ATP Alpha Research Articles

Analysis of P2-purinoceptor subtypes on the smooth muscle and endothelium of rabbit coronary artery.

We studied purinoceptor subtypes mediating constriction and dilatation in isolated rabbit coronary arteries with and without endothelium by comparing the effects of adenosine, ATP and the ATP analogues, alpha, beta-methylene ATP and 2-methylthio ATP. For contraction, the rank order of agonist potency was alpha, beta-methylene ATP > 2-methylthio ATP >> ATP; adenosine did not produce contraction. Removal of the endothelium did not affect these responses, but they were abolished by previous desensitisation with alpha, beta-methylene ATP. For relaxation, adenosine, ATP, and 2-methylthio ATP were equipotent, but alpha, beta-methylene ATP had no vasodilator effect and caused further contraction of the preparations. A transient contraction often preceded the relaxations to ATP at higher concentrations. The dilator responses to adenosine, ATP, and 2-methylthio ATP were significantly reduced but not abolished in preparations denuded of endothelium; previous desensitisation with alpha, beta-methylene ATP had no effect on the relaxant responses but abolished the initial transient contractions to ATP and 2-methylthio ATP. These results indicate the presence of vasodilator P1- and P2Y-purinoceptors on smooth muscle and, to a lesser extent, on endothelium and P2X-purinoceptors, mediating transient vasoconstriction on smooth muscle alone. The presence of smooth muscle P2Y-purinoceptors is unusual and has functional implications regarding the nature of the response of the coronary artery to ATP.

Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.

Mechanism of adenylate kinase. What can be learned from a mutant enzyme with minor perturbation in kinetic parameters?

The structural and functional roles of threonine-23 in the chicken muscle adenylate kinase (AK) were investigated by site-directed mutagenesis coupled with proton nuclear magnetic resonance (NMR) and phosphorus stereochemistry. The residue is potentially important because it is conserved among all types of AK and is part of the consensus P-loop sequence, 15GXPGXGKGT23. A mutant enzyme T23A (replacing threonine-23 with alanine) was constructed. Analyses of conformational stability and proton NMR indicate that the side chain of this residue contributes little to the structure of AK, which suggests that the side chain of Thr-23 does not play a structural role. The steady-state kinetic data of the mutant enzyme T23A showed no change in kcat and only 5-7-fold increases in Km and dissociation constants. Such minor changes in kinetic data are insufficient to suggest a functional role of Thr-23. However, two-dimensional NMR analyses of WT.MgAP5A and T23A.MgAP5A complexes indicated that the side chain of Thr-23 is in proximity to the adenine ring of the ATP moiety in the WT.MgAP5A complex in solution. In addition, T23A showed a significant perturbation in the stereospecificity toward the diastereomers of (Rp)- and (Sp)-adenosine 5'-(1-thiotriphosphate) (ATP alpha S), with the Rp/Sp ratio increased from < 0.02 in wild-type to 0.37 in T23A. Detailed 31P NMR analysis indicated that the stereospecificity at the AMP site was not perturbed. These results suggest that the side chain of Thr-23 is involved in catalysis, most likely via a hydrogen bonding interaction Thr-OH...O-P alpha(ATP).(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a receptor for ADP on blood platelets by photoaffinity labelling

