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M J Holtzman

Publications and source records attributed to M J Holtzman.

At least 55 records · Page 3Linked to original sources
PubMed1990

Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial cells distinct from leukocyte and platelet forms of the enzyme.

We examined the characteristics of an arachidonate 12-lipoxygenase in bovine tracheal epithelial cells in relation to the enzyme expressed in leukocytes and platelets. Homogenous preparations of intact or disrupted tracheal epithelial cells metabolized arachidonic acid predominantly to (12S)-hydroxyeicosatetraenoic acid, and subcellular fractionation by differential centrifugation demonstrated that the 12-lipoxygenase activity was localized predominantly to the 100,000 x g supernatant (cytosol fraction). Analysis of cytosolic enzymatic activity for pH dependence (maximum activity at pH 7.4-8.0), divalent cation effects (no dependence on cations), and kinetic characteristics (lag phase elimination by addition of hydroperoxide) exhibited similarity to leukocyte and platelet 12-lipoxygenases. Immunoprecipitation experiments demonstrated that the epithelial 12-lipoxygenase reacted with a monoclonal antibody (lox-2) directed against leukocyte 12-lipoxygenase but not with an antibody (HPLO-3) against the platelet enzyme. Immunoaffinity chromatography of the epithelial 100,000 x g supernatant fraction using lox-2 linked to Affi-Prep 10 yielded a single predominant protein band (Mr = 72,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identical in apparent mass to the bovine leukocyte lipoxygenase. Western blotting using a polyclonal antibody to leukocyte 12-lipoxygenase showed peroxidase staining of the same 72-kDa protein band. Activity assays of the purified enzymes demonstrated that substrate specificity for the epithelial 12-lipoxygenase was similar to that of the leukocyte enzyme, but the epithelial enzyme more efficiently converted 18-carbon fatty acids to the corresponding monohydroxylated conjugated dienes. We conclude that bovine tracheal epithelial cells express a 12-lipoxygenase that has immunological reactivity similar to leukocyte and distinct from platelet 12-lipoxygenase and possesses substrate specificity distinct from both enzymes. We further suggest that lipoxygenase heterogeneity may provide a basis for different functional roles for the enzyme in different cell types.

Animals↗
PubMed1990

Enhanced prostaglandin synthesis after ultraviolet injury is mediated by endogenous histamine stimulation. A mechanism for irradiation erythema.

Acute ultraviolet light B (UVB) injury is associated with dermal mast cell histamine release. The possibility that histamine-stimulated prostaglandin (PG) synthesis could be a mechanism for irradiation erythema was therefore examined using human skin explants. Explants responded to UV irradiation (120 mJ/cm2) with a fivefold increase in synthesis of prostaglandins E2, F2 alpha and 6-keto PGF1 alpha. Incubating explants with the H1 antihistamines brompheniramine (50 microM) or pyrilamine (30 microM) inhibited PG release from irradiated explants 63 +/- 4.9% (mean +/- SEM) 6 h after UV exposure. Antihistamines did not affect PG synthesis in control explants. Irradiation increased the histamine concentration in explant conditioned medium only 50% over basal values, suggesting that irradiation enhanced histamine responsiveness. Explants were therefore incubated with exogenous histamine. In irradiated explants, PG synthesis was stimulated threefold by 3 microM histamine. Unirradiated explants' PG synthesis was unaffected by histamine. Enhanced histamine sensitivity was also examined in epidermal cell cultures. In irradiated cultures, histamine sensitivity was again markedly potentiated: as little as 1 microM histamine stimulated significant PGE2 release and the response to 10-30 microM histamine was increased six to eight times compared with that of unirradiated cultures. These studies demonstrate that endogenous histamine stimulates PG synthesis in human skin after UV injury by potentiation of histamine-induced prostaglandin release. Potentiated agonist responses induced by UV exposure may contribute to the effects of UVB irradiation injury and in particular to irradiation erythema.

Animals↗
PubMed1989

Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation.

