Arachidonic acid regulation of steroid synthesis: new partners in the signaling pathway of steroidogenic hormones.

Although the role of arachidonic acid (AA) in trophic hormone-stimulated steroid production in various steroidogenic cells is well documented, the mechanism responsible for AA release remains unknown. We have previously shown evidence of an alternative pathway of AA generation in steroidogenic tissues. Our results are consistent with the hypothesis that, in steroidogenic cells, AA is released by the action of a mitochondrial acyl-CoA thioesterase (MTE-I). We have shown that recombinant MTE-I hydrolyses arachidonoyl-CoA to release free AA. An acyl-CoA synthetase specific for AA, acyl-CoA synthetase 4, has also been described in steroidogenic tissues. In the present study we investigate the new concept in the regulation of intracellular levels of AA, in which trophic hormones can release AA by mechanisms different from the classical PLA2-mediated pathway. Inhibition of ACS4 and MTE-I activity by triacsin C and NDGA, respectively results in a reduction of StAR mRNA and protein abundance. When both inhibitors are added together there is a synergistic effect in the inhibition of StAR mRNA, StAR protein levels and ACTH-stimulated steroid synthesis. The inhibition of steroidogenesis produced by the NDGA and triacsin C can be overcome by the addition of exogenous AA. In summary, results shown here demonstrate a critical role of the acyl-CoA synthetase and the acyl-CoA thioesterase in the regulation of AA release, StAR induction, and steroidogenesis. This further suggests a new concept in the regulation of intracellular distribution of AA through a mechanism different from the classical PLA2-mediated pathway that involves a hormone-induced acyl-CoA synthetase and a hormone-regulated acyl-CoA thioesterase.

Reduction of exercise-induced asthma oxidative stress by lycopene, a natural antioxidant.

BACKGROUND: Lycopene has previously been shown to have high antioxidative activity. In view of the controversy regarding the beneficial effect of antioxidants on asthma, the acute effects of lycopene (LYC-O-MATO) on airway hyperreactivity were assessed in patients with exercise-induced asthma (EIA). METHODS: Twenty patients with EIA participated in our study to verify the antioxidative effects. The test was based on the following sequence: measurement of baseline pulmonary function, 7-min exercise session on a motorized treadmill, 8-min rest and again measurement of pulmonary function, 1-week, oral, randomly administered, double-blind supplementation of placebo or 30 mg/day of lycopene (LYC-O-MATO), measurement of pulmonary function at rest, 7-min exercise session, and 8-min rest and again measurement of pulmonary function. A 4-week washout interval was allowed between each protocol. RESULTS: All patients given placebo showed significant postexercise reduction of more than 15% in their forced expiratory volume in 1 s (FEV1). After receiving a daily dose of 30 mg of lycopene for 1 week, 11 (55%) patients were significantly protected against EIA. Serum analyses of the patients by high-pressure liquid chromatography detected in the lycopene-supplemented patients an elevated level of lycopene compared to the placebo group, with no change in retinol, tocopherols, or in the other carotenoids. CONCLUSIONS: Our results indicate that a daily dose of lycopene exerts a protective effect against EIA in some patients, most probably through an in vivo antioxidative effect.

Regulation of arachidonic acid release in steroidogenesis: role of a new acyl-CoA thioestrase (ARTISt).

It has been well established that arachidonic acid (AA) and its metabolism to leukotrienes plays an obligatory role in steroid production. The release of AA is regulated by hormone stimulation and protein phosphorylation. We have cloned a cDNA of a phosphoprotein with a molecular mass of 43 kDa (p43), purified from the cytosol of stimulated adrenal glands. This protein acts as intermediary in the stimulation of steroid synthesis through AA release, and has been found to be a member of a recently described acyl-CoA thioesterase family. In view of the mandatory role of this protein in the activation of AA-mediated steroidogenesis, the term Arachidonic acid-Related Thioesterase Involved in Steroidogenesis (ARTISt), is proposed for p43. The present study describes the production of the recombinant protein by cDNA expression in Escherichia coli and its functional characterization. Recombinant acyl-CoA thioesterase was capable to release AA from the respective acyl-CoA, and this activity was affected by well-recognized inhibitors of AA release and metabolism: 4-bromophenacyl bromide (BPB) and nordihydroguariaretic acid (NDGA). In addition, the inhibition of acyl-CoA thioesterase activity by NDGA correlates with the inhibition of steroid synthesis produced by this compound in adrenal cortex cells. Moreover, the recombinant protein was phosphorylated in vitro by PKA. These results provide the first evidence linking acyl-CoA thioesterases with the regulation of steroidogenesis, and support a regulatory role for acyl-CoA thioesterases in steroidogenic tissues, suggesting an alternative pathway for AA release in signal transduction.

