scholarly journals Specific accumulation of 17α-hydroxyprogesterone in microsomal membranes during the process of cytochrome P-450(C-17)-catalysed androgen biosynthesis. A dynamic study of intermediate formation and turnover

1988 ◽  
Vol 256 (1) ◽  
pp. 53-59 ◽  
Author(s):  
N Kühn-Velten ◽  
M Lessmann ◽  
M E Förster ◽  
W Staib
Keyword(s):  
Direct Evidence ◽  
Intermediate Formation ◽  
Specific Accumulation ◽  
Membrane Compartment ◽  
Microsomal Membranes ◽  
Double Label ◽  
Double Substrate ◽  
Label Distribution ◽  
Cytochrome P 450 ◽  
Substrate State

A complete dynamic analysis of cytochrome P-450(C-17)-catalysed androgen biosynthesis from a single dose of progesterone and 17 alpha-hydroxyprogesterone in a double-label double-substrate experiment was performed in order to elucidate the controversial intermediacy of 17 alpha-hydroxyprogesterone. Label distribution within the steroid fractions as well as in the membrane and buffer compartments yields direct evidence that the endogenously formed 17 alpha-hydroxyprogesterone (which is in an ‘intermediate state’) accumulates to a higher degree in microsomal membranes than does the exogenously added 17 alpha-hydroxyprogesterone (which is in a ‘substrate state’) under certain conditions. It is also demonstrated that endogenously formed 17 alpha-hydroxyprogesterone may partly leave the membrane compartment (in terms of a ‘leakage’ or ‘overflow’ phenomenon) and is then able to equilibrate with the pool of exogenously added 17 alpha-hydroxyprogesterone. Since only the label distribution in the membrane-associated (but not always in the aqueous) 17 alpha-hydroxyprogesterone pool corresponds to the label distribution in the androgen fraction, it is concluded that only the membrane-associated 17 alpha-hydroxyprogesterone pool is directly accessible to cytochrome P-450(C-17)-catalysed conversion into androgens.

scholarly journals Loss of haem in rat liver caused by the porphyrogenic agent 2-allyl-2-isopropylacetamide

1971 ◽  
Vol 124 (4) ◽  
pp. 767-777 ◽  
Author(s):  
F. De Matteis
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Direct Evidence ◽  
Gradual Increase ◽  
Rapid Loss ◽  
Metabolizing Enzymes ◽  
Liver Microsomal Fraction ◽  
Green Pigments ◽  
Cytochrome P 450 ◽  
Increased Activity

1. The effect of a single dose of 2-allyl-2-isopropylacetamide on the cytochrome P-450 concentration in rat liver microsomal fraction was studied. The drug caused a rapid loss of cytochrome P-450 followed by a gradual increase to above the normal concentration. 2. The loss of cytochrome P-450 was accompanied by a loss of microsomal haem and by a brown–green discoloration of the microsomal fraction suggesting that a change in the chemical constitution of the lost haem had taken place. Direct evidence for this was obtained by prelabelling the liver haems with radioactive 5-aminolaevulate: the drug caused a loss of radioactivity from the haem with an increase of radioactivity in a fraction containing certain un-identified green pigments. 3. Evidence was obtained by a dual-isotopic procedure that rapidly turning-over haem(s) may be preferentially affected. 4. The loss of cytochrome P-450 as well as the loss of microsomal haem and the discoloration of the microsomal fraction were more intense in animals pretreated with phenobarbitone and were much less evident when compound SKF 525-A (2-diethylaminoethyl 3,3-diphenylpropylacetate) was given before 2-allyl-2-isopropylacetamide, suggesting that the activity of the drug-metabolizing enzymes may be involved in these effects. 5. The relevance of the destruction of liver haem to the increased activity of 5-aminolaevulate synthetase caused by 2-allyl-2-isopropylacetamide is discussed.

scholarly journals NADPH: cytochrome P-450 reductase in olfactory epithelium Relevance to cytochrome P-450-dependent reactions

1986 ◽  
Vol 240 (2) ◽  
pp. 585-592 ◽  
Author(s):  
C J Reed ◽  
E A Lock ◽  
F De Matteis
Keyword(s):  
Olfactory Epithelium ◽  
Direct Evidence ◽  
Reductase Activity ◽  
Electron Flow ◽  
Male Rats ◽  
Male And Female ◽  
Marked Activity ◽  
Nadh Cytochrome ◽  
Olfactory Tissue ◽  
Cytochrome P 450

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.

