Meclofenamate inhibits prostaglandin E binding and adenylyl cyclase activation in human myometrium.

The effect of sodium meclofenamate on the binding of [3H]prostaglandin E2 [( 3H]PGE2) to membranes from human myometrium was investigated. Meclofenamate inhibited the binding of [3H]PGE2 to high-affinity (dissociation constant 1.5 nmol/l) sites in a reversible dose-dependent manner (inhibition constant 11 mumol/l). The mechanism of inhibition was mainly competitive, but at high doses of meclofenamate (greater than or equal to 100 mumol/l) there was loss of PGE receptor sites. Of several PG synthesis inhibitors tested, only meclofenamate and, to a lesser extent, mefenamic acid had a significant inhibitory effect. PGE2 stimulated cyclic AMP generation in slices of human myometrium and this was inhibited by meclofenamate in a dose-dependent manner (50% inhibition occurred at 9 mumol/l). Again, this effect was specific for meclofenamate and fitted a competitive mechanism at doses in the range 1-10 mumol/l and a non-competitive mechanism at higher doses. The data show that meclofenamate, in addition to its traditional role as a PG synthesis inhibitor, affects directly PGE receptor binding and activation.     

Vitamin D-enhanced thyrotrophin release from rat pituitary cells: effects of Ca2+, dihydropyridines and ionomycin

Vitamin D may regulate pituitary function, as there are selective effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on gene expression in clonal pituitary tumour cells, and on TRH-induced TSH release in normal rat pituitary cells in vitro. The role of Ca2+ in 1,25-(OH)2D3-enhanced TSH release from primary rat pituitary cell cultures was investigated. Pretreatment with 10 nmol 1,25-(OH)2D3/l for 24 h augmented KCl (3-60 mmol/l)-induced TSH release over 1 h at all KCl concentrations greater than 7.5 mmol/l (P less than 0.001), with a 76% enhancement of TSH release induced by 30 mmol KCl/l (P less than 0.001). The Ca2+ channel antagonist nifedipine (10 nmol/l-10 mumol/l) caused a concentration-dependent inhibition of KCl (60 mmol/l)-induced TSH secretion. Pretreatment with 1,25-(OH)2D3 enhanced KCl-induced release at all concentrations of nifedipine (P less than 0.001). The Ca2+ selective divalent cation ionophore ionomycin (1 nmol/l-1 mumol/l), and the Ca2+ channel agonist BAY K 8644 (10 nmol/l-1 mumol/l) increased prolactin secretion but did not increase TSH release, and 1,25-(OH)2D3 had no effect. At an extracellular Ca2+ concentration of less than 500 nmol/l, TRH-induced TSH release was observed only after treatment with 1,25-(OH)2D3 (P less than 0.01). As the extracellular Ca2+ concentration was increased, greater increments of TRH-induced TSH release were observed following pretreatment with 1,25-(OH)2D3 (P less than 0.01). However, the effect of 1,25-(OH)2D3 in the thyrotroph was independent of the pretreatment extracellular Ca2+ concentration. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in-vitro responsiveness to TRH and KCl. These data suggest that the action of 1,25-(OH)2D3 in the thyrotroph is to enhance intracellular signal transduction. They further support a permissive or regulatory role of vitamin D in the normal pituitary gland.     

Inhibition of bovine adrenocortical steroidogenesis by benzodiazepines: a direct effect on microsomal hydroxylation or an inhibition of calcium uptake?

