cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.     

The impact of cold on photosynthesis in genotypes of Coffea spp.--photosystem sensitivity, photoprotective mechanisms and gene expression

Environmental constraints disturb plant metabolism and are often associated with photosynthetic impairments and yield reductions. Among them, low positive temperatures are of up most importance in tropical plant species, namely in Coffea spp. in which some acclimation ability has been reported. To further explain cold tolerance, the impacts on photosynthetic functioning and the expression of photosynthetic-related genes were analyzed. The experiments were carried out along a period of slow cold imposition (to allow acclimation), after chilling (4 °C) exposure and in the following rewarming period, using 1.5-year-old coffee seedlings of 5 genotypes with different cold sensitivity: Coffea canephora cv. Apoatã, Coffea arabica cv. Catuaí, Coffea dewevrei and 2 hybrids, Icatu (C. arabica × C. canephora) and Piatã (C. dewevrei × C. arabica). All genotypes suffered a significant leaf area loss only after chilling exposure, with Icatu showing the lowest impact, a first indication of a higher cold tolerance, contrasting with Apoatã and C. dewevrei. During cold exposure, net photosynthesis and Chl a fluorescence parameters were strongly affected in all genotypes, but stomatal limitations were not detected. However, the extent of mesophyll limitation, reflecting regulatory mechanisms and/or damage, was genotype dependent. Overnight retention of zeaxanthin was common to Coffea genotypes, but the accumulation of photoprotective pigments was highest in Icatu. That down-regulated photochemical events but efficiently protected the photosynthetic structures, as shown, e.g., by the lowest impacts on A_max and PSI activity and the strongest reinforcement of PSII activity, the latter possibly reflecting the presence of a photoprotective cycle around PSII in Icatu (and Catuaí). Concomitant to these protection mechanisms, Icatu was the sole genotype to present simultaneous upregulation of caCP22, caPI and caCytf, related to, respectively, PSII, PSI and to the complex Cytb₆/f, which could promote better repair ability, contributing to the maintenance of efficient thylakoid functioning. We conclude that Icatu showed the best acclimation ability among the studied genotypes, mostly due to a better upregulation of photoprotection and repair mechanisms. We confirmed the presence of important variability in Coffea spp. that could be exploited in breeding programs, which should be assisted by useful markers of cold tolerance, namely the upregulation of antioxidative molecules, the expression of selected genes and PSI sensitivity.     

Synthesis and biological evaluation of fatty acyl ester derivatives of 2',3'-didehydro-2',3'-dideoxythymidine

A number of 5′-O-fatty acyl derivatives of 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T) were synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cytotoxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these conjugates, 5′-O-12-azidododecanoyl derivative of d4T (2), displaying EC₅₀ = 3.1-22.4 μM, showed 4- to 9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through β-alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cellular uptake and anti-HIV activity of stavudine.     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

Dynamic response of UV-absorbing compounds, quantum yield and the xanthophyll cycle to diel changes in UV-B and photosynthetic radiations in an aquatic liverwort

We studied the diel responses of the liverwort Jungermannia exsertifolia subsp. cordifolia to radiation changes under laboratory conditions. The samples were exposed to three radiation regimes: P (only PAR), PA (PAR + UV-A), and PAB (PAR + UV-A + UV-B). The day was divided in four periods: darkness, a first low-PAR period, the high-PAR plus UV period, and a second low-PAR period. After 15 days of culture, we measured photosynthetic pigments, chlorophyll fluorescence and UV-absorbing compounds in the four periods of the day on two consecutive days. With respect to UV-absorbing compounds, we analyzed their global amount (as the bulk UV absorbance of methanolic extracts) and the concentration of seven hydroxycinnamic acid derivatives, both in the soluble (mainly vacuolar) and insoluble (cell wall-bound) fractions of the plant extracts. PAB samples increased the bulk UV absorbance of the soluble and insoluble fractions, and the concentrations of p-coumaroylmalic acid in the soluble fraction and p-coumaric acid in the cell wall. Most of these variables showed significant diel changes and responded within a few hours to radiation changes (more strongly to UV-B), increasing at the end of the period of high-PAR plus UV. F_v/F_m, Φ_PSII, NPQ and the components of the xanthophyll cycle showed significant and quick diel changes in response to high PAR, UV-A and UV-B radiation, indicating dynamic photoinhibition and protection of PSII from excess radiation through the xanthophyll cycle. Thus, the liverwort showed a dynamic protection and acclimation capacity to the irradiance level and spectral characteristics of the radiation received.     

Two-dimensional infrared correlation spectroscopy study of the interaction of oxidized and reduced cytochrome c with phospholipid model membranes

We have used two-dimensional infrared correlation spectroscopy (2D-IR) to study the interaction and conformation of cytochrome c in the presence of a binary phospholipid mixture composed of a zwitterionic perdeuterated phospholipid and a negatively-charged one. The influence of the main temperature phase transition of the phospholipid model membranes on the conformation of cytochrome c has been evaluated by monitoring both the Amide I′ band of the protein and the CH₂ and CD₂ stretching bands of the phospholipids. Synchronous 2D-IR analysis has been used to determine the different secondary structure components of cytochrome c which are involved in the specific interaction with the phospholipids, revealing the existence of a specific interaction between the protein with cardiolipin-containing vesicles but not with phosphatidic acid-containing ones. Interestingly, 2D-IR is capable of showing the existence of significant changes in the protein conformation at the same time that the phospholipid transition occurs. In summary, 2D-IR revealed an important effect of the phospholipid phase transition of cardiolipin on the secondary structure of oxidized cytochrome c but not to either reduced cytochrome c or in the presence of phosphatidic acid, demonstrating the existence of specific intermolecular interactions between cardiolipin and cytochrome c.     

Cytochrome bd-I in Escherichia coli is less sensitive than cytochromes bd-II or bo′' to inhibition by the carbon monoxide-releasing molecule,CORM-3: N-acetylcysteine reduces CO-RM uptake and inhibition of respiration

Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (K_m 0.24 μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance,stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots

The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H₂O₂/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe²⁺-dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.     

Fab1p Is Essential for PtdIns(3)P 5-Kinase Activity and the Maintenance of Vacuolar Size and Membrane Homeostasis

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH₂-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P₂. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1^tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P₂, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P₂ synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P₂ synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P₂. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P₂ by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P₂ has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P₂, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P₂.     

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.     

Endogenous abscisic acid is involved in methyl jasmonate-induced reactive oxygen species and nitric oxide production but not in cytosolic alkalization in Arabidopsis guard cells

We recently demonstrated that endogenous abscisic acid (ABA) is involved in methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we investigated whether endogenous ABA is involved in MeJA-induced reactive oxygen species (ROS) and nitric oxide (NO) production and cytosolic alkalization in guard cells using an ABA-deficient Arabidopsis mutant, aba2-2, and an inhibitor of ABA biosynthesis, fluridon (FLU). The aba2-2 mutation impaired MeJA-induced ROS and NO production. FLU inhibited MeJA-induced ROS production in wild-type guard cells. Pretreatment with 0.1 μM ABA, which does not induce stomatal closure in the wild type, complemented the insensitivity to MeJA of the aba2-2 mutant. However, MeJA induced cytosolic alkalization in both wild-type and aba2-2 guard cells. These results suggest that endogenous ABA is involved in MeJA-induced ROS and NO production but not in MeJA-induced cytosolic alkalization in Arabidopsis guard cells.     

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb^−/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A₂ in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb^−/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb^−/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb^−/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.     

(d)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Iα

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Iα inhibitors with K_i values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Iα and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.     

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation,platelet aggregation,and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.