microM ATP Research Articles

Abstract A180: Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity

Abstract Introduction> Cell cycle kinases have been recognized as potential anticancer targets, because loss of regulated cell cycle progression would lead to uncontrolled cell growth and development of cancer. Cdc7 kinase is involved in regulation of the cell cycle and plays important roles in DNA replication. Notably, inhibition of Cdc7 in cancer causes lethal S phase or M phase progression, whereas normal cells can survive, most likely through induction of cell cycle arrest at the DNA replication checkpoints. Therefore Cdc7 kinase has emerged as a promising therapeutic target for the treatment of cancers. Here we present a novel series of furanone derivatives with potent inhibitory effects on Cdc7 kinase. <Methods> To identify hit compounds that are insensitive or resistant to high ATP concentration, a high-throughput screening (HTS) was performed in the presence of 100 microM ATP which corresponds to 36-times higher than the Km value of ATP for Cdc7 kinase activity. To characterize inhibitors, ATP dependency and pre-incubation effects were examined. To determine the efficacy of Cdc7 inhibitors, phosphorylation of MCM2 protein in cells were analyzed by western blotting. Selected compounds were subjected to a kinase selectivity panel assay. <Results> Only a single hit compound was identified from the HTS, reflecting extremely low hit rate compared to that of other kinase targets in our past HTS. Subsequent hit to lead studies led to the identification of a novel furanone compound as a potential lead compound for further optimization study. Interestingly, these furanone derivatives showed slow-binding inhibition against Cdc7, and exhibited strong inhibitory activity for Cdc7 even in the presence of 1 mM ATP that was added after the pre-incubation of the kinase and compound. Notably this pre-incubation effect was not observed with other kinases tested. Detailed results using cancer cells will also be presented in this poster. Citation Format: Takayuki Irie, Tokiko Asami, Ayako Sawa, Chika Taniyama, Yoko Funakoshi, Yuko Uno, Hisao Masai, Masaaki Sawa. Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A180.

The ecto‐nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor‐dependent vasorelaxation

Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation. Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1(-/-) (lacking NTPDase1) mice. The absence of NTPDase1 in Entpd1(-/-) mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1(+/+) and Entpd1(-/-) mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole.kg(-1) both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1(-/-) mice. EC(50) values in Entpd1(+/+) versus Entpd1(-/-) aortic rings were 14 microM versus 0.35 microM for ADP, and 29 microM versus 1 microM for ATP. In Entpd1(-/-) aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPbetaS (P2Y1) and ATPgammaS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice. NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.

Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors

ATP is released from skeletal muscle during exercise, a discovery dating back to 1969. Surprisingly, few studies have examined the effects of extracellular ATP on mature mammalian skeletal muscle. This electrophysiological study examined the effects of extracellular ATP on fully innervated rat levator auris longus using two intracellular microelectrodes. The effects of ATP were determined by measuring the relative changes of miniature endplate potentials (mEPPs) and voltage responses to step current pulses in individual muscle fibres. Exposure to ATP (20 microm) prolonged the mEPP falling phase by 31 +/- 7.5% (values +/- s.d., n = 3 fibres). Concurrently, the input resistance increased by 31 +/- 2.0% and the time course of the voltage responses increased by 59 +/- 3.0%. Analogous effects were observed using 2 and 5 microm ATP, and on regions distal from the neuromuscular junction, indicating that physiologically relevant levels of ATP enhanced electrical signalling over the entire muscle fibre. The effects of extracellular ATP were blocked by 200 microm anthracene-9-carboxylic acid, a chloride channel inhibitor, and reduced concentrations of extracellular chloride, indicating that ATP inhibited chloride channels. A high affinity agonist for P2Y receptors, 2-methylthioadenosine-5-O-diphosphate (2MeSADP), induced similar effects to ATP with an EC(50) of 160 +/- 30 nm. The effects of 250 nm2MeSADP were blocked by 500 nmMRS2179, a specific P2Y(1) receptor inhibitor, suggesting that ATP acts on P2Y(1) receptors to inhibit chloride channels. The inhibition of chloride channels by extracellular ATP has implications for muscle excitability and fatigue, and the pathophysiology of myotonias.

