Localization of immunoreactive prolactin in ependyma and circumventricular organs of rat brain

Summary Immunoreactive prolactin (IMP) has been localized in the male rat brain using the soluble peroxidase-anti-peroxidase (PAP) technique. In normal untreated animals, reaction product was seen in choroid plexus (CP) and in ependymal cells of the ventricular lining with heaviest concentrations of positively staining cells in the 3rd ventricle near the subcommisural organ (SCO), in the lateral ventricles near the subfornical organ (SFO), and in the 4th ventricle near the area postrema (AP). IMP was also present in numerous ependymal cells resembling tanycytes in the cerebral aqueduct, central canal of the spinal cord at the level of the AP, the organum vasculosum of the lamina terminalis (OVLT) and the floor of the infundibular recess. Immunoreactive cells resembling neurons were localized within the substance of the AP, SCO, and OVLT. IMP was also present in fibers of the zona externa of the median eminence and infundibular stalk; a few cells of the pars tuberalis contained reaction product. Hypophysectomized rats and bromocriptine-treated rats exhibited a similar staining pattern except that bromocriptine treatment eliminated IMP from most CP cells. Hypophysectomy, bromocriptine or estrogen treatment enhanced staining for IMP in cells of the pars tuberalis; estrogen treatment or hypophysectomy produced an increase in the number and distribution of immunoreactive cells as well as increased density of reaction product in cells of the medial habenular nucleus. The functional relevance of prolactin in these locations in the brain, the possible routes of transport of prolactin from the pituitary gland to the central nervous system, and the strong suggestion of extra-pituitary sites of synthesis of a prolactin-like hormone are discussed.     

微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。     

Hypoxia Increases the Susceptibility to Oxidant Stress and the Permeability of the Blood-Brain Barrier Endothelial Cell Monolayer

Abstract: Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of a coculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxia-reoxygenation at the BBB level. The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. A decrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury. Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation.     

Ultrastructure of cerebrospinal fluid-contacting neurons immunoreactive to vasoactive intestinal peptide and properties of the blood-brain barrier in the lateral septal organ of the duck

Immuno-electron-microscopic investigations of cerebrospinal fluid (CSF)-contacting neurons immunoreactive to vasoactive intestinal peptide in the duck lateral septum have revealed that this cell type gives rise to an adventricular dendrite terminating with a bulbous swelling in the lateral ventricle. The swelling bears a cilium and contains mitochondria and immunolabeled dense-core vesicles. Two types of processes emerge from the basal part of the perikaryon. The first has a large diameter, contains diffusely distributed immunoreaction, and receives synaptic input, indicating that this process is a basal dendrite. The other type is of a beaded appearance, displays immunolabeled dense-core vesicles, and represents the axon of the CSF-contacting neuron. VIP-immunoreactive terminal formations are located within the neuropil of the lateral septum and the nucleus accumbens. Some of them form synaptic contacts with immunonegative profiles. No VIP-immunoreactive terminal formations are seen in the perivascular spaces of the lateral septum. Tracer experiments with horseradish peroxidase have revealed that the blood-brain barrier is lacking in the lateral septal organ and nucleus accumbens of the duck. Capillaries, arterioles, and venoles of this region are coated by nonfenestrated endothelial cells connected by leaky junctions, allowing the tracer to penetrate from the lumen into the perivascular space and further into the intercellular clefts of the neuropil. Our immuno-electron-microscopic investigations show that VIP-immunoreactive CSF-contacting neurons of the lateral septum closely resemble CSF-contacting neurons occurring in other brain regions, e.g., the hypothalamus. The arrangement of VIP-immunoreactive terminal formations suggests that, in the lateral septum, the VIP-like neuropeptide serves as a neurotransmitter (-modulator). The lack of a blood-brain barrier in the lateral septal organ and the nucleus accumbens raises the possibility that this region is a window in the avian brain allowing exchange of information between the central nervous system and the bloodstream; it thus resembles a circumventricular organ.     

Uptake of 36Cl and 22Na by the Brain-Cerebrospinal Fluid System: Comparison of the Permeability of the Blood-Brain and Blood-Cerebrospinal Fluid Barriers

