Kininases and converting enzyme in human placenta.

Homogenized tissue of the human placenta is capable of inactivating bradykinin. Ultracentrifuging of the homogenate has shown that the highest activity cosediments with ribosomal fraction. Partial purification of the enzyme by filtration on Sephadex G-200 has been performed. Three protein peaks of kininase and converting activities were obtained. The optimum pH of kininase activity of placenta was determined. An attempt to determine molecular weight was made with the use of Sephadex G-200.Presented results indicate that the placental tissue of a normal woman produces enzymes with kininase activity and activity converting angiotensinase I to angiotensinase II, which may have significance in mechanisms of the local autoregulation of the blood flow.     

Adenosine receptors in post-mortem human brain.

1. Adenosine A2-like binding sites were characterized in post-mortem human brain membranes by examining several compounds for their ability to displace [3H]-CGS 21680 (2[p-(2 carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) binding. 2. Two A2-like binding sites were identified in the striatum. 3. The more abundant striatal site was similar to the A2a receptor previously described in rat striatum, both in its pharmacological profile and striatal localization. 4. The less abundant striatal site had a pharmacological profile similar to that of the binding site characterized in the other brain regions examined. This was intermediate in character between A1 and A2 and may represent another adenosine receptor subtype. 5. The co-purification of [3H]-CGS 21680 binding during immunoisolation of human striatal cholinergic membranes was used to assess the possible cholinergic localization of A2-like binding sites in the human striatum. Only the more abundant striatal site co-purified with cholinergic membranes. This suggests that this A2a-like site is present on cholinergic neurones in the human striatum.     

Expression of acetoacetyl-CoA synthetase,a novel cytosolic ketone body-utilizing enzyme,in human brain

Acetoacetyl-CoA synthetase (AACS, acetoacetate-CoA ligase, EC 6.2.1.16) is a ketone body-utilizing enzyme, the physiological role of which remains unclear yet in mammals, particularly has never been studied in human. In order to investigate the tissue distribution of AACS in human, cDNA encoding AACS was isolated from HepG2 cells. Amino acid sequence of human AACS deduced from the open reading frame showed high homology (89.3%) with that of rat AACS and much less homology (43.7%) with that of bacterial AACS. The expression level of the AACS mRNA was high in kidney, heart and brain, but low in liver, and the expression profile of AACS in the human brain was quite similar to that of 3-hydroxy-3-methylglutaryl-CoA reductase.     

Quinonoid dihydropterin reductase--II. Regional and subcellular distribution of rat brain enzyme.

The regional and subcellular distribution of quinonoid dihydropterin reductase (DHPR, EC 1.6.99.7) was studied in the rat brain. In addition, the subcellular distribution of the enzyme was studied in the rat liver. The activity of rat brain DHPR was found to be lowest in the cerebral cortex (0.14 unit mg^?1) and highest in the posterior colliculus (2.35 units mg^?1). The distribution of brain DHPR correlated positively with the distribution of aromatic-l-amino acid decarboxylase but not with the distribution of tyrosine hydroxylase or tryptophan deearboxylase. However, this correlation was not observed in the caudate nucleus which contains low DHPR activity and high aromatic-l-amino acid decarboxylase and tyrosine hydroxylase activities. This finding is an indication that the amount of DHPR in the caudate nucleus may be rate limiting. The distribution profiles of brain and liver DHPR were found to be similar to that of lactate dehydrogenase; for both enzymes the greatest specific activity was found in the soluble fraction, indicating that DHPR is a cytoplasmic enzyme.     

Microsomal cytochrome P450 in human brain regions.

Cytochrome P450 (P450) levels were quantitated in microsomes from human brain regions obtained at autopsy. The reduced carbon monoxide binding spectra of cortical microsomes showed two absorption maxima at 449 and 425 nm. On solubilization of the microsomes, essentially a single peak was observed at 449 nm. The P450 levels in human brain cortical microsomes varied from 0.03 to 0.12 nmol/mg protein among the seven samples examined. The concentration of the hemeprotein present as nmol/g tissue was highest in the brain stem and cerebellum and lowest in the striatum and hippocampus.     