We have previously reported that benzodiazepines inhibit microsomal steroid hydroxylases. We have now studied their effects at much lower drug concentrations and have also addressed the suggestion that benzodiazepines alter cellular calcium metabolism. We investigated the in-vitro effects of midazolam on microsomal steroid hydroxylation by measuring basal and ACTH-stimulated cortisol and 17 alpha-hydroxyprogesterone (17-OHP) synthesis. Threshold inhibition of basal cortisol production was achieved by 3.4 mumol midazolam/l while ACTH-stimulated production required 13.6 mumol/l. This was accompanied by a biphasic response of 17-OHP production, rising to a maximum at 13.6 mumol midazolam/l for basal and 6.8 mumol midazolam/l for ACTH-stimulated synthesis suggesting a preferential inhibitory effect on 21-hydroxylase activity at < 6.8 mumol/l and additional effects on 17 alpha-hydroxylation at higher drug concentrations. This explains the inhibition of ACTH-stimulated cortisol synthesis by midazolam (50% inhibitor dose (IC50) 22 mumol/l). Using 21-deoxycortisol as substrate, we have demonstrated that midazolam is a competitive inhibitor of 21-hydroxylase (inhibitory constant (KI) 35 mumol/l). Both midazolam and diazepam inhibited K(+)-stimulated aldosterone synthesis, with IC50 values of 1.2 mumol/l and 0.8 mumol/l respectively, which are far lower than those observed for ACTH-stimulated cortisol synthesis. With 11 beta-hydroxyprogesterone as substrate, the KI for the inhibition of aldosterone synthesis by midazolam was 54 mumol/l. Potassium stimulates aldosterone biosynthesis at least partly by changing intracellular free calcium levels. To investigate possible antagonistic effects of benzodiazepines on calcium metabolism, we measured 45Ca uptake in the presence of midazolam.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of tumor necrosis factor-alpha and interferon-gamma on deoxyribonucleic acid and collagen synthesis by rat osteosarcoma cells (ROS 17/2.8)

Tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) have potent effects on bone resorption and collagen synthesis in cultured rat long bones. Since the effects of TNF alpha and IFN gamma may result from interaction with multiple cell types, we studied the effects of these cytokines on the synthesis of DNA and collagen in one cell type with osteoblast phenotype, cloned rat osteosarcoma cells (ROS 17/2.8). Recombinant human TNF alpha did not affect DNA synthesis after 48 h with concentrations of 10(-11)-10(-8) M and inhibited DNA synthesis slightly at 10(-6) M. Recombinant rat IFN gamma (5-500 U/ml) caused a dose-dependent inhibition of DNA synthesis. Coincubation with TNF alpha and IFN gamma inhibited DNA synthesis more than maximal doses of either cytokine alone. This enhanced inhibitory effect was due to the induction of a response to TNF alpha by IFN gamma, since preexposure of cells to IFN gamma for 24 h, followed by incubation with TNF alpha alone for an additional 48 h, also resulted in increased inhibition of DNA synthesis. Preexposure to TNF alpha for 24 h, followed by IFN gamma alone, did not increase the inhibition of DNA synthesis. Incubation with either IFN gamma (5-500 U/ml) or TNF alpha (10(-10)-10(-6) M) inhibited the incorporation of [3H]proline into collagen. Coincubation with intermediate concentrations of both cytokines resulted in an inhibitory effect greater than that produced by maximal concentrations of either alone. The results indicate that 1) IFN gamma and TNF alpha have direct actions on osteoblast-like cells in vitro; 2) IFN gamma modulates the DNA response to TNF alpha; and 3) the greater responses to combined cytokines than to high doses of either alone suggest that these cytokines act, at least in part, through different pathways.     

Hydrogen peroxide inhibits iodide influx and enhances iodide efflux in cultured FRTL-5 rat thyroid cells

This study describes the effects of hydrogen peroxide on the two iodide transport systems, I influx and I efflux, in the cultured FRTL-5 rat thyroid cells. I influx was measured by the amount of I taken up by the cells during incubation with Na125I and NaI for 7 min, and I efflux was measured by calculating the rate of 125I release from the 125I-loaded cells in the presence and absence of 5 mmol/l H2O2. Exposure to greater than 100 mumol/l H2O2 for 40 min caused a significant inhibition of I influx; the inhibition was reversible and non-competitive with iodide. Thyroid Na+K+ ATPase activity, a major mechanism to drive I influx, decreased by 40% after the cells were exposed to 5 mmol/l H2O2 for 10 min. H2O2 enhanced I efflux only when Ca2+ was present in the medium. The mechanism of an enhanced I efflux by H2O2 appears to be mediated through the elevation of free cytosolic Ca2+ concentration. Our data indicate that H2O2 can affect I transport by inhibiting I influx and enhancing I efflux.     

