O-dibutyryl Adenosine Research Articles

Research on the improvement of GABAergic differentiation in the bone marrow stromal cells with Hes1 gene silencing

Objective To study the influence of Hesl gene silencing on the GABAergic differentiation of bone marrow mesenchymal stem cells （ BMSCs ）. Methods BMSCs were isolated from 2-3 weeks SD rats and purified in vitro, then characterized by flow eytometry. After that, Hesl gene of BMSCs was inhibited by RNAi technology, then BMSCs （control group） and Hesl gene silencing BMSCs （experimental group） were induced by conditioned medium （10 pJnol of retinoic acid, 1 mmol NS, O2-dibutyryl adenosine 3＇： 5＇-cyclic monophosphate, 2% B27） respectively. Finally, immunocytoehemistry and Western blot were employed to analyze the differentiation potential of the induced cells. Results Experimental group and control group were induced by conditioned medium for 3-7 days, immunocytochemistry results showed that the percentage of GAD67-and NeuN- positive cells were （35. 7 ± 2. 8） % and （72. 4 ± 3. 2） % in the experimental group, which were more than the control group significantly （ P 〈 O. 05 ） ; Western blot results also showed that the protein gray value ratio of GAD67 or NeuN compared to the 13-aetin in the experiment was significantly higher than the control group through Bandscan software analysis （ P 〈0. 01 ）. Conclusions Hesl gene silencing can promote BMSCs to differentiate into GABAergic neurons. Key words: Bone marrow mesenchymal stem cells; Hesl; Differentiation

Effects of dibutyryl cAMP on growth performance and carcass traits in finishing pigs

This study was designed to investigate the effects of dietary supplementation with N6, 2′-O-dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP) on growth performance, carcass traits, histochemical characteristics and serum constituents in finishing pigs. Seventy-two Duroc×(Landrace×Large White) barrows (57.3±0.6kg) were randomly allotted to 3 treatments with 6 replicate pens/treatment (4 pigs/pen). The pigs were fed diets containing 0, 10 and 20mgdbcAMP/kg, respectively, until the final slaughter weight of approximately 90kg. There were no differences in growth performance among dietary treatments. Leaf fat proportion and first rib backfat thickness were reduced (P<0.05), whereas tenth rib backfat thickness tended to decrease (P=0.10), in pigs fed 10mgdbcAMP/kg. Lean percentage was greater (P<0.05) and longissimus muscle area tended to increase (P=0.10) in pigs fed 10mgdbcAMP/kg when compared to the control group, but hot carcass weight was not affected by dbcAMP. Growth rate of fat-free lean tissues tended to increase (P=0.09) in dbcAMP-supplemented pigs. Dietary dbcAMP decreased (P<0.05) adipocytes diameter in subcutaneous fat, whereas longissimus muscle fiber diameter tended to increase (P=0.06) with dbcAMP supplementation; however, no difference in longissimus muscle cell density was detected among treatments. Serum concentrations of total protein and 3′,5′-cyclic adenosine monophosphate increased (P<0.05) in response to dbcAMP, but concentrations of triglycerides, total cholesterol, glucose and urea in serum did not differ among dietary treatments. These results indicate that dbcAMP had a positive effect on carcass traits. Addition of 10mgdbcAMP/kg to the diet was beneficial for growth performance and lean percentage, as well as improving protein and fat metabolism.

Switching of G-protein Usage by the Calcium-sensing Receptor Reverses Its Effect on Parathyroid Hormone-related Protein Secretion in Normal Versus Malignant Breast Cells

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galphai in MMECs but coupled to Galphas in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.

