BACKGROUNDCyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.

METHODSIn an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.

RESULTSDose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP.

CONCLUSIONScAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Chloride Channels ; antagonists & inhibitors ; Ciliary Body ; drug effects ; physiology ; Colforsin ; pharmacology ; Cyclic AMP ; physiology ; Humans ; In Vitro Techniques ; Potassium Channel Blockers ; pharmacology ; Sodium Channel Blockers ; pharmacology

The study was aimed to investigate the possible effects of 8-chloroadenosine 3', 5'-monophosphate (8-Cl-cAMP) on the multiple myeloma cells. The multiple myeloma cell line RPMI8226 was used as in vitro models. The effect on growth inhibition of RPMI8226 cells was evaluated by cell growth and viability curve. DNA fragment was visualized by agarose gel electrophoresis. The amount of apoptosis cells was measured by flow cytometry. Meanwhile Western blot assay were used to detect the change of several key cell cycle regulatory proteins CDK2 and cyclin E in these cells before and after the treatment. The results showed that low dose 8-Cl-cAMP (1-30 micromol/L) inhibited the proliferation and viability of RPMI8226 cells significantly. Agarose gel electrophoresis of DNA revealed the apoptosis characteristic "ladder" pattern. Apoptosis was also confirmed by flow cytometry. In addition, 8-Cl-cAMP was able to inhibit the cell growth through modulating expression of cell cycle regulators CDK2 and cyclin E. It is concluded that 8-cl-cAMP inhibits the proliferation and induce apoptosis of multiple myeloma cells effectively.
8-Bromo Cyclic Adenosine Monophosphate ; analogs & derivatives ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Humans ; Multiple Myeloma ; pathology

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alpha v beta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alpha v beta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alpha;avbeta;b3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alpha v beta3 integrin expression during decidualization might influence on human implantation.
8-Bromo Cyclic Adenosine Monophosphate/*pharmacology ; Cell Size ; Cells, Cultured ; Decidua/*cytology/drug effects/metabolism ; Female ; Flow Cytometry ; Human ; Integrins/*analysis/metabolism ; Prolactin/analysis ; Stromal Cells/cytology/*drug effects/*metabolism

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^-1), whereas higher concentrations (>5 μmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca²⁺-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
8-Bromo Cyclic Adenosine Monophosphate/pharmacology* ; Acrosome/metabolism* ; Acrosome Reaction/drug effects* ; Calcimycin/pharmacology* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors* ; Exocytosis/drug effects* ; Guanine Nucleotide Exchange Factors/metabolism* ; Humans ; Male ; Protein Kinase Inhibitors/pharmacology* ; Signal Transduction/drug effects* ; Spermatozoa/metabolism* ; Thapsigargin/pharmacology*

OBJECTIVEThis study was designed to examine the in vitro effects of fenvalerate on steroid production and steroidogenic enzymes mRNA expression level in rat granulosa cells.

METHODSUsing primary cultured rat granulosa cells (rGCs) as model, fenvalerate of various concentrations (0, 1, 5, 25, 125, 625 micromol/L) was added to the medium for 24 h. In some cases, optimal concentrations of 22(R)-hydroxycholesterol (25 micromol/L), Follicle stimulating hormone (FSH, 2 mg/L), or 8-Bromo-cAMP (1 mmol/L) were provided. Concentrations of 17 beta-estradiol(E2) and progesterone (P4) in the medium from the same culture wells were measured by RIA and the steroidogenic enzyme mRNA level was quantified by semi-quantitative RT-PCR.

RESULTSFenvalerate decreased both P4 and E2 production in a dose-dependent manner while it could significantly stimulate rGCs proliferation. This inhibition was stronger in the presence of FSH. Furthermore, it could not be reversed by 22(R)-hydroxycholesterol or 8-Bromo-cAMP. RT-PCR revealed that fenvalerate had no significant effect on 3 beta-HSD, but could increase the P450scc mRNA level. In addition, 17 beta-HSD mRNA level was dramatically reduced with the increase of fenvalerate dose after 24 h treatment.

CONCLUSIONFenvalerate inhibits both P4 and E2 production in rGCs. These results support the view that fenvalerate is considered as a kind of endocrine-disrupting chemicals. The mechanism of its disruption may involve the effects on steroidogenesis signaling cascades and/or steroidogenic enzyme's activity.

3-Hydroxysteroid Dehydrogenases ; analysis ; metabolism ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Animals ; Base Sequence ; Cells, Cultured ; Dose-Response Relationship, Drug ; Estradiol ; analysis ; metabolism ; Female ; Follicle Stimulating Hormone ; pharmacology ; Granulosa Cells ; cytology ; drug effects ; metabolism ; Hydroxycholesterols ; pharmacology ; Nitriles ; pharmacology ; Progesterone ; analysis ; metabolism ; Pyrethrins ; pharmacology ; RNA, Messenger ; analysis ; metabolism ; Rats ; Steroids ; metabolism