Mildly oxidized low-density lipoprotein acts synergistically with angiotensin II in inducing vascular smooth muscle cell proliferation

OBJECTIVES: Considerable attention has been focused on both mildly oxidized low-density lipoprotein (mox-LDL) and highly oxidized LDL (ox-LDL) as important risk factors for cardiovascular disease. Further, angiotensin II (Ang II) appears to play a crucial role in the development of hypertension and atherosclerosis. We assessed the effect of oxidatively modified LDL and its major oxidative components, i.e., hydrogen peroxide (H2O2), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) and their interaction with Ang II on vascular smooth muscle cell (VSMC) DNA synthesis. METHODS: Growth-arrested rabbit VSMCs were incubated in serum-free medium with different concentrations of native LDL, mox-LDL, ox-LDL, H2O2, LPC, or HNE with or without Ang II. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. RESULTS: Ang II stimulated DNA synthesis in a dose-dependent manner with a maximal effect at a concentration of 1 micromol/l (173%). Ang II (0.5 micromol/l) amplified the effect of native LDL at 500 ng/ml, ox-LDL at 100 ng/ml, and mox-LDL at 50 ng/ml on DNA synthesis (108 to 234%, 124 to 399%, 129 to 433%, respectively). H2O2 had a maximal effect at a concentration of 5 micromol/l (177%), LPC at 15 micromol/l (156%), and HNE at 0.5 micromol/l (137%). Low concentrations of H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) also acted synergisitically with Ang II (0.5 micromol/l) in inducing DNA synthesis to 308, 304, or 238%, respectively. Synergistic interactions of Ang II (0.5 micromol/l) with mox-LDL, ox-LDL (both 50 ng/ml), H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) on DNA synthesis were completely reversed by the combined use of probucol (10 micromol/l), a potent antioxidant and candesartan (0.1 micromol/l), an AT1 receptor antagonist. CONCLUSIONS: Our results suggest that mox-LDL, ox-LDL, and their major components H2O2, LPC, and HNE act synergistically with Ang II in inducing VSMC DNA synthesis. A combination of antioxidants with AT1 receptor blockade may be effective in the treatment of VSMC proliferative disorders associated with hypertension and atherosclerosis.     

Prostaglandin E2 modulation of rheumatoid factor synthesis

We examined the influence of prostaglandin E2 (PGE2) on the in vitro synthesis of rheumatoid factor (RF) by purified human B and T lymphocytes stimulated with Staphylococcus aureus Cowan 1 or pokeweed mitogen (PWM). Supernatants were assayed for total IgM and RF. PGE2 at concentrations of 10(-7) M to 10(-9) M significantly inhibited RF and IgM secretion stimulated by S aureus Cowan 1, a cross-linker of B cell surface Ig. The magnitude of inhibition of RF production was significantly greater than that of total IgM at low PGE2 concentrations (P less than 0.05). In contrast, PWM-stimulated cultures were only minimally inhibited by PGE2 at all concentrations tested. Since cross-linking of surface Ig renders B cells more susceptible to inhibition by PGE2, heat-aggregated IgG (HAIgG) was added to the PWM-stimulated cultures in an attempt to increase the sensitivity of precursors of RF-secreting cells to the inhibitory effects of PGE2. Addition of HAIgG markedly increased PGE2-mediated inhibition of RF synthesis without significantly affecting IgM production. Inhibition could not be overcome by the addition of soluble T helper cell factors, indicating that PGE2-mediated suppression was not the result of an inhibitory action of T helper cells. When lymphocytes from patients with rheumatoid arthritis were examined, HAIgG was found to be unable to induce sensitivity to PGE2-mediated inhibition of responsiveness. These results suggest that down-regulation of RF synthesis requires both cross-linking of surface Ig and the influence of PGE2. Abnormalities in this immunoregulatory mechanism may explain the ongoing production of RF in patients with rheumatoid arthritis.     

Determination of free radical scavenging activity of quercetin, rutin, luteolin and apigenin in H2O2-treated human ML cells K562

We investigated protective effects of four flavonoids against H2O2- induced DNA damage in human myelogenous leukemia cells (K562) using the comet assay. The structural difference of studied flavonoids -- quercetin, rutin, luteolin and apigenin -- are characterized by the number of hydroxyl groups on the B ring. The presence of an o-dihydroxy structure on the B-ring confers a higher degree of stability to the flavonoid phenoxyl radicals by participating in electron delocalization and is, therefore, an important determinant for antioxidative potential. The results correlate with earlier published data obtained in murine leukemia cell line L1210. Hydrogen peroxide induced in human K562 cells a concentration-dependent increase of single cell DNA strand breaks. The strongest inhibition against H2O2-induced DNA damage (44%, 42%) was found in a range of luteolin and quercetin concentrations of 20-100 micromol/l. Protective effect of rutin (100 and 1000 micromol/l) was only marginal (8-10%). Apigenin had no protective effect on DNA single strand breaks induced by H2O2. Luteolin and quercetin are therefore effective in the protection of human single cell DNA from oxidative attack.     

