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Biomedical subjects

J R Falck

Publications and source records attributed to J R Falck.

At least 199 records · Page 11Linked to original sources
PubMed1992

Resolution of dihydroxyeicosanoates and of dihydroxyeicosatrienoates by chiral phase chromatography.

A chromatographic method is described for the direct enantiomeric characterization of 5,6-, 8,9-, 11,12-, and 14,15-vic-dihydroxyeicosatrienoic acids (DHETs), metabolites of the cytochrome P-450 arachidonate epoxygenase pathway, and of their corresponding saturated vic-dihydroxyeicosanoic acids. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral-phase chromatography utilizing a Chiralcel OC or OD column. This methodology will find analytical and preparative applications since it is simple and efficient and preserves, intact, the diol functionality.

8,11,14-Eicosatrienoic Acid↗
PubMed1992

Brain synthesis and cerebrovascular action of epoxygenase metabolites of arachidonic acid.

The purpose of this study was to determine if whole brain makes epoxygenase metabolites of arachidonic acid and, if so, whether they are vasoactive on the cerebral microcirculation. Blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, and the extracted metabolites were resolved by HPLC. Metabolite structures were confirmed by gas chromatography/mass spectrometry. In addition to prostaglandins, leukotriene B4, and hydroxyeicosatetraenoic acids, mouse brain metabolized arachidonic acid into several other compounds. Among them, we identified 5,6- and 14,15-epoxyeicosatrienoic acid. Next, we tested the effect of topical application of brain-synthesized 5,6-epoxyeicosatrienoic acid and synthetic epoxyeicosatrienoic acids on in vivo rabbit cerebral arteriolar diameter using the cranial window technique and in vivo microscopy. Brain-synthesized 5,6-epoxyeicosatrienoic acid caused a transient 28% arteriolar dilation, similar to that produced by 5 micrograms/ml of synthetic 5,6-epoxyeicosatrienoic acid. A concentration of synthetic 14,15- and 11,12-epoxyeicosatrienoic acid of 5 micrograms/ml CSF had little or no effect on diameter, whereas 8,9-epoxyeicosatrienoic acid caused a maximum dilation of 8%. These studies suggest that brain-synthesized 5,6-epoxyeicosatrienoic acid may play a role in the normal or pathophysiological regulation of the cerebral microcirculation.

8,11,14-Eicosatrienoic Acid↗
PubMed1992

Mechanism of action of cerebral epoxyeicosatrienoic acids on cerebral arterial smooth muscle.

Microsomal preparations of cat brain incubated with [14C]arachidonic acid produced epoxyeicosatrienoic acids (EETs) that eluted with the same retention times as synthetically prepared 5,6-, 8,9-, and 11,12-EETs. These compounds dilated serotonin-preconstricted, pressurized cat cerebral arteries in a dose-dependent fashion. Epoxide formation was not found in mitochondrial fractions and was dependent on the presence of NADPH. The maximum effects of 8,9-EET and 11,12-EET were greater than those of 5,6-EET. The cellular basis of this vasodilation was further investigated by examining the effects of 8,9-EET and 11,12-EET on K+ channel activity in vascular muscle cells freshly isolated from cat cerebral arteries. Both 8,9-EET and 11,12-EET increased the frequency of opening, mean open time, and open-state probability of a 98-pS K+ channel recorded in the cell-attached mode with 145 mM KCl in the pipette and 4.7 mM KCl in the bath. Blockade of K+ channel activity with tetraethylammonium attenuated the vasodilatory effects of 11,12-EET on serotonin-preconstricted cat cerebral arteries. These results suggest that endogenously formed EETs may participate in local regulation of cerebral blood flow by dilating cerebral arteries through a mechanism that involves activation of K+ channels.

8,11,14-Eicosatrienoic Acid↗
PubMed1992

Inhibition of renin secretion from rat renal cortical slices by (R)-12-HETE.

