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Biomedical subjects

A R Johnson

Publications and source records attributed to A R Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1989

Regulation of synthesis of prostacyclin and HETEs in human endothelial cells.

Human umbilical endothelial cells in culture synthesize prostacyclin (PGI2), 15-hydroxyeicosatetraenoic acid (15-HETE), and 12-hydroxyeicosatetraenoic acid (12-HETE). The synthesis of these eicosanoids was measured by specific radioimmunoassays after stimulation by arachidonic acid, A23187, bradykinin, melittin, or histamine. Under all conditions, the synthesis of PGI2 paralleled and exceeded the synthesis of 15-HETE and 12-HETE. Indomethacin inhibited arachidonic acid-stimulated PGI2 and 15-HETE synthesis but enhanced 12-HETE synthesis. Meclofenamate gave similar qualitative results. Drugs that act as inhibitors of lipoxygenase in some tissues, such as nordihydroguaiaretic acid (NDGA), caffeic acid, esculin, diethylcarbamazine, quercetin, and 5,8,11,14-eicosatetrayenoic acid (ETYA) were nonspecific in their inhibition of PGI2, 12-HETE, and 15-HETE synthesis. For example, NDGA inhibited arachidonic acid-stimulated release with a 50% inhibitory concentration (IC50) of 0.39 microM for PGI2, 0.25 microM for 15-HETE, and 0.10 microM for 12-HETE. These results show that endothelial cells metabolize both endogenous and exogenous arachidonic acid to PGI2, 15-HETE, and 12-HETE. These data also suggest, based on results with inhibitors, that PGI2 and 15-HETE are products of cyclooxygenase, whereas 12-HETE is produced via a different enzymatic pathway, most likely a lipoxygenase pathway.

6-Ketoprostaglandin F1 alpha↗
PubMed1988

Function of neutral endopeptidase on the cell membrane of human neutrophils.

Intact human neutrophils hydrolyzed N-formyl-Met-Leu-[3H]Phe (fMLP) and released Leu-[3H]Phe, cleaving 45-50% of the peptide within 20 min at 37 degrees C. The dipeptide after its release was then hydrolyzed to free amino acids by a dipeptidase (EC 3.4.13.11). This activity, present in plasma membrane-enriched fractions of neutrophil lysates, was also inhibited over 90% by phosphoramidon, an inhibitor of neutral endopeptidase (NEP, EC 3.4.24.11). Dithiothreitol and EDTA inhibited the activity to a comparable degree, suggesting the requirement for a heavy metal cofactor. Bestatin and amastatin, inhibitors of aminopeptidases (but not human kidney NEP), did not inhibit the rate of fMLP degradation but prevented the production of free phenylalanine and enhanced the accumulation of Leu-Phe. Of other inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin slightly enhanced the rate of fMLP hydrolysis by neutrophils, and others tested were ineffective. Rabbit antiserum to homogeneous human kidney NEP reacted specifically with a 100-kDa protein present in sodium dodecyl sulfate-solubilized neutrophils. The Mr of this protein was slightly larger than that of the kidney enzyme in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum incubated with intact cells specifically inhibited the degradation of fMLP over 70%. First, we confirm that NEP present on the plasma membrane cleaves fMLP at the Met-Leu bond; then the dipeptide Leu-Phe is cleaved by a dipeptidase. Finally, inhibition of NEP completely blocks fMLP-mediated chemotaxis. Thus, the enzyme may play an important role in modulating chemotactic responses.

Anti-Bacterial Agents↗
PubMed1988

Effect of platelet activating factor on leukocyte-endothelial cell interactions.

The proinflammatory effects of platelet activating factor (PAF) that impact upon tissue inflammation were studied in vitro using the adherence of human neutrophils to endothelium and the increase in macromolecule permeability of endothelial monolayers. PAF produced both a time- and dose-dependent increase in neutrophil-endothelial cell adhesion. The adhesion promoting properties observed were primarily due to an effect of PAF on endothelium and not on neutrophils and was independent of endothelial cell cyclooxygenase products. The PAF receptor antagonist kadsurenone inhibited the adhesion response suggesting endothelial surface PAF receptors are involved in these responses. Whereas PAF alone did not alter endothelial cell barrier properties, leukocyte activation (neutrophil and platelets) with PAF produced significant increases in 125I-albumin clearance across endothelial monolayers. These studies suggest that PAF has potent proinflammatory effects and that it can play a significant role in the endothelial response to injury.

