Ecto-nucleotidases terminate purinergic signalling in the cochlear endolymphatic compartment

There is strong evidence for a purinergic signalling system in the inner ear which regulates auditory sensitivity. This study describes the terminating mechanism for purinergic signalling in the cochlear endolymphatic compartment via ecto-nucleotidases. Exogenous ATP was introduced into the scala media (SM) of the isolated, perfused guinea-pig cochlea, and the effluent was assayed for the adenine nucleotide metabolites by reverse-phase HPLC. Tissue viability was confirmed by fluorescence imaging of cochlear tissues. Extracellular ATP degradation to adenosine was Ca2+/Mg2+ dependent, and was not affected by inhibitors of intracellular ATPases and non-specific alkaline phosphatase. High azide concentration (5 mM) and suramin produced an inhibitory effect on ATP hydrolysis, consistent with inhibition of E-type ATPase activity. The Vmax of ATP hydrolysis (2564 mumol min-1 SM-1) was indicative of high ecto-ATPase activity. Our results support the role of ecto-nucleotidases as a principal mechanism for termination of purinergic signalling within SM, a compartment of the cochlea showing considerable P2X receptor expression.     

Dual-mode cooperative binding of adenosine 5'-triphosphate to poly(L-lysine)

Equilibrium dialysis of adenosine triphosphate interacting with polylysine at low polymer concentration yield Scatchard exhibiting an apparent non-cooperative binding mode superimposed on a positively cooperative one. The same behavior has been reported in the literature for various mononucleotides and basic poly(amino acids). It is pointed out here that both modes must not be considered as independent but to be mutually exclusive. Applying a pertinent theoretical approach yields higher degrees of cooperativity than obtained for mutual independence. Another necessary modification in the analysis of data takes into account that one ligand interacts with more than one equivalent binding contact (i.e., an elementary charge here). This leads to a further enhancement of the actual cooperativity. Increasing the polymer concentration in the binding experiments reveals a cooperative effect also in the first binding mode. This is physically interpreted and theoretically analyzed in terms of bound dimers stabilized by base stacking.     

Metal ion/buffer interactions. Stability of binary and ternary complexes containing 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) and adenosine 5'-triphosphate (ATP)

The acidity constant of protonated 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)-1,3-propanediol (Bistris) has been measured. The influence of hydroxo groups on the basicity of Bistris and related bases is discussed. The interaction of Bistris with the metal ions (M2+) Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 1.0 M, KNO3; 25 degrees C) and the stability constants of the M(Bistris)2+ complexes were determined. Unexpectedly Ca(Bistris)2+ is the most stable among the alkaline earth ion complexes (log KCaCa(Bistris) = 2.25; the corresponding values for the Mg2+, Sr2+ and Ba2+ complexes are 0.34, 1.44 and 0.85, respectively). The ions of the 3d series follow the Irving-Williams sequence: log KMnMn(Bistris) = 0.70, for Cu2+, 5.27 and Zn2+ 2.38. Ternary complexes containing ATP4- as a second ligand were also investigated: the values for delta log KM (= log KM(ATP)M(ATP)(Bistris) -log KMM(Bistris) are in general negative (e.g. delta log KCa = -0.40 or delta log KCu = -1.65), thus indicating that the interaction of Bistris with M(ATP)2- is somewhat less pronounced tan with M2+. However, even in mixed-ligand systems, complex formation may still be considerable, hence great reservations should be exercised in employing Bistris as a buffer in systems containing metal ions. Moreover, in several cases delta log KM is relatively high [for Mg2+-ATP4- -Bistris even positive], indicating some cooperativity between the coordinated ligands, possibly hydrogen-bond formation. Distributions of the complexes in dependence on pH are given, and the structures of the binary M(Bistris)2+ and the ternary M(ATP) (Bistris)2- complexes are discussed. The participation of Bistris hydroxo groups in complex formation is evident.     

Effects of adenosine 5'-triphosphate, adenosine and acetylcholine in urinary bladder and colon muscles from streptozotocin diabetic rats

Bladder dysfunction and gastrointestinal disorders are common complications of diabetes mellitus and are attributed in part to peripheral neuropathy. Little is known of the mechanisms responsible for the bladder dysfunction and abnormality of the gastrointestinal tract. The effects of experimental diabetes on responses to adenosine, adenosine 5'-triphosphate (ATP) and acetylcholine (ACh) in rat bladder and colon were investigated 1 week and 8 weeks after i.v. injection of streptozotocin. Bladders from 8 week diabetic rats, but not from 1 week, were significantly larger than those from age matched controls. Relaxant and contractile responses of strips obtained from bladder body to these agonists were altered by diabetes. Relaxant responses to adenosine and ATP were also enhanced by diabetes. While maximum response and sensitivity to ATP relaxant effect were equivalent in 1 and 8 week diabetic rats as compared to age matched controls, both maximum responses to relaxant effect of adenosine and to contractile effect of ACh were reduced in colon preparations obtained from 8 week, but not from 1 week diabetic animals. The results suggest that changes in urinary bladder and colon smooth muscle function occur in diabetic rats and may contribute to the bladder dysfunction and colonic disorders seen in diabetes mellitus.     

