Effects Of Cyclic Adenosine Monophosphate Research Articles

Effect of Cyclic Adenosine Monophosphate on Connexin 37 Expression in Sheep Cumulus-Oocyte Complexes.

Gap junctional connection (GJC) in the cumulus-oocyte complex (COC) provides necessary support for message communication and nutrient transmission required for mammalian oocyte maturation. Cyclic adenosine monophosphate (cAMP) is not only a prerequisite for regulating oocyte meiosis, but also the key intercellular factor for affecting GJC function in COCs. However, there are no reports on whether cAMP regulates connexin 37 (Cx37) expression, one of the main connexin proteins, in sheep COCs. In this study, the expression of Cx37 protein and gene in immature sheep COC was detected using immunohistochemistry and PCR. Subsequently, the effect of cAMP on Cx37 expression in sheep COCs cultured in a gonadotropin-free culture system for 10 min or 60 min was evaluated using competitive ELISA, real-time fluorescent quantitative PCR (RT-qPCR), and Western blot. The results showed that the Cx37 protein was present in sheep oocytes and cumulus cells; the same results were found with respect to GJA4 gene expression. In the gonadotropin-free culture system, compared to the control, significantly higher levels of cAMP as well as Cx37 gene and protein expression were found in sheep COCs following treatment in vitro with Forskolin and IBMX (100 μM and 500 μM)) for 10 min (p < 0.05). Compared to the controls (at 10 or 60 min), cAMP levels in sheep COCs were significantly elevated as a result of Forskolin and IBMX treatment (p < 0.05). Following culturing in vitro for 10 min or 60 min, Forskolin and IBMX treatment can significantly promote Cx37 expression in sheep COCs (p < 0.05), a phenomenon which can be counteracted when the culture media is supplemented with RP-cAMP, a cAMP-specific competitive inhibitor operating through suppression of the protein kinase A (PKA). In summary, this study reports the preliminary regulatory mechanism of cAMP involved in Cx37 expression for the first time, and provides a novel explanation for the interaction between cAMP and GJC communication during sheep COC culturing in vitro.

A forskolin-mediated increase in cAMP promotes T helper cell differentiation into the Th1 and Th2 subsets rather than into the Th17 subset

The adenylyl cyclase (AC) signaling pathway is suggested to be a key regulator of immune system functions. However, specific effects of cyclic adenosine monophosphate (cAMP) on T helper (Th) cell differentiation and functions are unclear. The involvement of cAMP in the Th cell differentiation program, in particular the development of Th1, Th2, and Th17 subsets, was evaluated employing forskolin (FSK), a labdane diterpene well known as an AC activator. FSK mediated an elevation in Th1-specific markers reinforcing the Th1 cell phenotype. The Th2 differentiation was supported by FSK, though cell metabolism was negatively affected. In contrast, the Th17 immunophenotype was severely suppressed leading to the highly specific upregulation of CXCL13. The causality between FSK-elicited cAMP production and the observed reinforcement of Th2 differentiation was established by using AC inhibitor 2′,5′-dideoxyadenosine, which reverted the FSK effects. Overall, an FSK-mediated cAMP increase affects Th1, Th2 and Th17 differentiation and can contribute to the identification of novel therapeutic targets for the treatment of Th cell-related pathological processes.

Effects of cyclic adenosine monophosphate modulating agents during oocyte pre-maturation and the role of melatonin on in vitro maturation of bovine cumulus-oocyte complexes

This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10−6, 10−7 or 10−8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10−8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10−7 or 10−8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10−6 or 10−8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10−8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10−8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.

Arbutin effectively ameliorates the symptoms of Parkinson's disease: the role of adenosine receptors and cyclic adenosine monophosphate.