In vivo location and mechanism of EDHF-mediated vasodilation in canine coronary microcirculation

1999 ◽  
Vol 277 (3) ◽  
pp. H1252-H1259 ◽  
Author(s):  
Yasuhiro Nishikawa ◽  
David W. Stepp ◽  
William M. Chilian
Keyword(s):  
Nitric Oxide ◽  
Potassium Channels ◽  
Potassium Channel ◽  
Direct Evidence ◽  
Coronary Microcirculation ◽  
Combined Administration ◽  
Cytochrome P 450 ◽  
Coronary Arterioles

Responses of epicardial coronary arterioles to ACh were measured using stroboscopic fluorescence microangiography in dogs ( n = 38). ACh (0.1 and 0.5 μg ⋅ kg−1 ⋅ min−1ic) dilated small (<100 μm, 11 ± 2 and 19 ± 2%, respectively) and large (>100 μm, 6 ± 3 and 13 ± 3%, respectively) arterioles at baseline. Combined administration of N ω-monomethyl-l-arginine (l-NMMA; 1.0 μmol/min ic) and indomethacin (10 mg/kg iv) eliminated ACh-induced dilation in large coronary arterioles but only partially attenuated that in small arterioles. Suffusion of a buffer containing 60 mM KCl (high KCl) completely abolished cromakalim-induced dilation in arterioles and in combination with l-NMMA plus indomethacin completely blocked ACh-induced dilation in small arterioles. This indicated that the vasodilation to ACh that persists in small arterioles after administration of l-NMMA and indomethacin is mediated via a hyperpolarizing factor. The ACh-induced vasodilation remaining after l-NMMA and indomethacin was completely blocked by the large-conductance potassium-channel antagonist iberiotoxin or by epicardial suffusion of miconazole or metyrapone, inhibitors of cytochrome P-450 enzymes. These observations are consistent with the view that endothelium-derived hyperpolarizing factor (EDHF) is a product of cytochrome P-450 enzymes and produces vasodilation by the opening of large-conductance potassium channels. We conclude that ACh-induced dilation in large coronary arterioles is mediated mainly by nitric oxide (NO), whereas, in small arterioles both NO and EDHF mediate dilation to ACh. These data provide the first direct evidence for an in vivo role of EDHF in small coronary arterioles.

scholarly journals Neurotransmitter organization of the nucleus of Edinger–Westphal and its projection to the avian ciliary ganglion

1991 ◽  
Vol 6 (5) ◽  
pp. 451-472 ◽  
Author(s):  
Anton Reiner ◽  
Jonathan T. Erichsen ◽  
John B. Cabot ◽  
Craig Evinger ◽  
Malinda E. C. Fitzerald ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Sole Source ◽  
Ciliary Ganglion ◽  
Choroidal Blood Flow ◽  
Different Types ◽  
Double Label ◽  
Ciliary Ganglion Neurons ◽  
Immunohistochemical Techniques ◽  
Ganglion Neurons ◽  
Immunohistochemical Studies

AbstractTwo morphologically distinct types of preganglionic endings are observed in the avian ciliary ganglion: boutonal and cap-like. Boutonal endings synapse on ciliary ganglion neurons (called choroidal neurons) innervating choroidal blood vessels, while cap-like endings synapse on ciliary ganglion neurons (called ciliary neurons) controlling the lens and pupil. Some of both types of preganglionic endings contain the neuropeptides substance P (SP) and/or leucine-enkephalin (LENK). Although both types of preganglionic terminals are also known to be cholinergic, there has been no direct evidence that SP and LENK are found in cholinergic endings in the ciliary ganglion. The present studies in pigeons, which involved the use of single- and double-label immunohistochemical techniques, were undertaken to examine this issue, as well as to (1) determine the relative percentages of the boutonal and cap-like endings that contain SP, LENK, or both SP and LENK; and (2) determine if the two different types of terminals in the ciliary ganglion arise from different subdivisions of the nucleus of Edinger-Westphal (EW).Single- and double-label immunohistochemical studies revealed that all neurons of EW, regardless of whether they contained immunohistochemically detectible amounts of SP or LENK, are cholinergic. In the medial subdivision of EW (EWM), which was found to contain approximately 700 neurons, 20.2% of these neurons were observed to contain both SP and LENK, while 11.6% were observed to contain SP only and 10.7% were observed to contain LENK only. In contrast, in lateral EW (EWL), which was found to contain approximately 500 neurons, 16.2% of the neurons were observed to contain both SP and LENK, while 19.2% of the neurons were observed to contain SP only and 12.6% were observed to contain LENK only. Retrograde-labeling studies involving horseradish peroxidase injections into the ciliary ganglion revealed that EW was the sole source of input to the ciliary ganglion and all, or nearly all, neurons in EW innervate the ciliary ganglion.Immunohistochemical labeling of the ciliary ganglion neurons with an antiserum against choline acetyltransferase revealed that approximately 900 choroidal neurons and approximately 600 ciliary neurons are present in the ganglion, all of which receive cholinergic preganglionic endings. Of the choroidal neurons, 94% receive butonal terminals containing both SP and LENK, while only 2% receive SP+ only boutonal endings and 2% receive LENK+ only butonal endings. Of the ciliary neurons, 25% receive cap-like endings containing both SP and LENK, 30% receive cap-like endings containing only SP and 3% receive cap-like endings containing only LENK. Total unilateral lesions of EW resulted in the loss of all SP+ or LENK+ terminals in the ipsilateral ganglion. Subtotal EW lesions that spared either part of EWM or part of EWL revealed that boutonal endings arise from EWM neurons and cap-like endings from EWL neurons.The present results suggest that the choroidal neurons, which regulate choroidal blood flow, may be relatively uniform in their functional properties since they nearly all receive boutonal endings from EWM that co-contain SP, LENK, and acetylcholine. In contrast, the ciliary neurons, which receive their preganglionic input from EWL, may consist of at least three major functionally distinct subgroups: (1) those receiving SP/LENK/acetylcholine-containing cap-like endings; (2) those receiving SP/acetylcholine-containing cap-like endings; and (3) those receiving acetylcholine-containing cap-like endings. The functional diversity of ciliary neurons may in part be related to the fact that some ciliary neurons innervate the iris and others the ciliary body.