The synthesis of a new analogue of ADP, 2-(p-azidophenyl)-ethythioadenosine 5'-diphosphate (AzPET-ADP), is described. This compound contains a photolabile phenylazide group attached to the ADP molecule by a thioether link at the purine 2 position. It has been prepared in radioactive form with 32P in the beta-phosphate at a specific radioactivity of 100 mCi/mumol. The reagent activated platelets, causing shape change and aggregation, with somewhat lower affinity than ADP. On photolysis the affinity was increased. The reagent also inhibited platelet adenylate cyclase stimulation by prostaglandin E1, with considerably higher affinity than ADP. On photolysis the affinity was decreased. AzPET-ADP competitively inhibited the binding of 2-methylthio[beta-32P]ADP, a ligand for the receptor by which ADP causes inhibition of adenylate cyclase. In the dark, AzPET-[beta-32P]ADP bound reversibly and with high affinity to a single population of sites similar in number to the sites that bind 2-methylthio[beta-32P]ADP. Binding was inhibited by ADP and by ATP and by p-chloromercuribenzenesulphonic acid (pCMBS). On exposure to u.v. light in the presence of platelets, AzPET-[beta-32P]ADP was incorporated covalently but non-specifically into several platelet proteins, although prominent intracellular proteins were not labelled. Specific labelling was confined to a single region of SDS/polyacrylamide gels, overlying but not comigrating with actin. Incorporation of radioactivity into this region was inhibited by ADP and by ATP as well as by ADP beta S, ATP alpha S and pCMBS, but not by adenosine, GDP or AMP. Inhibition of AzPET-[beta-32P]ADP incorporation was closely correlated with inhibition of equilibrium binding of 2-methylthio[beta-32P]ADP. These results suggests that the labelled protein, which migrates with an apparent molecular mass of 43 kDa in reduced gels, is the receptor through which ADP inhibits adenylate cyclase.

DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product.

Genetic studies have previously demonstrated that the Saccharomyces cerevisiae CDC9 gene product, which is functionally homologous to mammalian DNA ligase I, is required for DNA replication and is also involved in DNA repair and genetic recombination. In the present study we have purified the yeast enzyme. When measured under denaturing conditions, Cdc9 protein has a polypeptide molecular mass of 87 kDa. The native form of the enzyme is an 80-kDa asymmetric monomer. Both estimates are in good agreement with the M(r) = 84,406 predicted from the translated sequence of the CDC9 gene. Cdc9 DNA ligase acts via the same basic reaction mechanism employed by all known ATP-dependent DNA ligases. The catalytic functions reside in a 70-kDa C-terminal domain that is conserved in mammalian DNA ligase I and in Cdc17 DNA ligase from Schizosaccharomyces pombe. The ATP analog ATP alpha S inhibits the ligation reaction, although Cdc9 protein does form an enzyme-thioadenylate intermediate. Since Cdc9 DNA ligase exhibited the same substrate specificity as mammalian DNA ligase I, this enzyme can be considered to be the DNA ligase I of S. cerevisiae. There is genetic evidence suggesting that DNA ligase may be directly involved in error-prone DNA repair. We examined the ability of Cdc9 DNA ligase to join nicks with mismatches at the termini. Mismatches at the 5' termini of nicks had very little effect on ligation, whereas mismatches opposite a purine at 3' termini inhibited DNA ligation. The joining of DNA molecules with mismatched termini by DNA ligase may be responsible for the generation of mutations.

Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling.

ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.

Transcription in maize mitochondria: effects of tissue and mitochondrial genotype.

Mitochondrial run-on assays were used to determine transcriptional rates for nine B37(N) maize mitochondrial genes. Quantitation by radiographic imaging detected a 15-fold range in transcriptional rates; the order of apparent promoter strength was rps12 greater than rrn26 greater than atp6 greater than rrn18 greater than cox2 greater than atp alpha greater than atp9 greater than cox3 greater than cob. By probing single-stranded DNAs of both polarities with the run-on-products we showed that gene-specific antisense transcription did not occur. We also tested whether relative transcriptional rates were dependent on either the mitochondrial genotype or the tissue from which the mitochondria were isolated. Although tissue-specific differences in transcriptional rates were not detected, significant variation in apparent promoter strength for at least one gene, rps12, was dependent on the cytoplasmic genotype; rps12 had a five-fold reduced transcriptional rate in B37(T), the Texas male cytoplasmic strain of maize. Pulse-chase experiments suggested that differential transcript stability was not a major determinant of steady state mitochondrial RNA levels. These results indicate not only that promoter strength is an important component of the regulation of transcript levels in maize mitochondria, but also that the strength of a specific gene promoter can be dependent on the cytoplasmic genotype. Finally, the high transcriptional rate of both ribosomal RNA genes and the one mitochondrially encoded ribosomal protein gene studied suggests coordinate transcriptional regulation of both RNA and protein components of the mitochondrial ribosome.

Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing.

We have investigated the function of ATP hydrolysis in mammalian pre-mRNA in vitro splicing using adenosine phosphorothioates (ATP alpha S and ATP tau S) known to affect the activity of a number of ATP-requiring enzymes. Spliceosome assembly, but neither one of the two transesterification reactions involved in splicing, occurs with ATP alpha S suggesting that at least two types of ATP-requiring factors are brought into play. ATP alpha S has no effect in the presence of normal ATP and, therefore, spliceosomes assembled in the presence of ATP alpha S remain competent for splicing when supplied with normal ATP. ATP tau S noticeably and irreversibly inhibits the second transesterification reaction, i.e. at a time when most of the analog has been hydrolyzed and regenerated to normal ATP by creatine phosphate. This indicates that the inhibition results from an earlier event, most likely the thiophosphorylation of spliceosomal proteins. Under this assumption, the inhibition could be due to the failure of the thiophosphorylated proteins to be dephosphorylated. Indeed, okadaic acid, a potent inhibitor of protein phosphatases, inhibits the second step of a reaction in the presence of normal ATP. We propose that some splicing factors undergo phosphorylation-dephosphorylation cycles during spliceosome assembly and splicing, while others that could be the mammalian equivalents of the RNA helicase-like proteins recently discovered in yeast most likely bind and hydrolyze ATP.

Error in the calibration of the MgATP chemical-shift limit: effects on the determination of free magnesium by 31P NMR spectroscopy.

The measurement of free intracellular magnesium (Mg2+) using the 31P chemical shifts of ATP requires the use of appropriate calibration solutions to determine the chemical-shift limits delta ATP alpha beta and delta MgATP alpha beta. Solutions containing excess Mg2+ contain significant amounts of Mg2ATP and yield positive errors in the value of delta MgATP alpha beta. For physiological applications this may overestimate free intracellular Mg2+ by as much as 300%. This error may be minimized if appropriate mole ratios of Mg2+/ATP are used to calibrate delta MgATP alpha beta.

Low structural specificity for nucleoside triphosphates as antagonists of ADP-induced platelet activation.

Although the platelet ADP receptor is thought to exhibit a high degree of structural selectivity, adenosine 5'-O-(thiotriphosphate) (ATP alpha S) is a potent inhibitor of ADP-induced platelet activation and has been recently shown to bind with high affinity (Kd 3 +/- 0.1 nM) to formaldehyde-fixed platelets and to be photoincorporated into an 18-kDa fragment beginning at Tyr-198 of glycoprotein (GP) IIb alpha (Greco, N. J., Yamamoto, N., Jackson, B. W., Tandon, N. N., Moos, M., Jr., and Jamieson, G. A. (1991) J. Biol. Chem. 266, 13627-13633). Further studies have now shown that guanosine 5'-O-(thiotriphosphate) (GTP alpha S) also binds to high affinity sites (Kd 4.7 +/- 0.9 nM; 13,600 +/- 1,140 sites/platelet) and to low affinity sites (Kd 470 +/- 85 nM; 135,900 +/- 19,400 sites/platelet). Competition binding studies showed that all GTP alpha S binding sites were accessible to ADP and vice versa. The corresponding pyrimidine nucleotide cytidine 5'-O-(thiotriphosphate) (CTP alpha S) was found to be similarly effective in competing in the binding of ADP and both 5'-O-(thiotriphosphates) as well as uridine 5'-O-(thiotriphosphate) (UTP alpha S) were potent inhibitors of platelet shape change and aggregation. Ultraviolet irradiation of platelets in the presence of either [35S]GTP alpha S or [35S]UTP alpha S resulted in their specific incorporation into the alpha chain of GPIIb as previously shown with [35S]ATP alpha S. These results show that the structure of the nucleotide base has little influence on its ability to occupy the ADP-binding site on platelets, to function as an inhibitor of ADP-induced activation or to be photoincorporated into GPIIb alpha.