An antagonistic communication exists between adenosinergic and dopaminergic signaling in the basal ganglia, which suggests that the suppression of adenosine A2A receptors-cyclic adenosine monophosphate pathway may be able to restore the disrupted dopamine transmission that results in motor symptoms in Parkinson's disease (PD). Arbutin is a natural glycoside that possesses antioxidant, anti-inflammatory, and neuroprotective properties. The purpose of this study was to investigate whether arbutin could ameliorate the symptoms of PD and to examine the underlying mechanism. In this study, Swiss albino mouse models of PD were established by the intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine for 4 successive days, with the concurrent intraperitoneal administration of arbutin (50 and 100 mg/kg) for 7 days. The results showed that arbutin significantly reduced lipid peroxidation, total nitrite levels, and inflammation in the substantia nigra and striatum of PD mouse models. In addition, arbutin decreased the activity of endogenous antioxidants, reduced the levels of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and γ-aminobutyric acid, and minimized neurodegeneration in the striatum. Arbutin also reduced the abnormal performance of PD mouse models in the open field test, bar test, pole test, and rotarod test. The therapeutic efficacy of arbutin was similar to that of madopar. The intraperitoneal injection of the A2AR agonist CGS21680 (0.5 mg/kg) attenuated the therapeutic effects of arbutin, whereas the intraperitoneal injection of forskolin (3 mg/kg) enhanced arbutin-mediated improvements. These findings suggest that arbutin can improve the performance of PD mouse models by inhibiting the function of the A2AR and enhancing the effects of cyclic adenosine monophosphate. This study was approved by the Institutional Animal Ethics Committee (1616/PO/Re/S/12/CPCSEA) on November 17, 2019 (approval No. IAEC/2019/010).

Persistent effects of cyclic adenosine monophosphate are directly responsible for maintaining a neural network state

Network states are often determined by modulators that alter the synaptic and cellular properties of the constituent neurons. Frequently neuromodulators act via second messengers, consequently their effects can persist. This persistence at the cellular/molecular level determines the maintenance of the state at the network level. Here we study a feeding network in Aplysia. In this network, persistent modulation supports the maintenance of an ingestive state, biasing the network to generate ingestive motor programs. Neuropeptides that exert cyclic adenosine monophosphate (cAMP) dependent effects play an important role in inducing the ingestive state. Most commonly, modulatory effects exerted through cAMP signaling are persistent as a consequence of PKA activation. This is not the case in the neurons we study. Instead maintenance of the network state depends on the persistence of cAMP itself. Data strongly suggest that this is a consequence of the direct activation of a cyclic nucleotide gated current.

Cyclic Adenosine Monophosphate Promotes Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via Suppression of Transforming Growth Factor Beta 1 Signaling

IntroductionStem cells from the apical papilla (SCAPs) possess strong odonto/osteogenic differentiation potential. This study investigated the effect of cyclic adenosine monophosphate (cAMP) on odonto/osteogenic differentiation of SCAPs and the underlining interplay between cAMP and transforming growth factor beta 1 (TGF-β1). MethodsSCAPs were stimulated with an activator of cAMP (forskolin) in the presence of either TGF-β1 or a TGF-β1 inhibitor. The amounts of calcium mineral deposition and alkaline phosphatase activity were determined. Quantitative real-time polymerase chain reaction was performed to elucidate cAMP on the TGF-β1–mediated odonto/osteogenic differentiation of SCAPs. The effect of cAMP on the phosphorylation of Smad2/Smad3 and extracellular-regulated kinase (ERK)/P38 induced by TGF-β1 was analyzed by Western blotting. ResultsCotreatment with forskolin and a TGF-β1 inhibitor enhanced alkaline phosphatase activity and deposition of calcium minerals in SCAPs. Moreover, the TGF-β1 inhibitor synergized the effect of forskolin on the expression of type I collagen and runt-related transcription factor 2. The results of Western blotting revealed that forskolin attenuated the unregulated expression of the phosphorylation of Smad3 and ERK induced by TGF-β1, and a cAMP inhibitor (H89) antagonized this effect. ConclusionsThis study showed that cAMP signaling exerts its up-regulating effects on the odonto/osteogenic differentiation of SCAPs by interfering with TGF-β1 signaling via inhibiting Smad3 and ERK phosphorylation.

Effect of bucladesine, pentoxifylline, and H-89 as cyclic adenosine monophosphate analog, phosphodiesterase, and protein kinase A inhibitor on acute pain.