Glucocorticoid regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts: evidence for a receptor-mediated mechanism involving effects on specific de novo protein synthesis

Human skin fibroblast cultures synthesize and accumulate glycosaminoglycan (GAG). This laboratory has reported recently that the synthetic rate of hyaluronate (HA), the most abundant GAG produced by these cells, is inhibited by glucocorticoids. The purpose of these studies was to characterize further that hormonal response. Effects of potent glucocorticoid agonists dexamethasone (Dex) and RU28362 on GAG synthesis were studied. Both steroids inhibited [3H]GAG synthesis when added 24 hours before labeling with [3H]acetate. RU28362 inhibited HA synthesis by 58% and Dex by 60%. The half-maximal effect for each was achieved at a concentration of 0.5 nmol/L and maximal effects at 10 nmol/L. The recently described glucocorticoid antagonist RU38486 failed to alter [3H]GAG synthesis at concentrations up to 1,000 nmol/L. However, this compound did block the inhibition of the agonists. At concentrations of RU28362 and Dex that maximally inhibited [3H]GAG synthesis (10 nmol/L), RU38486 attenuated the glucocorticoid effect in a dose-dependent manner, with half-maximal inhibition at 5 nmol/L and maximal inhibition at 100 nmol/L. The antagonist blocked as much as approximately 80% of the Dex inhibition and approximately 70% of the RU28362 inhibition. Cycloheximide (25 micrograms/mL) inhibited [3H]HA synthesis by 55% within four hours of its addition to the culture. When cultures were pretreated with cycloheximide without or with Dex (100 nmol/L) for three hours and then treated with actinomycin D (2 micrograms/mL) followed by [3H]acetate labeling, Dex pretreated cultures synthesized 33% less [3H]GAG, implying that Dex had decreased the abundance of a translatable mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cultured human cells with a high content of metallothionein show resistance against gold-chloride

Resistance to gold(III)chloride was tested in cultures of human epithelial cells derived from normal skin. Two cell strains were used, a 'wild type' (HE) and a substrain, previously made resistant to 100 mumol/l of CdCl2 (HE100), with a high content of the cysteine-rich cytoplasmic protein, metallothionein. Cell growth was studied for a period of 4 days during exposition to gold-chloride (100-400 mumol/l of HAuCl4 X 4H2O). Gold-chloride produced a dose-dependent inhibition of cell proliferation of both cell strains. Compared with untreated control cells, however, the percentage survival after 4 days' treatment with 400 mumol/l was only 11% of HE cells, vis-à-vis 44% of the HE100 cells. After 24 hours' exposition to 200 mumol gold-chloride/l, the subcellular distribution of gold was determined by gel-filtration of cytosols, and subsequent analyses for Au by flame atomic absorption spectrophotometry. In the HE100 cells 30% of the cytosolic gold co-eluted with the metallothionein. In the HE cells, which do not contain detectable amounts of metallothionein, only traces of gold were found in the corresponding eluate fractions. The total amount of intracellular gold was 15% higher in HE100 than in HE cells. This study renders it probable that the binding of gold to pre-existing metallothionein in HE100 cells affords protection against otherwise lethal gold concentrations. The significance of the results is briefly discussed in relation to therapeutic use of gold substances.     

Acute ethanol dosage reduces the synthesis of smooth muscle contractile proteins in the small intestine of the rat.

