scholarly journals Human CD4+T lymphocytes with increased intracellular cAMP levels exert regulatory functions by releasing extracellular cAMP

2006 ◽  
Vol 80 (4) ◽  
pp. 880-888 ◽  
Author(s):  
Silvia Vendetti ◽  
Mario Patrizio ◽  
Antonella Riccomi ◽  
Maria Teresa De Magistris
Keyword(s):  
T Lymphocytes ◽  
Intracellular Camp ◽  
Regulatory Functions ◽  
Extracellular Camp ◽  
Camp Levels

Ammonia promotes accumulation of intracellular cAMP in differentiating amoebae of Dictyostelium discoideum

Development ◽  
1990 ◽  
Vol 109 (3) ◽  
pp. 715-722
Author(s):  
B.B. Riley ◽  
S.L. Barclay
Keyword(s):  
Dictyostelium Discoideum ◽  
Active Species ◽  
Spore Formation ◽  
Intracellular Camp ◽  
Simultaneous Exposure ◽  
High Ph ◽  
Camp Hydrolysis ◽  
Extracellular Camp ◽  
Camp Levels

We used sporogenous mutants of Dictyostelium discoideum to investigate the mechanism(s) by which exogenous NH4Cl and high ambient pH promote spore formation during in vitro differentiation. The level of NH4Cl required to optimize spore formation is correlated inversely with pH, indicating that NH3 rather than NH4+ is the active species. The spore-promoting activity of high ambient pH (without exogenous NH4Cl) was eliminated by the addition of an NH3-scavenging cocktail, suggesting that high pH promotes spore differentiation by increasing the ratio of NH3:NH4+ secreted into the medium by developing cells. High ammonia levels and high pH stimulated precocious accumulation of intracellular cAMP in both sporogenous and wild-type cells. In both treatments, peak cAMP levels equaled or exceeded control levels and were maintained for longer periods than in control cells. In contrast, ammonia strongly inhibited accumulation of extracellular cAMP without increasing the rate of extracellular cAMP hydrolysis, indicating that ammonia promotes accumulation of intracellular cAMP by inhibiting cAMP secretion. These results are consistent with previous observations that factors that raise intracellular cAMP levels increase spore formation. Lowering intracellular cAMP levels with caffeine or progesterone inhibited spore formation, but simultaneous exposure to these drugs and optimal concentrations of NH4Cl restored both cAMP accumulation and spore formation to normal levels. These data suggest that ammonia, which is a natural Dictyostelium morphogen, favors spore formation by promoting accumulation or maintenance of high intracellular cAMP levels.

Transmembrane Calcium Influx Associated with von Willebrand Factor Binding to GP Ib in the Initiation of Shear-Induced Platelet Aggregation

1993 ◽  
Vol 69 (05) ◽  
pp. 496-502 ◽  
Author(s):  
Yasuo Ikeda ◽  
Makoto Handa ◽  
Tetsuji Kamata ◽  
Koichi Kawano ◽  
Yohko Kawai ◽  
...  
Keyword(s):  
Shear Force ◽  
Calcium Influx ◽  
Fold Increase ◽  
Intracellular Camp ◽  
Recombinant Fragment ◽  
Transmembrane Flux ◽  
Irreversible Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Camp Levels

SummaryWe found that the binding of multimeric vWF to GP Ib under a shear force of 108 dynes/cm2 resulted in the transmembrane flux of Ca2+ ions with a two-to three-fold increase in their intracellular concentration ([Ca2+]i). The blockage of this event, obtained by inhibiting the vWF-GP Ib interaction, suppressed aggregation. In contrast, the blockage of vWF binding to GP IIb-IIIa, as well as the prevention of activation caused by increased intracellular cAMP levels, inhibited aggregation but had no significant effect on [Ca2+]i increase. A monomeric recombinant fragment of vWF containing the GP Ib-binding domain of the molecule (residues 445-733) prevented all effects mediated by multimeric vWF but, by itself, failed to support the increase in [Ca2+]i and aggregation. These results suggest that the binding of multimeric vWF to GP Ib initiates platelets aggregation induced by high shear stress by mediating a transmembrane flux of Ca2+ ions, perhaps through a receptor-dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of GP IIb-IIIa; the latter receptor then binds vWF and mediates irreversible aggregation.

scholarly journals Novel Template Plasmids pCyaA’-Kan and pCyaA’-Cam for Generation of Unmarked Chromosomal cyaA’ Translational Fusion to T3SS Effectors in Salmonella

2021 ◽  
Vol 9 (3) ◽  
pp. 475
Author(s):  
Paulina A. Fernández ◽  
Marcela Zabner ◽  
Jaime Ortega ◽  
Constanza Morgado ◽  
Fernando Amaya ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Eukaryotic Cells ◽  
Intracellular Camp ◽  
Gene Fusions ◽  
Secretion Systems ◽  
Translational Fusion ◽  
Flp Recombinase ◽  
Recombination System ◽  
Camp Levels

