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PubMed1991

Carbachol induced release of diadenosine polyphosphates--Ap4A and Ap5A--from perfused bovine adrenal medulla and isolated chromaffin cells.

The diadenosine polyphosphates--Ap4A and Ap5A--were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds (Ap4A and Ap5A) were identified by the retention time in H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands stimulated with 100 microM carbachol released 0.47 +/- 0.12 nmol/gland of Ap4A and 1.11 +/- 0.26 nmol/gland of Ap5A. Isolated bovine chromaffin cells after 100 microM carbachol, as secretagogue, released 11.1 +/- 0.8 pmol/10(6) cells of Ap4A and 15.8 +/- 1.1 pmol/10(6) cells of Ap5A. The ratio of these compounds with respect to the exocytotically released ATP and catecholamines was in the same order as that found in isolated chromaffin granules.

Adenosine Triphosphate↗
PubMed1996

Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis.

Ionic currents evoked by alpha, omega-adenine dinucleotides (ApXA; X = 2-6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P1 and P2 purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sensitive P2 purinoceptor which showed an agonist potency order (at 10 microM): Ap4A > ATP > Ap3A > > Ap5A, while Ap2A and Ap6A were inactive. Outward currents were mediated by a novel theophylline-sensitive P1 purinoceptor which showed an agonist potency order (at 10 microM): Ap2A > ATP > > Ap4A = Ap5A = Ap6A > Ap3A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of dinucleotide cleavage, indicating that currents evoked by dinucleotides resulted from a direct activation of oocyte P1 and P2 purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P4 purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.

Animals↗
PubMed1995

Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: a fluorimetric study.

This article presents a fluorimetric study of the main properties of the enzymes dinucleoside tetraphosphate (asymmetrical) hydrolase or dinucleoside tetraphosphatase (Ap4Aase, EC 3.6.1.17) and dinucleoside triphosphate hydrolase or dinucleoside triphosphatase (Ap3Aase, EC 3.6.1.29), both present in adrenal medulla cytosolic extracts. Diethenoadenosine polyphosphates, epsilon-(ApnA), are used as artificial fluorogenic substrates. Ap4Aase exhibits a molecular mass around 20 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ and preferentially hydrolyzes substrates with four phosphate groups. Km for epsilon-(Ap4A) is 1.3 microM and Ki for Ap4A and Gp4G are 1 and 0.2 microM respectively. Km for Ap4A determined by HPLC is 1.6 microM. epsilon-(Ap5A) and epsilon-(Ap6A) are hydrolyzed at reduced rates. This enzyme is inhibited by Zn2+, F- and very strongly by Ap4 and epsilon-Ap4. Ca2+ cannot replace Mg2+, but behaves as inhibitor in its presence. The substrate analogs dinucleoside triphosphates Ap3A, G;3G, m7Gp3G and m7Gp3A and the periodate-oxidized nucleotides o-(Ap4A), o epsilon-(Ap4A), o-Ap4 and o epsilon-Ap4 behave as inhibitors. Ap3Aase exhibits a molecular mass around 30 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ or Ca2+, but retains a low measurable activity around 10% in the absence of these divalent cations. It only hydrolyzes substrates with three phosphate groups. Km for epsilon-(Ap3A) is 11 microM and Ki for Ap3A and Gp3G are 20 and 22 microM, respectively. Km for Ap3A determined by HPLC is 16 microM. m7Gp3G and m7Gp3A are also good substrates for triphosphatase.

Acid Anhydride Hydrolases↗
PubMed1989

Formation of inositol polyphosphates in cultured human sweat duct cells in response to cholinergic stimulation.