Activation of a thioesterase specific for very-long-chain fatty acids by adrenergic agonists in perfused hearts.

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.

Prevention of exercise-induced asthma by a natural isomer mixture of beta-carotene.

BACKGROUND: The unicellular alga Dunaliella bardawil was previously shown to contain very high concentrations of beta-carotene composed of equal amounts of the all-trans and 9-cis stereoisomers which differ in their physicochemical features and antioxidative activity. Due to the controversy regarding the beneficial effect of antioxidants on asthma, the acute effects of beta-carotene of Dunaliella was assessed on airway hyperreactivity in patients with exercise-induced asthma (EIA). METHODS: Thirty-eight patients with EIA participated in our study to verify the antioxidative effect. The test was based on the following sequence: baseline pulmonary function, 7 minutes exercise session on a motorized treadmill, 8 minutes rest, 1-week oral random, double-blind supplementation of placebo or 64 mg/day beta-carotene, pulmonary functions at rest, 7 minutes exercise session, 8 minutes rest and again pulmonary functions. RESULTS: All patients given placebo showed a significant postexercise reduction of more than 15% in their forced expiratory volume in one second (FEV1). Of the 38 patients who received a daily dose of 64 mg of beta-carotene for 1 week, 20 (53%) were protected against EIA. CONCLUSIONS: Our results indicate that a daily dose of Dunaliella beta-carotene exerts a protective effect against EIA in some patients most probably through in vivo antioxidative effect.

An adrenocorticotropin-regulated phosphoprotein intermediary in steroid synthesis is similar to an acyl-CoA thioesterase enzyme.

We have previously reported the purification of a phosphoprotein (p43) intermediary in steroid synthesis from adrenal zona fasciculata [Paz C., Dada, L. A., Cornejo Maciel, M. F., Mele, P. G., Cymeryng, C. B., Neuman, I., Mendez, C. F., Finkielstein, C. V., Solano, A. R., Park, M., Fischer, W. H., Towbin, H., Scartazzini, R. & Podestá, E. J. (1994) Eur J. Biochem. 224, 709-716]. Here, we describe the cloning and sequencing of a cDNA encoding p43 as well as the hormonal regulation of the p43 transcript. The protein resulted homologous to a very recently described mitochondrial peroxisome-proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I). The deduced amino acid sequence of the protein shows consensus sites for phosphorylation by different protein kinases, and a lipase serine motif. Antibodies raised against a synthetic peptide that includes the lipase serine motif and against the N-terminal region of p43 block the action of the protein. The transcript of p43 was detected in ovary of pseudopregnant rats, rat adrenal zona fasciculata and glomerulosa, mouse Leydig tumor cell line (MA-10), rat brain and human placenta. Inhibition of adrenocorticotropin hormone (ACTH) release and steroid synthesis by dexamethasone produced a dose-dependent decrease in the abundance of the adrenal transcript. The transcript was induced by in vivo stimulation of the adrenals with ACTH. The effect had a rapid onset (5 min), reached maximal stimulation (62%) at 15 min, and returned to basal levels at 30 min. The effect of ACTH on the p43 transcript was inhibited by actinomycin D and enhanced by cycloheximide. Our results provide the first evidence linking acyl-CoA thioesterases with very-long-chain specificities, and a protein intermediary in steroid synthesis, thereby supporting a regulatory role for acyl-CoA thioesterases in steroidogenic tissues.