Regulation of cyclooxygenase expression was studied in homogenous preparations of epithelial cells isolated from sheep tracheal mucosa. Cellular capacity to generate cyclooxygenase-derived arachidonate metabolites (predominantly prostaglandin E2) increased markedly in cultured compared to freshly isolated cells. A 70 kDa cyclooxygenase protein and corresponding 2.8 kb mRNA were coordinately expressed but their levels did not increase proportionately to the increase in cyclooxygenase activity. Rehybridization of Northern blots at lower stringency revealed the presence of a new tissue-specific 4.0 kb mRNA species exhibiting increased expression during cell culture. Hybridization of the 4.0 kb mRNA with two nonoverlapping cDNA probes at only low stringency conditions suggests that it is derived from a distinct gene. Its relatedness to cyclooxygenase and its increase in parallel with enzymatic activity further suggest that the larger mRNA may encode for a cyclooxygenase.

Animals↗
PubMed1989

Heterogeneity of cellular expression of arachidonate 15-lipoxygenase: implications for biological activity.

Comparison of relative arachidonate 15-lipoxygenase activities in different types of human cell reveals striking differences in levels of product generation. Among available human cell types, the tracheal epithelial cell and the eosinophil have markedly higher levels of activity than other cell types with reported activity. Theories of functional significance for this enzymatic pathway will need to account for the selective expression of high levels of activity in a highly limited number of cell types.

Arachidonate 15-Lipoxygenase↗
PubMed1989

Arachidonate 12-lipoxygenase and cyclooxygenase: PGE isomerase are predominant pathways for oxygenation in bovine tracheal epithelial cells.

The capacity of bovine tracheal epithelial cells to convert arachidonic acid to oxygenation products with potential biologic activity was studied in homogeneous preparations of isolated cells. Purified epithelial cell suspensions were incubated with radiolabeled arachidonic acid, and oxygenated metabolites were identified using high-pressure liquid chromatography and gas chromatography-mass spectrometry. The cells released predominantly two products during incubation with 0.3 to 150 microM arachidonic acid for 1 to 60 min at 37 degrees C: prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). Concentration-response curves for the two products yielded half-maximal effects at 2 and 45 microM arachidonic acid, respectively. Stereochemical analysis by chiral-phase high-pressure liquid chromatography demonstrated that the epithelial 12-HETE consisted exclusively of the 12(S) isomer, providing supporting evidence that it was derived from an arachidonate 12-lipoxygenase. Epithelial cells prelabeled with arachidonic acid and incubated with 5 microM A23187 to stimulate endogenous arachidonic acid metabolism also released two predominant products with the chromatographic properties of PGE2 and 12-HETE. The findings demonstrate that bovine tracheal epithelial cells express both a cyclooxygenase:PGE isomerase and a 12-lipoxygenase pathway and therefore implicate this pathway as a new source of epithelial cell mediators.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

A regiospecific monooxygenase with novel stereopreference is the major pathway for arachidonic acid oxygenation in isolated epidermal cells.

We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects. Cell incubation with AA (0.3-150 microM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry. Maximal amounts of 12-HETE were 126 +/- 21 pmol/10(6) cells (+/- SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 microM AA. Two epoxyeicosatrienoic acids derived from AA were also identified. Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects. Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that greater than 99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets. Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions. The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells.

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.

Animals↗
PubMed1987

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects.

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.

Arachidonate 5-Lipoxygenase↗
PubMed1987

Preferential human eosinophil chemotactic activity of the platelet-activating factor (PAF) 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC).

The chemotactic responses of human blood neutrophils and of eosinophils of two different densities, which were resolved by centrifugation on gradients of polyvinylpyrrolidone-coated silica gel (Percoll), were quantified in modified Boyden micropore filter chambers using highly purified synthetic 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC or PAFacether) and leukotriene B4 (LTB4) as stimuli. Maximal chemotactic responses of the densest eosinophils, less dense eosinophils, and neutrophils were evoked by 1 nM, 100 nM, and 1 microM PAFacether, respectively, and by 30-100, 30-100, and 10 nM LTB4. The magnitude of the maximal chemotactic response to PAFacether of the densest eosinophils was significantly greater than that of neutrophils. The eosinophil responses to PAFacether were chemotactic, as distinguished from chemokinetic, and were not influenced by the percentage of contaminating neutrophils. PAFacether is a more potent chemotactic factor for eosinophils than neutrophils and selectively attracts the densest population of human blood eosinophils.