Effect of extracellular adenosine 5'-triphosphate on principal neurons of the rat ventral tegmental area

Intracellular recordings were made in a midbrain slice preparation of the rat brain containing the ventral tegmental area (VTA). Dopaminergic principal cells were identified by their electrophysiological properties and their hyperpolarizing responses to dopamine. Superfusion with dopamine (100 microM) caused hyperpolarization and a decrease of the apparent input resistance. By contrast, two structural analogues of ATP, 2-methylthio ATP (2-MeSATP; 10 microM) and alpha,beta-methylene ATP (alpha, beta-meATP; 30 microM) had no effect, when added to the superfusion medium. Pressure applied dopamine also hyperpolarized the membrane, while both 2-MeSATP and alpha,beta-meATP were ineffective. Hence, dopaminergic principal neurons of the VTA do not possess somatic P2 purinoceptors present on peripheral and central noradrenergic neurons.     

Extracellular adenosine 5'-triphosphate induces Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchange mechanism

In the present study, we examined the effect of extracellular adenosine 5'-triphosphate (ATP) on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. ATP at 1 mM caused a release of 3.6+/-0.08% of the total cellular content. The 45Ca2+ efflux from the cells was also stimulated by adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma s), alpha, beta-methylene-ATP and adenosine 5'-diphosphate (ADP), but not by adenosine 5'-monophosphate (AMP) or adenosine. The effect of ATP was inhibited by a known purinergic P2-receptor antagonist, but not by a P1-receptor antagonist. From these results, it is conceivable that the effect of ATP on Ca2+ efflux from cardiomyocytes is mediated through P2-purinoceptors. It was also observed that ATP caused a rise in [Ca2+]i to almost 200 nM. The ATP-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. Moreover, ATP caused a 22Na+ influx into the cells of about 2.0-fold over the basal value. These result suggest that ATP stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane P2-purinoceptors which may couple to Na+/Ca2+ exchange.     

Improved radioisotopic assay for cytidine 5'-triphosphate synthetase (EC 6.3.4.2)

An improved radioassay for cytidine 5-triphosphate synthetase is reported which employs thin-layer chromatographic methods and provides a number of advantages over previously available techniques. (i) The method resolves the nucleotides and the degradation products generated during the time course of the enzymatic reaction by ascending chromatography employing polyethyleneimine cellulose plastic-backed sheets. (ii) Determinations of CTP formed and all nucleotide pairs generated during kinetic analysis of CTP synthetase are greatly simplified, further facilitating the detection of extraneous enzymatic activities. (iii) The sensitivity of the assay is enhanced and as little as 50 pmol of product formed was readily detected in supernatant fluids. This was made possible, in part, by the addition of NaF and phosphoenolpyruvate which together maintain the nucleotide triphosphates in the reaction mixture. (iv) A large number of samples can be handled at one time with highly reproducible results. The synthesis of CTP from UTP by enzyme preparations from rat liver, hepatomas, and Salmonella typhimurium LT2 was quantitated with this method.     

Effects of suramin on contractions of the guinea-pig vas deferens induced by analogues of adenosine 5'-triphosphate