Deletion of Phe508 in the first nucleotide‐binding domain of the cystic fibrosis transmembrane conductance regulator increases its affinity for the heat shock cognate 70 chaperone

The primary cause of cystic fibrosis (CF), the most frequent fatal genetic disease in Caucasians, is deletion of phenylalanine at position 508 (F508del), located in the first nucleotide-binding domain (NBD1) of the CF transmembrane conductance regulator (CFTR) protein. F508del-CFTR is recognized by the endoplasmic reticulum quality control (ERQC), which targets it for proteasomal degradation, preventing this misfolded but partially functional Cl(-) channel from reaching the cell membrane. We recently proposed that the ERQC proceeds along several checkpoints, the first of which, utilizing the chaperone heat shock cognate 70 (Hsc70), is the major one directing F508del-CFTR for proteolysis. Therefore, a detailed characterization of the interaction occurring between F508del-CFTR and Hsc70 is critical to clarify the mechanism that senses misfolded F508del-CFTR in vivo. Here, we determined by surface plasmon resonance that: (a) F508del-murine (m)NBD1 binds Hsc70 with higher affinity (K(D), 2.6 nm) than wild-type (wt) mNBD1 (13.9 nm); (b) ATP and ADP dramatically reduce NBD1-Hsc70 binding; (c) the F508del mutation increases by approximately six-fold the ATP concentration required to inhibit the NBD1-Hsc70 interaction (IC(50); wt-mNBD1, 19.7 microm ATP); and (d) the small molecule CFTR corrector 4a (C4a), but not VRT-325 (V325; both rescuing F508del-CFTR traffic), significantly reduces F508del-mNBD1 binding to Hsc70, by approximately 30%. Altogether, these results provide a novel, robust quantitative characterization of Hsc70-NBD1 binding, bringing detailed insights into the molecular basis of CF. Moreover, we show how this surface plasmon resonance assay helps to elucidate the mechanism of action of small corrective molecules, demonstrating its potential to validate additional therapeutic compounds for CF.

Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells

P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 microM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by alphabetamethylene ATP (10 microM, 94% cells, n= 31) and was antagonized in LAD2 cells by NF 449 (1 microM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 microM). A P2X7-like non-desensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n= 24; 54% HLMCs, n= 24) which was antagonized by AZ11645373 (1 microM). P2X7-like responses were also evoked in LAD2 cells by 2'(3')-0-(4-benzoylbenzoyl)ATP (300 microM). A P2X4-like current was evoked by 100 microM ATP (80% LAD2, n= 10; 21% HLMCs, n= 29), the amplitude and duration of which was potentiated by ivermectin (3 microM). Our data confirmed the presence of functional P2X1, P2X4 and P2X7 receptors in LAD2 cells and HLMCs.

Effects of Magnesium Lithospermate B and its Analogues on Ca2+Homeostasis in Cultured Rat Thoracic Aorta Vascular Smooth Muscle Cells

Magnesium lithospermate B (MLB), sodium rosmarinate (SR), and magnesium lithospermate (ML) are biologically active components isolated from aqueous extracts of the Chinese medicine Danshen, the dried root and rhizome of SALVIA MILTIORRHIZA Bunge (Labiatae). These compounds share similar chemical structures of polyphenols and oligomer condensates with caffeic acid. In this study, we compared the effects of MLB, ML, and SR on intracellular Ca (2+) concentrations ([Ca (2+)] (i)) in cultured rat thoracic aorta vascular smooth muscle cells (VSMCs), using the Ca (2+)-sensitive dye, Fluo-3, as an indicator. In our experiments, MLB, ML, and SR did not affect the basal value of [Ca (2+)] (i) in VSMCs. In the absence of extracellular Ca (2+), the [Ca (2+)] (i) increase in VSMCs induced by 20 microM ATP was attenuated by MLB, ML, and SR in a dose-dependent manner. Moreover, under this condition, MLB suppressed the increase in [Ca (2+)] (i) in VSMCs induced by 1 microM thapsigargin, but not ML or SR. In the presence of extracellular Ca (2+), the [Ca (2+)] (i) increase in VSMCs induced by 60 mM KCl was attenuated by MLB and SR in a dose-dependent manner, but not by ML. These results suggest that MLB, ML and SR regulate Ca (2+) homeostasis in VSMCs via different pathways. Our findings should aid in elucidating the mechanisms of the vasodilator action of aqueous extracts of SALVIA MILTIORRHIZA.