The effects of an acute dose of ethanol (75 mmol/kg body weight; ip) on protein synthesis were investigated in the small intestine of the rat (n = 6). Control rats (n = 6) were injected with isovolumetric 0.15 mol/l NaCl, ip. After 2.5 h, fractional rates of protein synthesis (defined as the percentage of tissue protein renewed each day by synthesis and RNA efficiencies (defined as the amount of protein synthesised per unit RNA) were measured with a large flooding dose (0.3 Ci/mol; 150 mmol/l; 150 mumol/100 g body weight; iv) of [4(3)H]-phenylalanine. Rats were killed 10 minutes after injection of the isotope and portions of the small intestine were rapidly dissected and frozen. Tissues and plasma were processed for phenylalanine specific radioactivities to obtain fractional rates of protein synthesis or protein synthesis rates relative to RNA. Rates of protein synthesis in mixed tissue proteins fell approximately 15-25% (p ranged from less than 0.005 to greater than 0.05), in response to acute ethanol dosage. The decrease in the synthesis rates of the cytoplasmic protein fraction was similar (p less than 0.025). Proteins extracted from the smooth muscle contractile apparatus, however, showed a greater response to ethanol--that is, 40-50% inhibition in protein synthesis (p less than 0.001). It is therefore possible that the functional disturbances in the ethanol-exposed gut may be because of changes in smooth muscle protein turnover with decreased amounts of contractile apparatus.     

Selenium has a protective role in caspase-3-dependent apoptosis induced by H2O2 in primary cultured pig thyrocytes

OBJECTIVE: Hydrogen peroxide (H2O2), necessary for thyroid hormonogenesis, is produced at the apical surface of the thyroid follicular epithelium. Excess H2O2 is potentially cytotoxic and may contribute to the development of hypothyroidism, e.g. in severe selenium deficiency. Yet it is unclear how H2O2 contributes to thyroid cell death. DESIGN AND METHODS: H2O2-induced apoptosis and necrosis were studied in primary cultured pig thyroid cells. Glutathione peroxidase (GPx) activity was altered by culture in low serum with or without selenite substitution. Apoptosis was evaluated by spectrofluorometric measurement of caspase-3-specific substrate cleavage, and by analysis of DNA fragmentation by agarose gel electrophoresis. Necrosis was detected by 51Cr release from prelabeled cells. RESULTS: Exogenous H2O2 dose-dependently (100-400 micromol/l) activated caspase-3 within 3-12 h, and DNA degradation was observed after 24 h. The potency of H2O2 to induce apoptosis was low compared with that of staurosporine, a strong proapoptotic agent. H2O2-treated cells with reduced GPx activity showed increased caspase-3 activation. Incubation of serum-starved cells with selenite (10-100 nmol/l) normalized the GPx activity and reduced the activation of caspase-3 by H2O2. High H2O2 concentrations (400-800 micromol/l) were required to obtain necrosis. The H2O2-induced necrosis was exaggerated by both low GPx activity and catalase inhibition. CONCLUSIONS: Cytotoxic effects of H2O2 on thyroid cells include caspase-3-dependent apoptosis that occurs at H2O2 concentrations insufficient to induce necrosis. Selenium deficiency aggravates the apoptotic response, probably due to impaired capacity of GPx to degrade H2O2.     

2'-Deoxycytidine protects normal human bone marrow progenitor cells in vitro against the cytotoxicity of 3'-azido-3'-deoxythymidine with preservation of antiretroviral activity

Bone marrow cytotoxicity of 3'-azido-3'-deoxythymidine (AZT), an anti- human immunodeficiency virus (anti-HIV) drug, has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined, in vitro, the effect of high, but clinically achievable and nontoxic, concentrations of 2'-deoxycytidine (dCyd) (greater than or equal to 100 mumol/L) on high-dose AZT mediated growth inhibition and intracellular biochemical perturbations in normal bone marrow progenitor cells. Colony formation by bone marrow progenitor cells in semisolid medium was significantly protected by dCyd against the inhibitory effects of co-administered, high concentrations of AZT (10 mumol/L). Also, dCyd significantly corrected AZT mediated depletion of intracellular thymidine triphosphate (dTTP) and dCyd triphosphate (dCTP) levels in normal bone marrow mononuclear cells (BMMC). Moreover, dCyd reduced the intracellular accumulation of AZT triphosphate (AZT-TP) and its DNA incorporation in BMMC. In contrast, co-administration of dCyd (100 mumol/L to 1 mmol/L) did not reverse AZT (10 mumol/L) mediated suppression of HIV infectivity in HUT-102 cells in culture, although a partial reduction in intracellular AZT-TP pools and its DNA incorporation as well as a correction of AZT mediated depletion of dTTP and dCTP pools was observed in these cells. These studies suggest that dCyd at high concentrations might ameliorate the bone marrow cytotoxicity of high-dose AZT without impairing its anti-HIV effect.     