Abstract: Transport and permeability properties of the blood-brain and blood-CSF barriers were determined by kinetic analysis of radioisotope uptake from the plasma into the CNS of the adult rat. Cerebral cortex and cerebellum uptake curves for ³⁶Cl and ²²Na were resolved into two components. The fast component (t_½ 0.02–0.05 h, fractional volume 0.04–0.08) is comprised of the vascular compartment and a small perivascular space whereas the slow component (t_½ 1.06–1.69 h, fractional volume 0.92–0.96) represents isotope movement across the blood-brain barrier into the brain extracellular and cellular compartments. Uptake curves of both ³⁶Cl and ²²Na into the CSF were also resolved into two components, a fast component (t_½ 0.18 h, fractional volume 0.24) and a slow component (t_½ 1.2 h, fractional volume 0.76). Evidence suggests that the fast component represents isotope movement across the blood-CSF barrier, i.e., the choroid plexuses, whereas the CSF slow component probably reflects isotope penetration primarily from the brain extracellular fluid into the CSF. The extracellular fluid volume of the cerebral cortex and cerebellum was estimated as ?13% from the initial slope of the curve of brain space versus CSF space curve for both ³⁶Cl and ²²Na. Like the choroid plexuses, the glial cell compartment of the brain appears to accumulate Cl from 2 to 6 times that predicted for passive distribution. The relative permeability of the blood-CSF and blood-brain barriers to ³⁶Cl, ²²Na, and [³H]mannitol was determined by calculating permeability surface-area products (PA). Analysis of the PA values for all three isotopes indicates that the effective permeability of the choroidal epithelium (blood/CSF barrier) is significantly greater than that of the capillary endothelium in the cerebral cortex and cerebellum (blood-brain barrier).     

Blood-Brain Transfer of Galactose in Experimental Galactosemia, with Special Reference to the Competitive Interaction Between Galactose and Glucose

The interaction between glucose and galactose during transport across the cerebral capillary endothelium was studied in anesthetized rats. Although galactose is present in the diet of suckling mammals and is a potential substrate for brain metabolism in adult mammals, its effect on glucose transport in adult rats is unknown. A kinetic model was formulated to analyze the effect of chronically elevated galactose levels on glucose transport in adult rats. The analysis indicated that galactose and glucose compete for the same transport mechanism in the cerebral capillary endothelium. The Tmax of glucose and galactose were both about 380 mumol 100 g-1 min-1 and the Kt of galactose (30 mM) was about three times that of glucose (10 mM). During prolonged galactosemia in adult rats, neither the Tmax, nor the Kt of either competitor changed substantially when compared with rats subjected to acute galactosemia. At 10 mM galactose in plasma in rats with acute galactosemia, the inhibition of glucose transport, simulated a 25% reduction of plasma glucose, and in rats with chronic galactosemia a 20% reduction. This moderate effect is in contrast to the effect of galactose in suckling rats in which 10 mM galactose in plasma reduced the glucose transport to a level corresponding to a 50% reduction of the plasma glucose concentration.     

Expression of cytochrome P-450lpr Is developmentally regulated and limited to house fly

Expression of house fly cytochrome P-450_lpr was examined using immunoblotting in male and female adult LPR house flies, mixed sex adult house flies at 12 different ages, larvae, and pupae. P-450_lpr was expressed in both male and female adult house flies. P-450_lpr was clearly present in all adult stages examined, was barely detectable in pupae, and could not be detected in larvae. Thus, cytochrome P-450_lpr is developmentally regulated and present in both sexes of house fly. Expression of cytochrome P-450, immunologically homologous to house fly cytochrome P-450_lpr was examined in other species using immunoblot analysis. Eleven animal species were tested in the orders Diptera, Hymenoptera, Lepidoptera, Orthoptera, Acari, and Rodentia, using microsomes in some species from both induced and noninduced animals or insecticide-resistant and susceptible strains. P-450_lpr appears to be restricted to house flies, as none of these species contained cytochrome P-450 that reacted with antiserum to cytochrome P-450_lpr.     

Effects of the Binding of Imipramine to Erythrocytes and Plasma Proteins on Its Transport Through the Rat Blood-Brain Barrier

Brain extraction of a tricyclic antidepressant, imipramine, was investigated using the carotid injection technique in the rat. The extent to which drug binding to plasma proteins and erythrocytes could inhibit the brain extraction was measured. Equilibrium dialysis showed that imipramine is highly bound to human serum albumin (HSA), alpha 1-acid glycoprotein (AAG), lipoproteins, and erythrocytes. The free dialyzable drug fraction was inversely related to the protein concentration. Despite this degree of binding, no significant reduction in the brain extraction of the drug was observed in the presence of HSA, lipoprotein, or erythrocytes. Only AAG reduced the brain transport of this drug in a ratio related to the protein concentration. However, the rat brain extraction was higher than expected from the in vitro measurement of the dialyzable fraction. These data indicate that the amount of circulating imipramine available for penetration in brain exceeds widely the dialyzable fraction of the drug as measured in vitro.     

Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats

The brain uptake and brain to blood efflux transport of (14)C-GABA were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats using 20 min bilateral in situ brain perfusion in rats anesthetized using urethane. The volume of distribution (Vd) of (14)C-GABA into cerebrospinal fluid (CSF) and brain regions (cortex, diencephalon, cerebellum, and brain stem) was significantly greater in SHR than in the corresponding regions in WKY rats (p<0.05). The estimated Vd value of (14)C-GABA in CSF of SHR was 3.4 fold greater than that in WKY. Also compared to WKY, the Vd of (14)C-GABA into cerebellum and cortex of SHR was 15.3 fold and 19.4 fold greater, respectively. Although the study of blood-brain barrier (BBB) integrity using (3)H-mannitol revealed increased paracellular permeability at the brain capillaries of SHR when compared to WKY rats, this was found to be only partially responsible for the increased (14)C-GABA uptake. The study of brain to blood efflux transport of (14)C-GABA (after loading of brain with (14)C-GABA by vascular perfusion) revealed that the half-time of elimination was significantly shorter in SHR (5.35+/-0.66 min) than in WKY rats (14.83+/-1.94 min), (p<0.001). HPLC analysis revealed that GABA concentrations in brain extracts and CSF of SHR were similar to those in WKY rats (p>0.05). The faster efflux in SHR might be, at least partially, responsible to compensate for increased uptake of this neurotransmitter and to preserve the protective function of BBB towards GABA. The protective function of the BCSFB towards GABA appears to be also preserved, since systemic infusion of GABA within a wide range of administered doses (0.004-5.00 mg/kg) produced an increase in GABA CSF concentration from around 0.5 microM to only 11 microM, and the obtained pattern of CSF GABA concentrations under these conditions did not differ between SHR and WKY rats, as revealed by HPLC.     

Localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats An immunohistochemical study

Summary Immunohistochemical localization of cytochrome P-450 in the colonic mucosa of 3-methylcholanthrene-pretreated and untreated rats was studied by indirect fluorescent antibody staining technique. A polyclonal antibody for cytochrome P-450_MC purified from hepatic microsomes of 3-methylcholanthrene-pretreated rats was used for this experiment. A strong immunofluorescence was found to be localized in the cytoplasm of the surface epithelium of the mucosa in the colon of 3-methylcholanthrene-pretreated rats. A faint immunofluorescence was also observed in the epithelium of untreated rats. 7-Ethoxycoumarin O-deethylase activity of colonic microsomes was significantly enhanced by 3-methylcholanthrene-pretreatment in parallel with an increase in the intensity of immunostaining for cytochrome P-450_MC in Western blotting analysis. This is the first report on the localization of cytochrome P-450 in the colonic mucosa.     

Induced tolerance of neonate Heliothis zea to host plant allelochemicals and carbaryl following incubation of eggs on foliage of Lycopersicon hirsutum f. glabratum

Summary Incubation of Heliothis zea (Boddie) eggs on foliage of Lycopersicon hirsutum f. glabratum C.H. Mull (accession PI 134417) results in neonates with elevated levels of tolerance to the toxic effects of PI 134417 foliage attributable to 2-tridecanone found in the glandular trichomes which abound on that foliage. The neonates from such eggs are also shown to have elevated levels of tolerance to the carbamate insecticide carbaryl. Incubation of eggs in an atmosphere containing 2-tridecanone similarly produced elevated levels of tolerance to 2-tridecanone among resulting neonates, indicating that 2-tridecanone is the likely inducing agent and that exposure to 2-tridecanone vapor, which is known to emanate from PI 134417 foliage, is sufficient for induction. Analysis of the cytochrome P-450 content in gut microsomes of fifth instar larvae indicated that exposure of larvae to 2-tridecanone in artificial diet or to PI 134417 foliage resulted in significantly elevated levels of cytochrome P-450 relative to larvae fed diet without 2-tridecanone or foliage of L. esculentum which contains no 2-tridecanone. In addition, removal of the glandular trichomes from PI 134417 foliage eliminated the ability of that foliage to induce elevated levels of cytochrome P-450. These results provide circumstantial evidence that cytochrome P-450 may be involved in the induced tolerance to xenobiotics among neonates from eggs exposed to 2-tridecanone or PI 134417 foliage.Support for this research was provided by the USDA Competitive Research Grants Program in Biological Stress under Grant No. 83-CRCR-1-1241 and Grant No. 85-CRCR-1-1615, and the North Carolina Agricultural Research Service. Paper No. 10856 of Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC, USA 27650. Use of trade names does not imply endorsement of products named nor criticisms of similar ones not mentioned     

Comparative Effects of Dietary Fat Types on Hepatic Enzyme Activities Related to the Synthesis and Oxidation of Fatty Acid and to Lipogenesis in Rats