Effect of 100 mT homogeneous static magnetic field on [Ca2+]c response to ATP in HL‐60 cells following GSH depletion

Calcium is an important molecule in a number of biological systems. Often these systems are signal transduction cascades involving molecules such as ATP. ATP activates second messengers which can interact with ion channels on the endoplasmic/sarcoplasmic reticulum resulting in the emptying of the intracellular calcium stores and an increase in cytosolic free calcium concentration ([Ca2+]c). Changes in [Ca2+]c can be influenced by external factors such as a static magnetic field (SMF). One hypothesis suggests that a SMF affects the cells through the radical pair mechanism. By reducing the number of antioxidant molecules like glutathione (GSH), the proportion of free radicals in the cells is increased and may lead to a greater probability of a biological response to a SMF. The purpose of this study was to determine if the [Ca2+]c response to ATP was affected by depletion of GSH by diethylmaleate (DEM) and the absence or presence of a 100 mT homogeneous SMF. Undifferentiated HL-60 cells were loaded with fura-2 AM. [Ca2+]c was measured in real time using a ratiometric fluorescence spectroscopy system. Various (DEM) ranging from 1 to 15 mM were added to deplete GSH. Cells were either exposed to sham or magnetic field (100 mT) for 13 min (780 s) and challenged with 1 microM ATP. The data show that [Ca2+]c was elevated following treatment with DEM with greater [Ca2+]c at higher [DEM]. The [Ca2+]c response to ATP was decreased as the DEM concentration increased. However, there was no effect of a 100 mT SMF on the average [Ca2+]c peak following ATP activation or the full width at half maximum (FWHM) of the [Ca2+]c response and recovery after ATP activation.

Solution Structure of the DNA Binding Domain of Rice Telomere Binding Protein RTBP1,

RTBP1 is a rice telomeric protein that binds to the duplex array of TTTAGGG repeats at chromosome ends. The DNA binding domain of RTBP1 contains a Myb-type DNA binding motif and a highly conserved C-terminal Myb extension that is unique to plant telomeric proteins. Using an electrophoretic mobility shift assay, we identified the C-terminal 110-amino acid region (RTBP1(506-615)) as the minimal telomeric DNA binding domain, suggesting that the Myb extension is required for binding plant telomeric DNA. Like other telomeric proteins such as human TRF1 and yeast Rap1, RTBP1 induced a DNA bending in the telomeric repeat sequence, suggesting that RTBP1 may play a role in establishing and/or maintaining an active telomere configuration in vivo. To elucidate the DNA binding mode of RTBP1, we determined the three-dimensional structure of RTBP1(506-615) in solution by NMR spectroscopy. The overall structure of RTBP1(506-615) is composed of four alpha-helices and stabilized by three hydrophobic patches. The second and third helices in RTBP1 form a helix-turn-helix motif that interacts directly with DNA. The fourth helix located in the Myb extension is essential for binding to telomeric DNA via stabilization of the overall structure of the RTBP1 DNA binding domain. When DNA bound to RTBP1(506-615), large chemical shift perturbations were induced in the N-terminal arm, helix 3, and the loop between helices 3 and 4. These results suggest that helix 3 functions as a sequence-specific recognition helix while the N-terminal arm stabilizes the DNA binding.