Inositol phosphate formation in response to cholinergic stimulation was studied in cultured human sweat duct cells, prelabelled with myo-[2-3H]inositol. Formation of inositol mono-, bis-, tris- and tetrakisphosphates was increased after 15 min stimulation by 30 microM carbachol. Formation of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate was significantly increased within 1 min at carbachol concentrations between 10 microM and 100 microM. No detectable increase in inositol 1,4,5-trisphosphate formation was observed at 15 s or 1 min, but an increase was observed after 15 min at a carbachol concentration of 30-100 microM. The data are consistent with an involvement of inositol polyphosphates in the biphasic response of ion transport, to cholinergic stimulation in these cells (see Pederson, P.S. (1986) 6th Professional Conference "Broken Arrow 1986". Genetic and Eptihelial Dysfunction in Cystic Fibrosis (Riordan, J.R. and Buchwalds, M., eds.), Alan Liss, New York and Pedersen, P.S. (1987) Med. Sci. Res. 15, 769-770) and suggest a different pattern of metabolism from exocrine acinar cells.

Calcium↗
PubMed1992

Presence of diadenosine polyphosphates--Ap4A and Ap5A--in rat brain synaptic terminals. Ca2+ dependent release evoked by 4-aminopyridine and veratridine.

The study of the adenine nucleotides in middle brain synaptosomes from rat showed the presence of two diadenosine polyphosphates, Ap4A and Ap5A. HPLC techniques and phosphodiesterase digestion were employed in order to characterize and quantify the dinucleotides. The Ap4A content per mg of protein was 169 +/- 25 pmol and 159 +/- 22 pmol for Ap5A. The study of the exocytotic release of these compounds was carried out with 100 microM 4-aminopyridine or 10 microM veratridine in the presence and in the absence of calcium. 4-Aminopyridine released 14.5 +/- 3.0 pmol/mg protein of Ap4A and 11.6 +/- 2.4 pmol/mg protein of Ap5A in a calcium dependent process. Veratridine in the presence of calcium released 19.9 +/- 3.0 and 16.6 +/- 2.8 pmol/mg of protein of Ap4A and Ap5A respectively. The ratios of exocytosis were close to 7-9% and 10-12% of the total synaptosomal content in the presence of 4-aminopyridine and veratridine, respectively.

4-Aminopyridine↗
PubMed1994

Effects of diadenosine polyphosphates on sodium nitroprusside-induced soluble guanylate cyclase activity in rat cerebellum.

Diadenosine polyphosphates (ApnAs,n = 1-6 phosphates) have been shown to interact with heme containing proteins and in the present study we determined for guanylate cyclase (GC), a heme containing enzyme whose activity is modulated by adenine nucleotides and nitric oxide, the degree to which ApnAs affect sodium nitroprusside (SNP, a nitric oxide donor)-induced GC activity using cytosolic fractions of rat cerebellum and radioimmunoassays for cGMP. SNP-stimulated GC activity was inhibited by up to 60% by ApnAs; rank order of potency was Ap6A > Ap5A > Ap4A > Ap3A = Ap2A. At concentrations ranging from 0.01 to 100 microM, only Ap6A potentiated in a concentration-dependent manner (apparent EC50 value 1 microM), by up to two-fold, SNP-induced GC activity. The effects of Ap6A on GC activity were neither to changes in SNP degradation nor due to metabolites of Ap6A. These results suggest that ApnAs through its effects on GC activity may play a role in regulating a variety of CNS functions.

Adenine Nucleotides↗
PubMed1989

Developmental aspects of muscarinic-induced inositol polyphosphate accumulation in rat cerebral cortex.