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) inhibits renin secretion both in vivo and in vitro, but the enzymatic origin of the 12-HETE responsible for renin inhibition is unknown. These studies examined the effect of the 12-HETE stereoisomers (R)-12-HETE (a product of the cytochrome P-450 monooxygenase enzyme system) and (S)-12-HETE (a product of the lipoxygenase enzyme system) on basal and stimulated renin secretion from superficial cortical slices in the rat. First, rat cortex was shown to produce 12-HETE; chiral-phase high-performance liquid chromatography revealed that cortex produced 81% (S)-12-HETE and 19% (R)-12-HETE. (R)-12-HETE reduced basal renin release by 28 +/- 4% to 49 +/- 5% at concentrations of 10(-9) to 10(-7) M (P < 0.05 to 0.01). (S)-12-HETE did not significantly affect renin release. (R)-12-HETE also blunted isoproterenol-stimulated renin secretion (P < 0.05) at a concentration of 10(-6) M. 20-HETE, another cytochrome P-450 product, did not exert a significant effect on renin release. In summary, both (R)-12-HETE and (S)-12-HETE are synthesized by renal cortical tissue. Only (R)-12-HETE directly inhibits in vitro renin release. These findings suggest that the renal cytochrome P-450 enzyme system is capable of directly influencing local renin secretion.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Cyclooxygenase dependency of the renovascular actions of cytochrome P450-derived arachidonate metabolites.

The renovascular effects of cytochrome P450-dependent arachidonic acid (P450-AA) metabolites synthesized by rat and rabbit kidneys were studied in the rabbit isolated kidney under conditions of constant flow and examined for their dependency on cyclooxygenase relative to their expression of vasoactivity. Kidneys were perfused with Krebs-Henseleit solution, and perfusion pressure was raised to levels of 90 to 110 mm Hg with the addition of 2 to 3 microM phenylephrine to the perfusate. Close arterial injection of 1 to 20 micrograms of 5,6-, 8,9- and 11,12-epoxyeicosatrienoic acid (EET) dose-dependently decreased perfusion pressure. The 5,6-EET was the most potent and the only epoxide dependent on cyclooxygenase for expression of vasoactivity, being inhibited by indomethacin (2.8 microM). In contrast, 14,15-EET resulted in dose-dependent increases in perfusion pressure. The vasodilator effects of the omega- and omega-1 oxidation products, 20-hydroxyeicosatetraenoic acid (HETE) and the stereoisomers of 19-HETE, were also inhibited by indomethacin. Furthermore, the renal vasodilator responses to 5,6-EET were not inhibited by either superoxide dismutase (10 U) or catalase (40 U) and, therefore, were unrelated to the formation of oxygen radicals generated during transformation of the epoxide by cyclooxygenase. As 5,6-EET and 19- and 20-HETE are synthesized by the renal tubules and can affect movement of salt and water, expression of vasoactivity by P450-dependent arachidonic acid metabolites, and after release from a nephron segment, may represent a mechanism that couples altered renal tubular function to appropriate changes in local blood flow.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

Metabolism of 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) in corneal tissues: formation of novel metabolites.

12(R)-Hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], a cytochrome P450 arachidonate metabolite, is metabolized by corneal tissues via three distinct metabolic pathways: beta-oxidation, omega-hydroxylation, and keto-reduction. The major metabolite released from the intact rabbit corneal epithelium or cultured cells was identified by mass spectrometric analysis as 8-hydroxy-4,6,10-hexadecatrienoic acid, the tetranor metabolite derived following two steps of beta-oxidation from the carboxy terminus. The beta-oxidation pathway was expressed in both microsomes and mitochondria isolated from bovine corneal epithelium and was dependent on the addition of oxidizing equivalents. The major metabolite of 12(R)-HETE in subcellular fractions of bovine corneal epithelial cells was a dihydro compound, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). This derivative is presumably formed by an oxidation of the hydroxyl group followed by two keto-reduction steps, since its formation was accompanied by the appearance of a keto metabolite identified as 12-oxo-5,8,14-eicosatrienoic acid. The omega-hydroxylation, in contrast to other cell types, was a minor route for 12(R)-HETE metabolism in these tissues. Since 12(R)-HETE has been implicated as a modulator of Na(+)-K(+)-ATPase activity and its related functions in ocular tissues, these findings raise the possibility that the newly described metabolites may be involved in regulating corneal functions. In addition, the presence of a keto reductase in the cornea may be of great importance following injury since 12(R)-HETrE resulting from 12(R)-HETE by this activity is a potent ocular proinflammatory compound.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

Detection of 20-hydroxyeicosatetraenoic acid in rat urine.

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonate metabolite of the cytochrome P450 omega hydroxylase, was detected in rat urine by gas chromatography-mass spectrometric techniques. The concentration of 20-HETE in urine from 7-week-old hypertensive and normotensive rats was 2.1 and 1.3 nM, respectively. This is the first demonstration of 20-HETE urinary excretion and thus calls attention to the possibility that 20-HETE participates in the regulation of renal function via its effect on vascular tone and ion transport processes.