The type III secretion systems (T3SS) encoded in pathogenicity islands SPI-1 and SPI-2 are key virulence factors of Salmonella. These systems translocate proteins known as effectors into eukaryotic cells during infection. To characterize the functionality of T3SS effectors, gene fusions to the CyaA’ reporter of Bordetella pertussis are often used. CyaA’ is a calmodulin-dependent adenylate cyclase that is only active within eukaryotic cells. Thus, the translocation of an effector fused to CyaA’ can be evaluated by measuring cAMP levels in infected cells. Here, we report the construction of plasmids pCyaA’-Kan and pCyaA’-Cam, which contain the ORF encoding CyaA’ adjacent to a cassette that confers resistance to kanamycin or chloramphenicol, respectively, flanked by Flp recombinase target (FRT) sites. A PCR product from pCyaA’-Kan or pCyaA’-Cam containing these genetic elements can be introduced into the bacterial chromosome to generate gene fusions by homologous recombination using the Red recombination system from bacteriophage λ. Subsequently, the resistance cassette can be removed by recombination between the FRT sites using the Flp recombinase. As a proof of concept, the plasmids pCyaA’-Kan and pCyaA’-Cam were used to generate unmarked chromosomal fusions of 10 T3SS effectors to CyaA’ in S. Typhimurium. Each fusion protein was detected by Western blot using an anti-CyaA’ monoclonal antibody when the corresponding mutant strain was grown under conditions that induce the expression of the native gene. In addition, T3SS-1-dependent secretion of fusion protein SipA-CyaA’ during in vitro growth was verified by Western blot analysis of culture supernatants. Finally, efficient translocation of SipA-CyaA’ into HeLa cells was evidenced by increased intracellular cAMP levels at different times of infection. Therefore, the plasmids pCyaA’-Kan and pCyaA’-Cam can be used to generate unmarked chromosomal cyaA’ translational fusion to study regulated expression, secretion and translocation of Salmonella T3SS effectors into eukaryotic cells.

scholarly journals Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 107-117
Author(s):  
Qi Yang ◽  
Katherine A Borkovich
Keyword(s):  
Signal Transduction ◽  
Neurospora Crassa ◽  
Signal Transduction Pathway ◽  
Sexual Cycle ◽  
Intracellular Camp ◽  
Wild Type ◽  
Independent Functions ◽  
Aerial Hyphae ◽  
Mutational Activation ◽  
Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

scholarly journals Anti-Allergic and Anti-Inflammatory Effects of Undecane on Mast Cells and Keratinocytes

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1554
Author(s):  
Dabin Choi ◽  
Wesuk Kang ◽  
Taesun Park
Keyword(s):  
Mast Cells ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Allergic Diseases ◽  
Intracellular Camp ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Skin Conditions ◽  
Factor Α ◽  
Camp Levels

The critical roles of keratinocytes and resident mast cells in skin allergy and inflammation have been highlighted in many studies. Cyclic adenosine monophosphate (cAMP), the intracellular second messenger, has also recently emerged as a target molecule in the immune reaction underlying inflammatory skin conditions. Here, we investigated whether undecane, a naturally occurring plant compound, has anti-allergic and anti-inflammatory activities on sensitized rat basophilic leukemia (RBL-2H3) mast cells and HaCaT keratinocytes and we further explored the potential involvement of the cAMP as a molecular target for undecane. We confirmed that undecane increased intracellular cAMP levels in mast cells and keratinocytes. In sensitized mast cells, undecane inhibited degranulation and the secretion of histamine and tumor necrosis factor α (TNF-α). In addition, in sensitized keratinocytes, undecane reversed the increased levels of p38 phosphorylation, nuclear factor kappaB (NF-κB) transcriptional activity and target cytokine/chemokine genes, including thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and interleukin-8 (IL-8). These results suggest that undecane may be useful for the prevention or treatment of skin inflammatory disorders, such as atopic dermatitis, and other allergic diseases.

scholarly journals PGE2-mediated inhibition of T cell p59fynis independent of cAMP

1999 ◽  
Vol 277 (2) ◽  
pp. C302-C309 ◽  
Author(s):  
Mashkoor A. Choudhry ◽  
Zulfiqar Ahmed ◽  
Mohammed M. Sayeed
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adenylate Cyclase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Prostaglandin E ◽  
Intracellular Camp ◽  
Sprague Dawley ◽  
Cell Functions ◽  
Camp Levels

We recently observed that prostaglandin E2(PGE2)-mediated suppression of T cell functions could result from an attenuation of p59fynprotein tyrosine kinase activity. The present study evaluated the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), as well as those of cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cell p59fynkinase activity. The study allowed us to assess whether PGE2-mediated activation of adenylate cyclase by itself or the elevation in intracellular cAMP levels is an integral event in the modulation of anti-CD3-linked p59fynactivation in T cells. The experiments were carried out with splenic T cells from male Sprague-Dawley rats. A 30–50% suppression in the autophosphorylation and the kinase activity of p59fynin T cells incubated with PGE2or forskolin was observed. Pretreatment of T cells with SQ-22536 prevented significant PGE2-mediated inhibition of T cell p59fynkinase activity. In contrast, no change in p59fynautophosphorylation and kinase activity in T cells treated with cAMP analogues was observed. These data suggest that PGE2-mediated suppression of p59fynautophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.

scholarly journals Erythromycin shortens neutrophil survival by accelerating apoptosis.