The ability of carbachol to stimulate phosphoinositide hydrolysis in developing brain was examined by assaying [3H]inositol phosphates in the presence and absence of lithium. Lithium (5 mM) enhanced carbachol-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulations at every age tested but the enhancement of both [3H]inositol phosphates was greater at 7 days than at 40 days. A marked, time-dependent inhibition of [3H]inositol trisphosphate and [3H]inositol tetrakisphosphates accumulations, i.e. 29-33 and 76-79%, respectively, was produced by lithium at every age tested. Lithium also inhibited both [3H]inositol-1,3,4-trisphosphate and [3H]inositol-1,4,5-trisphosphate by 29-38%. There were no developmental differences in the EC50 values for lithium-induced potentiations of [3H]inositol mono- and bisphosphate accumulations (i.e. 0.4-0.6 and 4-6 mM, respectively). Similarly, negligible changes in the EC50 values for carbachol-induced [3H]inositol mono- and bisphosphate accumulations were observed in the presence or absence of lithium at every age tested. Models of receptor coupling and the sensitivity of inositol polyphosphate dephosphorylation to lithium block during development are considered.

Animals↗
PubMed2005

The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2.

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.

Animals↗
PubMed2005

Vancomycin release behaviour from amorphous calcium polyphosphate matrices intended for osteomyelitis treatment.

Calcium polyphosphate (CPP) antibiotic delivery matrices were prepared using a unique processing technique involving the exposure of antibiotic-loaded CPP pastes to high humidity for 0, 5, or 24 h. After the designated gelling period, samples were dried for a minimum of 24 h. At several time points out to 130 h, the elution medium was monitored for vancomycin, Ca2+ ion and ortho and poly phosphate release levels. Vancomycin activity was also assessed after 1, 24 and 130 h, while solution 31P-NMR was used to monitor changes in chain length within a 24 hr gelled VCM disc throughout the elution process. The gelling and drying process significantly reduced the rate of vancomycin release during the initial 2-4 h of elution, while extending the effective antibiotic release period by an additional 80 h. The mild conditions associated with matrix fabrication readily allowed for vancomycin incorporation within an environment that did not disrupt antibiotic activity. Throughout the elution process, all sample groups experienced considerable swelling followed by some apparent bulk erosion. Phosphate chain lysis was clearly observed by the end of the elution period. Generally, no strong or consistent correlation existed between matrix degradation and antibiotic release for the treatment groups investigated. An ability to delay antibiotic release using CPPs in conjunction with this protocol supports further investigations into the potential of this matrix as a localized drug delivery system.

Anti-Bacterial Agents↗
PubMed2004

Synthesis of novel fluorescent-labelled dinucleoside polyphosphates.

A novel tandem synthetic-biosynthetic procedure is described for the synthesis of four new fluorescent dinucleoside polyphosphates: mant-Ap4A, mant-AppCH2ppA, TNP-Ap4A and TNP-AppCH2ppA. These compounds are expected to supplement the existing etheno (epsilon) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) labelled derivatives, being the fluorescent probes of choice to investigate polyphosphate/enzyme binding behaviour.

Boron Compounds↗
PubMed2005

Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1.

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k(cat) = 0.29+/-0.05 s(-1)) the transfer of AMP from the E-AMP-ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra- or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.

Adenosine Monophosphate↗
PubMed2005

Polyphosphate kinase: a new colonization factor of Helicobacter pylori.

In order to elucidate the role of polyphosphate kinase (PPK) during the course of an infection by Helicobacter pylori, PPK deficient mutants were constructed using two genetic backgrounds: Hp141v and X47-2AL. The efficiencies of the parental strains and the derivative mutants at colonizing the gastric mucosa of mice were compared. When animals received the Hp141v and the X47-2AL parental strains, 100% of the mice remained colonized for the duration of the 45 days experiment. In contrast, none of the mice that were given the PPK deficient X47-2AL derivative strain had a detectable bacterial load in their gastric mucosa, while the deficient Hp141v derivative strain was detected in 100%, 20% and 40% of the mice at days 3, 15 and 45 post-inoculation (p.i.), respectively. The absence of PPK expression did not impair the in vitro growth of the ppk mutants. However, the reduced ability of the ppk defective mutants to colonize mice was associated with a significant decrease in both motility and in an accumulation of polyP in the bacterial cells. These results are consistent with an essential role of PPK during the initial steps of colonisation of the mouse gastric mucosa and confirm that PPK may act on the virulence of H. pylori partly through an energy dependent mechanism.