U50,488H Inhibits Effects of Norepinephrine in Rat Cardiomyocytes—Cross-Talk between κ-opioid and β-adrenergic Receptors

In order to determine the effect ofκ-opioid receptor agonist on theβ1-adrenoceptor stimulation in the heart, the effects of norepinephrine (NE), aβ1-adrenoceptor agonist, on contraction and electrically induced intracellular calcium ([Ca2+]i) transient in the single rat ventricular myocyte pretreated with aκ-opioid receptor agonist, trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide(U50,488H), at 0.01–1μMwere studied with a video edge tracker method and a spectrofluorometric method using fura-2 as calcium indicator, respectively. NE at 0.01–10μMaugmented both twitch amplitude and electrically induced [Ca2+]itransient dose-dependently, which were abolished by propranolol at 1μm, aβ-adrenoceptor antagonist. The effects of NE on both contraction and [Ca2+]itransient were attenuated in a dose-dependent manner by U50,488H at 0.01–1μM, which itself had no effect at all. The maximum response (Emax) was decreased, while the concentration that produces 50% of the maximum response (EC50) was enhanced, by U50,488H. The inhibitory effects of U50,488H onβ-adrenoceptor stimulation were completely blocked by pretreatment with norbinaltorphimine, a specificκ-opioid receptor antagonist at 1μM, or preincubation with pertussis toxin (PTX) at 200 ng/ml for 6 h. On the other hand, the inhibition on NE-induced augmentation in electrically induced [Ca2+]itransient by U50,488H was not affected by pretreatment with U73122, a specific inhibitor of phospholipase C (PLC), at 10μmfor 30 min. U50,488H attenuated the augmentation of the electrically stimulated [Ca2+]itransient induced by forskolin at 0.1 and 0.5μM. It did not, however, affect the augmentation of the electrically induced [Ca2+]itransient by N6,2′-O-dibutyryl adenosine cyclic monophosphate (DB-cAMP). The results suggest thatκ-opioid receptor stimulation by U50,488H at 10−6Mor lower may inhibit the effects ofβ-adrenoceptor stimulation by acting at a PTX-sensitive G-protein and AC, but not via the phosphoinositol pathway.

Catecholamines increase monocyte TNF receptors and inhibit TNF through beta 2-adrenoreceptor activation.

Postinjury deficits in monocyte tumor necrosis factor receptors (moTNFR) activity may alter beneficial functions during an inflammatory response. Several counter-regulatory hormones elicited during inflammation may modulate tumor necrosis factor (TNF) activity, but little is known about their influence on moTNFR. Also, catecholamines inhibit TNF production, but the adrenoreceptor mechanism of this effect has not been fully clarified. To determine the effect of catecholamines and corticosteroids on moTNFR, whole blood was coincubated for up to 8 (moTNFR) or 24 h (cytokines) in the presence of lipopolysaccharide (100 ng/ml) and 1) epinephrine (Epi, 10(-6) M), dexamethasone (Dex, 10(-6) M) or both (EpiDex, 10(-6) M) to assess the expression of total moTNFR, moTNFR-I, and moTNFR-II. 2) Epi and norepinephrine (EpiNE, 10(-6) M) and the alpha 1 + 2-, beta 1 + 2-, beta 1-, or beta 2-adrenergic antagonists were used to assess the role of such adrenoreceptors on total moTNFR and TNF production, and N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) alone or in combination with the phosphodiesterase inhibitor Ro-20-1724/000, to study the cAMP-dependent pathway on total moTNFR. We found that Epi upregulated total moTNFR and moTNFR-II. Dex did not significantly influence total moTNFR or moTNFR-II. Also, EpiNE increased total moTNFR and inhibited TNF by a beta 2-dependent mechanism. DBcAMP (10(-5) M) modestly enhanced total moTNFR. This suggests a common mechanism for acutely enhancing moTNFR and attenuation of soluble TNF appearance during conditions of severe stress.

Visceral adiposity, increased adipocyte lipolysis, and metabolic dysfunction in obese postmenopausal women.