1995 ◽  
Vol 39 (4) ◽  
pp. 872-877 ◽  
Author(s):  
K Aoshiba ◽  
A Nagai ◽  
K Konno
Keyword(s):  
Inflammatory Diseases ◽  
Clinical Effectiveness ◽  
Maximum Effect ◽  
Intracellular Camp ◽  
Diffuse Panbronchiolitis ◽  
Dose Dependent Fashion ◽  
Anti Inflammatory ◽  
Neutrophil Survival ◽  
Camp Levels ◽  
Inflammatory Action

Erythromycin is reported to have an anti-inflammatory action, which may account for its clinical effectiveness in the treatment of chronic inflammatory diseases such as diffuse panbronchiolitis. To evaluate the anti-inflammatory action of erythromycin, we examined the survival of isolated neutrophils with and without erythromycin. Erythromycin shortened neutrophil survival in a dose-dependent fashion, with a maximum effect at 10 micrograms/ml [corrected] and above. Survival at 24 h was 63.4% in medium with 10 micrograms of erythromycin per ml compared with 82.7% in control medium (P < 0.01). This shortening of survival was brought about by acceleration of apoptosis, as evidenced by transmission electron microscopy. In a manner similar to that of erythromycin, other macrolide antibiotics, i.e., clarithromycin, roxithromycin, and midecamycin, also shortened neutrophil survival, but neither the beta-lactams ampicillin and cefazolin nor the aminoglycoside gentamicin affected their survival. Erythromycin increased intracellular levels of cyclic AMP (cAMP) to 150% of control levels in neutrophils. Forskolin, rolipram, and dibutyryl-cAMP, which are known to increase intracellular cAMP levels, also shortened neutrophil survival. H-89, an inhibitor of cAMP-dependent protein kinase A, partially blocked the survival-shortening effect of erythromycin. Our findings suggest that erythromycin shortens neutrophil survival at least in part through elevation of intracellular cAMP levels.

Involvement of glycinergic neurons in the diminished surround activity of ganglion cells in the dark-adapted rabbit retina

1991 ◽  
Vol 6 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Ralph J. Jensen
Keyword(s):  
Retinal Ganglion Cells ◽  
Dopaminergic Neurons ◽  
Dark Adaptation ◽  
Ganglion Cells ◽  
Dopamine Antagonist ◽  
Intracellular Camp ◽  
Rabbit Retina ◽  
Retinal Ganglion ◽  
Camp Levels ◽  
Dependent Mechanism

AbstractPrevious studies have reported that the surround responses of retinal ganglion cells weaken or disappear upon dark adaptation. The mechanism(s) by which this occurs is largely unknown, although changes in activity of retinal dopaminergic neurons have been implicated. In the light-adapted rabbit retina, the surround ON responses of OFF-center ganglion cells have been shown to be markedly reduced or abolished by a dopamine antagonist. This effect of a dopamine antagonist was recently shown to be reversed by the glycine antagonist strychnine and by compounds that elevate intracellular cAMP levels. The present study was conducted to determine whether strychnine and cAMP-elevating compounds could bring out the surround ON responses in OFF-center ganglion cells that are diminished upon dark adaptation. Extracellular recordings of OFF-center brisk ganglion cells were made from isolated, superfused retinal preparations. During the course of dark adaptation, the surround On responses of many cells decreased markedly.Application in both brisk-transient and brisk-sustained OFF-center ganglion cells. The center OFF responses of these cells, on the other hand, were not enhanced by strychnine. Of the cAMP-elevating compounds tested, 8-(4-chlorophenylthio) cyclic AMP was the most effective in bringing out the surround ON responses in dark-adapted OFF-center ganglion cells. The findings from this study suggest that under dark-adapted conditions glycinergic neurons inhibit the surround component of OFF-center ganglion cells. The release of glycine from these neurons is suggested to be regulated by a cAMP-dependent mechanism.

Chloroquine stimulates absorption and inhibits secretion of ileal water and electrolytes

1982 ◽  
Vol 243 (2) ◽  
pp. G117-G126
Author(s):  
R. Fogel ◽  
G. W. Sharp ◽  
M. Donowitz
Keyword(s):  
Cholera Toxin ◽  
Calcium Content ◽  
Intracellular Camp ◽  
Rabbit Ileum ◽  
Camp Content ◽  
Serosal Surface ◽  
Ileal Absorption ◽  
Camp Levels

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.

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