Characterization of the biological effects of 2‐methylthio‐ATP on rat hepatocytes: clear‐cut differences with ATP

1. In several tissues, 2-methylthio adenosine triphosphate (2MeSATP) is a very potent P2y-purine agonist. In rat hepatocytes, 2MeSATP half-maximally activated glycogen phosphorylase at 20 nM and was therefore about 25 times more effective than ATP (Ka 0.5-0.8 microM). This strong glycogenolytic potency of 2MeSATP suggests on its own the presence of P2Y-purinoceptors in liver. 2. Displacement of the radioligand ATP alpha[35S] from its receptor however occurred at much higher concentrations of 2MeSATP than was anticipated on the basis of its glycogenolytic potency. 3. The interaction of 2MeSATP with the receptor, characterized with ATP alpha[35S] as radioligand, cannot be considered as a pure competitive interaction. 4. 2MeSATP did not share the ability of ATP to counteract the effect of glucagon on the adenosine 3':5'-cyclic monophosphate levels. 5. 2MeSATP barely increased the levels of inositol trisphosphate (IP3). 6. The glycogenolytic effect of 2MeSATP was completely abolished by pretreatment of the hepatocytes with phorbol myristic acetate. 7. It is tentatively concluded that 2MeSATP and ATP are interacting with different P2 purinoceptors.

DNA ligases from rat liver. Purification and partial characterization of two molecular forms

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.

Covalent incorporation of 3'-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts.

ATP, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a photoaffinity analog of ATP, and several other ATP analogs induced an increase in plasma membrane permeability to monovalent ions and normally impermeant metabolites, including nucleotides, in transformed 3T6 mouse fibroblasts. The rank order of agonist potency for induction of nucleotide channels was BzATP (EC50 = 15 microM) greater than ATP (EC50 = 50 microM) approximately adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) greater than 2-methylthio-ATP (EC50 = 75 microM) approximately 3'-amino-3'-deoxy-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) (EC50 = 175 microM). Long wavelength UV illumination of 3T6 cells in the presence of greater than or equal to 20 microM BzATP at 4 degrees C, a nonpermeabilizing temperature, followed by removal of unbound BzATP, resulted in the efflux of 86Rb+ and the release of a prelabeled pool of cytoplasmic nucleotides when the temperature was shifted to 37 degrees C. Photoincorporation of BzATP was inhibited by ATP, ATP alpha S, ATP gamma S, and other ATP analogs that induced an increase in plasma membrane permeability to nucleotides in 3T6 cells under nonphotoactivating conditions. GTP, ITP, UTP, adenosine, and ATP analogs that did not alter plasma membrane permeability to nucleotides under nonphotoactivating conditions also had no effect on BzATP photoincorporation. Photoincorporation of BzATP occurred optimally between pH 6.6 and pH 8.2 but was inhibited at pH 6.0. Photoincorporation of BzATP was also modulated by the osmolarity and the divalent cation concentration of the assay medium. The increase in plasma membrane permeability to nucleotides induced by photoincorporated BzATP occurred at the same rate and had the same temperature, pH, ionic strength, and divalent cation requirements as the increase in plasma membrane permeability to nucleotides induced by ATP and BzATP under nonphotoactivating conditions. These findings support the hypothesis that BzATP can be covalently incorporated into a P2 purinoceptor in 3T6 cells that is coupled to plasma membrane channels for ions and other metabolites.

