Search

Mononuclear phagocytes play a pivotal role in the progression of septic shock by producing tumor necrosis factor-alpha (TNF-alpha) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram-negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon-gamma (IFN-gamma), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human monocytic cell line MonoMac 6, the production of TNF-alpha and interleukin-6 (IL-6) was approximately five times greater in cells primed with IFN-gamma than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole-1-yl)heptane phosphonate) and YM 50201 (3-hydroxyethyl 5,3'-thiophenyl pyridine) strongly inhibited (up to 90%) TNF-alpha and IL-6 production in response to LPS in both unprimed MonoMac-6 cells and in cells primed with IFN-gamma. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence-based amplification methods showed that SK&F 98625 inhibited TNF-alpha production by decreasing TNF-alpha mRNA levels in MonoMac-6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.

A novel human homologue of Escherichia coli, yeast and plant 1-acylglycerol-3-phosphate acyltransferase has been isolated from U937 cell cDNA. Expression of the cloned sequence in 1-acylglycerol-3-phosphate acyltransferase-deficient E. coli resulted in increased incorporation of oleic acid into cellular phospholipids. Membranes made from COS7 cells transfected with the cDNA exhibited higher acyltransferase activity towards a range of donor fatty acyl-CoAs and lysophosphatidic acid. Northern-blot analysis of the cDNA sequence indicated high levels of expression in immune cells and epithelium. Rapid amplification of cDNA ends revealed differentially expressed splice variants, which suggests regulation of the enzyme by alternative splicing. This cDNA therefore represents the first described sequence of a mammalian gene homologous to non-mammalian lysophosphatidic acid acyltransferases.

When human peripheral blood neutrophils were stimulated with various agonists which activate and/or prime neutrophils, we found that Cl − efflux was enhanced with a dramatic (50%) loss of intracellular Cl − . Interestingly, the Cl − efflux was enhanced by both agonists which induce a rapid transient increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [class I, e.g. N -formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL8), platelet-activating factor, leukotriene B 4 and C5a] and those which do not induce such an [Ca 2+ ] i elevation (class II, e.g. tumor necrosis factor α-(TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. The time course of agonist-stimulated Cl − efflux differed depending on the agonist. Class I agonists such as IL8 and fMLP exhibited a 1 min lag phase before the onset of Cl − efflux; class II agonists such as GM-CSF and TNF displayed a 2 and 5 min lag phase, respectively. Both IL8 (class I)- and TNF (class II)-stimulated CF efflux exhibited similar sensitivity to inhibition by different types of ion transport inhibitors [ethacrynic acid (EA), amiloride, 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid, anthracene-9-carboxylic acid, and 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid). On the other hand, natural Cl − efflux, which is thought to be mainly mediated by Cl − /Cl − self exchange, was not inhibited by EA (0.5 mM) or amiloride (0.3 mM). These results imply that both class I and class II agonist-stimulated Cl − efflux occurs via a common Cl − transporter which is different from that reported previously in resting human neutrophils. Although all agonists which induced a Cl − efflux also induced shape change of neutrophils, there did not appear to be a causal relationship between shape change and agonist-stimulated Cl − efflux. However, a temporal correlation was found to exist between agonist-stimulated Cl − efflux and intracellular alkalinization following agonist stimulation. Agonist-stimulated Cl − efflux therefore seems to be a common phenomenon activated by several agonists which act through different signal transduction pathways.