N-acetyl-L-asparagine in human brain

A substance identical with N-acetyl-L-asparagine was isolated in pure form from an aqueous extract of human brain. The substance was isolated by a combination of an ion exchange and paper chromatography. The substance reacts avidly with chlorine-starch-iodide but does not react with ninhydrin. Upon acid hydrolysis it yields only aspartic acid and ammonia. An acetyl hydrazide was identified by paper chromatography. The unknown had the L-configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for authentic N-acetyl-L-asparagine.     

Angiotensin-converting enzyme: presence of high activity in choroid plexus of mammalian brain.

The activity of angiotensin-converting enzyme in rat choroid plexus was higher than that of any other organ, being 6--7 times higher than that in lung and more than 50 times higher than in any other region of brain. Rabbit choroid plexus also had high activity of enzyme while that of human choroid plexus was relatively low. The enzyme in rat choroid plexus showed similar biochemical properties to that in other tissues; it was inhibited by the nonapeptide SQ 20,881, by (Sar1-Ala8)-angiotensin II and by EDTA, and required chloride ions for activity. As in other tissues, the choroid plexus enzyme was associated with particulate fractions after differential centrifugation. The corpus striatum and substantia nigra had the highest activities in the various brain regions examined.     

Functional pharmacology in human brain

Most neurological and psychiatric disorders involve selective or preferential impairments of neurotransmitter systems. Therefore, studies of functional transmitter pathophysiology in human brain are of unique importance in view of the development of effective, mechanism-based, therapeutic modalities. It is well known that central nervous system functional proteins, including receptors, transporters, ion channels, and enzymes, can exhibit high heterogeneity in terms of structure, function, and pharmacological profile. If the existence of types and subtypes of functional proteins amplifies the possibility of developing selective drugs, such heterogeneity certainly increases the likelihood of interspecies differences. It is therefore essential, before choosing animal models to be used in preclinical pharmacology experimentation, to establish whether functionally corresponding proteins in men and animals also display identical pharmacological profiles. Because of evidence that scaffolding proteins, trafficking between plasma membrane and intracellular pools, phosphorylation and allosteric modulators can affect the function of receptors and transporters, experiments with human clones expressed in host cells where the environment of native receptors is rarely reproduced should be interpreted with caution. Thus, the use of neurosurgically removed fresh human brain tissue samples in which receptors, transporters, ion channels, and enzymes essentially retain their natural environment represents a unique experimental approach to enlarge our understanding of human brain processes and to help in the choice of appropriate animal models. Using this experimental approach, many human brain functional proteins, in particular transmitter receptors, have been characterized in terms of localization, function, and pharmacological properties.     

Dopamine metabolites in human brain

The relationship of human brain levels of 3,4-dihydroxyphenylacetic acid (DOPAC) to cerebrospinal fluid levels of this dopamine (DA) metabolite was studied. The effect of postmortem delay was evaluated in the rat. DOPAC was resistant to postmortem changes in brain kept in situ. The level of DOPAC (free and conjugated) determined in DA-rich areas of six human brains amounted to only a fraction of the homovanillic acid (HVA) found in the same regions. The DOPAC/HVA ratio in human brain was similar to that found in CSF. We conclude that HVA is the major DA metabolite in human brain and that DA metabolite levels in CSF reflect DA metabolite levels in brain.     

Glutathione metabolizing enzyme activities in human thyroid

Glutathione peroxidases, glutathione transferase, glutathione reductase and gamma-glutamyl transpeptidase activities were analyzed in human thyroid tissues obtained from 17 patients undergoing resectional surgery because of a malignancy. It was deduced, from measurements of glutathione peroxidase activity with both H202 and cumene hydroperoxide, that thyroid contains only the selenium enzyme. The absence of selenium independent glutathione peroxidase activity in thyroid was confirmed with gel filtration experiments. An interindividual variation of about 28-fold was found measuring glutathione transferase activity with 1-chloro-2,4-dinitrobenzene. Subjecting a fraction of human thyroid cytosols partially purified by G-100 Sephadex column to isoelectricfocusing run, a single peak of glutathione transferase activity centered at pH 4.6 was obtained. An adequate level of glutathione reductase and gamma-glutamyl transpeptidase activities was also found in all specimens investigated.     