The aim of this study was to determine the effects of cyclic adenosine monophosphate (cAMP) and its dependent pathway on thermal nociception in a mouse model of acute pain. Here, we studied the effect of H-89 (protein kinase A inhibitor), bucladesine (Db-cAMP) (membrane-permeable analog of cAMP), and pentoxifylline (PTX; nonspecific phosphodiesterase (PDE) inhibitor) on pain sensation. Different doses of H-89 (0.05, 0.1, and 0.5 mg/100 g), PTX (5, 10, and 20 mg/100 g), and Db-cAMP (50, 100, and 300 nm/mouse) were administered intraperitoneally (I.p.) 15 min before a tail-flick test. In combination groups, we injected the first and the second compounds 30 and 15 min before the tail-flick test, respectively. I.p. administration of H-89 and PTX significantly decreased the thermal-induced pain sensation in their low applied doses. Db-cAMP, however, decreased the pain sensation in a dose-dependent manner. The highest applied dose of H-89 (0.5 mg/100 g) attenuated the antinociceptive effect of Db-cAMP in doses of 50 and 100 nm/mouse. Surprisingly, Db-cAMP decreased the antinociceptive effect of the lowest dose of H-89 (0.05 mg/100 g). All applied doses of PTX reduced the effect of 0.05 mg/100 g H-89 on pain sensation; however, the highest dose of H-89 compromised the antinociceptive effect of 20 mg/100 g dose of PTX. Co-administration of Db-cAMP and PTX increased the antinociceptive effect of each compound on thermal-induced pain. In conclusion, PTX, H-89, and Db-cAMP affect the thermal-induced pain by probably interacting with intracellular cAMP and cGMP signaling pathways and cyclic nucleotide-dependent protein kinases.

Cyclic AMP signaling enhances lipopolysaccharide sensitivity and interleukin-33 production in RAW264.7 macrophages.

While it has been suggested that IL-33 plays pathogenic roles in various disorders, the factors that stimulate IL-33 production are poorly characterized. In the present study, the effect of cyclic adenosine monophosphate (cAMP) signaling on IL-33 production in RAW264.7 macrophages in response to various doses of LPS was examined. High-dose LPS treatment induced IL-33 and TNF protein production in RAW264.7 macrophages. In contrast, low-dose LPS failed to induce IL-33 production while significantly inducing TNF production. In the presence of the membrane-permeable cAMP analog 8-Br-cAMP, low-dose LPS induced vigorous IL-33 production. This phenomenon was consistent with amounts of mRNA. Similarly, the cAMP-increasing agent adrenaline also enhanced the sensitivity of RAW264.7 macrophages to LPS as demonstrated by IL-33 production. The protein kinase A (PKA) inhibitor H89 blocked the effects of 8-Br-cAMP and adrenaline on IL-33 production, suggesting that PKA is involved in IL-33 induction. Taken together, cAMP-mediated signaling pathway appears to enhance the sensitivity of RAW264.7 macrophages to LPS with respect to IL-33 production. Our findings suggest that stress events and the subsequent secretion of adrenaline enhance macrophage production via IL-33; this process may be associated with the pathogenesis of various disorders involving IL-33.

In Pursuit of Genes of Glucose Metabolism

If you don't know where you're going, you might wind up someplace else—Yogi Berra My professional journey, once I accepted the fact that I would not play first base for the Chicago Cubs, was not accomplished in a straight line. I entered medical school intending to become a family physician but soon became interested in research. I took an internal medicine residency at the University of California, San Francisco (UCSF), then a long research fellowship in the nascent field of molecular biology at the National Institutes of Health (NIH), and followed that with training in clinical endocrinology at the University of Wisconsin. I spent the next fifteen years dividing my time between the clinic and laboratory at the University of Iowa. Finally, I decided to concentrate on research and spent the next twenty-three years at Vanderbilt University. For the past eleven years, I have been at the University of Iowa, where I helped establish a new diabetes research center. My research journey also had several different directions. Upon reflection, I guess I ended up “someplace else” by choice, and it certainly has been an interesting trip. It is one I unreservedly recommend.

