Involvement of human cytochrome P450 3A4 in reduced haloperidol oxidation

Objective: The present study was conducted to identify in vitro the cytochrome P450(CYP) isoform involved in the metabolic conversion of reduced haloperidol to haloperidol using microsomes derived from human AHH-1 TK +/− cells expressing human cytochrome P450s. The inhibitory and/or stimulatory effects of reduced haloperidol or haloperidol on CYP2D6-catalyzed carteolol 8-hydroxylase activity were also investigated. Results: The CYP isoform involved in the oxidation of reduced haloperidol to haloperidol was CYP3A4. CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 2E1 were not involved in the oxidation. The k_M value for the CYP3A4 expressed in the cells was 69.7 μmol · l⁻¹, and the V_max was 4.87 pmol · min⁻¹ · pmol⁻¹ P450. Troleandomycin, a relatively selective probe for CYP3A enzymes, inhibited the CYP3A4-mediated oxidation of reduced haloperidol in a dose-dependent manner. Quinidine and sparteine competitively inhibited the oxidative reaction with a k_i value of 24.9 and 1390 μmol · l⁻¹, respectively. Carteolol 8-hydroxylase activity, which is a selective reaction probe for CYP2D6 activity, was inhibited by reduced haloperidol with a k_i value of 4.3 μmol · l⁻¹. Haloperidol stimulated the CYP2D6-mediated carteolol 8-hydroxylase activity with an optimum concentration of 1 μmol · l⁻¹, whereas higher concentrations of the compound (>10 μmol · l⁻¹) inhibited the hydroxylase activity. Conclusion: It was concluded that CYP3A4, not CYP2D6, is the principal isoform of cytochrome P450 involved in the metabolic conversion of reduced haloperidol to haloperidol. It was further found that reduced haloperidol is a substrate of CYP3A4 and an inhibitor of CYP2D6, and that haloperidol has both stimulatory and inhibitory effects on CYP2D6 activity. Received: 10 April 1997 / Accepted in revised form: 16 December 1997     

New metrics for analysis of dendritic branching patterns demonstrating similarities and differences in ON and ON-OFF directionally selective retinal ganglion cells.

The morphology and dendritic branching patterns of retinal ganglion cells have been studied in Golgi-impregnated, whole-mount preparations of rabbit retina. Among a large number of morphological types identified, two have been found that correspond to the morphology of ON and ON-OFF directionally selective (DS) ganglion cells identified in other studies. These two kinds of DS ganglion cell are compared with each other, as well as with examples of class I, class II, and class III cells, defined here with reference to our previous studies. Cell body, dendritic field size and branching pattern are analyzed in this paper and levels of dendritic stratification are examined in the following paper. ON DS ganglion cells are about 10% larger in soma size and about 5 times the dendritic field area of ON-OFF DS ganglion cells, when compared at the same retinal location. These two morphological types of ganglion cell can be said to define the upper and lower bounds of an intermediate range of cell body and dendritic field sizes within the whole population of ganglion cells. Nevertheless, in previous physiological studies receptive field sizes of the two types were shown to be similar. This discrepancy between morphological and physiological evidence is considered in the Discussion in terms of a model of the excitatory receptive field of ON-OFF DS ganglion cells incorporating starburst amacrine cells. A new set of metrics is introduced here for the quantitative analysis and characterization of the branching pattern of neuronal arborizations. This method compares the lengths of terminal and preterminal dendritic branches (treated separately), as a function of the distances of their origins from the soma, viewed graphically in a two-dimensional scatter plot. These values are derived from computer-aided 3D logging of the dendritic trees, and distance from the soma is measured as the shortest distance tracked along the dendritic branches. From these metrics of the "branch length distributions," scale-independent branching statistics are derived. These make use of mean branch lengths and distances, slopes of lines fitted to the distributions, and elliptical indices of scatter in the distributions. By these measures, ON and ON-OFF DS ganglion cells have similar branching patterns, which they share to varying degrees with functionally unrelated class III.1 ganglion cells. The scale of the branching patterns of ON and ON-OFF DS cells and their degree of uniformity are different, however.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional brain kinetics of 6-fluoro-(β-11C)-L-dopa and (β-11C)-L-dopa following COMT inhibition. A study in vivo using positron emission tomography