This study determines whether there are regional differences in lipolysis and whether adipocyte lipolysis is associated with the degree of visceral adiposity and its metabolic complications in 32 obese (28-37 kg/m2), nondiabetic, postmenopausal women. In vitro lipolysis was measured in the basal state and after addition of epinephrine (Epi), Epi plus yohimbine, Epi plus propranolol, and N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) in abdominal (ABD) and gluteal (GLT) adipocytes. Upper body obese [UBO, waist-to-hip ratio (WHR) > or = 0.80, n = 19] women had a greater intra-abdominal fat area (IA, 199 +/- 50 vs. 142 +/- 28 cm2) and Epi-stimulated lipolysis (ABD: 1.60 +/- 1.10 vs. 0.95 +/- 0.54 mumol glycerol.10(6) cells-1.2h; GLT: 1.14 +/- 0.70 vs. 0.66 +/- 0.42 mumol glycerol.10(6) cells-1.2 h) than lower body obese (LBO, WHR < 0.80, n = 13) women. The UBO women also had lower high-density lipoprotein cholesterol (1.39 +/- 0.40 vs. 1.64 +/- 0.39 mmol/l, P < 0.05), higher plasma triglycerides (1.89 +/- 0.48 vs. 1.44 +/- 0.56 mmol/l, P < 0.05), and higher fasting insulin levels (154 +/- 57 vs. 118 +/- 33 pmol/l, P < 0.05) than LBO women. Basal, adrenergic receptor-mediated, and DBcAMP-stimulated lipolytic rates in ABD and GLT adipocytes were positively correlated with IA (r = 0.44-0.76, P < 0.05, n = 28). In both UBO and LBO women, Epi-stimulated lipolysis was higher (+30%, P < 0.05) in ABD than GLT adipocytes. These results show that, in postmenopausal women, visceral obesity is associated with increased rates of lipolysis in both ABD and GLT subcutaneous adipocytes. The findings also indicate that Epi-stimulated lipolysis is greater in ABD than GLT adipocytes regardless of fat distribution.

Local exposure to salbutamol or Bt 2 cyclic AMP inhibits pleural exudation and leukocyte influx caused by antigen in rats

The local effect of salbutamol and N 6,2′- O-dibutyryl adenosine 3′:5′-cyclic monophosphate (Bt 2 cyclic AMP) on the rat pleural inflammation caused by allergen was investigated. Antigen (ovalbumin, 12 μg/cavity) intrathoracically administered to immunized rats led to a marked pleural protein extravasation and leukocyte infiltration, as attested by the quantification of protein and enumeration of leukocytes recovered from the pleural cavity. Salbutamol (10–40 μg/cavity) and the cell-permeable cyclic AMP analogue, Bt 2 cyclic AMP (20–160 μg/cavity), injected 1 h and 5 min before the antigen, respectively, inhibited the exudation occurring within 30 min, and neutrophil and eosinophil accumulation ocurring 4 and 24 h, respectively. The late eosinophilia was also markedly attenuated by salbutamol administered 10 min post-challenge, when mast cells had already been degranulated. Pretreatment with the β-adrenoceptor antagonist propranolol (1 mg/kg, i.v.) failed to modify the inhibitory effect of Bt 2 cyclic AMP, but abolished the blockade caused by salbutamol of leukocyte infiltration under conditions where the salbutamol anti-exudatory activity was impaired to about 80%. In another set of experiments, salbutamol (20 and 40 μg/cavity) markedly inhibited the exudation caused by histamine and 5-hydroxytryptamine (5-HT) which, though to a lesser extent, was also sensitive to Bt 2 cyclic AMP (80 μg/cavity). As observed with allergic pleurisy, propranolol impaired the inhibition by salbutamol of histamine- and 5-HT-induced exudation, whereas the Bt 2 cyclic AMP inhibition was not affected. We conclude that salbutamol and Bt 2 cyclic AMP share the ability to inhibit pleural exudation and leukocyte recruitment caused by allergen in immunized rats, suggesting that the anti-inflammatory effect of salbutamol may be mediated by a cyclic AMP signaling pathway, probably via β 2-adrenoceptor activation.