The effects of the application of a nitric oxide generating acidified nitrite cream comprising sodium nitrite and citric acid, on the healing of incisional wounds in mice, has been investigated. The effects of acidified nitrite on wound healing were critically dependent on the time of application after wounding. Application of acidified nitrite starting on the day of wounding and on consecutive days thereafter significantly inhibited both half time to closure and extent of wound closure. Conversely, application starting on days 1-4 after wounding and on consecutive days thereafter significantly augmented the rate and extent of wound healing. Optimal effects on improving wound healing were observed with cream concentrations of 3.0% (w/v) sodium nitrite and 4.5% (w/v) citric acid. Starting application on day 5 after wounding had no effect on the rate or extent of wound healing. In diabetic Lepr db/db mice, starting treatment at day 2 after wounding, acidified nitrite at 3.0% (w/v) sodium nitrite and 4.5% (w/v) citric acid significantly increased the rate and extent of wound healing. This suggests that acidified nitrite is effective in improving wound healing against a diabetic background. The present data shows that acidified nitrite cream, a clinically effective means of topically delivering nitric oxide, augments the wound healing process and may be of clinical benefit.

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of Superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 μM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg 10 6 cells in untreated cells to background levels in forskolin-treated cells (110 pg 10 6 cells). Also, the reduction of cytochrome c by Superoxide anions fell from 4.2 solnmol 10 6 cells in the absence of forskolin to 2.0 nmol 10 6 cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.

A dimethylated chlorocyclodiene epoxide (DME) ∗ is metabolized by adult male rat liver enzymes to produce two g.l.c. detectable metabolites M1 and M2. Liver enzymes from adult female rats metabolize DME producing only metabolite M1 in detectable quantities. This apparent qualitative sex difference in the metabolism of DME is first manifest between the ages of 35 and 45 days. Liver enzymes from rats of both sexes younger than 35 days metabolize DME in a qualitatively similar manner. The actions of the testes in the development of this apparent qualitative sex difference are exerted between the ages of 7 and 14 days. Castration of male rats at and before the age of 7 days results in the expression of a typically feminine pattern of metabolism of DME by liver enzymes in adult life. Castration of male rats after the age of 14 days does not prevent adult liver enzymes from exhibiting a basically masculine pattern of DME metabolism. At ages between 7 and 14 days castration has a graded effect on the metabolism of DME displayed by liver enzymes in adult life. These results suggest that testicular androgens do not play a direct role at puberty and in adulthood in the expression of the ability of liver enzymes to produce metabolite M2, but that the apparent qualitative sex dependent difference in DME metabolism is determined primarily by neonatal imprinting by testicular androgens.

The effects of the neonatal administration of reserpine, chlorpromazine, phenobarbitone and morphine on the development of sex differences in hepatic drug metabolism in the rat have been investigated. Treatment of neonatal male rats with reserpine or chlorpromazine for the first two weeks post-partum significantly inhibited the development of sex differences in drug metabolism in adult life. Similar treatment of neonatal female rats with reserpine or chlorpromazine had no effect on the development of hepatic drug metabolism in adulthood. Morphine or phenobarbitone treatment of neonatal rats of either sex had no effect on the development of sex differences in hepatic drug metabolism in adult life.

A sensitive fluorimetric assay to determine both Phase 1 (oxidation) and Phase 2 (conjugation) drug metabolism in epidermal cells isolated from hairless mice, using ethoxycoumarin as a model substrate, is described. Ethoxycoumarin was metabolized by isolated epidermal cells via dealkylation to 7-hydroxycoumarin (7-OHC) and subsequent conjugation. Phase 1 metabolites were extracted in ether from the aqueous incubation media, back extracted into sodium hydroxide and determined fluorimetrically. Conjugated metabolites remaining in the aqueous phase were hydrolysed by the action of beta-glucuronidase and extracted and determined in a similar manner. The production of free 7-OHC by isolated epidermal cells was biphasic at all substrate concentrations tested, exhibiting an initial linear increase followed by a plateau phase. The plateau phase was attributable to the conjugation of 7-OHC produced in situ. Metabolism was inhibited by SKF 525A, carbon monoxide, and alpha-naphthoflavone. Endogenous supplies of reducing equivalents in the form of NADPH were adequate to attain maximal rates of metabolism. With human hair follicles both Phase 1 and Phase 2 activity was detectable in 7 out of 11 subjects. The assay has the advantages of being sensitive, producing single defined metabolites from both Phase 1 and Phase 2 metabolism; is readily adaptable to human skin samples.