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence

Does heparin ablate the advantageous effects of cyclic adenosine mono-phosphate (cAMP) modulators during pre-in vitro maturation (IVM) and have a deleterious effect in standard oocyte IVM? Heparin interrupts energy metabolism and meiotic progression and adversely affects subsequent development of oocytes under conditions of elevated cAMP levels in cumulus-oocyte complexes (COCs) after pre-IVM treatment with forskolin. In animal IVM studies, artificial regulation of meiotic resumption by cAMP-elevating agents improves subsequent oocyte developmental competence. Heparin has no effect on spontaneous, FSH- or epidermal growth factor (EGF)-stimulated meiotic maturation. An in vitro cross-sectional study was conducted using immature mouse and human COCs. Depending on individual experimental design, COCs were treated during pre-IVM with or without heparin, in the presence or absence of forskolin and/or 3-isobutyl-1-methylxanthine (IBMX), and then COC function was assessed by various means. Forty-two women with polycystic ovaries (PCOs) or polycystic ovarian syndrome (PCOS) donated COCs after oocyte retrieval in a non-hCG-triggered IVM cycle. COCs were collected in pre-IVM treatments and then cultured for 40 h and meiotic progression was assessed. COCs from 21- to 24-day-old female CBA F1 mice were collected 46 h after stimulation with equine chorionic gonadotrophin. Following treatments, COCs were checked for meiotic progression. Effects on mouse oocyte metabolism were measured by assessing oocyte mitochondrial membrane potential using JC-1 staining and oocyte ATP content. Post-IVM mouse oocyte developmental competence was assessed by in vitro fertilization and embryo production. Blastocyst quality was evaluated by differential staining of inner cell mass (ICM) and trophectoderm (TE) layers. In the absence of heparin in pre-IVM culture, the addition of cAMP modulators did not affect human oocyte MII competence after 40 h. In standard IVM, heparin supplementation in pre-IVM did not affect MII competence; however, when heparin was combined with cAMP modulators, MII competence was significantly reduced from 65 to 15% (P < 0.05). In mouse experiments, heparin alone in pre-IVM significantly delayed germinal vesicle breakdown (GVBD) so that fewer GVBDs were observed at 0 and 1 h of IVM (P < 0.05), but not by 2 or 3 h of IVM. Combined treatment with IBMX and forskolin in the pre-IVM medium produced a large delay in GVBD such that no COCs exhibited GVBD in the first 1 h of IVM, and the addition of heparin in pre-IVM further significantly delayed the progression of GVBD (P < 0.05), in a dose-dependent manner (P < 0.01). Combined IBMX and forskolin treatment of mouse COCs during pre-IVM significantly increased mitochondrial membrane potential and ATP production in the oocyte at the end of pre-IVM (P < 0.05), and significantly improved fertilization, embryo development and quality (P < 0.05). However, heparin abolished the IBMX + forskolin-stimulated increase in mitochondrial membrane potential and ATP production (P < 0.05), and adversely affected embryonic cleavage, development rates and embryo quality (P < 0.05). This latter adverse combinational effect was negated when mouse COCs were collected in heparin and IBMX for 15 min, washed and then cultured for 45 min in IBMX and forskolin without heparin. Experiments in mice found that heparin ablation of the advantageous effects of cAMP modulators during pre-IVM was associated with altered oocyte metabolism, but the mechanism by which heparin affects metabolism remains unclear. This study has revealed a novel and unexpected interaction between heparin and cAMP modulators in pre-IVM in immature mouse and human oocytes, and established a means to collect oocytes using heparin while modulating oocyte cAMP to improve developmental potential.

Selective inhibition of cell death in malignant vs normal B-cell precursors: implications for cAMP in development and treatment of BCP-ALL

AbstractB-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most commonly occurring pediatric cancer. Despite its relatively good prognosis, there is a steady search for strategies to improve treatment effects and prevent the undesired side effects on normal cells. In the present paper, we demonstrate a differential effect of cyclic adenosine monophosphate (cAMP) signaling between normal BCPs and BCP-ALL blasts, pointing to a potential therapeutic window allowing for manipulation of cAMP signaling in the treatment of BCP-ALL. By studying primary cells collected from pediatric BCP-ALL patients and healthy controls, we found that cAMP profoundly decreased basal and DNA damage-induced p53 levels and cell death in malignant cells, whereas normal BCP counterparts displayed slightly augmented cell death when exposed to cAMP-increasing agents. We did not find evidence for a selection process involving generation of increased basal cAMP levels in BCP-ALL cells, but we demonstrate that paracrine signaling involving prostaglandin E2-induced cAMP generation has the potential to suppress p53 activation and cell death induction. The selective inhibitory effect of cAMP signaling on DNA damage-induced cell death in BCP-ALL cells appears to be an acquired trait associated with malignant transformation, potentially allowing the use of inhibitors of this pathway for directed killing of the malignant blasts.