Metal ion dependence of phosphorothioate ATP analogs in the Bacillus stearothermophilus tyrosyl-tRNA synthetase

Pre-steady-state kinetic analyses on the formation of tyrosyl adenylate from tyrosine and each of the four diastereomers of alpha- and beta-phosphorothioate adenosine triphosphates [ATP alpha S and ATP beta S; Eckstein, F., & Goody, R. (1976) Biochemistry 15, 1685-1691; Yee, D., Armstrong, V. W., & Eckstein, F. (1979) Biochemistry 18, 4116-4123] were performed in the presence of Mg2+, Co2+, and Cd2+ as the divalent metal ion cofactor. A modest preference of 5.5-fold in kappa 3/KA' (where kappa 3 is the rate constant for tyrosyl adenylate formation and KA' is the dissociation constant for ATP, or phosphorothioate ATP, from the E.Tyr.metal.ATP complex) for the Sp ATP alpha S diastereomer and the absence of an inversion of preference when the metal ion is changed suggest that there is a stereospecific enzyme-alpha-phosphate interaction and that there is no direct metal ion interaction with the alpha-phosphate. The extent of reaction of the ATP alpha S diastereomers (30-50%) implies that these analogues are more susceptible to the hydrolytic site reaction previously reported for this enzyme [Wells, T. N. C., & Fersht, A. R. (1986) Biochemistry 25, 1881-1886]. The strong preference in kappa 3/KA' for the RP ATP beta S diastereomer (16-fold for Mg2+ and 50-fold for Co2+) is indicative of a stereospecific interaction with the pro SP beta oxygen of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Intralenticular Water Interactions with Phosphates in the Intact Crystalline Lens

Since the ATP molecule is associated with the intracellular milieu, which consists mainly of water, this study suggests an intimate intracellular association with water molecules. Intact canine crystalline lenses (n = 6) were incubated at 37 degrees C in a buffer containing a pure D2O solvent and examined by [31P]-nuclear magnetic resonance (NMR) spectroscopy. Control lenses (n = 6) from contralateral eyes were incubated in a pure H2O-based buffer. During the first 4 h of a 24-hour incubation, the D2O-incubated lens maintained its phosphorus metabolic profile, indicating that the tissue maintains its metabolic health. During this period, however, the spectral signal widths of ATP narrowed progressively. Spectral-simulation analysis of ATP alpha-, beta- and gamma-phosphate signals revealed that the gamma-phosphate signal was narrowed significantly in contrast to the alpha-phosphate signal. Such differential signal narrowing within a single NMR spectrum indicates that the principal intracellular site of water hydrogen binding to the ATP molecule is at the gamma-group phosphate.

In vivo T1 values of phosphorus metabolites in human liver and muscle determined at 1.5 T by chemical shift imaging.

A procedure for obtaining T1 values for phosphorous metabolites in localized regions of human subjects, using a standard 1.5 T MR imager, is described. 31P spectra and T1 values localized to the liver, and to abdominal and calf muscle of healthy volunteers were obtained by means of a multi-slice spectroscopy technique, consisting of a chemical shift imaging (CSI) sequence with a B1-insensitive excitation and one dimension of phase encoding, used in conjunction with a surface coil. An examination consisting of proton imaging, shimming and collection of 31P progressive saturation spectroscopic data for T1 determination required 1 h to perform. Shimming on the signal from the body region detected by the surface coil gave spectra of excellent spectral resolution. Quantification of all peaks in the localized 31P spectra was carried out with the PIQABLE algorithm, and T1 values were calculated for inorganic phosphate (Pi), the phosphodiester region, and the ATP alpha-, beta- and gamma peaks of liver, and for calf muscle Pi, phosphocreatine (PCr), and the three ATP peaks. The precisions of the measurement and of the entire process for obtaining and quantifying localized spectra by one-dimensional CSI were determined, and the accuracy of T1 values obtained by this means was verified. The temporal variation in T1 values obtained in a series of examinations of a single normal subject was also assessed. The consistency of the T1 values obtained in this study with in vivo T1 values obtained by other techniques is a stringent test of accuracy of localized spectra obtained with CSI.

