Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

Cyclic AMP modulates insulin binding and induces post-receptor insulin resistance of glucose transport in isolated rat adipocytes

The effect of cAMP on insulin binding and insulin stimulation of glucose transport was investigated in isolated rat adipocytes. Preincubation for 30 min in medium containing 16 mmol/l glucose and either db-cAMP or bromo-cAMP in concentrations of 10(-4)-10(-3) M inhibited high affinity binding of insulin by 15 to 30% and glucose transport by 30 to 50%. Preincubation with IBMX (10(-4)-10(-3) M) reduced insulin binding by 25% and glucose transport by 70%. Closer analysis of these data indicated that preincubation with these compounds caused not only a decrease in insulin binding but also a post-receptor resistance. High intracellular cyclic AMP-levels seem therefore to induce insulin resistance at both receptor and post-receptor levels.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Protonic and cationic changes during cyclical cation transport driven by electron transfer

A method is described for the subcellular fractionation of goldfish xanthophores. The procedure produces relatively pure fractions of caroteniod droplets, pterinosomes, cytosol and what appears to be plasma membrane. The presence of a distinct pattern of proteins is shown to be associated with the carotenoid droplets. Treatment of the xanthophores with ACTH affects the buoyant density of some carotenoid droplets and stimulates the phosphorylation of a polypeptide associated with the carotenoid droplets.     

Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Diacylglycerol, 1-oleoyl-2-acetyl-glycerol,stimulates superoxide-generation from human neutrophils

1-Oleoyl-2-acetyl-glycerol which activates Ca²⁺-activated phospholipid-dependent protein kinase, induced the superoxide-production of human neutrophils, while other diacylglycerols did not. The induction was independent of extracellular calcium and did not accompany the increase of the intracellular free calcium. The superoxide-release by the diacylglycerol was inhibited by retinal, the inhibitor of the protein kinase. The diacylglycerol stimulated the phosphorylation of at least 4 proteins in intact neutrophils, the phosphorylation of which was stimulated by phorbol 12-myristate 13-acetate, the activator of the protein kinase. These observations indicate the possible involvement of the kinase in the induction process.     

IL-32θ negatively regulates IL-1β production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation

It has been well known that IL-32 exerts pro-inflammatory effects on the various inflammatory diseases in clinical studies. Here, we confirmed that IL-32θ, a new isoform of IL-32, decreased the phorbol 12-myristate 13-acetate (PMA)-induced IL-1β expression in THP-1 human myelomonocyte. We previously reported that the IL-32 isoforms control expressions of other cytokines via novel PKCs. Likewise, IL-32θ interacted with PKCδ, and consequently inhibited PKCδ-mediated phosphorylation of PU.1. Moreover, IL-32θ attenuated the localization of PU.1 into the IL-1β promoter region. These findings reveal that IL-32θ reduces PKCδ-mediated phosphorylation of PU.1, resulting in attenuation of IL-1β production.     

Cooperative non-specific DNA binding of the N-terminal core of the cyclic AMP receptor protein of Escherichia coli and its modulation by cyclic AMP

The non-specific DNA binding of CRP and its N-terminal core, alpha CRP, to a 298 base pair DNA fragment, in the presence and absence of cAMP, has been studied using the nitrocellulose filter binding technique and analysed quantitatively using the theory of Clore et al. [J. Mol. Biol. (1982) 155, 447-466]. It is shown that both CRP and alpha CRP bind cooperatively to DNA. At an ionic strength of 100 mM and pH 7.5, the intrinsic equilibrium association constant for the binding of alpha CRP to DNA is approximately 10-times smaller than that for CRP, but the cooperativity parameter is approximately 17-times larger for alpha CRP than CRP. cAMP exerts its effect solely on the intrinsic equilibrium constant and does not alter the cooperativity. In the case of alpha CRP, cAMP reduces the intrinsic equilibrium association constant by a factor of 3, in contrast to the case of CRP where cAMP increases it by a factor of 3. The possible location of the DNA binding site present in the N-terminal core of CRP is discussed in the light of crystallographic data on the cAMP . CRP complex [McKay et al. (1982) J. Biol. Chem. 257, 9518-9524].     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

Crystal structure and substrate specificity of ExoY,a unique T3SS mediated secreted nucleotidyl cyclase toxin from Pseudomonas aeruginosa

Background

The nucleotidyl cyclase toxin ExoY is an important virulence determinant of Pseudomonas aeruginosa that causes severe acute and chronic infections in immune-compromised individuals. Additionally, this unique T3SS effector shows a striking preference for cUMP, a newly identified non-canonical secondary messenger. Thereby, ExoY is also considered as a potential tool to study unexplored cUMP signaling pathways.

Oxidation state of binuclear iron site in azidosemimethemerythrin

Electron paramagnetic resonance spectra of azidosemimethemerythrin (from Phascolopsis gouldii) have been integrated to find the total number of spins per monomer unit. The value observed, 1.0 +/- 0.1 spins per Fe2 pair, confirms the assignment of a hybrid oxidation state, FeIIFeIII, to each site.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.