Subclassification of release-regulating alpha 2-autoreceptors in human brain cortex.

1. Release-regulating alpha 2-autoreceptors in human brain were characterized pharmacologically in cortical slices from patients undergoing neurosurgery to remove subcortical tumours; the slices were prelabelled with [3H]-noradrenaline ([3H]-NA) and stimulated electrically (3 Hz, 2 ms, 24 mA) under superfusion conditions. 2. The stimulus-evoked tritium overflow was almost totally Ca(2+)-dependent and tetrodotoxin-sensitive. 3. Clonidine and oxymetazoline 0.01 to 1 microM inhibited in a concentration-dependent manner the evoked overflow of tritium. The two drugs were equipotent (EC50 = 0.03 microM) and their maximal effect was approx. 45%. Phenylephrine and methoxamine, up to 1 microM, did not affect tritium overflow. 4. Yohimbine (0.01-0.1 microM) shifted the concentration-response curve of clonidine to the right. The calculated pA2 value was 8.29. 5. Prazosin and 2-[2-[4-(o-methoxyphenyl)piperazine-1-yl]ethyl]-4,4- dimethyl-1,3(2H,4H)-isoquinolinedione (AR-C 239), tested at 0.3 microM, did not modify the concentration-response curve of clonidine. 6. The effect of clonidine was antagonized by (+)-mianserin (pA2 = 7.74), but not by up to 0.3 microM of the (-)-enantiomer. The concentration-response curve of clonidine was shifted to the right by the novel alpha 2-adrenoceptor antagonist, 5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-ami ne (Z)-2-butenedioate (1:1) salt (ORG 20350) (pA2 = 7.55). 7. Yohimbine, (+)-mianserin and ORG 20350, but not prazosin and (-)-mianserin, increased the electrically-evoked tritium overflow, suggesting that autoreceptors may be tonically activated by endogenous NA. 8. Desipramine (1 microM) increased evoked tritium overflow from human cortex slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of aldehyde reductase isoenzymes in human and rat brain.

Anticonvulsant drugs, such as barbiturates, open-chained analogues of barbiturates, glutethimide, succinimides and hydantoins have been tested in vitro as inhibitors of the isoenzymes of aldehyde reductase from human and rat brain. One major isoenzyme of both species is highly sensitive to the ionizable forms of these drugs containing the CONHCO grouping and a minimal lipophilic substitution. Differences between the isoenzymes were observed with respect to the absolute configuration of various succinimides as inhibitors. One isoenzyme of both species exerts activity with NADH as well as NADPH. The NADH-dependent activity of the human enzyme is inhibited in a noncompetitive way by NADP with a K_i-intercept of 2.2 × 10^?7 M. A mixed inhibition is obtained with the biogenic acid, 4-hydroxyphenylacetic acid, as inhibitor of the main human isoenzyme. The inhibition is uncompetitive up to [I] = 1 × 10^?3 M and yields a K_i value of 4.2 × 10^?4M.     

Effects of azapropazone on pain-related brain activity in human subjects.

1. The dose-related effects of azapropazone on (i) event-related and spontaneous EEG-activity and (ii) the subjects' pain ratings were investigated using an experimental human pain model based on both chemo-somatosensory event-related potentials (CSSERP) and subjects' pain ratings. 2. Healthy subjects (n = 20) participated in a placebo-controlled, randomized, double-blind, four-way cross-over study. Single doses of azapropazone (300 mg, 600 mg and 1200 mg) and placebo were administered intravenously. Each experiment consisted of five sessions (before and 1, 2, 4 and 8 h after administration of the medication). Each session lasted for approximately 40 min. In the first 20 min, pain was induced by short CO2-stimuli presented to the right nostril (phasic pain; interstimulus interval 30 s) and EEG was recorded from five positions. CSSERPs were obtained in response to painful CO2-stimuli. In the following 20 min period, tonic pain was induced by a constant stream of dry air introduced in the left nostril. Subjects rated the intensity of both phasic and tonic pain by means of a visual analogue scale. Additionally, a frequency analysis of the spontaneous EEG was performed. 3. Azapropazone reduced the pain-related CSSERP-amplitudes at frontal and parietal recording positions. This topographical pattern was observed in previous studies with opioids, while NSAIDs such as flurbiprofen and ketoprofen exerted effects at frontal and central positions. In contrast to other NSAIDs, administration of azapropazone resulted in a reduction of the frequency bands alpha 1, delta and theta of the spontaneous EEG. At the subjective level, analgesic effects of azapropazone were observed in the ratings of tonic pain. 4. Analgesic properties of azapropazone were demonstrated in man. The topographical pattern of the changes in the CSSERPs and the effects on EEG background activity suggest a central component of the analgesic action of azapropazone.     