Summary The regional brain kinetics of (-11C)-L-dopa and 6-fluoro-(-11C)-L-dopa was measured in six Rhesus monkeys using positron emission tomography (PET). Radioactivity accumulated specifically in the striatal region and the increase in L-dopa-derived radioactivity utilization with time was calculated using surrounding brain as a reference area, this being devoid of dopaminergic activity. The rate constant for selective striatal utilization i.e. grossly decarboxylation was 0.0110 ± 0.0007 (S.D) and 0.0057 ± 0.0006 min1 for (-11C)-L-dopa and 6-fluoro-(-11C)-L-dopa, respectively. After pre-treatment of the monkeys with the peripherally and centrally active catecholamine-O-methyl transferase (COMT) inhibitor Ro 40-7592 10 mg/kg, the decarboxylation rate remained unchanged (0.0112 ± 0.0015 min-1) for (11C)-L-dopa, whereas an increase in rate was measured for 6-fluoro-(-11C)L-dopa (0.0092 ± 0.0015 min–1). Differences in the distribution of radiolabelled metabolites i.e. the corresponding O-methyl-L-dopa in the reference area is most probably the reason for the difference in calculated decarboxylation rate seen between the radiotracers. The higher decarboxylation rate measured for 6-fluoro-(-11C)-L-dopa after blockade of COMT shows that the radiolabelled metabolites i.e. 6-fluoro-O-methyl-(-11C)-L-dopa significantly contributes to background radioactivity.     

GD3 expression by cultured human tumor cells of neuroectodermal origin

Summary Seven monoclonal antibodies (mAbs) reactive with ganglioside II³(NeuAc)₂-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV³(NeuAc)₂nLcOse₄Cer(3,8-LD1), and very weakly with IV³(NeuAc)₂II³NeuAc-GgOse₄Cer (GTla), but not with II³NeuAc-LacCer (GM3), II³NeuAcGgOse₃Cer(GM2), II³NeuAc-GgOse₄Cer(GM1), II³NeuAc, IV³NeuAcGgOse₄Cer (GD1a), II³(NeuAc)₂GgOse₃(GD2), II³(NeuAc)₂GgOse₄Cer (GD1b), IV³NeuAcII³(NeuAc)₂, GgOse₄Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc2-8NeuAc2-3Gal1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF. There was no detectable GD3 found in gangliosides isolated from cell lines U-373 MG, D-54 MG, TE-671, and PA-1, which were negative for both DMAb-7 and DMAb-8 by CS-RIA and IF assay. Our results provide evidence that GD3 is expressed extensively with significant quantitative heterogeneity on cultured human neuroectodermal tumor cells including glioma, medulloblastoma, neuroblastoma, and melanoma.Supported by NIH grants R37 CA11898, NS 20023, and CA32672 and by grants from the Swedish Medical Research Council (project no. 03X-627), Swedish Cancer Society (project no. 2260-B88-01X) and the National Swedish Board for Technical Development (project no. 84-4667)     

Transient GABA immunoreactivity in cranial nerves of the chick embryo

The distribution and time course of gamma-aminobutyric acid (GABA) immunoreactivity was investigated in the cranium of the chick embryo from 2 to 16 days of incubation (E2-16). A fraction of nerve fibers transiently stains GABA-positive in all cranial motor nerves and in the vestibular nerve. Cranial motor nerves stain GABA-positive from E4 to E11, including neuromuscular junctions at E8-11; labeled fibers are most frequent in the motor trigeminal root (E6-9.5). Substantial GABA staining is present from E4 to E10 in a subpopulation (1-2%) of vestibular ganglion cells. Their peripheral processes are labeled in the vestibular endorgan, predominantly in the posterior crista. Some GABA-positive fibers are present in the olfactory nerve (after E5) and in the optic nerve (after E9.5); their immunoreactivity persists throughout the period investigated. Transient GABA immunoreactivity follows "pioneer" fiber outgrowth and coincides with the formation of early synaptic contacts. GABA-containing neurons may change their neuronal phenotype (loss of GABA expression) or they may be eliminated by embryological cell death. Periods of cell death were determined in cranial ganglia and motor nuclei by aggregations of pycnotic cells in the same embryonic material. The periods of embryonic cell death partly coincide with transient GABA immunoreactivity. The function(s) of transient GABA expression is unknown. Some lines of evidence suggest that GABA has neurotrophic functions in developing cranial nerves or their target tissue. In the developing neuromuscular junction, GABA may be involved in the regulation of acetylcholine receptors.     