The inflammatory dose-response to topical anthralin and whole skin aryl hydrocarbon hydroxylase (AHH) activity were measured before and 24 h after application of a coal tar solution to the uninvolved skin of patients with psoriasis. The inflammatory response to anthralin decreased and total AHH activity increased after the tar treatment. A possible explanation is that anthralin or an irritant product is metabolized by AHH activity in the skin. Induction of AHH by coal tar increases its removal and reduces anthralin irritancy.

Aryl hydrocarbon hydroxylase (AHH) has been measured in the skin, jejunum and liver of normal and psoriatic individuals. We have been unable to confirm previous reports of an abnormality in AHH activity in patients with psoriasis. Re-examination of the laboratory records on which the original reports were based leads us to doubt their veracity and validity.

SUMMARY Aryl hydrocarbon hydroxylase (AHH) activity has been measured in full-thickness biopsies of human skin from patients with and without psoriasis. Basal levels of enzyme activity varied over a fivefold range and were not related to age, sex or clinical condition. Induction of AHH activity by the application of coal tar resulted in a two-to fivefold increase in activity above basal levels, which was not related to the presence or absence of psoriasis. Separation of human skin into dermis and epidermis showed that human skin AHH activity is predominantly dermal.

The role of the pituitary in the control of the neonatally imprinted sex difference in the metabolism of a dimethylated chlorocyclodiene epoxide (DME)∗ was investigated. Hypophysectomy of adult female rats resulted in an effective masculinization of the hepatic metabolism of DME. Adrenalectomy, thyroidectomy, castration or hypophysectomy of adult male rats did not prevent liver enzymes from displaying a basically masculine pattern of DME metabolism. However, castration of male rats at one day old resulted in a basically feminine pattern of metabolism being displayed by liver enzymes in adult life. The effects of adult castration on the metabolism of DME and ethylmorphine by rat liver enzymes were completely reversed by testosterone propionate. The effects of combined hypophysectomy and castration on the hepatic metabolism of these substrates, however, were not reversed by testosterone treatment. These results suggest that sex dependent differences in the metabolism of foreign compounds by adult rat liver enzymes are mediated directly by the pituitary gland.

The effect of the topical application of clobetasol propionate on cutaneous ethoxycoumarin O'dealkylation (EOD) has been studied in various strains of mice. Clobetasol propionate markedly increased cutaneous EOD activity in adult hairless mice only. Similar treatment of adult haired C57BL/6J mice, or adult haired DBA/2J mice had no significant effect on cutaneous EOD activity. In contrast 3 methylcholanthrene induced cutaneous EOD activity in both hairless and C57 strains to a far greater extent than in the DBA strain. EOD activity in hairless mice non-responsive to polycyclic hydrocarbons, derived by selective breeding of hairless and DBA strains was induced by clobetasol propionate to a similar extent to that observed in responsive hairless strains. Hepatic EOD activity was not induced by clobetasol propionate in any of the strains tested. Strain differences in the induction of EOD by clobetasol propionate were not related to differences in either the concentration of cytosolic glucocorticoid receptor in the skin, the dissociation constant of the cytosolic receptor, or differences in percutaneous absorption. Polycyclic hydrocarbons did not compete with triaminolone acetonide for binding to the cytosolic glucocorticoid receptor. Strain differences in the induction of EOD activity by clobetasol propionate appear therefore not to be related to strain differences in either the Ah receptor or the glucocorticoid receptor, but to be regulated by the hr locus.

Dithranol (anthralin) inflammation of forearm skin was completely inhibited by various scavengers of free radicals of the oxygen species. It is concluded that dithranol inflammation is initiated by formation of free radicals; these may act through lipid peroxidation and production of inflammatory endoperoxides or by a more direct mechanism.