Gastrin secretion from primary cultures of rabbit antral G cells: Stimulation by inflammatory cytokines

BACKGROUND & AIMS: In Helicobacter pylori-induced gastritis, local production of cytokines may favor hypergastrinemia as an endocrine link between H. pylori-induced gastritis and duodenal ulcer. The aim of this study was to characterize cytokine effects on cultured rabbit antral G cells. METHODS: Monolayers (14.2% +/- 2.9% G cells) were studied after 48 hours in primary culture. RESULTS: Interleukin (IL) 1 beta (50% effective concentration [EC50], 5.3 +/- 0.4 ng/mL) and tumor necrosis factor (TNF) alpha (EC50, 5.5 +/- 0.5 ng/mL) stimulated gastrin release to 50% of the maximal response to 10(-9) mol/L neuromedin C. Stimulation by the maximally effective concentration of IL-1 beta (10 ng/mL) was inhibited by the human IL-1 receptor antagonist (100 ng/mL; inhibitory constant, 23.0 ng/mL), which prefers type I over type II IL- 1 receptors. The response to the maximally effective concentration of TNF-alpha (10 ng/mL) was markedly inhibited by monoclonal antibody H398, an antagonist at TNF P55 receptors (inhibitory constant, 1.7 micrograms/mL), whereas monoclonal antibody utr1, an antagonist at TNF P75 receptors, was ineffective. Stimulation by IL-1 beta and TNF-alpha was additive to the responses to neuromedin C and O2-dibutyryl adenosine 3',5'-cyclic monophosphate. IL-6 and IL-8 (0.1-50 ng/mL) were ineffective. CONCLUSIONS: IL-1 beta and TNF-alpha stimulate gastrin secretion via receptors potentially residing on rabbit antral G cells themselves. We speculate that G cells express type I IL-1 receptors and TNF P55 but not TNF P75 receptors. (Gastroenterology 1996 Jan;110(1):147-54)

Differential regulation of L-arginine transport and inducible NOS in cultured vascular smooth muscle cells

Experiments were performed to characterize the uptake of L-arginine in rat aortic smooth muscle cells (SMC) and to examine whether inducers of nitric oxide synthase (NOS) could regulate the transport of L-arginine into these cells. L-Arginine transport by SMC was saturable, Na+ independent, strongly inhibited in the presence of other cationic amino acids, and could be stimulated by preloading the cells with cationic amino acids. Kinetic studies revealed the presence of both a high [Michaelis constant (Km) approximately 125 microM] and low (Km approximately 1.4 mM) affinity L-arginine transporter. Treatment of vascular SMC with interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) resulted in parallel increases in L-arginine transport and nitric oxide (NO) synthesis, as measured by nitrite production. Increasing the concentration of IL-1 beta and TNF-alpha caused a progressive elevation in nitrite production but did not further stimulate L-arginine uptake. Treatment of SMC with the combination of TNF-alpha and interferon-gamma (IFN-gamma) synergistically enhanced nitrite release without having any additional effect on L-arginine transport. Both induction of L-arginine transport and NOS activity by these cytokines were blocked by cycloheximide. Finally, treatment of SMC with interferon-gamma and N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate selectively stimulated the formation of nitrite by SMC but had no effect on L-arginine transport. These results demonstrate that L-arginine transport by cultured vascular SMC is mediated by the system y+ carrier and that inducers of NOS differentially regulate the activity of this transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic regulation by N6, 2′- O-dibutyryl adenosine 3′, 5′-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N 6, 2′- O-dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12- O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10 −11M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10 −5 and 10 −3 M dbcAMP, respectively. The stimulatory effect of 10 −11 M dbcAMP was not blocked by 10 −5 M H-8 (PKA inhibitor), but the inhibitory effect of 10 −5 or 10 −3 M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.

Cholera toxin-induced G sα down-regulation in neural tissue: studies on the pineal gland

Cholera toxin (CT) treatment (50 μg/ ml) was used to down regulate the α subunit of the stimulatory guanine nucleotide binding protein (Gsα) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces G sα by ≈ 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of Gβ, G iα or G oα. This effect on G sα was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of G sα. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP ( N 6,2′-O-dibutyryl adenosine 3′,5′monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced α- or β-adrenergic stimulation. α 1-Adrenergic activation of the pineal gland elevates [Ca 2+] i, which potentiates effects of cyclic AMP; in these studies the response to α-adrenergic activation was markedly increased in 48-h CT-treated glands, reflecting Ca 2+ potentiation of the effects of elevated levels of cyclic AMP. In contrast, the effects of the selective β-adrenergic agonist isoproterenol was reduced by ≈ 75%. These studies not only establish CT-induced G sα down-regulation as a new tool for the study of adrenergic signal transduction in the pineal gland, but indicate that this paradigm is probably useful in all neural tissue.