Animals↗
PubMed1991

Modulation of erythropoiesis by novel human bone marrow cytochrome P450-dependent metabolites of arachidonic acid.

In the hematopoietic system the adherent stromal cells produce cytokines necessary for proliferation and differentiation of hematopoietic cells. In the present study, we showed the ability of adherent stromal cells to generate novel metabolites of arachidonic acid via the NADPH-cytochrome P450-dependent monooxygenase system. These metabolites were recovered in the incubation media, suggesting their release from cells. The formation of arachidonic acid metabolites was inhibited by 7-ethoxyresorufin and SKF-525A, but not by indomethacin or BW-755C. By using two-step high-pressure liquid chromatography (HPLC), bone marrow-adherent stromal cells and incubation media showed the presence of metabolites in a peak eluted at 19 to 20 minutes. The isolated HPLC peak was used to measure its effect on colony-forming unit-erythroid (CFU-E) growth and compare it with that of synthetic cytochrome P450 arachidonate metabolites, 19- and 20-hydroxyeicosatetraenoic (HETE) acid. These bone marrow cytochrome P450 arachidonic acid metabolites at picomolar concentration potentiated erythropoietin (Epo)-induced CFU-E growth by fourfold to sixfold. Addition of 19- and 20-HETE to the bone marrow culture resulted in a potentiating effect on CFU-E number in a dose-dependent manner. 20-HETE was much more potent in stimulating CFU-E growth than 19-HETE at a similar concentration of 10(-11) mol/L. The potentiating effect of 20-HETE resulted in a shifting to the left of the dose-response curve to Epo. To substantiate the finding of an active NADPH-dependent cytochrome P450-metabolizing system, we further examined the ability of adherent cells to metabolize exogenous pharmacologic compounds such as benzo(a)pyrene, a substrate for the heme-cytochrome P450 system, aryl hydrocarbon hydroxylase. The adherent stromal cytochrome P450 metabolizes benzo(a)pyrene at comparable levels to blood vessel endothelial cells. These novel observations underscore the importance of adherent stromal cytochrome P450 to metabolize endogenous substrates, including arachidonic acid, to compounds that may interact in a paracrine manner with Epodependent hematopoietic cells.

Arachidonic Acid↗
PubMed1991

Endogenous epoxyeicosatrienoyl-phospholipids. A novel class of cellular glycerolipids containing epoxidized arachidonate moieties.

By chromatographic and mass spectral techniques we document in rat liver the presence of new classes of glycerophospholipids which contain an epoxyeicosatrienoate moiety, esterified to the glycerol-sn-2 position. These novel lipids are formed in vivo from endogenous precursors and under physiological conditions. Chromatographic resolution followed by hydrolysis and regioisomeric analysis showed that they consist of the 8,9-, 11,12-, and 14,15-epoxyeicosatrienoyl derivatives of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Their relative concentrations (micromoles of oxidized lipid/mol of phospholipid) were 70, 85, and 106 for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, respectively. Chiral analysis of the fatty acids at sn-2 revealed an enantioselective preference for 8(S),9(R)-, 11(S),12(R)-, and 14(R),15(S)-epoxyeicosatrienoates in all three lipid classes. The data suggest a multienzyme process initiated by cytochrome P-450 epoxidation of arachidonic acid followed by ATP-dependent activation to epoxyeicosatrienoyl-CoA derivatives and stereoselective lysolipid acylation. These results provide a molecular basis for a potential physiological role of cytochrome P-450 in the biosynthesis of unique cellular glycerolipids and, consequently, in the control of cell membrane structure and function.

Acylation↗
PubMed1991

Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle.

In the medullary segment of the thick ascending limb of the loop of Henle (mTALH), arachidonic acid (AA) is metabolized by a cytochrome P450-dependent monooxygenase to products that affect ion transport. The linkage between changes in ion transport and AA metabolism in isolated cells of the mTALH was examined. AA produced a concentration-dependent inhibition of 86Rb uptake--an effect that was prevented by selective blockade of cytochrome P450 monooxygenases. Inhibition by cytochrome P450 blockade of the effect of AA on 86Rb uptake could be circumvented by addition of the principal products of AA metabolism in the mTALH.