Animals↗
PubMed1988

Neither laminin nor prior optic nerve section are essential for the regeneration of adult mammalian retinal ganglion cell axons in vitro.

Retinal explants obtained from normal adult rats and from operated animals in which the optic nerve had been sectioned 10 days previously were cultured in either serum-containing or serum-free medium on poly-L-lysine and laminin substrata. Regenerating ganglion cell axons growing from these explants have been identified using monoclonal antibodies against Thy-1.1 cell surface glycoprotein and the 200-kDa subunit neurofilament protein. Irrespective of substratum or medium composition, axons regenerated from 28-49% of normal rat retinal explants. This percentage increased to 60-84% of explants from operated rats. There were no significant differences in percentages of explants from normal or operated rats showing neurite outgrowth when substrata of either poly-L-lysine or laminin were compared in serum-free medium. In serum-containing medium the results were less easily interpreted due to the presence of an outgrowth of non-neuronal (glia and mesenchymal) 'flat cells', which served as a preferred axonal substratum in many cases. Thus we show that adult rat retinal ganglion cell axons will regrow in vitro, and that a 'priming' optic nerve section will increase this response. In neither case is the response laminin-dependent.

Animals↗
PubMed1988

Cultured bovine coronary arterial endothelial cells synthesize HETEs and prostacyclin.

Arachidonic acid metabolism was examined in endothelial cells cultured from bovine coronary arteries. In culture, these cells exhibit specific characteristics of endothelial cells. They form a contact-inhibited monolayer with a cobblestone appearance, contain immunoreactive von Willebrand's factor antigen, and have angiotensin I converting enzyme activity. Prostacyclin was the major prostaglandin synthesized from exogenous and endogenous arachidonic acid in these cells. In addition, exogenous arachidonic acid was metabolized to small amounts of prostaglandin E2 (PGE2) and several relatively nonpolar metabolites including 12-, 15-, and 11-hydroxyeicosatetraenoic acids (12-, 15-, and 11-HETE). Histamine, bradykinin, and thrombin increased PGI2 synthesis in these bovine coronary endothelial cells. Of these agonists, bradykinin was the most potent, increasing basal PGI2 release by fourfold. More vigorous stimulation of the cells with mechanical disruption of the cell monolayer, melittin, or A23187 resulted in release of both PGI2 and PGE2. Pretreatment of cells with exogenous arachidonic acid (10(-5) M) abolished their responsiveness to subsequent stimulation by arachidonic acid or vasoactive agents, but not PGH2. Furthermore, treatment of cells with 15-HPETE (10(-7)-10(-4) M), but not 15-HETE, specifically inhibited basal as well as A23187-stimulated PGI2 release. PGE2 release was increased slightly after 15-HPETE treatment. These studies indicate that bovine coronary endothelial cells can metabolize arachidonic acid to several biologically active products and that PGI2 synthesis by these cells is specifically related to the type of vasoactive agent employed. Both the qualitative pattern and quantity of eicosanoids synthesized by bovine coronary endothelial cells differ substantially from endothelial cells isolated from noncardiac vascular beds.

Animals↗
PubMed1988

Histamine stimulation of prostaglandin and HETE synthesis in human endothelial cells.

Endothelial cells (EC) cultured from human umbilical artery (UA) and vein (UV) metabolized [14C]arachidonic acid to prostaglandins (PGs), monohydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs). Major radioactive products were identified as 6-keto-PGF1 alpha, PGE2, PGF2 alpha, 12-hydroxy heptadecatrienoic acid, 15-HETE, and 11-HETE. In addition, extracts from UV ECs contained 12-HETE, 5-HETE, 14,15-EET, and 5,6-EET as minor products, whereas extracts from UA ECs contained only 12-HETE as a minor product. UA ECs also produced metabolites comigrating with 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. Histamine increased the release of [14C]PGs and [14C]HETEs from [14C]arachidonic acid-labeled ECs. Indomethacin, aspirin, and nordihydroguauretic acid completely inhibited synthesis of both [14C]PGs and [14C]HETEs from exogenous [14C]arachidonic acid in these cells. Microsomes metabolized [14C]arachidonic acid to the same [14C]PGs and [14C]HETEs as intact cells. Pretreatment of microsomes with indomethacin completely inhibited formation of these products. These data indicate that UA ECs and UV ECs metabolize endogenous and exogenous arachidonic acid to both PGs and HETEs. Also 15-HETE and 11-HETE appear to be synthesized by a microsomal enzyme with the properties of cyclooxygenase.