Cyclosporin H is a potent and selective formyl peptide receptor antagonist. Comparison with N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L- leucyl-L-phenylalanine and cyclosporins A, B, C, D, and E.

The cyclic undecapeptide, cyclosporin (Cs) H, is a potent inhibitor of FMLP-induced superoxide anion (O2-) formation in human neutrophils. We studied the effects of CsH in comparison with those of N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine (BocPLPLP), a well known formyl peptide receptor antagonist, and of other Cs on activation of N6,2'-O-dibutyryl adenosine 3:5'-monophosphate-differentiated HL-60 cells and human erythroleukemia cells (HEL cells). CsH inhibited FMLP binding in HL-60 membranes with a Ki (inhibition constant) of 0.10 microM. CsH inhibited activation by FMLP of high affinity GTPase (the enzymatic activity of alpha-subunits of heterotrimeric regulatory guanine nucleotide-binding proteins) in HL-60 membranes with a Ki of 0.79 microM. CsH inhibited the stimulatory effects of FMLP on cytosolic Ca2+ concentration ([Ca2+]i), O2- formation, and beta-glucuronidase release with Ki values of 0.08, 0.24, and 0.45 microM, respectively. BocPLPLP was 14-fold less potent than CsH in inhibiting FMLP binding and 4- to 6-fold less potent than CsH in inhibiting FMLP-induced GTP hydrolysis, rises in [Ca2+]i, O2- formation, and beta-glucuronidase release. CsA reduced FMLP-induced O2- formation by 20%, but CsB, CsC, CsD, and CsE did not. CsA, CsB, CsC, CsD, and CsE did not affect FMLP-induced rises in [Ca2+]i. BocPLPLP inhibited leukotriene B4-induced rises in [Ca2+]i with a Ki of 0.33 microM, whereas CsH showed no inhibitory effect. CsH and BocPLPLP did not inhibit the rises in [Ca2+]i induced by several other stimuli in HL-60 cells and HEL cells. Our results show that 1) CsH is a more potent formyl peptide receptor antagonist than BocPLPLP; 2) unlike BocPLPLP, CsH is selective; and 3) N-methyl-D-valine which is present at position 11 of the amino acid sequence of CsH but not of other Cs is crucial for FMLP antagonism.

Differential inhibition and potentiation by cell-permeant analogues of cyclic AMP and cyclic GMP and NO-containing compounds of exocytosis in human neutrophils

The chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), complement C5a and platelet-activating factor (PAF), induce beta-glucuronidase release and aggregation and an increase in cytosolic Ca2+ [Ca2+]i in human neutrophils. We studied the roles of cAMP and cGMP in neutrophil avtivation, using their cell-permeant analogues, N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) and N2,2'-O-dibutyryl guanosine 3':5'-cyclic monophosphate (Bt2cGMP) and the NO-containing compounds, sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and its prodrug, molsidomine (SIN-10). Bt2cAMP, Bt2cGMP, SIN-1 and SIN-10 but not SNP inhibited exocytosis induced by fMet-Leu-Phe. Superoxide dismutase potentiated the inhibitory effect of SIN-1. Bt2cGMP and SNP potentiated C5a-induced beta-glucuronidase release, Bt2cAMP, KCN, SIN-1 and SIN-10 being ineffective. KCN partially reversed the stimulatory effect of SNP, and in the presence of superoxide dismutase, SIN-1 potentiated C5a-induced exocytosis. PAF-induced beta-glucuronidase release was not affected by Bt2cAMP, Bt2cGMP, SNP and SIN-1. Bt2cGMP was more effective than Bt2cAMP to inhibit aggregation and the increase in [Ca2+]i induced by fMet-Leu-Phe at submaximally effective concentrations. C5a-induced rises in [Ca2+]i were not affected by Bt2cAMP and Bt2cGMP. Bt2cAMP but not Bt2cGMP inhibited the effect of PAF at submaximally effective concentrations on [Ca2+]i. Our data suggest (I) that Bt2cGMP and Bt2cAMP differentially modulate neutrophil activation, that (II) NO-containing compounds partially mimic the effects of Bt2cGMP on exocytosis and that (III) cGMP plays an inhibitory role in fMet-Leu-Phe- and a stimulatory role in C5a-induced beta-glucuronidase release.

Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes.

To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.

Differential regulation of chemoattractant-induced superoxide formation in human neutrophils by cell-permeant analogues of cyclic AMP and cyclic GMP.

Conference Article| February 01 1991 Differential regulation of chemoattractant-induced superoxide formation in human neutrophils by cell-permeant analogues of cyclic AMP and cyclic GMP Jürgen Ervens; Jürgen Ervens 1Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69/73, D-1000 Berlin 33, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Gunter Schultz; Gunter Schultz 1Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69/73, D-1000 Berlin 33, F.R.G. Search for other works by this author on: This Site PubMed Google Scholar Roland Seifert Roland Seifert * *To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (1): 59–63. https://doi.org/10.1042/bst0190059 Article history Received: October 10 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Jürgen Ervens, Gunter Schultz, Roland Seifert; Differential regulation of chemoattractant-induced superoxide formation in human neutrophils by cell-permeant analogues of cyclic AMP and cyclic GMP. Biochem Soc Trans 1 February 1991; 19 (1): 59–63. doi: https://doi.org/10.1042/bst0190059 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: fMet-Leu-Phe, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, Bt2cAMP, N6,2′-O-dibutyryl adenosine 3′:5′-cyclic monosphosphate, Bt2cGMP, N2,2′-O-dibutyryl guanosine 3′:5′-cyclic monophosphate, R-NO, reactive nitrogen oxide, O−2, superoxide anion, G-protein, guanine nucleotide-binding protein This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

Cyclic AMP as a modulator of NaCl transport in gills of the euryhaline Chinese crabEriocheir sinensis

Experiments on the effect of cyclic AMP on Na+ and Cl− transport pathways were performed on isolated, perfused preparations of the posterior, osmoregulating gills of the euryhaline Chinese crabEriocheir sinensis. The crabs were caught during the autumn of 1987 in freshwater lakes near Emden (Federal Republic of Germany). N6-2′-O-dibutyryl adenosine 3′:5′-cyclic monophosphate (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and 7β-acetoxy-8,13-epoxy-1α, 6β, 9α-trihydroxy-labd-14-ene-11-one (forskolin) induce an increase in the influx of both Na+ and Cl− in the gills. They also induce significant hyperpolarization. This last result indicates that the increase in NaCl uptake is related to an activation of both the Na+/K+ pump and the Cl−channels located at the serosal side of the gill epithelium. The possibility that cAMP acts as a second messenger, mediating the effects of different neuroendocrine factors involved in ionic and osmotic regulation in crustaceans is discussed.

Histamine-producing cell-stimulating activity. Interleukin 3 and granulocyte-macrophage colony-stimulating factor induce de novo synthesis of histidine decarboxylase in hemopoietic progenitor cells.

Both interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce increased histamine production by murine hemopoietic cells. Histidine-free culture conditions or addition of alpha-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, completely abrogate this phenomenon, indicating that increased histamine levels result from an augmentation of the rate of its synthesis. L-Histidine decarboxylase (HDC) (EC 4.1.1.22) activity is detected in normal bone marrow cell lysates. It is markedly increased following incubation of the cells with IL-3 or GM-CSF. The cells responding by the most important enhancement of HDC activity are located in the less dense layers of a discontinuous Ficoll gradient containing the majority of the hemopoietic progenitor cell types, such as colony-forming units (spleen), granulocyte-macrophage colony-forming cells, and mast cell precursors. In comparison with other HDC-containing cell populations tested, the enzymatic activity contained in these cells is particularly high after IL-3 or GM-CSF treatment and similar to the HDC levels observed in murine fetal liver. The time course of IL-3 and GM-CSF-induced HDC activation at comparable concentrations is slightly different. In response to GM-CSF, HDC activation is more rapid, with a significant enhancement after 4 hr of incubation, as compared with IL-3-induced HDC activation. Moreover, in the latter case the activation increases more progressively up to 48 hr of incubation, whereas GM-CSF-induced increase of HDC activity reaches a plateau more rapidly. In addition, maximal increase in histamine production in response to IL-3 is always higher than in response to GM-CSF. Moreover, the simultaneous presence of both factors at optimal concentration induces only a partially cumulative effect. These results suggest that IL-3 and GM-CSF induce HDC activation in two distinct ways, possibly reflecting the involvement of distinct target cells. However, both mediators act by inducing the transcription of the HDC gene and de novo synthesis of this enzyme since actinomycin D or cycloheximide abolish GM-CSF-or IL-3-induced histamine-producing cell-stimulating activity. This synthesis is independent from cell proliferation as demonstrated by the lack of effect of bone marrow cell irradiation. Finally, the observation that cholera toxin, prostaglandin E2, and N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate mimic the effects of IL-3 and GM-CSF on bone marrow cell HDC suggests an involvement of cyclic adenosine monophosphate in factor-induced histamine-producing cell-stimulating activity.