Animals↗
PubMed1991

Synthesis, structural identification and biological activity of 11,12-dihydroxyeicosatetraenoic acids formed in human platelets.

An enantiospecific route for the synthesis of 11,12-dihydroxyeicosatetraenoic acids was developed and used to synthesize 11,12-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acids. The 11,12-DHETEs were synthesized with the stereochemistry of the hydroxyl group being 11(R),12(S) and 11(S),12(S). The synthetic compounds were used to elucidate the structure of 11,12-DHETEs formed in human platelets by comparison of the chromatographic retention time in HPLC and GC as well as their ion fragmentation pattern in GC-MS. The major 11,12-DHETE formed in human platelets was found to be identical with 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid. Two more compounds were tentatively identified as 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid and 11,12-dihydroxy-5(E),7(E),9(E),14(Z)-eicosatetraenoic acid. Furthermore, the 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid was found to possess biological activity on neutrophil functional responses. However, the major compound, 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid, formed in platelets lacks biological activity in the test systems used. The present data further support that 11,12-dihydroxy-eicosatetraenoic acids are formed in human platelets via a leukotriene like mechanism presumably by the 12-lipoxygenase. Furthermore, the biological effects of one of the compounds showed a unique activity profile compared to other lipoxygenase products.

Blood Platelets↗
PubMed1991

12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea.

Human and bovine corneal epithelial cytochrome P450 convert arachidonic acid to compound D [12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid], a metabolite with inflammatory properties including vasodilatation and breakdown of the blood-aqueous barrier. Angiogenic properties of the endogenous compound D and the synthetic enantiomers DR and DS were examined using the corneal micropocket technique. The synthetic compound DR was as active as the endogenously formed compound D. Neovascularization of the cornea was found in all the implants containing as little as 0.5 micrograms of compound DR. In contrast, the stereoisomer DS at the same concentration (0.5 micrograms) was inactive. Since angiogenesis can be secondary to a local inflammatory response, we evaluated the effects of compound DR and its stereoisomer DS on human neutrophil chemotaxis by using a modified Boyden chamber technique. DR, but not DS, was found to be a potent chemotactic factor, exhibiting dose-dependent neutrophil chemotaxis with significant responses observed at doses as low as 10(-11) M, a concentration at which leukotriene B4 does not exhibit significant chemotactic activity. Therefore, compound D produced by the cornea may qualify as an intrinsic corneal angiogenic factor which, in association with other inflammatory mechanisms, account for the growth of new vessels in the cornea that appear in chronic inflammation or in the reparative stages of an acute process.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

Enhanced synthesis of epoxyeicosatrienoic acids by cholesterol-fed rabbit aorta.

Arachidonic acid metabolism via cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase was investigated in thoracic aortic tissue obtained from rabbits fed either standard rabbit chow or chow containing 2% cholesterol. Aortic strips were incubated with [14C]arachidonic acid and A23187. Metabolites from extracted media were resolved by high-pressure liquid chromatography (HPLC). Normal and cholesterol-fed rabbit aortas synthesized prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs). The major cyclooxygenase products were 6-keto-PGF1 alpha and PGE2. Basal aortic 6-keto-PGF1 alpha production was slightly reduced in cholesterol-fed compared with normal rabbits. 12(S)- and 15(S)-HETE were the major aortic lipoxygenase products from both normal and cholesterol-fed rabbits. The structures were confirmed by gas chromatography-mass spectrometry (GC-MS). Only cholesterol-fed rabbit aortas metabolized arachidonic acid via cytochrome P-450 epoxygenase to the epoxyeicosatrienoic acids (EETs). 14,15-, 11,12-, 8,9-, and 5,6-EET were identified based on comigration on HPLC with known 14C-labeled standards and typical mass spectra. Incubation of normal aorta with 14,15-EET decreased the basal synthesis of 6-keto-PGF1 alpha. The other EETs were without effect. The four EET regioisomers relaxed the norepinephrine-precontracted normal and cholesterol-fed rabbit aorta. The relaxation response to 14,15-EET was greater in aortas from cholesterol-fed rabbits. These studies demonstrate that hypercholesterolemia, before the development of atherosclerosis, alters arachidonic acid metabolism via both the cyclooxygenase and epoxygenase pathways.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

Glomerular stereospecific synthesis and hemodynamic actions of 8,9-epoxyeicosatrienoic acid in rat kidney.