ATP and NO dually control migration of microglia to nerve lesions

Microglia migrate rapidly to lesions in the central nervous system (CNS), presumably in response to chemoattractants including ATP released directly or indirectly by the injury. Previous work on the leech has shown that nitric oxide (NO), generated at the lesion, is both a stop signal for microglia at the lesion and crucial for their directed migration from hundreds of micrometers away within the nerve cord, perhaps mediated by a soluble guanylate cyclase (sGC). In this study, application of 100 microM ATP caused maximal movement of microglia in leech nerve cords. The nucleotides ADP, UTP, and the nonhydrolyzable ATP analog AMP-PNP (adenyl-5'-yl imidodiphosphate) also caused movement, whereas AMP, cAMP, and adenosine were without effect. Both movement in ATP and migration after injury were slowed by 50 microM reactive blue 2 (RB2), an antagonist of purinergic receptors, without influencing the direction of movement. This contrasted with the effect of the NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide), which misdirected movement when applied at 1 mM. The cPTIO reduced cGMP immunoreactivity without changing the immunoreactivity of eNOS (endothelial nitric oxide synthase), which accompanies increased NOS activity after nerve cord injury, consistent with involvement of sGC. Moreover, the sGC-specific inhibitor LY83583 applied at 50 microM had a similar effect, in agreement with previous results with methylene blue. Taken together, the experiments support the hypothesis that ATP released directly or indirectly by injury activates microglia to move, whereas NO that activates sGC directs migration of microglia to CNS lesions.

Identification, Characterization and Initial Hit-to-Lead Optimization of a Series of 4-Arylamino-3-Pyridinecarbonitrile as Protein Kinase C theta (PKCθ) Inhibitors

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.

Metal complexation chemistry used for phosphate and nucleotide determination: an investigation of the Yb3+-pyrocatechol violet sensor.

Metal complexation reactions can be used effectively as sensors to measure concentrations of phosphate and phosphate analogs. Recently, a method was described for the detection of phosphate or ATP in aqueous solution based on the displacement by these ligands of pyrocatechol violet (PV) from a putative 2:1 (Yb3+)2PV complex. We have not been able to reproduce this stoichiometry and report this work in order to correct the coordination chemistry upon which sensor applications are based. In our work, colorimetric and spectrophotometric detection of phosphate was confirmed qualitatively (blue PV+Yb3+; yellow+Pi); however, the sequence of visual changes on the titration of PV with 2 equiv. of Yb3+ and back titration with ATP as described previously could not be reproduced. In contrast to the linear response to Pi that was reported previously, the absorbance response at 443 or 623 nm was found to be sigmoidal using the recommended 2:1 Yb3+:PV solution (100 microM:50 microM, pH 7, HEPES). Furthermore, both continuous variation titration and molar ratio analysis (Job plot) experiments are consistent with 1:1, not 2:1, YbPV complex stoichiometry at pH 7 in HEPES buffer, indicating that the deviation from linearity is produced by excess Yb3+. Indeed, using a 1:1 Yb3+:PV ratio produces a linear response in DeltaAbs at 443 or 623 nm on back titration with analyte (phosphate or ATP). In addition, speciation analysis of the Yb-ATP system demonstrates that a 1:1 complex containing Yb3+ and ATP predominates in solution at microM metal ion and ATP concentrations. Paramagnetic 1H NMR spectroscopy directly establishes the formation of Yb3+-solute complexes in dilute aqueous solution. The 1:1 YbPV complex can be used for the colorimetric measurement of phosphate and ATP concentrations from approximately 2 microM.

Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate

The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 microM oxyluciferin; 0.0025 to 1.25 microM L-AMP) has been measured in 50 mM Hepes buffer (pH=7.5), 10 nM Luc, 250 microM ATP and D-Luciferin (from 3.75 up to 120 microM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (Ki=0.50+/-0.03 microM) while L-AMP act as a tight-binding competitive inhibitor (Ki=3.8+/-0.7 nM). The Km values obtained both for oxyluciferin and L-AMP were 14.7+/-0.7 and 14.9+/-0.2 microM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.

Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis

Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.