Arachidonic Acids↗
PubMed1987

Prostaglandin synthesis in bovine coronary endothelial cells: comparison with other commonly studied endothelial cells.

Comparison of arachidonic acid metabolism by bovine coronary artery endothelial cells, bovine aortic endothelial cells and human umbilical endothelial cells indicated potentially important differences in relative amounts of the different prostaglandins produced. Bovine coronary endothelial cells converted 14C-arachidonic acid to radioactive 6-keto PGF1 alpha (the stable metabolite of PGI2) and to a lesser extent PGE2. Bovine aortic cells synthesized 6-keto PGF1 alpha and 6,15-diketo PGF1 alpha as the major products. PGE2, 6-keto PGE1, PGF2 alpha and PGD2 were minor metabolites. By comparison, endothelial cells isolated from human umbilical artery or vein formed mainly 6-keto PGF1 alpha and substantial amounts of PGF2 alpha, PGE2 and PGD2. Basal concentrations of 6-keto PGF1 alpha were two-fold higher in bovine coronary cells than in bovine aortic endothelial cells, but seven-fold less than in endothelial cells cultured from human umbilical vessels. Histamine, bradykinin and thrombin stimulated PGI2 synthesis in both coronary endothelial cells and human umbilical cells, but only bradykinin stimulated PGI2 synthesis in bovine aortic cells. This comparative study indicates that endothelial cells vary in the metabolites of arachidonic acid that they produce depending upon the vascular origin of the cells. Also, endothelial cells from different vascular beds respond differently to specific vasoactive agents.

Animals↗
PubMed1987

Steroid 5 alpha-reductase activity in endothelial cells from human umbilical cord vessels.

The metabolism of radiolabeled progesterone and androstenedione was evaluated in endothelial cells from human umbilical cord vein and arteries maintained in culture. The predominant metabolite of progesterone was 5 alpha-pregnane-3,20-dione and that of androstenedione was 5 alpha-androstane-3,17-dione. Thus, the major pathway of progesterone and androstenedione metabolism within these cells is via steroid 5 alpha-reductase. The rate of formation of 5 alpha-pregnane-3,20-dione from progesterone by venous endothelial cells was linear with incubation time up to 4 h and with cell number up to 1.6 X 10(6) cells/ml. The apparent Km of 5 alpha-reductase for progesterone was 0.4 microM; and, the Vmax was 55 pmol 5 alpha-pregnane-3,20-dione formed/mg protein X h. The rate of 5 alpha-androstane-3,17-dione formation from androstenedione also was linear with incubation time up to 4 h. In addition to 5 alpha-androstane-3,17-dione, the metabolism of androstenedione by either venous or arterial cells resulted in the formation of various minor metabolites, including testosterone and 5 alpha-reduced steroids, viz. 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, and 5 alpha-androstane-3 beta, 17 beta-diol. Estrogens (i.e. estradiol-17 beta and estrone) were not detected as products of androstenedione metabolism. The formation of these metabolites are indicative that the steroid-metabolizing enzymes present in endothelial cells are: 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase, 3 alpha-hydroxysteroid oxidoreductase, and 3 beta-hydroxysteroid oxidoreductase.

5-alpha-Dihydroprogesterone↗
PubMed1986

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells.

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.

Animals↗
PubMed1986

Androstenedione metabolism in human lung fibroblasts.

Human lung fibroblasts in culture metabolized [3H]androstenedione to a number of different compounds, including testosterone, 5 alpha-androstanedione, androsterone, 5 alpha-dihydrotestosterone, isoandrosterone, and 5 alpha-androstane-3 alpha,-17 beta-diol. The major products were 5 alpha-androstanedione and testosterone. Estrone, estradiol-17 beta and 5 beta-reduced steroids were not formed. The production rates of testosterone and 5 alpha-androstanedione from [3H]androstenedione by lung fibroblasts were studied both as a function of incubation time and substrate concentration. The rates of formation of testosterone and 5 alpha-androstanedione remained linear with time up to 4 h. The apparent Km of human lung fibroblast 5 alpha-reductase was 1 microM, and that of 17 beta-hydroxysteroid oxidoreductase was 11 microM. The findings of this study suggest that mesenchyma may contribute to the metabolism of androstenedione in human lung tissue.