Renal glomerular and cortical metabolism of endogenous arachidonic acid by cytochrome P-450 epoxygenase yields 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EET). Using gas chromatography-mass spectrometry, we measured the synthesis of 8,9-EET from an endogenous pool of arachidonic acid in normal rat kidney. The (8S,9R) isomer was favored over the (8R,9S) isomer in a ratio (%) of 59 to 41 in isolated glomeruli and 68 to 32 in cortex tissue. (8S,9R)- but not (8R,9S)-EET elicited dose-dependent vasoconstriction on intrarenal administration in the euvolemic Munich-Wistar rat. Micropuncture measurements of glomerular dynamics revealed that (8S,9R)-EET increased afferent arteriolar resistance (RA) leading to reductions in single-nephron plasma flow rate (QA), net transcapillary hydraulic pressure difference (delta P), and consequently single-nephron glomerular filtration rate (SNGFR). There was no significant change in the value of the glomerular capillary ultrafiltration coefficient (Kf). In the presence of a cyclooxygenase inhibitor, indomethacin, the effects of 8,9-EET were reversed. RA fell leading to increases in QA and delta P, with resultant augmentation of SNGFR. Under these conditions, a modest reduction if Kf was noted. Thus (8S,9R)-EET is a stereoselective renal vasoconstrictor, preferentially generated over its optical isomer, (8R,9S)-EET, suggesting that it is biologically relevant and implying specific structural requirements for EET receptor activation. The principal mechanism of action of 8,9-EET is preglomerular vasoconstriction. The vasoconstrictor effect of 8,9-EET is CO dependent.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

Metabolism of arachidonic acid by rat adrenal glomerulosa cells: synthesis of hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids.

Metabolites of arachidonic acid have been implicated in the regulation of aldosterone release. To form a basis for further investigations in this area, the present study has isolated and identified the metabolites formed from exogenous arachidonic acid by adrenal zona glomerulosa cells and characterized the effects of several inhibitors on the synthesis of these eicosanoids. Rat adrenal glomerulosa cells metabolized exogenous [14C]arachidonic acid to products comigrating with the prostaglandins (PGs), hydroxyeicosatatraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs). The metabolites were found in the cells and the incubation media; however, none of the metabolites were found esterified to cellular lipids. The major metabolites were identified as 6-keto PGF1 alpha, PGE2, PGF2 alpha, PGD2, 12(S)-HETE, 15(S)-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. The identities of the HETEs and EETs were confirmed by gas chromatography/mass spectrometry. There was no evidence for the synthesis of leukotrienes. The cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitors, nordihydroguaiaretic acid, baicalein and AA861, and the combined cyclooxygenase/lipoxygenase inhibitors, BW755C and eicosatetrayenoic acid, inhibited the formation of the [14C]PGs, the [14C]HETEs, and the [14C]EETs. Metyrapone and clotrimazole, inhibitors of cytochrome P450, increased the synthesis of [14C]PGs and [14C]HETEs and reduced the synthesis of [14C] EETs. Superoxide dismutase did not alter arachidonic acid metabolism. In contrast, arachidonic acid metabolism was increased in cells pretreated with catalase. These data indicate that adrenal glomerulosa cells metabolize exogenous arachidonic acid to a number of oxygenated metabolites including PGs, HETEs, and EETs. From studies with inhibitors, the EETs appear to be synthesized by a cytochrome P450 epoxygenase and the HETEs by lipoxygenases.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

14, 15-cis-episulfide-eicosatrienoic acid, an 'epoxygenase' eicosanoid analog, inhibits ionophore- but not thrombin-induced platelet aggregation.

An 'epoxygenase' eicosanoid analog, 14, 15-cis-episulfide-eicosatrienoic acid, has several unique pharmacological effects on platelets. These include (i) inhibition of ionophore A23187- but not thrombin-induced activation, (ii) inhibition of thromboxane B2 biosynthesis derived from endogenous but not exogenous arachidonic acid, and (iii) attenuation of ionophore-mediated increases in cytosolic Ca2+ when extracellular or membrane Ca2+ is available but not when these pools are excluded. Neither elevation of cyclic AMP levels, a potent inhibitory process, nor direct antagonism of the prostaglandin H2/thromboxane A2 receptor is responsible for the actions of 14, 15-cis-episulfide-eicosatrienoic acid. These properties distinguish 14, 15-cis-episulfide-eicosatrienoic acid from other antiaggregatory substances.

8,11,14-Eicosatrienoic Acid↗