Effect of inhibition of DNA synthesis on recovery of X-irradiated L5178Y-S cells, II. 9-beta-D-arabinofuranosyladenine

The effects of 9-beta-D-arabinofuranosyladenine (araA) on radiosensitivity, postirradiation changes in the cell cycle distribution and DNA synthesis in mouse L5178Y-S lymphoma cells are described. Increase in the araA concentration (from 25 to 200 mumol/l) was is correlated with reduction in clonogenic survival except in the low concentration range. where resistance was observed. AraA treatment (200 mumol/l for 2 h) temporarily inhibited progression through the G1/S boundary. AraA (200 mumol/l applied immediately after X-irradiation (1 Gy) for 2 h gave a radiosensitizing effect with only slight changes in postirradiation cell cycle distribution; at higher doses (4 and 6 Gy) the effect was less pronounced. AraA slowed down progression through the cell cycle after irradiation and enhances the lethal effect of radiation. The phenomenon was dependent on the time between irradiation and araA treatment. The differences in dose-related radiosensitization created araA + X-ray survival curves with extrapolation numbers lower than 1. This may reflect the existence of relatively radioresistant and radiosensitive subpopulations in the araA-treated population.     

Histamine H1 receptors on adherent rheumatoid synovial cells in culture: demonstration by radioligand binding and inhibition of histamine-stimulated prostaglandin E production by histamine H1 antagonists.

Histamine H1 receptors have been demonstrated on adherent rheumatoid synovial cells using biochemical and radioligand binding assays in vitro. The addition of histamine (17.8 mumol/l) to nine primary cultures of adherent rheumatoid synovial cells resulted in a two- to 21-fold increase in the production of prostaglandin E (PGE). This increase was inhibited by three H1 receptor antagonists (mepyramine, tripelennamine, and chlorpheniramine) in a dose related manner at concentrations below 10(-6) mol/l. Competitive binding assays with [3H]mepyramine gave ED50 values of approximately 10(-5) mol/l for the three H1 antagonists. H2 receptor antagonists (cimetidine and ranitidine) did not inhibit the histamine induced increase in PGE and did not compete effectively with the binding of H1 antagonists.     

Stimulation of glycoprotein and protein synthesis in isolated pig gastric mucosal cells by prostaglandins.

The purpose of this study is to evaluate the effects of different prostaglandin derivatives on protein and glycoprotein synthesis and secretion in isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into acid insoluble macromolecules (AIM). PGE2 and 16,16-dimethyl-PGE2 enhanced the incorporation of the amino sugar into cellular (EC50 8 and 75 nmol/l) and secreted (EC50 30 and 270 nmol/l) AIM in a concentration dependent manner during a 20 hours incubation. After incubation for eight hours or more they also stimulated the incorporation of [3H]L-leucine into cellular AIM. PGF2 alpha was considerably less potent (EC50 greater than 1 mumol/l) than the E-type prostaglandins. Iloprost, a stable prostacyclin analogue, was ineffective.     

Occurrence of (E)-4-hydroxy-2-nonenal in plasma and synovial fluid of patients with rheumatoid arthritis and osteoarthritis.

(E)-4-Hydroxy-2-nonenal (HNE), a cytotoxic propagation product of lipid peroxidation, is present in the synovial fluid (0.54 (0.19) mumol/l; mean (SE), n = 9) and plasma (0.34 (0.09) mumol/l, n = 9) of patients with rheumatoid arthritis. This compound was also found in the synovial fluid (0.24 (0.19) mumol/l, n = 9) and plasma (0.09 (0.03) mumol/l, n = 9) of patients with osteoarthritis. The concentration of HNE in the plasma of patients with rheumatoid arthritis was significantly greater than in patients with osteoarthritis.     