Effect of cyclic adenosine monophosphate and prostaglandin E2 on multinucleated giant cell formation from human monocytes in sarcoidosis

Multinucleated giant cells(MGC) are characteristic cells in granulomatous disorders and derived from monocyte-macrophage lineage cells. The exact mechanisms of MGC formation are not determined. Cyclic adenosine monophosphate(cAMP) is the intracellular second messenger for immunoregulatory mediators and plays a key role in regulation of monocyte-macrophage-mediated inflammatory responses. Here we examined the effects of cAMP on MGC formation. When monocytes were cultured with ConA-stimulated mononuclear cell supernatants(CS), MGC were formed on day 3. MGC formation was significantly suppressed by adding phosphodiesterase inhibitors(PDEI). MGC formation is a complex event involving plasma membrane receptors that are responsible for adhesion, activation and the fusion process. Monocytes cultured with CS highly expressed CD11a and CD54. The addition of PDEI decreased these expressions. Sarcoidosis is a granulomatous disorder with MGC appearance. To investigate the relevance of cAMP in sarcoidosis, we measured serum cAMP level and intracellular cAMP concentration. There was no significant difference in the serum cAMP level between healthy controls(HC) and sarcoidosis patients(SP). On the other hand, there was a significant difference in the intracellular cAMP concentration in monocytes. Because prostaglandin E2(PGE2) affected the cAMP level, we determined the localization of PGE2 in the skin lesion of sarcoidosis. PGE2 was not detectable in the sarcoidal lesion. Furthermore, there was no significant difference in the serum levels of PGE2 of HC and SP. These findings indicate that up-regulation of cAMP in human monocytes inhibited the MGC formation, suggesting that cAMP is a mediator of the granuloma formation. However, PGE2 is not related to this mechanism in sarcoidosis. JSID AbstractsJournal of Dermatological ScienceVol. 69Issue 2Preview Full-Text PDF

Effects of cyclic AMP on development and secondary metabolites of Monascus ruber M-7

To study effects of cyclic adenosine monophosphate (cAMP) on development and secondary metabolites of Monascus ruber M-7. Plate culture, liquid-state fermentation (LSF) and solid-state fermentation (SSF) were used to evaluate effects of cAMP on colonial growth, spore formation and polyketide production of Strain M-7. The results revealed that the variation trends of colonial sizes, numbers of sexual spores and red pigment contents of M-7 were in a dose-dependent manner. And generally they increased and decreased with cAMP concentrations in the ranges of low cAMP concentrations and high cAMP concentrations, respectively. But the variation trends of numbers of asexual spores and citrinin production in both LSF and SSF were opposite to those of colonial sizes, sexual sporulation and red pigment. The regulation of cAMP on development and secondary metabolites in Strain M-7 was in a dose-dependent pattern. And red pigment might convert to citrinin under changing cAMP concentrations. The effects of cAMP on Strain M-7 in SSF give a new clue to enhance beneficial polyketides and reduce citrinin produced by M. ruber.

The effect of sildenafil on the elasticity of erythrocytes in homozygous sickle cell disease

One of the features of homozygous sickle cell disease (HbSS) is the impaired elasticity of the erythrocyte membrane that could impede microcirculatory blood flow and cause hypoxia and tissue damage. We investigated the effect of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor that inhibits the breakdown of cyclic guanosine monophosphate (cGMP) resulting in vasodilatation, on the elasticity of HbSS erythrocyte. Blood samples from ten HbSS patients in steady state was exposed to different doses (5, 10, 20, and 40 μg/mL) of sildenafil and the elasticity of the erythrocytes measured at native hematocrit with the BioProfiler. An equal number of subjects with normal hemoglobin (HbAA) served as the control group. There was a marginal increase in elasticity with 5 μg/mL of the drug and this became significant (P<0.05) with the 10 μg/mL dose. Thereafter, gradual nonsignificant decreases were observed with the 20 and 40 μg/mL doses. A similar trend was observed for the control group. The elasticity values for the HbSS subjects at native hematocrit were significantly (P<0.05) less when compared with the corresponding concentrations for the HbAA controls. This was reversed at a corrected hematocrit of 45%. The result of this study shows that sildenafil caused an initial increase in erythrocyte membrane elasticity in both HbSS and HbAA subjects, and this later decreased with increasing concentration of the drug possibly due to the dual effect of cyclic adenosine monophosphate (cAMP).