Genetic transformation via somatic embryogenesis to establish herbicide-resistant opium poppy

A reliable genetic transformation protocol via somatic embryogenesis has been developed for the production of fertile, herbicide-resistant opium poppy plants. Transformation was mediated by Agrobacterium tumefaciens using the pCAMBIA3301 vector, which harbors the phosphinothricin acetyltransferase (pat) gene driven by a tandem repeat of the cauliflower mosaic virus (CaMV) 35S promoter and the beta-glucuronidase (gus) structural gene driven by a single copy of the CaMV 35S promoter between left- and right-border sequences. Co-cultivation of explants and A. tumefaciens was performed in the presence of 50 microM ATP and 50 microM MgCl(2). Root explants pre-cultured on callus induction medium were used for transformation. Herbicide-resistant, proliferating callus was obtained from explants on a medium containing both 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Globular embryogenic callus, induced by removal of the BA from the medium, was placed on a hormone-free medium to form somatic embryos, which were converted to plantlets under specific culture conditions. Plantlets with roots were transferred to soil, allowed to mature and set seed. Both pat and gus gene transcripts, and PAT and GUS enzyme activities were detected in the transgenic lines tested. Histochemical localization of GUS activity in T(1) opium poppy plants revealed transgene expression in most tissues of all plant organs. The protocol required 8-12 months to establish transgenic T(1) seed stocks and was developed using a commercial opium poppy cultivar that produces high levels of pharmaceutical alkaloids.

Functional expression of ionotropic purinergic receptors on mouse taste bud cells

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

Stimulation of rat erythrocyte P2X7 receptor induces the release of epoxyeicosatrienoic acids

Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation. Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry. EETs were released from rat RBCs: 14,15-, 11,12-, 8,9- and 5,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.66+/-0.14 ng per 10(9) RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X(7) receptor antagonists. 5 microM ATP increased release of EETs over 20% to 0.83+/-0.15 ng per 10(9) RBCs; 10 microM BzATP tripled the amount of EET release to 1.87+/-0.20 ng per 10(9) RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs. EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, pannexin-1 and CFTR.

Extracellular Nucleotides Block Bone Mineralization in Vitro: Evidence for Dual Inhibitory Mechanisms Involving Both P2Y2 Receptors and Pyrophosphate

Extracellular nucleotides, signaling through P2 receptors, may act as local regulators of bone cell function. We investigated the effects of nucleotide agonists [ATP, ADP, uridine triphosphate (UTP), and uridine diphosphate] and pyrophosphate (PPi, a key physiological inhibitor of mineralization) on the deposition and mineralization of collagenous matrix by primary osteoblasts derived from rat calvariae. Our results show that extracellular ATP, UTP, and PPi strongly and selectively blocked the mineralization of matrix nodules; ADP and uridine diphosphate were without effect. Significant inhibition of mineralization occurred in the presence of relatively low concentrations of ATP, UTP, or PPi (1-10 microm), without affecting production of fibrillar or soluble collagen. In cultures treated with 10 microm ATP or UTP, the expression and activity of alkaline phosphatase, which promotes mineralization by hydrolyzing PPi, was inhibited. The potent inhibitory actions of ATP and UTP on bone mineralization are consistent pharmacologically with mediation by the P2Y(2) receptor, which is strongly expressed by mature osteoblasts. In support of this notion, we found 9-17% increases in bone mineral content of hindlimbs of P2Y(2)-deficient mice. We also found that osteoblasts express ectonucleotide phosphodiesterase/pyrophosphatase-1, an ectonucleotidase that hydrolyzes nucleotide triphosphates to yield PPi, and that addition of 10 microm ATP or UTP to osteoblast cultures generated 2 microm PPi within 10 min. Thus, a component of the profound inhibitory action of ATP and UTP on bone mineralization could be mediated directly by PPi, independently of P2 receptors.

Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins.