Narrow-band red light phototherapy in perennial allergic rhinitis and nasal polyposis.

BACKGROUND: Allergic rhinitis and nasal polyposis are common nasal diseases, but the available treatment modalities have only limited success. OBJECTIVE: To assess the therapeutic effect of low-energy narrow-band red light phototherapy on nasal clinical symptoms of allergic rhinitis and nasal polyposis. METHODS: In a double-blind randomized prospective study, 50 patients with allergic rhinitis and 10 with nasal polyposis received intranasal illumination at 660 nm for 4.4 minutes three times a day for 14 days (total dose 6 joules per day). Twenty-nine rhinitic patients and one patient with polyposis received equivalent sham illumination as placebo. Evaluation was based on symptom scores and a clinical assessment that included pre-treatment and post-treatment videotaped rigid and flexible nasendoscopy. RESULTS: Following treatment, improvement of symptoms was reported by 72% of the allergic rhinitis patients and objective improvement was endoscopically demonstrated in 70% of them as compared with 24% and 3%, respectively, in the placebo group. These differences were significant. No improvement was obtained in any of the patients with polyposis. CONCLUSIONS: Allergic rhinitis, if uncomplicated by polyps or chronic sinusitis, can be effectively treated by narrow-band red light illumination of the nasal mucosa at 660 nm, with marked alleviation of clinical symptoms. Whenever possible, candidates for phototherapy should be selected by endoscopic examination.

Blocking effect of vitamin C in exercise-induced asthma.

OBJECTIVE: To determine if vitamin C (ascorbic acid) has a protective effect on the hyperreactive airways of patients with exercise-induced asthma (EIA). DESIGN: All the patients underwent pulmonary function tests at rest, before and 1 hour after receiving 2 g of oral ascorbic acid. They were then randomly assigned in a double-blind manner to receive 2 g of ascorbic acid or a placebo 1 hour before a 7-minute exercise session on a treadmill. Pulmonary function tests were performed after an 8-minute rest. This procedure was repeated 1 week later, with each patient receiving the alternative medication. SETTING: A university hospital. PARTICIPANTS: Twenty patients with asthma (13 males and 7 females), with ages ranging from 7 to 28 years (mean, 13.8 years). All patients who had a decline of at least 15% in their forced expiratory volume in 1 second after a standard exercise test on a motorized treadmill received a diagnosis of EIA. MAIN-OUTCOME MEASURES: All patients were advised to stop using their regular asthma medication or bronchodilator 12 hours before the test. Pulmonary function tests were performed in the same ambient conditions on all patients. RESULTS: All patients received a diagnosis of EIA. Ascorbic acid administration did not change the results of pulmonary functions at rest after 1 hour. In 9 patients, a protective effect on exercise-induced hyperreactive airways was documented. Four of 5 patients who received ascorbic acid and documented a protective effect on EIA continued to receive ascorbic acid, 0.5 g/d, for 2 more weeks with the same protective effect. CONCLUSIONS: The efficacy of vitamin C in preventing EIA cannot be predicted. However, vitamin C may have a protective effect on airway hyperreactivity in some patients with EIA.

Involvement of arachidonic acid and the lipoxygenase pathway in mediating luteinizing hormone-induced testosterone synthesis in rat Leydig cells.

Evidence has been introduced linking the lipoxygenase products and steroidogenesis in Leydig cells, thereby supporting that this pathway may be a common event in the hormonal control of steroid synthesis. On the other hand, it has also been reported that lipoxygenase products of arachidonic acid (AA) may not be involved in Leydig cells steroidogenesis. In this paper, we investigated the effects of PLA2 and lipoxygenase pathway inhibitors on steroidogenesis in rat testis Leydig cells. The effects of two structurally unrelated PLA2 inhibitors (4-bromophenacyl bromide (BPB) and quinacrine) were determined. BPB blocked the LH- and Bt2cAMP-stimulated testosterone production but had no effect on 22(4)-OH-cholesterol conversion to testosterone. Quinacrine caused a dose-dependent inhibition of LH- and Bt2cAMP-induced steroidogenesis. The effects of different lipoxygenase pathway inhibitors (nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), caffeic acid and esculetin) have also been determined. Both NDGA and ETYA inhibited LH- and Bt2cAMP-stimulated steroid synthesis in a dose-related manner. Furthermore caffeic acid and esculetin also blocked the LH-stimulated testosterone production. Moreover, exogenous AA induced a dose-dependent increase of testosterone secretion which was inhibited by NDGA. Our results strongly support the previous concept that the lipoxygenase pathway is involved in the mechanism of action of LH on testis Leydig cells.