Chemotactic Factors, Eosinophil↗
PubMed1986

Phospholipid fatty acid composition of pulmonary airway epithelial cells: potential substrates for oxygenation.

To determine possible substrates for airway epithelial lipoxygenase and cyclooxygenase activities, we examined the amounts and distributions of fatty acids in phospholipids of human, dog and sheep airway epithelial cells. We found that the cells contained significant levels of n-6 and n-3 fatty acids in species-specific amounts: dog cells were relatively enriched in the n-6 series and poor in n-3, while sheep cells were enriched in the n-3 series and poor in n-6. Despite differences in fatty acid content, cells from each species expressed a constant phospholipid composition and distributed their n-6 and n-3 fatty acids in a stereotyped fashion among phospholipid classes. The analysis shows that the species differences in oxygenation activities reported previously are paralleled by heterogeneity in potential substrates.

Animals↗
PubMed1986

O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans.

The increase in airway responsiveness induced by O3 exposure in dogs is associated with airway epithelial inflammation, as evidenced by an increase in the number of neutrophils (polymorphonuclear leukocytes) found in epithelial biopsies and in bronchoalveolar lavage fluid. We investigated in 10 healthy, human subjects whether O3-induced hyperresponsiveness was similarly associated with airway inflammation by examining changes in the types of cells recovered in bronchoalveolar lavage fluid obtained after exposure to air or to O3 (0.4 or 0.6 ppm). We also measured the concentrations of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in lavage fluid. We measured airway responsiveness to inhaled methacholine aerosol before and after each exposure and performed bronchoalveolar lavage 3 h later. We found more neutrophils in the lavage fluid from O3-exposed subjects, especially in those in whom O3 exposure produced an increase in airway responsiveness. We also found significant increases in the concentrations of prostaglandins E2, F2 alpha, and thromboxane B2 in lavage fluid from O3-exposed subjects. These results show that in human subjects O3-induced hyperresponsiveness to methacholine is associated with an influx of neutrophils into the airways and with changes in the levels of some cyclooxygenase metabolites of arachidonic acid.

Adult↗
PubMed1985

An anti-inflammatory drug (BW755C) inhibits airway hyperresponsiveness induced by ozone in dogs.

To follow up our previous observation that airway hyperresponsiveness induced by ozone is linked to airway inflammation, we investigated the effect of BW755C, an anti-inflammatory drug, on ozone-induced hyperresponsiveness in dogs. Airway responsiveness was assessed with dose-response curves of acetylcholine aerosol versus pulmonary resistance in two sets of experiments. In one set (placebo treatment), five dogs were given only saline solution treatment and were studied before treatment or ozone exposure and then after treatment both before and after ozone (3.0 ppm, 2 hours); in another set (BW755C treatment), the same dogs were studied before BW755C treatment or ozone and then after treatment (10 mg/kg intravenously) both before and after ozone. When the dogs were given no BW755C treatment, ozone induced a marked increase in airway responsiveness to acetylcholine. When the dogs were given BW755C, responsiveness was no different during treatment than before treatment but, more importantly, responsiveness did not increase significantly after ozone. We conclude that BW755C markedly inhibits ozone-induced airway hyperresponsiveness in dogs, probably by inhibiting the formation of oxygenation products of arachidonic acid.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1985

Predominant generation of 15-lipoxygenase metabolites of arachidonic acid by epithelial cells from human trachea.