Evidence that growth hormone depletion and uncoupling of the regulatory protein of adenylate cyclase (Ns) both contribute to the desensitization of growth hormone responses to growth hormone-releasing factor

Recent data suggest that the response of GH to GH-releasing factor (GRF) is reduced following prior exposure to high concentrations of GRF. However, it is unknown whether this is due to alterations in GRF receptors, adenylate cyclase activity or the size of a GRF-releasable storage pool of GH. In order to clarify these questions we have compared the effects of pretreatment with GRF (10 nmol/l every 2 h for 12 h) with those of pretreatment with somatostatin (SRIF; 1 mumol/l), forskolin (10 mumol/l) and GRF plus SRIF (10 nmol/l and 1 mumol/l added together) on the subsequent responses of GH to GRF (1 pmol/l-10 nmol/l), cholera toxin (10 nmol/l), 3-isobutyl-l-methylxanthine (IBMX) (100 mumol/l) and forskolin (10 mumol/l). Experiments were performed on 4-day monolayer cultures of rat anterior pituitary cells. The cells were pretreated with test substances every 2 h for 12 h and incubated with GRF or forskolin for 3 h. Per cent maximal (Bmax) GH responses to GRF (10 nmol/l) were reduced after pretreatment with both GRF (control, 173% of basal; GRF, 25% of basal; P less than 0.001) and forskolin (98% of basal; P less than 0.001), but were increased after pretreatment with SRIF (246% of basal; P less than 0.02). However, GH responses after pretreatment with GRF plus SRIF were not significantly different from those of the control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the viscosity of hyaluronic acid after exposure to a myeloperoxidase-derived oxidant

Both purified hyaluronic acid (HA) and bovine synovial fluid react with OCI-, the major oxidant produced by the myeloperoxidase (MPO)/H2O2/CI- system, resulting in a decrease in their specific viscosity. This reaction is inhibited in the presence of excess methionine. H2O2 alone decreases the viscosity of HA, presumably by the Fenton reaction, in the absence (but not in the presence) of the iron chelator, diethyltriaminepentacetic acid (DETAPAC). In the presence of DETAPAC, incubation of HA with the complete MPO/H2O2/CI- system lowered the viscosity of HA. Analysis of 3H-HA exposed to OCI- by gel filtration chromatography indicated that cleavage of HA occurred only at higher OCI- concentrations. We suggest that the reduction in viscosity of HA by the MPO/H2O2/CI- system may be due to a combination of oxidative cleavage and changes in the conformation of the molecule. We speculate that the changes in the molecular size of rheumatoid synovial fluid HA may be due to the action of the neutrophil MPO/H2O2/CI- system.     

Adrenal androgens and illness

We have studied the adrenal androgen status of medically ill patients, patients before and after cholecystectomy and during recovery from burns injury. In patients ill for less than 2 weeks, serum androstenedione concentrations (mean +/- SEM) were raised (7.94 +/- 0.98 nmol/l) as compared with a control group (4.83 +/- 0.38 nmol/l, P less than 0.005) or with patients ill for more than 2 weeks (5.21 +/- 0.46 nmol/l, P less than 0.02); serum dehydroepiandrosterone sulphate (DHAS) levels were lower in patients ill for more than 2 weeks (1.21 +/- 0.42 mumol/l) than in either the acutely ill group (5.98 +/- 1.06 mumol/l, P less than 0.001) or the control ill group (5.56 +/- 0.59 mumol/l, P less than 0.001). In post-operative patients serum DHAS levels fell to below pre-operative levels reaching a nadir at day 8 (0.54 +/- 0.19 vs 1.66 +/- 0.56 mumol/l, P less than 0.02). In burned patients serum cortisol levels were increased on admission (661 +/- 91 vs 359 +/- 30 nmol/l, P less than 0.005) and remained high over the study period. Serum androstenedione concentrations were also high on admission (7.5 +/- 1.0 vs 3.9 +/- 0.3 nmol/l, P less than 0.02). Serum DHAS concentrations were similar to control values on admission (6.8 +/- 1.2 vs 5.2 +/- 0.7 mumol/l), fell to low levels thereafter reaching a nadir during week 3 (1.6 +/- 0.6 mumol/l, P less than 0.001). Steroid synthesis in times of chronic illness may be diverted from adrenal androgen to corticosteroid pathways ensuring maintained secretion of cortisol, which is essential to the health of ill patients.