diet led to an increase in the (n--6)/(n--3) molar ratio in whole brain membranes. Na+/K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain. In rats fed sunflower oil diet, the ouabain titration displayed three inhibitory processes with markedly different affinities: low affinity (alpha 1); high affinity (alpha 2); and very high affinity (alpha 3). Membranes of rats fed soya oil diet exhibited only two inhibitory processes, i.e., low affinity (likely alpha 1+ alpha 2) and high affinity (likely alpha 2+ alpha 3) with the low affinity form intermediate between the sunflower alpha 1 and alpha 2 forms, and the high affinity form intermediate between the sunflower alpha 2 and alpha 3 forms. In fact, the Na+ response shows that the three isoenzymes have different Na+ sensitivities. Regardless of the diet, alpha 1 has a similar Na+ sensitivity (less than 1 mM), whilst alpha 2 and alpha 3 are more sensitive in soya oil membranes compared to sunflower oil membranes (5.1 vs. 7.2 mM and about 11 vs. 22.5 mM, respectively). Thus, sodium appears to be a better criterion of heterogeneity than ouabain.

Heterogeneous Na+ Sensitivity of Na+,K+‐ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na+ sensitivity of whole brain membrane Na+,K+‐ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10−5 and 10−7M), we were able to discriminate Na+ sensitivity of Na+, K+‐ATPase isoenzymes using nonlinear regression. The ouabain low‐affinity isoform, α1, exhibited high Na+ sensitivity [Ka of 3.88 ± 0.25 mMNa+ and a Hill coefficient (n) of 1.98 ± 0.13]; the ouabain high‐affinity isoform, α2, had two Na+ sensitivities, a high (Ka of 4.98 ± 0.2 mM Na+ and n of 1.34 ± 0.10) and a low (Ka of 28 ± 0.5 mM Na+ and an n of 1.92 ± 0.18) Na+ sensitivity activated above a thresh old (22 ± 0.3 mM Na+); and the ouabain very‐high‐affinity isoform, α3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.

Sprague–Dawley rats display metabolism-mediated sex differences in the acute toxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)

The use of the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has been associated with unexplained deaths. Male humans and rodents are more sensitive to acute toxicity than are females, including a potentially lethal hyperthermia. MDMA is highly metabolized to five main metabolites, by the enzymes CYP1A2 and CYP2D. The major metabolite in rats, 3,4-methylenedioxyamphetamine (MDA), also causes hyperthermia. We postulated that the reported sex difference in rats is due to a sexual dimorphism(s). We therefore determined (1) the LD50 of MDMA and MDA, (2) their hyperthermic effects, (3) the activities of liver CYP1A2 and CYP2D, (4) the liver microsomal metabolism of MDMA and MDA, (5) and the plasma concentrations of MDMA and its metabolites 3 h after giving male and female Sprague-Dawley (SD) rats MDMA (5 mg.kg(-1) sc). The LD50 of MDMA was 2.4-times lower in males than in females. MDMA induced greater hyperthermia (0.9 degrees C) in males. The plasma MDA concentration was 1.3-fold higher in males, as were CYP1A2 activity (twice) and N-demethylation to MDA (3.3-fold), but the plasma MDMA concentration (1.4-fold) and CYP2D activity (1.3-fold) were higher in females. These results suggest that male SD rats are more sensitive to MDMA acute toxicity than are females, probably because their CYP1A2 is more active, leading to higher N-demethylation and plasma MDA concentration. This metabolic pathway could be responsible for the lethality of MDMA, as the LD50 of MDA is the same in both sexes. These data strongly suggest that the toxicity of amphetamine-related drugs largely depends on metabolic differences.

Ventilatory effects of low-dose paraoxon result from central muscarinic effects.

Paraoxon induces respiratory toxicity. Atropine completely reversed parathion- and paraoxon-induced respiratory toxicity. The aim of this study was to assess the peripheral or central origin of ventilatory effects of low-dose paraoxon. Male Sprague-Dawley rats were given paraoxon 0.215 mg/kg subcutaneously and treated with either atropine (10 mg/kg sc) or ascending doses of methylatropine of 5.42 (equimolar to that of atropine), 54.2, and 542 mg/kg administered subcutaneously 30 min after paraoxon. Ventilation at rest was assessed using whole-body plethysmography and rat temperature using infra-red telemetry. Results are expressed as mean+/-SE. Statistical analysis used two-way ANOVA for repeated measurements. Paraoxon induced a significant decrease in temperature 30 min after injection lasting the 90 min of the study period. This effect was partially corrected by atropine, but not by methylatropine whatever the dose. Paraoxon induced a decrease in respiratory rate resulting from an increase in expiratory time associated with an increase in tidal volume. Atropine completely reversed the ventilatory effects of low-dose paraoxon while the equimolar dose of methylatropine had no significant effects. The 54.2 and 542 mg/kg doses of methylatropine had no significant effects. Atropine crosses the blood-brain barrier and reverses peripheral and central muscarinic effects. In contrast, methylatropine does not cross the blood-brain barrier. Atropine completely reversed the ventilatory effects of low-dose paraoxon, while methylatropine had no significant effects at doses up to 100-fold the equimolar dose of atropine. We conclude that the ventilatory effects of low-dose paraoxon are mediated by disrupted muscarinic signaling in the central nervous system.

Sprague–Dawley rats display sex-linked differences in the pharmacokinetics of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA)

The use of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has increased in recent years; it can lead to life-threatening hyperthermia and serotonin syndrome. Human and rodent males appear to be more sensitive to acute toxicity than are females. MDMA is metabolized to five main metabolites by the enzymes CYP1A2, CYP2D and COMT. Little is presently known about sex-dependent differences in the pharmacokinetics of MDMA and its metabolites. We therefore analyzed MDMA disposition in male and female rats by measuring the plasma and urine concentrations of MDMA and its metabolites using a validated LC-MS method. MDA AUC(last) and C(max) were 1.6- to 1.7-fold higher in males than in females given MDMA (5 mg/kg sc), while HMMA C(max) and AUC(last) were 3.2- and 3.5-fold higher, respectively. MDMA renal clearance was 1.26-fold higher in males, and that of MDA was 2.2-fold higher. MDMA AUC(last) and t(1/2) were 50% higher in females given MDMA (1 mg/kg iv). MDA C(max) and AUC(last) were 75-82% higher in males, with a 2.8-fold higher metabolic index. Finally, the AUC(last) of MDA was 0.73-fold lower in males given 1 mg/kg iv MDA. The volumes of distribution of MDMA and MDA at steady-state were similar in the two sexes. These data strongly suggest that differences in the N-demethylation of MDMA to MDA are major influences on the MDMA and MDA pharmacokinetics in male and female rats. Hence, males are exposed to significantly more toxic MDA, which could explain previously reported sexual dysmorphism in the acute effects and toxicity of MDMA in rats.