Characterization of the cDNA corresponding to a phosphoprotein (p43) intermediary in the action of ACTH.

We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal peptides of p43.

Cytosolic and mitochondrial proteins as possible targets of cycloheximide effect on adrenal steroidogenesis.

It is well accepted that protein(s) with a short half-life are required in the pathway leading to steroid synthesis following stimulation by trophic hormones. A correlation between the disappearance of several proteins in different subcellular compartments and the inhibition of steroid synthesis produced by cycloheximide (CHx) has also been shown. In the present report we describe the effect of CHx in the stimulation of steroid synthesis using a cell-free assay. Mitochondrial progesterone (P4) production was studied by recombination of the different subcellular fractions of adrenal zona fasciculata and determined by radioimmunoassay. Soluble factors from ACTH-treated adrenals produced a four-fold stimulation of mitochondrial steroidogenesis (3.0 +/- 0.6 vs. 13.3 +/- 0.5 ng P4/tube for control and ACTH-treated adrenals respectively). Mitochondria obtained from CHx-ACTH-treated adrenals fail to respond to soluble ACTH-dependent factors. A permeable analogue of cholesterol (22(R)-OH cholesterol) could overcome the inhibition imposed by CHx, confirming the role of mitochondrial proteins in intramitochondrial cholesterol transport. The treatment of the adrenals with CHx 10 minutes before ACTH administration abolished also the stimulation induced by the cytosol on control mitochondria (2.6 +/- 0.5 vs. 13.0 +/- 1.0 ng P4/tube for CHx-ACTH-treated cytosol vs. ACTH-treated cytosol). Arachidonic acid (AA) added to CHx-ACTH-treated cytosol subdued this inhibition (10.3 +/- 1.2 ng P4/tube). CHx treatment had no effect on the stimulation by ACTH of the cAMP-dependent protein kinase. These results indicate the involvement of a cycloheximide-sensitive protein in the release of AA in adrenal steroidogenesis.

Purification of a novel 43-kDa protein (p43) intermediary in the activation of steroidogenesis from rat adrenal gland.

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five-step procedure that includes DEAE-cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43-kDa that retains the capacity to stimulate steroid synthesis in an in vitro recombination assay. This activity is inhibited by the use of phospholipase A2 inhibitors. Antipeptide antibodies against the N-terminal region recognize p43 as a double band on SDS/PAGE that resolves in different spots on two-dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearance of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N-terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein. We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin-induced stimulation of arachidonic acid release and steroid synthesis.

[Mechanical lithotripsy of a gastric bezoar]. / Mechanická litotripse bezoáru zaludku.

At the First Medical Clinic of the Faculty Hospital Prague Motol the authors detected during a routine gastroscopic examination of a 71-year-old female patient the presence of a gastric bezoar. The examination was indicated on account of dyspeptic complaints. With regard to the size of the bezoar (50 x 20 mm) the authors decided for mechanical lithotripsy. It proved possible to reduce the concrement during the first session and during subsequent sessions it was broken down to small size and the fragments were eliminated per vias naturales. Several fragments were extracted. Chemical analysis provided evidence of the origin of the concrement in the biliary pathways--the concrement was pure cholesterol. ERCP did not reveal an artificial communication between the biliary system and the stomach or duodenum. It did not prove possible to visualize the gallbladder by ultrasonography. The selected therapeutic procedure was successful.