The purification and functional characterization of protein kinase A catalytic subunit (PKAcat) from bovine lens cytosol has been described. Purification to homogeneity has been achieved by using 100 kDa cut-off membrane filtration followed by Sephacryl S-300 chromatography and finally fractionating on High Q anion exchange column. The purified protein migrates as a single band of molecular mass approximately 41 kDa on 12.5% SDS-PAGE. Proteomic data from ion trap LC-MS when analyzed through NCBI blast program reveals significant homology (52%) with bovine zeta-crystallin and also some homology with pig casein kinase I alpha chain (38%) and SLA-DR1 beta 1 domain (38%). The search does not indicate homology with any known catalytic subunit of PKA. Inspite of the significant homology with the zeta-crystallin, our protein is different from it in terms of molecular mass. pI value of the kinase (5.3) obtained from 2D analysis is also different from zeta-crystallin (8.5). The protein is found to contain 17% alpha-helix, 26.5% beta-sheet, 21.4% turn and 34.7% random coil. The active catalytic subunit of the bovine lens cAMP-dependent kinase belongs to Type I Calpha subtype. The enzyme shows maximum activity at 30 min incubation in presence of 5 mM MgCl(2 )and 50 microM ATP. The kinase shows broad substrate specificity. It prefers Ser over Thr as phosphorylating residue. Phosphorylation of crystallin proteins, major protein fraction of bovine lens and phosphorylation of chaperone protein alpha crystallin by the kinase suggests that the kinase plays some crucial role in regulation of chaperone function within lens.

Modulation of the Permeability Transition Pore by Inhibition of the Mitochondrial KATP Channel in Liver vs. Brain Mitochondria

Inhibition of the mitochondrial K(ATP) (mitoK(ATP)) channel abrogates the beneficial effects of preconditioning induced by a brief episode of sublethal ischemia. We studied the effect of 5-hydroxydecanoate, a well-known inhibitor of the mitoK(ATP) channel, on swelling of isolated liver and brain mitochondria. Volume changes were determined by measurement of light absorbance at 540 nm. Mitochondrial swelling induced by adding Ca(2+ )ions correlated with opening of the permeability transition pore as shown by modulation by 1 microM cyclosporin A. In brain mitochondria, 5-hydroxydecanoate did not significantly affect Ca(2+)-induced swelling. In contrast, 50 or 500 microM 5-hydroxydecanoate increased swelling of liver mitochondria by 9.7 +/- 5.1% (n = 6, P = 0.057) and 29.4 +/- 1.4% (n = 5, P < 0.0001), respectively. The effect of 5-hydroxydecanoate was blocked by cyclosporin A and was dependent on the presence of potassium in the medium. In medium containing 200 microM ATP to inhibit the mitoK(ATP )channel, 5-hydroxydecanoate did not further increase Ca(2+)-induced swelling. We conclude that inhibition of the mitoK(ATP) channel exerts its detrimental effect by facilitation of permeability transition pore opening.

An Accelerated State of Myosin-Based Actin Motility

We use an in vitro motility assay to determine the biochemical basis for a hypermotile state of myosin-based actin sliding. It is widely assumed that the sole biochemical determinant of actin-sliding velocities, V, is actin-myosin detachment kinetics (1/tauon), yet we recently reported that, above a critical ATP concentration of approximately 100 microM, V exceeds the detachment limit by more than 2-fold. To determine the biochemical basis for this hypermotile state, we measure the effects of ATP and inorganic phosphate, Pi, on V and observe that at low [ATP] V decreases as ln [Pi], whereas above 100 microM ATP the hypermotile V is independent of Pi. The ln [Pi] dependence of V at low [ATP] is consistent with a macroscopic model of muscle shortening, similar to Hill's contractile component, which predicts that V varies linearly with an internal force (Hill's active state) that drives actin movement against the viscous drag of myosin heads strongly bound to actin (Hill's dashpot). At high [ATP], we suggest that the hypermotile V is caused by shear thinning of the resistive population of strongly bound myosin heads. Our data and analysis indicate that, in addition to contributions from tauon and myosin's step size, d, V is influenced by the biochemistry of myosin's working step as well as resistive properties of actin and myosin.