1. Adenosine 5'-triphosphate (ATP) and some of its analogues contract the guinea-pig vas deferens, acting via receptors which have been classified as P2X-purinoceptors. We have recently shown, however, that the effects of ATP are enhanced, rather than inhibited, by the non-selective P2 antagonist, suramin, and that this enhancement could not easily be explained in terms of inhibition by suramin of the breakdown of ATP. We therefore investigated the effects of suramin on contractions induced by ATP analogues, to define the structure-activity relationships of the suramin-resistant response. 2. In the absence of suramin, the order of potency for ATP analogues was adenosine 5'-(alpha,beta-methylene)triphosphonate (AMPCPP) = P1,P5-diadenosine pentaphosphate (Ap5A) = adenosine 5'-tetraphosphate (Ap4) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) = adenylyl 5'-(beta,gamma-methylene) diphosphonate (AMPPCP) > P1,P5-diadenosine tetraphosphate (Ap4A) > adenosine 5'-O-(2- thiodiphosphate) (ADP beta S) > 2-methylthioadenosine 5'-triphosphate (MeSATP) > or = ATP > adenosine 5'-diphosphate (ADP). This is generally in agreement with previously reported structure-activity relationships in this tissue. 3. In the presence of suramin (1 mM), responses to Ap5A, Ap4A, AMPPCP, ADP beta S and ADP were abolished or greatly reduced, and contractions induced by AMPCPP, Ap4 and ATP gamma S were inhibited. Contractions induced by MeSATP however, like those induced by ATP itself, were not reduced, but at concentrations above 100 microM were enhanced. In the presence of suramin (1 mM) the order of potency of analogues was therefore AMPCPP = Ap4> ATP = MeSATP> ATP gamma S, with all other analogues tested being essentially inactive at concentrations up to 500 microM.4. Contractile responses of the vas deferens to transmural nerve stimulation (1-50 Hz) in the presence of the alpha-adrenoceptor antagonist, phentolamine (10 microM), were abolished by suramin (1 mM). This is in agreement with previous reports that suramin inhibits the excitatory junction potential, a response thought to be mediated by P2 purinoceptors. It is however hard to reconcile the evidence implicating ATP as the non-adrenergic transmitter responsible for this response with the failure of suramin to inhibit the contractions induced by ATP itself while abolishing nerve-mediated contractions.5. In conclusion, these results confirm our previous findings of a suramin-resistant component to the ATP-induced contraction in the guinea-pig vas deferens, and show that the structure-activity relationships of this response are not identical to those of any known P2-purinoceptor subclass. Although the inhibition by suramin of the breakdown of ATP may contribute to the suramin-resistance of some of the ATP analogues, it does not appear to provide the full explanation.     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Beta-adrenoceptor mediated facilitation of noradrenaline and adenosine 5'-triphosphate release from sympathetic nerves supplying the rat tail artery

1. The effects of prejunctional beta-adrenoceptor activation on electrically evoked noradrenaline (NA) and adenosine 5'-triphosphate (ATP) were studied by use of continuous amperometry and conventional intracellular recording techniques. Excitatory junction potentials (e.j.ps) were used as a measure of ATP release, and NA-induced slow depolarizations and oxidation currents as measures of NA release, from postganglionic sympathetic nerves innervating the rat tail artery in vitro. 2. Isoprenaline (0.1 microM) increased the amplitude of e.j.ps, slow depolarizations and oxidation currents evoked by short trains of stimuli at 1 to 4 Hz. The facilitatory effect of isoprenaline on e.j.ps and oxidation currents was most pronounced on responses evoked by the first stimulus in a train. 3. Isoprenaline (0.1 microM) did not detectably alter the amplitude-frequency distribution of spontaneous e.j.ps. 4. The facilitatory effect of isoprenaline on e.j.ps, slow depolarizations and oxidation currents was abolished by the beta-adrenoceptor antagonist, propranolol (0.1 microM). Propranolol alone had no effect on e.j.ps, slow depolarizations or oxidation currents. 5. Thus, activation of prejunctional beta-adrenoceptors increases the release of both NA and ATP from postganglionic sympathetic nerves. The findings are consistent with the hypothesis that NA and ATP are released from the same population of nerve terminals and presumably from the same vesicles.     

2',3'-O-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)--a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels

1. P2X receptor activation by alpha,beta-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2. The selective P2X1 and P2X3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 microM alpha,beta-meATP (an approximately EC90 concentration) with an IC50 of approximately 2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X1 receptors. 3. TNP-ATP inhibited alpha,beta-meATP induced contractions with an IC50 of approximately 30 microM and had non-specific effects on smooth muscle contraction. 4. The reduced potency of TNP-ATP in whole tissue experiments probably reflects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.     

Progression of cochlear and retinal degeneration in the tubby (rd5) mouse

Various molecules expressed on the surface of platelets have been shown to mediate the protective or deleterious role of these cells in immuno-inflammatory mechanisms. Increasing evidence points to the involvement of the cell adhesion molecules, gpIIb-IIIa, P-selectin, CD31, LFA-1, and CD36 in the interaction between platelets and endothelial cells as well as other cell types. The possible role of these molecules in the ability of platelets to support endothelium and to protect against tumour necrosis factor mediated cytolysis or parasitic invasion are reviewed. The involvement of platelets as effectors of tissue damage in cerebral malaria, lipopolysaccharide induced pathology, and pulmonary fibrosis is also discussed. This has then been extended to include the intercellular mechanisms underpinning their pathogenic role in metastasis, transplant rejection, stroke, brain hypoxia, and related conditions. A better understanding of the complex regulation and hierarchical organisation of these various platelet adhesion molecules may prove useful in the development of new approaches to the treatment of such diseases.     