Evidence against adenosine 3′,5′-monophosphate as a mediator of fever in the brain

The aim of this study was to test the hypothesis that increased concentrations of adenosine 3',5'-monophosphate (cyclic AMP) in the pyrogen-sensitive preoptic/anterior hypothalamic region (PO/AH) mediate fever. Micro-injection of N 6-2'- O-dibutyryl adenosine 3',5'-monophosphate (db cyclic AMP) into the preoptic/anterior hypothalamic region in rats produced a dose-dependent fall in body temperature which is inconsistent with the proposal that the nucleotide mediates fever. Hyperthermia was observed in some rats in response to large doses of db cyclic AMP, but this response was associated with convulsions. Endogenous concentrations of cyclic AMP in the preoptic/anterior hypothalamic region, as well as in the cerebral cortex, liver, spleen, thymus, white fat and plasma were unaffected by the febrile response to the subcutaneous injection of yeast in rats. A rise in levels of cyclic AMP was observed in the skeletal muscle of rats treated with yeast. The data presented do not indicate that cyclic AMP is involved in the neuronal events mediating fever in the rat.

Cyclic AMP levels and cellular kinetics during maturation of human promyelocytic leukemia cells.

Differentiation of human promyelocytic leukemia cells (HL-60) in response to several classes of inducing agents is characterized by the sequential appearance of granulocytic or monocytic markers. Compounds that increase intracellular cAMP in HL-60 cells induce a program of maturation in which cells demonstrate early functional phagocytic properties. Cyclic nucleotide metabolism was studied during monocytic and granulocytic differentiation of the HL-60 cell line. In synchronous and nonsynchronous cell cultures, cyclic AMP levels were raised 300-fold or 25-fold in response to N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphate or the combination of prostaglandin E2 and theophylline, respectively. No reproducible changes in intracellular adenosine 3':5'-cyclic monophosphate levels occurred in response to retinoic acid or dimethyl sulfoxide, suggesting that changes in adenosine 3':5'-cyclic monophosphate levels alone do not mediate cell maturation induced by these compounds. Agents that increased intracellular cyclic AMP in HL-60 cells slowed the progress through the S and G2-M phase of the cell cycle, whereas other agents such as dimethyl sulfoxide stimulated cells through this same period.

The anti-ulcerogenic activity of the unripe plantain banana (Musa species).

Various preparations of dried unripe plantain banana were found to be anti-ulcerogenic against aspirin-induced ulceration in the rat and were effective both as a prophylactic treatment and in healing ulcers already induced by aspirin. Ripe fruit bananas were inactive. The active factor(s) were water soluble and were concentrated by extraction to approximately three hundred times that in the dried banana powder. The anti-ulcerogenic action of banana preparations appears to be due to their ability to stimulate the growth of gastric mucosa. Aluminium hydroxide, cimetidine, prostaglandin E2, N6, O2-dibutyryl adenosine 3',5' cyclic monophosphate but not 5-hydroxytryptamine were also anti-ulcerogenic when used prophylactically in rats but were ineffective in healing ulcers already formed by aspirin. These substances did not stimulate the growth of gastric mucosa.