Epithelial cells of 99% purity and 92% viability were isolated from human tracheas obtained post mortem, and the cellular pathways for lipoxygenation of arachidonic acid were examined in vitro. The lipoxygenase metabolites were identified by comparison with synthetic standards during reversed-phase and straight-phase high-pressure liquid chromatography, UV spectroscopy, and gas chromatography/mass spectrometry. Epithelial cells incubated without arachidonic acid failed to generate detectable quantities of metabolites, while cells incubated with arachidonic acid at 1-50 micrograms/mf for 1-30 min invariably generated predominantly 15-lipoxygenase products, including 15-hydroxyicosatetraenoic acid (15-HETE), four isomers of 8,15-dihydroxyicosatetraenoic acid (two 8,15-diHETES and two 8,15-leukotrienes), at least one isomer of 14,15-dihydroxyicosatetraenoic acid, and smaller amounts of 12-HETE and 8-HETE, but little or no detectable 5-HETE or 5,12-diHETEs. The capacity of epithelial cells from human pulmonary airway to selectively generate 15-lipoxygenase metabolites of arachidonic acid suggests a potential role for the products as mediators of airway epithelial function.

Arachidonate Lipoxygenases↗
PubMed1985

Leukotriene B4 induces airway hyperresponsiveness in dogs.

We studied the effect of leukotriene B4 aerosols on airway responsiveness to inhaled acetylcholine aerosols and on the cellular components and cyclooxygenase metabolites in bronchoalveolar lavage fluid in dogs. Inhalation of leukotriene B4 aerosols had no effect on resting total pulmonary resistance but increased airway responsiveness, an effect that was maximum in 3 h and that returned to control levels within 1 wk. Three hours after leukotriene B4, the number of neutrophils and the concentration of thromboxane B2 recovered in lavage fluid increased markedly. Pretreatment with the thromboxane synthase inhibitor OKY-046 prevented the increases in airway responsiveness and in thromboxane B2 but did not alter neutrophil chemotaxis. Thus we speculate that leukotriene B4 causes neutrophil chemotaxis and release of thromboxane B2, which increases airway responsiveness.

6-Ketoprostaglandin F1 alpha↗
PubMed1984

Prostaglandin F2 alpha increases responsiveness of pulmonary airways in dogs.

We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on the responsiveness of pulmonary airways in dogs. Airway responsiveness was assessed by determining the bronchoconstrictor response to increasing concentrations of acetylcholine aerosol delivered to the airways. In each of five dogs, we determined responsiveness during treatment with physiologic saline, histamine, or PGF2 alpha aerosols. The doses of histamine and PGF2 alpha were determined by establishing the largest dose of each which could be given to the dog without causing bronchoconstriction (subthreshold doses). We found that airway responsiveness was not significantly different during histamine treatment than after saline, however, responsiveness increased during treatment with PGF2 alpha. In addition, the hyperresponsiveness induced by PGF2 alpha was prevented by pretreatment with the ganglion blocking drug hexamethonium (5 mg/kg given intravenously). The results show that PGF2 alpha specifically increases the responsiveness of pulmonary airways in doses that do not cause bronchoconstriction, and suggest that the hyperresponsiveness involves a neural mechanism such as increased responsiveness of airway sensory nerves.

Acetylcholine↗
PubMed1984

Control of neurotransmission by prostaglandins in canine trachealis smooth muscle.

Contractile responses of canine tracheal smooth muscle to electrical field stimulation diminished over a 2-h period of incubation. However, addition of indomethacin (10(-5) M) for a similar time not only prevented this inhibition of contractile response, but actually markedly increased the response to electrical field stimulation, suggesting that prostaglandins were responsible for the time-dependent inhibition. Measured prostaglandin E2 increased in the tissue bath over 2 h in control tissues. Addition of prostaglandin E2 to the tissue produced similar inhibition of contractile responses to electrical field stimulation in a concentration-dependent manner. In contrast, incubation alone, treatment with indomethacin, or addition of prostaglandin E2 had little, if any, effect on contractions induced by acetylcholine. We conclude that the release of prostaglandins from canine tracheal smooth muscle that occurs with time has a predominantly inhibitory effect on cholinergic neurotransmission at a prejunctional site.

Animals↗