scholarly journals Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes.

1978 ◽  
Vol 78 (2) ◽  
pp. 503-519 ◽  
Author(s):  
S Matsuura ◽  
Y Fujii-Kuriyama ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Control Level ◽  
Rat Hepatocytes ◽  
Present Observation ◽  
Membrane Structures ◽  
Rat Liver Cells ◽  
Microsomal Membranes ◽  
Almost All ◽  
Cytochrome P 450

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.

Studies on the Nature of the Action of Organic Accelerators of Vulcanization. The Action of Organic Accelerators on Rubber Sols I–III

1933 ◽  
Vol 6 (2) ◽  
pp. 273-277
Author(s):  
Keiichi Shimada
Keyword(s):  
Direct Evidence ◽  
Active Form ◽  
Intermediate Formation ◽  
Difference Of Opinion ◽  
Vulcanization Process

Abstract While there is a difference of opinion as to the exact mechanism involved, it is generally agreed that the action of organic accelerators in hastening the vulcanization process is usually attributed to the conversion of the sulfur into an active form, probably through the intermediate formation of a compound capable of yielding “nascent” sulfur. But some workers, (Ames, J. Soc. Chem. Ind., 43, 117T (1924); Kirchhof, Kolloid-Z.,, 14, 35 (1914); Twiss, J. Soc. Chem. Ind., 35, 782 (1917); Ditmar, Z. angew. Chem., 34, 465 (1921)) have suggested that the action of organic accelerators might be on the rubber hydrocarbon itself. This theory has not been generally developed, though it has received some small amount of evidence through the works of Le Blanc and Kröger (Z. Elektrochem., 27, 335 (1921)) and Naunton (J. Soc. Chem. Ind., 44, 243T (1925)). These investigators found that the viscosity of rubber solutions decreased with the introduction of an organic accelerator, and referred this phenomenon as a “depolymerization” of rubber. These observations possibly suggest that the influence of accelerators is exerted upon the rubber and constitute the only direct evidence in favor of this view.

scholarly journals N-alkylation of exogenous haem analogues caused by drugs in isolated hepatocytes. Structural isomerism and chirality of the resulting porphyrins

1986 ◽  
Vol 238 (1) ◽  
pp. 263-268 ◽  
Author(s):  
F De Matteis ◽  
C Harvey ◽  
S R Martin
Keyword(s):  
Direct Evidence ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isomeric Composition ◽  
Isolated Rat Hepatocytes ◽  
Structural Isomerism ◽  
Cytochrome P 450 ◽  
Isolated Rat ◽  
Suicide Substrates

Isolated rat hepatocytes incubated with two suicide substrates of cytochrome P-450, 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine(4-ethyl-DD C), convert exogenous mesohaem and deuterohaem into N-alkylated mesoporphyrins and deuteroporphyrins respectively. The N-alkylated mesoporphyrins can be separated by h.p.l.c. from the corresponding N-alkylated protoporphyrins originating from endogenous haem; in this way the contribution of both endogenous and exogenous pools of haem can be studied in the same experiment. N-Alkylated mesoporphyrin exhibits chiral properties, and its isomeric composition and/or amount are dependent on the particular cytochrome P-450 enzyme predominating in the cell. These findings provide additional and more direct evidence that exchangeable haem is taken up by cytochrome P-450 before being N-alkylated.

Oxygenated cytochrome P-450-CAM and chloroperoxidase: direct evidence for sulfur donor ligation trans to dioxygen and structural characterization using EXAFS spectroscopy

1986 ◽  
Vol 108 (25) ◽  
pp. 8114-8116 ◽  
Author(s):  
John H. Dawson ◽  
Lung Shan. Kau ◽  
James E. Penner-Hahn ◽  
Masanori. Sono ◽  
Kim Smith. Eble ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Direct Evidence ◽  
Exafs Spectroscopy ◽  
Cytochrome P 450 ◽  
Sulfur Donor
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