Androstenedione↗
PubMed1986

Adrenal masses differentiated by MR.

Magnetic resonance imaging was performed in 30 patients with adrenal masses. The abnormalities included adrenal adenomas (n = 10), carcinomas (n = 2), pheochromocytomas (n = 12), and adrenal metastases (n = 6). By the ratio of the signal intensity of the adrenal mass to that of the liver, adenomas could be distinguished from adrenal metastases, adrenal carcinomas, and pheochromocytomas. Metastases and pheochromocytomas could generally be differentiated.

Adenoma↗
PubMed1986

Inhibition of prostaglandin synthesis by glucocorticoids in human endothelial cells.

The vasodilator prostaglandins (PGs), prostacyclin (PGI2) and PGE2, may contribute to the inflammatory response. Because glucocorticosteroids reduce inflammation, possibly through inhibition of arachidonic acid release, we examined the influence of dexamethasone on PG formation in cultures of human endothelial cells. Binding of [3H]dexamethasone by intact cells was competed by unlabeled steroids and was half-maximal at 1.2 X 10(-8) M. A cytosolic fraction complexed with [3H]dexamethasone and migrated on sucrose density gradient centrifugation with a sedimentation coefficient of 8S. 3H-steroid binding was diminished by unlabeled steroid. Histamine, bradykinin, and the ionophore, A23187, stimulated release of PGI2 and PGE2 to as much as 25 times basal release. Dexamethasone (10(-11) to 10(-7) M) reduced PG formation in cells that were stimulated by histamine, bradykinin, calcium ionophore, or mechanical agitation. The inhibitory effect required at least 4 h to develop, was maximal at 24 h, and persisted after the steroid was removed. Hydrocortisone and triamcinolone had similar effects but were less potent than dexamethasone. Testosterone and progesterone did not affect PG generation. Both arachidonic acid and PGH2 augmented formation of PGs but were not inhibited by dexamethasone. Cortisol-21-mesylate, an antagonist of glucocorticoid receptors, blocked the effects of dexamethasone on PG formation, as did treatment of the cells with cycloheximide. We conclude that glucocorticoids inhibit PG production in endothelial cells by interaction with specific steroid receptors. The steroid-mediated inhibitory effect occurs at the level of arachidonic acid release and depends upon protein synthesis.

6-Ketoprostaglandin F1 alpha↗
PubMed1986

Tailgut cyst: diagnosis with CT and sonography.

Tailgut cyst is a rare congenital lesion that presents as a presacral mass, found in infancy or persisting undetected into adulthood, and characterized by multiple cysts lined with a gastrointestinal type of epithelium. In a retrospective review of five patients whose ages ranged from 1 month to 42 years--three cases with CT and two with sonograms--tailgut cyst appeared as a complex mass on both procedures, representing its composition of multiple small cysts filled with keratinous material and other debris. Other CT and sonographic features include well-defined borders, and, in one case, presentation as a mass lateral to the midline. CT also shows preservation of adjacent fat planes. Tailgut cyst should be included in the differential diagnosis of a retrorectal mass at any age.

Adolescent↗
PubMed1985

Synthesis of dihomoprostaglandins from adrenic acid (7,10,13,16-docosatetraenoic acid) by human endothelial cells.