Biomonitoring: cadmium deteriorates electro-orientation performance in catfish.

1. Exposure of catfish, Ictalurus nebulosus, to sublethal concentrations of cadmium deteriorates electro-orientation performance. 2. Cadmium, at a concentration of 40 micrograms/l, doubles the behavioural threshold for electric stimuli within 48 hr of exposure; both prolonged exposure and higher concentrations result in higher thresholds. The effect is reversible. 3. Electro-orientation performance can be used to monitor the quality of surface water.

The action of luteinizing hormone on the testis.

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.

Astemizole in perennial allergic rhinitis with seasonal exacerbations: a placebo-controlled double-blind study.

Astemizole is a nonsedative H1-antagonist. It was used in a randomized double-blind, placebo-controlled in-season trial to assess its efficacy in patients with perennial allergic rhinitis showing seasonal exacerbations. By the last (6th) week of the study, the mean overall symptomatology, as rated by a global composite score, was significantly mitigated in the astemizole-treated group (18 patients) compared with their first week's global composite score (P less than .01). No such improvement was observed in the placebo-treated group (18 patients). Side effects were not appreciable throughout this short-term study. We conclude that astemizole is effective in the treatment of patients with perennial allergic rhinitis showing seasonal enhancement of symptoms.

Variations in skin tests before and after immunotherapy in allergic asthma.

The clinical relevance of positive intradermal and prick skin tests were evaluated on 195 patients with allergic asthma subjected to prick test and intradermal tests before and after 3 years of immunization. Satisfactory clinical improvement followed immunotherapy based on the results of intradermal skin tests. Skin reactivity may change in time with or without immunotherapy.

The kinin system in exercise-induced asthma.

Seventeen asthmatic children, nine with and eight without exercise-induced asthma (EIA), and nine control non-asthmatic children were studied in an attempt to discern possible associations between the activity of the kinin system and EIA. Pulmonary function tests and clinical check-up were performed before and after 6 min of free-range running. Concomitant blood tests revealed a consistent elevation of the kallikrein levels following the exercise challenge in all experimental groups. However, only in the EIA positive group did this elevation exceed the normal laboratory range (16 +/- 7.0 mu/ml). Prekallikrein levels both before and after exercise did not exceed in any group the normal laboratory values. The findings thus suggest that provocation of EIA is associated with a certain threshold of kallikrein level below which no such symptoms are observed. EIA may be triggered only when kallikrein levels are in excess.

Hyperreactivity to bee stings: reevaluation.

(a) A survey of beekeepers in Israel did not show that they differed allergy-wise from the general population. (b) Twenty-eight non-beekeeper systemic reactors to bee stings were not necessarily more allergic or more exposed to stings than the others. These systemic reactors had a spontaneous kinin system hyperreactivity and in some of them changes in the immune system and a complement defect were detected. We think that predisposition may enhance the likelihood of a systemic reaction to bee stings. Thus the common denominator for the hyperreactivity to bee stings is not their exposure to stings nor their allergic background but rather the spontaneous kinin activity and a possible defect in their immunological system.

Differences between swimming and running as stimuli for exercise-induced asthma.

Thirteen children each exercised for 6 min by running on a treadmill and by tethered swimming, breathing air at room temperature and either 8% or 99% relative humidity continuously. Ventilation, gas exchange and heart rate were closely matched in all four tests in each child, with a mean oxygen consumption of 32.3 +/- 1.7 ml x min-1 x kg-1. The post-exercise fall in FEV1 expressed as a percentage of the baseline FEV1 (delta FEV1) was significantly greater after running compared with swimming breathing either humid or dry air. The delta FEV1 was also related to respiratory heat loss (RHL) calculated from measurements of inspired and expired gas temperature and humidity. At a standardised RHL, the difference between running and swimming was highly significant [delta FEV1 (%) +/- SE = 39 +/- 5 and 28 +/- 4 respectively, p less than 0.01]. These experiments suggest that the type of exercise influences the severity of exercise-induced asthma even under conditions of the same metabolic stress and respiratory heat loss.