Animals↗
PubMed2006

Lily pollen alkaline phytase is a histidine phosphatase similar to mammalian multiple inositol polyphosphate phosphatase (MINPP).

Phytic acid is the most abundant inositol phosphate in cells; it constitutes 1-5% of the dry weight of cereal grains and legumes. Phytases are the primary enzymes responsible for the hydrolysis of phytic acid and thus play important roles in inositol phosphate metabolism. A novel alkaline phytase in lily pollen (LlALP) was recently purified in our laboratory. In this paper, we describe the cloning and characterization of LlALP cDNA from lily pollen. Two isoforms of alkaline phytase cDNAs, LlAlp1 and LlAlp2, which are 1467 and 1533 bp long and encode proteins of 487 and 511 amino acids, respectively, were identified. The deduced amino acid sequences contains the signature heptapeptide of histidine phosphatases, -RHGXRXP-, but shares < 25% identity to fungal histidine acid phytases. Phylogenetic analysis reveals that LlALP is most closely related to multiple inositol polyphosphate phosphatase (MINPP) from humans (25%) and rats (23%). mRNA corresponding to LlAlp1 and LlAlp2 were expressed in leaves, stem, petals and pollen grains. The expression profiles of LlAlp isoforms in anthers indicated that mRNA corresponding to both isoforms were present at all stages of flower development. The expression of LlAlp2 cDNA in Escherichia coli revealed the accumulation of the active enzyme in inclusion bodies and confirmed that the cDNA encodes an alkaline phytase. In summary, plant alkaline phytase is a member of the histidine phosphatase family that includes MINPP and exhibits properties distinct from bacterial and fungal phytases.

6-Phytase↗
PubMed2001

Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1321N1 cells.

The selectivities of the diadenosine polyphosphates (Ap(n)As, n=2-6) at the human P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(11) receptors stably expressed in 1321N1 human astrocytoma cells was determined using a Fluorescence Imaging Plate Reader (FLIPR) to measure intracellular Ca(2+) mobilisation. The rank order of agonist potencies at P2Y(1) were: ADP>P(1),P(3)-diadenosine triphosphate (Ap(3)A)>P(1),P(3)-diadenosine hexaphosphate (Ap(6)A)=P(1),P(3)-diadenosine diphosphate (Ap(2)A)>>P(1),P(3)-diadenosine pentaphosphate (Ap(5)A). P(1),P(3)-diadenosine tetraphosphate (Ap(4)A) was inactive up to 1 mM. The rank order of agonist potencies at P2Y(2) were: UTP>Ap(4)A>>Ap(6)A>Ap(5)A>Ap(3)A>>Ap(2)A. The Ap(4)A concentration response curve appeared to be bi-phasic. At P2Y(4) all the Ap(n)As tested were inactive as agonists. At P2Y(6), only Ap(3)A and Ap(5)A showed significant agonist activity. At P2Y(11), only Ap(4)A showed significant agonist activity. Ap(n)As were inactive as antagonists of the P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(11) receptors. At P2Y(4), however, the Ap(n)As potentiated the UTP response.

Adenosine Diphosphate↗
PubMed2003

Ryanodine receptor modulation by diadenosine polyphosphates in synaptosomal and microsomal preparations of rat brain.

Diadenosine polyphosphates (Ap(n)As) are transmitter-like substances that act intracellularly via unclear mechanisms. Here we tested hypotheses that diadenosine tetraphosphate (Ap(4)A) modulates ryanodine binding in microsomal and synaptosomal fractions of rat brain, and that Ap(4)A affects modulation of ryanodine binding by divalent cations and caffeine. Using [3H]ryanodine-binding assays, we showed that Ap(4)A produced significant and concentration-dependent increases in [3H]ryanodine binding in microsomes and these actions were reduced by Mg(2+) and potentiated by caffeine. In synaptosomal subfractions, effects of Ap(4)A on [3H]ryanodine binding were most profound in subfractions enriched in synaptic vesicle-associated protein synaptophysin. These results suggest that Ap(n)As and ryanodine receptors are well placed to modulate Ca(2+)-dependent synaptic processes.