Orf186 represents a new member of the Nudix hydrolases, active on adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH

orf186, a new member of the Nudix hydrolase family of genes, has been cloned and expressed, and the protein has been purified and identified as an enzyme highly specific for compounds of ADP. Its three major substrates are adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH, all implicated in a variety of cellular regulatory processes, supporting the notion that the function of the Nudix hydrolases is to monitor the concentrations of reactive nucleoside diphosphate derivatives and to help modulate their accumulation during cellular metabolism.     

Development of cochlear function in the neonate Mongolian gerbil (Meriones unguiculatus).

Studied auditory sensitivity and changes of selected structures of the external, middle, and inner ear in 31 Mongolian gerbil neonates. Data demonstrate an improvement in sensitivity to sound associated with postnatal changes in the morphology of the ear. Cochlear potentials and a concomitant reflex response to sound were observed 14 days after birth. At this state of postnatal development the organ of Corti appeared mature, the external auditory canal was open, but mesenchyme was present within the tympanic bulla. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Probing the nucleotide binding sites of axonemal dynein with the fluorescent nucleotide analogue 2'(3')-O-(-N-Methylanthraniloyl)-adenosine 5'-triphosphate

MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] was employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically with enhanced fluorescence (approximately 2.2-fold), homogeneous lifetime (8.4 ns), and high anisotropy (r approximately 0.38) to dynein and can be displaced by ATP and ADP added to the medium. The association constants of mantATP complexed with dynein were determined from anisotropy titration data. Using a multiple stepwise equilibrium model, the average values of the first two association constants are K1 = 2.7 x 10(5) M-1 and K2 = 1.8 x 10(4) M-1. This value of K1 is 7-8 times higher than that found previously for unsubstituted ATP, whereas K2 is little changed [Mocz and Gibbons (1996) Biochemistry 35, 9204-9211]. The lower-affinity binding sites, K3 and K4, observed previously could not be studied with mantATP within the available protein concentrations. The alpha and beta heavy chain subfractions have binding parameters similar to those of intact dynein. Formation of the stable ternary complex of mantATP with dynein and monomeric vanadate is accompanied by only a moderate increase in the binding affinities. Oligomeric vanadate reduces the binding affinities by approximately 50%. Addition of TritonX-100, methanol, or various salts changes the binding affinities by up to 50%, suggesting that the microenvironment of the nucleotide-binding sites involves significant contributions from both polar and apolar interactions. The distinct affinities of the individual binding sites are consistent with a physiological role in regulating nucleotide binding.     

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.     

Unique inhibitory effect of 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil 5'-triphosphate on Epstein-Barr virus and human DNA polymerases

1-(2'-Deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (L-FMAU) was shown to have potent antiviral activity against Epstein-Barr virus (EBV) without any cellular toxicity at concentrations up to 200 microM (Yao et al., Biochem Pharmacol 51: 941-947, 1996). The 5'-triphosphate of L-FMAU was not a substrate for EBV or cellular DNA polymerases, but could inhibit the elongation reaction, 3'-to-5' exonuclease activity, and nucleotide turnover catalyzed by EBV DNA polymerase. DNA synthesis catalyzed by human DNA polymerases was inhibited to a lesser extent. The inhibition pattern of EBV DNA polymerase by L-FMAU-5'-triphosphate (L-FMAU-TP) was consistent with an uncompetitive mechanism when dNTP or template-primer were used as the variable substrates. The Ki values were 38+/-10 microM for the elongation reaction, and about 50+/-10 microM for both nucleotide exchange and 3'-to-5' exonuclease reactions, values that were 10-20 times less than that for GMP. L-FMAU-TP is the first nucleoside 5'-triphosphate shown to have such unique behavior toward DNA polymerases. EBV DNA polymerase could be one of the targets for the inhibitory effect of L-FMAU-TP on EBV replication.     

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets

We investigated the effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) on intracellular Ca2+ concentration ([Ca2+]i) and cell length in isolated and field-stimulated rat cardiomyocytes. [Ca2+]i and cell length of field-stimulated cells were determined simultaneously by confocal laser scan microscopy by using the fluorescent Ca2+ dye Fluo-3. PAF (10(-12)-10(-8) M) inhibited systolic [Ca2+]i increase in a time- and concentration-dependent manner. Maximal effects were observed after an incubation time of 6-8 min, resulting in a 17% (10(-12) M), 41% (10(-10) M), and 52% (10(-8) M PAF) inhibition of systolic [Ca2+]i increase. A time- and concentration-dependent decrease in simultaneously measured cell shortening also was demonstrated. Cell shortening was inhibited by 10% (10(-12) M), 32% (10(-10) M), and 50% (10(-8) M) after an incubation time of 8 min. The effects of PAF could be antagonized by the PAF-receptor antagonist WEB 2170. These data demonstrate that PAF receptor-dependently induces a negative inotropic effect, which is correlated with a decrease in systolic [Ca2+]i and is most likely not due to a decrease in myofilament sensitivity.