Nucleoside-catabolizing enzyme activities in primary rabbit kidney cells and human skin fibroblasts.

Primary rabbit kidney (PRK) and human skin fibroblast (HSF) cell cultures, two cell systems which are regularly employed in our laboratory to explore the antiviral properties of nucleoside analogues, were analyzed for the specific activities of the following nucleoside catabolizing enzymes: cytidine deaminase (EC 3.5.4.5), pyrimidine nucleoside phosphorylases (uridine phosphorylase: EC 2.4.2.3, and 2′-deoxythymidine phosphorylase: EC 2.4.2.4), purine nucleoside phosphorylase (EC 2.4.2.1) and adenosine deaminase (EC 3.5.4.4). No cytidine deaminase activity was detected in either PRK or HSF cells. Likewise, no 2′-deoxythymidine phosphorylase activity could be demonstrated in PRK and HSF cells. PRK cells contained low levels of uridine phosphorylase (3–;5 U/mg protein) and high levels of purine nucleoside phosphorylase (～ 100 U/mg protein). For both PRK and HSF cells, relatively high adenosine deaminase activities were recorded (at an average 20 and 36 U/mg protein, respectively). Infection of the PRK or HSF cell cultures with either vaccinia virus, herpes simplex virus (type 1 or 2) or vesicular stomatitis virus at high multiplicity of infection (MOI ～ 1) did not bring about marked changes in any of the enzymatic activities tested. For uridine phosphorylase (from PRK cells) and adenosine deaminase (from both PRK and HSF cells) the substrate specificities were determined with various uridine and adenosine analogues.     

The effect of angiotensin-converting enzyme inhibitors on human neutrophil chemotaxis in vitro.

1. Myocardial 'reperfusion injury' has been partly attributed to the production of free radicals which are cytotoxic towards cells. Neutrophils are recruited by ischaemic tissue and are one source of free radicals. Angiotensin-converting enzyme (ACE) inhibitors can reduce 'reperfusion injury' and we decided to determine if ACE inhibitors might contribute to this effect by inhibiting neutrophil chemotaxis. 2. The effects of captopril (a thiol containing ACE inhibitor) and enalaprilat (a nonthiol ACE inhibitor) and N-mercaptopropionyl glycine (MPG) (a simple thiol) on neutrophil chemotaxis were tested in an in vitro Boyden chamber assay. 3. The chemotactic response of human neutrophils to fMLP was reduced by 27.6% with MPG (n = 8; P < 0.05), by 13.2% with enalaprilat (n = 8; P = 0.075) and by 5.2% with captopril (n = 8; P = 0.66) at 5 microM (therapeutic concentration.) 4. Neutrophil chemotaxis was significantly decreased with 50 microM and 500 microM MPG and enalaprilat and 500 microM captopril. 5. Supratherapeutic concentrations of ACE inhibitors can reduce neutrophil chemotaxis at high concentrations and this effect does not appear to be -SH dependent.     

人胎肾上腺,肝和肾内谷胱甘肽相关酶活性

目的：通过谷胱甘肽相关酶活性及其在亚细胞分布，了解胎肾上腺在发育期间对活性代谢产物的解毒处理能力。方法：制备肾上腺、肝亚细胞组分。测定谷胱甘肽转移酶（ＧＳＴ）、还原酶、过氧化物酶。结果：ＧＳＴ在胎肾上腺微粒体、线粒体、胞浆中含量分别是肝各亚细胞组分中的３７３％、２７０％和民６７％、肾上腺微粒体ＧＳＴ活性与细胞色素Ｐ－４５０、与氨基比林脱甲基酶活性皆呈正相关，肾上腺线粒体谷胱甘肽还原酶、过氧化物分别