Effects of cAMP and β-Adrenergic Receptor Antagonists on the Function of Peripheral T Helper Lymphocytes in Patients with Heart Failure

Objective: To observe the effects of cyclic adenosine monophosphate (cAMP) in peripheral lymphocytes on T helper 1 (TH1)/TH2 cytokine ratios and to investigate the potential impact and mechanism of β-adrenergic receptor antagonists on immune function in patients with chronic heart failure (CHF). Methods: Sixty-nine patients with New York Heart Association functional class II–IV CHF and a radionuclide left ventricular ejection fraction of less than 45% received carvedilol or metoprolol in a randomized fashion. Thirty healthy persons were studied as controls. Interferon (IFN)-γ and interleukin (IL)-10 in CD4+ T lymphocytes were quantified by three-color flow cytometry. cAMP levels in peripheral blood mononuclear cells were tested by radioimmunoassay at baseline and 6 months after treatment. Results: The levels of lymphocyte cAMP in CHF patients were significantly lower than in normal controls (p < 0.01). The IFN-γ/IL-10 ratio was significantly higher in CHF patients (p < 0.01). The levels of cAMP, IFN-γ and IL-10 among CHF patients with different pathogenic factors displayed no significant differences (p > 0.05). The lymphocyte cAMP level was negatively correlated with the IFN-γ/IL-10 ratio (p < 0.01). After treatment with metoprolol and carvedilol, the IFN-γ/IL-10 ratio in the CHF patients was dramatically decreased (p < 0.01 for each drug) and lymphocyte cAMP was remarkably increased (p < 0.01 for each drug). Conclusion: These results suggest that the decrease of cAMP affects the TH1/TH2 phenotype in peripheral lymphocytes of CHF patients. β-Blocking medications appear to have a beneficial effect on the TH1/TH2 balance and on the immune system through increasing production of cAMP, offering further evidence to support the use of β-adrenergic receptor blockers to treat CHF.

Zinc inhibits calcium-mediated and nitric oxide-mediated ion secretion in human enterocytes

Zn 2+ is effective in the treatment of acute diarrhea, but its mechanisms are not completely understood. We previously demonstrated that Zn 2+ inhibits the secretory effect of cyclic adenosine monophosphate but not of cyclic guanosine monophosphate in human enterocytes. The aim of the present study was to investigate whether Zn 2+ inhibits intestinal ion secretion mediated by the Ca 2+ or nitric oxide pathways. To investigate ion transport we evaluated the effect of Zn 2+ (35 μM) on electrical parameters of human intestinal epithelial cell monolayers (Caco2 cells) mounted in Ussing chambers and exposed to ligands that selectively increased intracellular Ca 2+ (carbachol 10 − 6 M) or nitric oxide (interferon-γ 300 UI/ml) concentrations. We also measured intracellular Ca 2+ and nitric oxide concentrations. Zn 2+ significantly reduced ion secretion elicited by carbachol (− 87%) or by interferon-γ (− 100%), and inhibited the increase of intracellular Ca 2+ and nitric oxide concentrations. These data indicate that Zn 2+ inhibits ion secretion elicited by Ca 2+ and nitric oxide by directly interacting with the enterocyte. They also suggest that Zn 2+ interferes with three of the four main intracellular pathways of intestinal ion secretion that are involved in acute diarrhea.