[3H]5-HT binding in post-mortem human cerebral cortex: methodological considerations

Summary The effects of different membrane preparations and assay conditions on [³H]5-HT binding to post-mortem human cortical tissue was studied. Optimal binding necessitated thorough removal of endogenous 5-HT and this was achieved either by hypotonic lysis or by preincubation of the membranes at 37C. Calcium chloride (4 mM) increased specific [³H]5-HT binding. The further addition of ascorbic acid (5.7 mM) or ascorbic acid and clorgyline (10 M) reduced specific [³H]5-HT binding.     

“Occult” hydrocephalus in children

The authors describe 32 children between 2 and 15 years of age who had hydrocephalus that was only clinically manifest late in life. The clinical picture of these children did not suggest an obvious increase in intracranial pressure; instead, the presenting signs were rather nonspecific and included macrocrania, mild psychomotor retardation, unsteady gait, increased muscle tone and deep tendon reflexes in the lower limbs, impaired ocular movement, epilepsy, and endocrine dysfunction. Their histories suggest the possible causes of the ventricular dilation in about one third of the cases were: perinatal hemorrhage, leptomeningitis, neurofibromatosis, and untreated aneurysm of the great vein of Galen. In 20 patients, however, no positive anamnestic findings were reported. CT scan revealed triventricular dilation in more than half of the cases; tetraventricular dilation was present in 6 patients, and biventricular dilation in the remaining subjects. All children underwent CSF shunting, which resulted in complete recovery in all but 2 cases. The most frequently recorded surgical complication was postoperative subdural effusion (7 subjects), which required surgical treatment in only 2 cases.Presented at the 15th Annual Scientific Meeting of the International Society for Pediatric Neurosurgery, New York, 1987     

Differential regulation of cytokine genes in gingival epithelial cells challenged by Fusobacterium nucleatum and Porphyromonas gingivalis

IL-8 mRNA in human gingival epithelial cells (HGECs) is up-regulated by Fusobacterium nucleatum, and up-/down-regulated by Porphyromonas gingivalis in a complex interaction in the early stages (< or = 4 h) after infection. The mechanisms involved in this regulation in response to F. nucleatum and/or P. gingivalis infection, and identification of co-regulated cytokine genes, are the focus of this investigation. Heat, formalin or protease treatment of F. nucleatum cells attenuated the IL-8 mRNA up-regulation. NF-kappaB, mitogen-activated protein kinase (MAPK) p38 and MAPK kinase/extracellular signal-regulated kinase (MEK/ERK) pathways were involved in IL-8 mRNA induction by F. nucleatum. Pretreatment of P. gingivalis with heat, formalin or protease enhanced IL-8 mRNA induction. NF-kappaB, MARK p38, and MEK/ERK pathways were also involved in this induction. In contrast, down-regulation of IL-8 mRNA by P. gingivalis involved MEK/ERK, but not NF-kappaB or MAPK p38 pathways. cDNA arrays analysis revealed that mRNA down-regulation by P. gingivalis is a specific reaction that only a number of genes, e.g. IL-1beta, IL-8, macrophage inflammatory protein-2alpha, and migration inhibitory factor-related protein-14, are affected based on examination of 278 cytokine/receptor genes. These data indicate that F. nucleatum and P. gingivalis trigger specific and differential gene regulation pathways in HGECs.     

Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.