Human umbilical vein endothelial cells were found to contain adrenic acid (22:4) in their cellular lipids. Since this fatty acid may be metabolized by cyclooxygenase in the kidney, the metabolism of adrenic acid was studied in endothelial cell cultures. [14C]Adrenic acid was metabolized to several more polar metabolites. Two of these metabolites co-migrated on HPLC with 1 alpha,1 beta-dihomo-8-ketoprostaglandin F1 alpha (the metabolite of 1 alpha, 1 beta-dihomoprostaglandin I2) and 1 alpha,1 beta-dihomoprostaglandin E2. Indomethacin (10(-5) M) inhibited the synthesis of these metabolites. When cells were treated with adrenic acid (3 X 10(-5) M), a peak that co-migrated with dihomo-8-ketoprostaglandin F1 alpha was detected by radioimmunoassay using an antiserum directed against 6-ketoprostaglandin F1 alpha. The presence of dihomo-8-ketoprostaglandin F1 alpha was confirmed by gas chromatography-mass spectrometry. Immunoreactive peaks that co-migrated with dihomoprostaglandins E2 and F2 alpha were identified with antisera against prostaglandin E2 and prostaglandin F2 alpha, respectively. [14C]Arachidonic acid was metabolized to [14C]prostaglandin F2 alpha, 6-keto[14C]prostaglandin F1 alpha, and [14C]prostaglandin E2. Similar results were found with unlabelled arachidonic acid using specific antisera. When the two fatty acids were combined, adrenic acid reduced the metabolism of arachidonic acid. The culture media from endothelial cells inhibited thrombin-induced platelet aggregation, an effect blocked by aspirin. The inhibitory activity of the media was enhanced when arachidonic acid was added to the cells, but it was reduced by adrenic acid. Both prostaglandin I2 and dihomoprostaglandin I2 inhibited platelet aggregation, but prostaglandin I2 was 100-times more potent. We conclude that adrenic acid is metabolized in human endothelial cells to 1 alpha, 1 beta-dihomoprostaglandins and can compete with endogenous arachidonic acid for conversion by cyclooxygenase. These findings suggest that adrenic acid may reduce the formation of prostaglandin I2 by the blood vessel.

Arachidonic Acid↗
PubMed1985

Rapid method for quantitation of androgen binding protein in Sertoli cell cultures and its use for measurement of binding kinetics.

The accurate measurement of the kinetics of binding of 5 alpha-dihydrotestosterone to the Sertoli cell specific protein, androgen binding protein (ABP), has been frustrated by the extremely rapid rate of dissociation of the ABP-dihydrotestosterone complex. We describe a rapid and highly sensitive assay suitable for ABP quantitation which utilizes DEAE Bio-Gel and [3H]dihydrotestosterone. The assay has been used to accurately measure the rate of dissociation (8.25 X 10(-4) s-1, t1/2 14 min) and the rate of association (2.04 X 10(5) M s-1) of the binding of [3H]dihydrotestosterone to rat ABP. The ratio of these rate constants is in perfect agreement with the equilibrium dissociation constant determined by Scatchard analysis (4.0 nM). This multipoint assay is extremely rapid such that binding can be measured at equilibrium, it has high precision (coefficient of variation 3%), and is particularly useful at low protein concentrations (50 ng/ml); furthermore, the assay background of nonspecific 3H-binding is extremely low (0.2%). Since at such low protein concentrations a 10 point Scatchard analysis can be performed on 1 ml culture medium containing as little as 3 fmol ABP, the assay is suitable for monitoring changes in ABP secretion resulting from manipulations of cells in culture. The assay which utilizes DEAE Bio-Gel A is compared to five alternative methods: the standard method of steady state gel electrophoresis, Dextran-coated charcoal assay, hydroxylapatite assay, DEAE filter assay, and radioimmunoassay. The DEAE Bio-Gel assay has advantages over all of these alternative methods. In summary, this new assay is particularly useful for monitoring temporal changes in the secretion of ABP, and the method is equally effective in quantitating ABP in rat, rabbit and hamster Sertoli cell cultures.

Adsorption↗
PubMed1985

Effect of aging and cold temperature storage of hamster ova as assessed in the sperm penetration assay.

The penetration of zona pellucida-free hamster ova by human spermatozoa has been used to quantitate sperm penetration potential. However, since mammalian eggs in vitro have limited viability, the effect of in vitro aging on the ability of hamster ova to be penetrated by human spermatozoa was examined. Zona-free ova maintained at room temperature (25 degrees C) lost their ability to be subsequently penetrated with a half-life of 50.1 +/- 8.8 minutes. This was partly the result of removing the zona pellucida by trypsin digestion, since zona-free oocytes in the presence of trypsin inhibitor or zona pellucida-intact oocytes had half-lives of 99.1 +/- 15.2 and 120.5 +/- 17.4 minutes, respectively. Reduction in penetration rates associated with ovum aging did not appear to be due to loss of viability and could be completely prevented by maintaining the ova on ice (4 degrees C). In the presence of TEST-yolk buffer at 4 degrees C, ova retained (100%) their ability to be penetrated for up to 24 hours and were morphologically indistinguishable from fresh ova. These observations show that ovum aging in vitro at 25 degrees C is much greater than previously anticipated. This may result in artifactually low and variable scores in the penetration bioassay.

Animals↗