The Effect of Cyclic Adenosine Monophosphate (cAMP) Modulators on the Activity of Selected Anti-Malaria Drugs

The Effect of Cyclic Adenosine Monophosphate (cAMP) Modulators on the Activity of Selected Anti-Malaria Drugs

Relationship between cyclic adenosine monophosphate and aquaporin 8 expression and distribution in human amnion-derived cells

To study the effect of cyclic adenosine monophosphate on aquaporin 8 (AQP8) expression and distribution in human amnion-derived WISH cells. Human amnion-derived WISH cells were cultured. The cells were divided into control group and study group at random. The study group was established by exposure to various concentrations of 8-Br-cAMP. Western analysis was used to quantify AQP8 expression levels. RT-PCR was used to quantify AQP8 mRNA expression levels. Immunofluorescence was used to determine the localization of AQP8 in WISH cells. With increase of cAMP, AQP8 mRNA and protein expressions significantly increased in WISH cells in vitro. When the concentration of cAMP reached 200 micromol/L, AQP8 mRNA and protein expressions were highest. Incubation with cAMP (200 micromol/L) for 2 hours resulted in a 2-fold increase in AQP8 mRNA level, and incubation for 8 hours resulted in a peak. After incubation for 16 hours AQP8 mRNA level began to descend, and after 24 hours it decreased to baseline. Incubation with cAMP for 8 hours AQP8 protein level began to increase. Incubation for 24 hours resulted in a peak in AQP8 protein level, and after incubation for 48 hours it began to decline. By immunofluorescence microscopy after incubation with cAMP (200 micromol/L) cells, AQP8 labeling in plasma membrane was enhanced and intracellular AQP8 labeling was decreased. The cAMP triggers translocation of AQP8 from cytosol to the plasma membrane via vesicle-transporting related protein instead of AQP8 itself. cAMP may upregulate the transcription of target gene protein kinase A. The cAMP may be the critical regulatory medium of AQP8 in WISH cells.

Study of ATP-binding Cassette Transporter A1 (ABCA1)-mediated Cellular Cholesterol Efflux in Diabetic Golden Hamsters

The effects of cyclic adenosine monophosphate (cAMP) and atorvastatin on macrophage adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux were investigated in a diabetic animal model. Golden hamsters were fed a high-fat diet which resulted in insulin resistance. Diabetes was induced by a single intraperitoneal injection of streptozotocin (30 mg/kg). Normal golden hamsters were used as controls. Peritoneal macrophages were incubated with apolipoprotein A-1 (apoA-1), 8-bromoadenosine-3',5'-cyclic monophosphate (8-br-cAMP), and atorvastatin in vitro. Intracellular cholesterol accumulation was greater in the diabetic animals than in the insulin-resistant animals. Expression of ABCA1 mRNA in macrophages from diabetic animals was upregulated by 8-br-cAMP and atorvastatin. ApoA-1 caused a time-dependent cellular cholesterol efflux. Both atorvastatin and 8-br-cAMP significantly facilitated ABCA1-mediated cellular cholesterol efflux, with the maximal cholesterol efflux rate observed in the macrophages from diabetic animals. Accumulation of cholesterol in the macrophages of diabetic animals can be significantly alleviated by atorvastatin or 8-br-cAMP through improving ABCA1-mediated cellular cholesterol efflux.

Increased glutamate uptake and GLAST expression by cyclic AMP in retinal glial cells

The aim was to investigate the effect of cyclic adenosine monophosphate (cAMP) on glutamate uptake and glial glutamate aspartate transporter (GLAST) expression in cultured rat retinal glial cells. Retinal glial cell cultures were prepared from 1- to 3-day-old Sprague-Dawley rats. Cultures were treated with dibutyryl-cAMP (dBcAMP) for 24 h. Glutamate uptake was measured as 3H-glutamate content of the lysates as determined by scintillation counting. The expression of GLAST in dBcAMP-treated cells was examined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Cells were identified by immunocytochemistry using antibodies against vimentin and carbonic anhydrase C. Treatment of cultured cells with dBcAMP significantly increased glutamate uptake after 60 min compared with untreated cells (p < 0.01). Maintenance of cells in medium supplemented with dBcAMP increased GLAST mRNA and protein levels. GLAST mRNA levels were elevated 2.9-fold 24 h after treatment. GLAST protein levels were elevated 2.5-fold 24 h after treatment. These results indicate that treatment with cAMP increases glutamate uptake through upregulated GLAST activity in cultured retinal glial cells. These findings suggest that retinal glial cells may regulate extracellular glutamate concentration and protect retinal neurons against glutamate excitotoxicity.