Previous reports raised question as to whether 8-chloro-cyclic adenosine 3,5-monophosphate (8-Cl-cAMP) is a prodrug for its metabolite, 8-Cl-adenosine which exerts growth inhibition in a broad spectrum of cancer cells. The present study was carried out to clarify overall cellular affects of 8-Cl-cAMP and 8-Cl-adenosine on SK-N-DZ human neuroblastoma cells by ystematically characterizing gene expression using radioactive human cDNA microarray. Microarray was prepared with PCR-amplified cDNA of 2,304 known genes spotted on nylon membranes, employing (1)P-labeled cDNAs of SK-N-DZ cells as a probe. the expression levels of approximately 100 cDNAs, representing about 8% of the total DNA elements on the array, were altered in 8-Cl-adenosine- or 8-Cl-cAMP-treated cells, respectively. The genome-wide expression of the two samples exhibited partial overlaps; different sets of up-regulated genes but the same set of down-regulated genes. 8-Cl-adenosine treatment up- egulated genes involved in differentiation and development (LIM protein, connexin 26, neogenin, neurofilament triplet L protein and p21( WAF1/CIP1)) and immune response such as natural killer cells protein 4, and down-regulated ones involved in proliferation and transformation (transforming growth factor-beta, DYRK2, urokinase-type plasminogen activator and proteins involved in transcription and translation) which were in close parallel with those by 8-Cl-cAMP. Our results indicated that the two drugs shared common genomic pathways for the down-regulation of certain genes, but used distinct pathways for the up-regulation of different gene clusters. Based on the findings, we suggest that the anti-cancer activity of 8-Cl-cAMP results at least in part through 8-Cl-adenosine. Thus, the systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in anticancer activities of chemotherapeutics.
2-Chloroadenosine/*analogs & derivatives/chemistry/*pharmacology ; 8-Bromo Cyclic Adenosine Monophosphate/*analogs & derivatives/chemistry/*pharmacology ; Antineoplastic Agents/chemistry/*pharmacology ; Blotting, Western ; *Gene Expression Profiling ; Gene Expression Regulation, Neoplastic/*drug effects ; Genome, Human ; Human ; Neuroblastoma/*genetics ; Oligonucleotide Array Sequence Analysis ; Reproducibility of Results ; Tumor Cells, Cultured ; Up-Regulation/drug effects

OBJECTIVETo observe the effects of ATP-binding cassette A1 (ABCA1) on intercellular cell adhesion molecule type 1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and interleukin-1beta (IL-1beta) in THP-1 macrophages stimulated with 8-Br-cAMP to identify a possible new mechanism that ABCA1 contributes to atherosclerogenesis (AS).

METHODSMonocytic THP-1 cells were cultured in the presence of 100 nmol/L phorbol myristate acetate (PMA) for 72 h to transform the cells into THP-1 macrophages. After the macrophages were stimulated with 8-Br-cAMP (final concentration 0.5 mmol/L) for 3, 6, 12 and 24 h respectively, the amounts of ABCA1, ICAM-1 and MCP-1 mRNA were examined by real-time fluorescent quantitative RT-PCR, and the protein amounts of ABCA1, ICAM-1, MCP-1 and IL-1beta were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Phosphorothioate antisense oligonucleotides of ABCA1 were add into the culture media at a final concentration of 100 nmol/L and the experiments were repeated.

RESULTSABCA1, ICAM-1 and MCP-1 mRNA and protein and IL-1beta protein were increased in the macrophages after stimulation with 8-Br-cAMP for 6 and 12 h. The mRNA expressions of ABCA1, ICAM-1 and MCP-1 were decreased significantly at 3 and 6 h (P<0.01), and the protein expressions of ABCA1, ICAM-1, MCP-1 and IL-1beta declined significantly at 12 and 24 h (P<0.01) after transfection of the macrophages with antisense oligonucleotides of ABCA1.

CONCLUSIONABCA1 can increase the expressions of the inflammatory cytokines in THP-1 macrophages stimulated by 8-Br-cAMP and plays a role in the pathogenesis of AS.

8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; biosynthesis ; genetics ; Cell Line ; Chemokine CCL2 ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Interleukin-1beta ; genetics ; metabolism ; Macrophages ; cytology ; drug effects ; metabolism ; Monocytes ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.

METHODSHERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.

RESULTSElevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.

CONCLUSIONSPKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.

1-Methyl-3-isobutylxanthine ; pharmacology ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Adenylyl Cyclases ; metabolism ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Cation Transport Proteins ; Cell Line ; Colforsin ; pharmacology ; Cyclic AMP ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA-Binding Proteins ; ERG1 Potassium Channel ; Enzyme Activation ; drug effects ; Ether-A-Go-Go Potassium Channels ; Female ; Humans ; Membrane Potentials ; drug effects ; Microinjections ; Oocytes ; Patch-Clamp Techniques ; Phenethylamines ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; Phosphoric Diester Hydrolases ; drug effects ; metabolism ; Phosphorylation ; Potassium Channels ; genetics ; metabolism ; physiology ; Potassium Channels, Voltage-Gated ; RNA, Complementary ; administration & dosage ; genetics ; Sulfonamides ; pharmacology ; Trans-Activators ; Transcriptional Regulator ERG ; Xenopus laevis