The effects of different types of dietary fat on the activities of hepatic enzymes related to fatty acid synthesis {glucose-6-phosphate dehydrogenase (G6PDH) and acetyl-CoA carboxylase ACC)}, oxidation {acyl-CoA synthetase (AST), carnitine palmitoyl transferase (CPT), and peroxisomal β-oxidation (P βOX)}, and lipogenesis {phosphatidate phosphohydrolase (PAP), diacylglycerol acyltransferase (DGAT), and phosphocholine diacylglycerol transferase (PCDGT)}, and plasma and liver lipid levels were investigated in male Wistar rats. The animals were 6 weeks old and about 120 g of body weight, and were fed on test diets containing 20% of a mixture of tripalmitin, tristearin and corn oil (SFA), olive oil (OLI), sunflower oil (SUN), linseed oil (LIS), and sardine oil (SAR) for 2 weeks. The concentrations of plasma total cholesterol (T-CHOL), high-density lipoprotein-cholesterol (HDL-CHOL), triacylglycerol (TG) and phospholipid (PL) were generally higher in the rats fed on SEA and OLI than in those given SUN, LIS and SAR. The rats fed on OLI had a higher level of liver T-CHOL than those fed on the other fats. The liver TG content was nearly higher from the intake of SFA and OLI than from SUN, LIS and SAR, although the liver PL level was not affected by the type of dietary fat. The SFA and OLI groups had the highest activities of hepatic G6PDH and ACC, and the SAR group, the lowest activities. The activities of AST and CPT, and peroxisomal P βOX in the liver were higher in the rats fed on the LIS and SAR diets than in those given the other diets. The hepatic PAP activity was higher from the intake of OLI and SUN, and tended to be higher from SFA than from LIS and SAR. The activity of liver DGAT was higher from SFA and inclined to be higher from OLI, SUN, and LIS than from SAR, while the PCDGT activity in the liver was not effected by the type of dietary fat. The concentrations of plasma and liver TG were generally positively correlated with the activities of liver enzymes related to the synthesis of fatty acids and lipids, and negatively with those involved in fatty acid oxidation. Based on these results, it is suggested that the levels of plasma and liver TG were controlled by different types of dietary fat through changes in the hepatic enzyme activities related to fatty acid synthesis, lipogenesis, and fatty acid oxidation.     

Constituents from the Roots of Actinidia chinensis and Their Cytochrome P450 Enzyme Inhibitory Activities

A newly discovered triterpenoid, (2α,3β)‐2,3,23‐trihydroxyurs‐13(18)‐en‐28‐oic acid ( 1 ), along with twelve known compounds ( 2  –  13 ), were isolated from the roots of Actinidia chinensis Planch (Actinidiaceae). Their chemical structures were determined by 1D‐ and 2D‐NMR spectra and mass spectrometry (MS). The crude extracts and six main constituents ( 8  –  13 ) were tested for cytochrome P450 (CYPs) enzyme inhibitory activity. The results showed that, except for compound 8 , compounds 9  –  13 had different inhibitory effects on the cytochrome P450 (CYPs) enzyme, and compound 9 significantly inhibited the catalytic activities of CYP3A4 to < 10% of its control activities.     

Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes

The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography–tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.     

Differential Responses of Soil Organic Carbon Fractions and Carbon Turnover Related Enzyme Activities to Wheat Straw Incorporation in Subtropical China

Soil organic carbon (SOC) fractions and C turnover related enzyme activities are essential for nutrient cycling. This is because they are regarded as important indicators of soil fertility and quality. We measured the effects of wheat straw incorporation on SOC fractions and C turnover related enzyme activities in a paddy field in subtropical China. Soil samples were collected from 0–10 cm and 10–20 cm depths after rice harvesting. The total SOC concentrations were higher in the high rate of wheat straw incorporation treatment (NPKS2) than in the not fertilized control (CK) (P < 0.05). The concentrations of labile C fractions [i.e., water soluble organic C (WSOC), hot-water soluble organic C (HWSOC), microbial biomass C (MBC), and easily oxidizable C (EOC)], were higher in the moderate NPKS1 and NPKS2 treatments than in CK and the fertilized treatment without straw (NPK) (P < 0.05). The geometric means of labile C (GMC) and C pool management index (CPMI) values were highest in NPKS2 (P < 0.05). The SOC concentrations correlated positively with the labile C fractions (P < 0.05). Soil cellulase activity and the geometric mean of enzyme activities (GMea) were higher in NPKS2 than in CK in all soil layers (P < 0.05), and the invertase activity was higher in NPKS2 than in CK in the 0–10 cm layer (P < 0.05). Stepwise multiple linear regression indicated that the formation of the SOC, WSOC, HWSOC, MBC, and EOC was mostly enhanced by the cellulase and invertase activities (P < 0.05). Therefore, the high rate of wheat straw incorporation may be recommended to increase soil C pool levels and soil fertility in subtropical paddy soils.     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.