Animals↗
PubMed1997

Diadenosine polyphosphates inhibit adenosine kinase activity but decrease levels of endogenous adenosine in rat brain.

Findings in peripheral tissues that diadenosine polyphosphates (Ap(n)As) activate 5'-nucleotidase activity and inhibit adenosine kinase activity in vitro led us to test the hypothesis that Ap(n)As and analogues thereof, through such actions on purine enzymes, increase brain levels of endogenous adenosine in vivo. Accordingly, we tested Ap(n)As for their effects on the in vitro activities of adenosine kinase, adenosine deaminase, AMP deaminase and 5'-nucleotidase and, following unilateral microinjections in rat striatum, on in vivo levels of endogenous adenosine. Adenosine kinase activity was not affected significantly by 5',5'''-P1,P2-diadenosine pyrophosphate (Ap2A) or by 5',5'''-P1,P3-diadenosine triphosphate (Ap3A), but was inhibited by 5',5'''-P1,P4-diadenosine tetraphosphate (Ap4A), 5',5'''-P1,P5-diadenosine pentaphosphate (Ap5A) and 5',5'''-P1,P6-diadenosine hexaphosphate (Ap6A); apparent IC50 values were 5.0, 3.3 and 500 microM, respectively. Inhibition of adenosine kinase activity by Ap4A and the four metabolically stable analogues of Ap4A tested was uncompetitive. Following unilateral intrastriatal injections, adenosine levels, relative to uninjected contralateral striatum, were decreased significantly (P < 0.05) by 48% with Ap4A and by 37% with AppCH2ppA, a metabolically stable analogue of Ap4A. Striatal levels of adenosine were not affected significantly by Ap5A or Ap6A. Cytosolic, but not particulate 5'-nucleotidase activity was inhibited and AMP deaminase activity was increased by some Ap(n)As. Although adenosine kinase inhibitors increase levels of endogenous adenosine and we showed here that Ap(n)As were potent inhibitors of this enzyme, these particular actions of Ap(n)As were not consistent with their effects on levels of endogenous adenosine.

5'-Nucleotidase↗
PubMed1999

Selectivity of diadenosine polyphosphates for rat P2X receptor subunits.

The pharmacological activity of diadenosine polyphosphates was investigated at three recombinant P2X receptors (rat P2X1, rat P2X3, rat P2X4) expressed in Xenopus oocytes and studied under voltage-clamp conditions. For the rat P2X1 receptor, only P1,P6-diadenosine hexaphosphate (Ap6A) was a full agonist yet 2-3 folds less potent than ATP. At rat P2X3, P1,p4-diadenosine tetraphosphate (Ap4A), P1,P5-diadenosine pentaphosphate (Ap5A) and Ap6A were full agonists and more potent than ATP. Ap4A alone was equipotent with ATP at rat P2X4, but only as a partial agonist. Compared to known data for rat P2X2 and human P2X1 receptors, our findings contrast with rat P2X2 where only Ap4A is a full agonist although four folds less potent than ATP. At rat and human orthologues of P2X1, Ap5A was a partial agonist with similar potency. These data provide a useful basis for selective agonists of P2X receptor subunits.

Adenine Nucleotides↗
PubMed2003

The importance to chondrocyte differentiation of changes in expression of the multiple inositol polyphosphate phosphatase.

It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP(5) and InsP(6). We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates-both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP(6) levels in ATDC5 cells. However, InsP(5) levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP(5) and InsP(6) levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP(5) and InsP(6). Thus, substantial changes in MINPP expression can occur without a net effect upon InsP(5) and InsP(6) turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.

Alkaline Phosphatase↗