A Permissive Role of Pertussis Toxin Substrate G-protein in P2-purinergic Stimulation of Phosphoinositide Turnover and Arachidonate Release in FRTL-5 Thyroid Cells: Cooperative mechanism of signal transduction systems

Abstract Extracellular ATP and other purinergic agonists were found to inhibit cAMP accumulation by depressing adenylate cyclase as an and/or to stimulate arachidonate release in association with phospholipase C or A2 activation and Ca2+ mobilization as in FRTL-5 cells. The stimulatory actions of a group of P2-agonists represented by ATP were partially inhibited by the pretreatment of the cells with islet-activating protein (IAP), pertussis toxin, even when an about 41-kDa membrane protein(s) was completely ADP-ribosylated. Only the IAP-sensitive part of the stimulatory actions was antagonized by 1,3-diethyl-8-phenylxanthine (DPX), an adenosine antagonist. GTP and 8-bromoadenosine 5'-triphosphate (Br-ATP) at two to three orders of higher concentrations than ATP also exerted the stimulatory actions, although they were entirely insensitive to both IAP and DPX. Ligand binding experiments with, [35S]ATP gamma S and [3H]DPX showed that ATP occupies both DPX-sensitive and insensitive receptor sites, whereas GTP does only ATP-displaceable DPX-insensitive sites. Thus, lack of sensitivity of GTP action to DPX was associated with its inability to occupy the DPX-sensitive sites. Adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) and P1-agonists such as AMP and N6-(L-2-phenylisopropyl-adenosine (PIA) did not show any stimulatory action. Nevertheless, the agonists remarkably enhanced the stimulatory actions of GTP or Br-ATP. Such permissive actions of PIA and others were sensitive to both IAP and DPX, as were shown for a part of the stimulatory actions of ATP as well as the of both PIA and ATP. We conclude that an IAP substrate G-protein(s) which mediates the inhibitory action of purinergic agonists via a DPX-sensitive purinergic receptor(s) may not directly link to the phospholipase C or A2 system but enhance the system which links to a DPX-insensitive P2-receptor, in an indirect or permissive manner.

Potential role of mitochondrial calcium metabolism during reperfusion injury.

Ischemia-reperfusion injury has been associated with intracellular H2O2 and superoxide radical production from accumulated hypoxanthine (HX) and xanthine oxidase (XO). The effect of H2O2 and superoxide radical on mitochondrial Ca2+ efflux was characterized in isolated renal mitochondria using a HX-XO system. Mitochondria were suspended in buffered medium containing 200 microM HX. Extramitochondrial Ca2+ was monitored kinetically at 660-685 nm using the Ca2+ indicator arsenazo III. After preloading mitochondria with 18-25 nmol Ca2+/mg protein, addition of XO to the medium caused a rapid oxidation of mitochondrial NAD(P)H followed by Ca2+ release. Ca2+ efflux was attributed to mitochondrial metabolism of H2O2 because efflux could be prevented with catalase but not superoxide dismutase. The Ca2+ efflux rate (r = 0.995) and lag time to Ca2+ efflux (r = 0.987) both correlate well with the NAD(P)H oxidation rate. Exogenous ATP prevents Ca2+ efflux in a dose-dependent fashion (Km = 35 microM ATP) without affecting NAD(P)H oxidation; ATP plus oligomycin, however, had no effect. The protective effect of ATP on Ca2+ efflux was diminished by ruthenium red (RR). XO-induced Ca2+ efflux increased state 4 respiration 148% via a futile Ca2+ cycle involving the Ca2+ uniport. The increase in state 4 respiration could be reversed with RR (alpha less than 0.001) or ATP (alpha less than 0.01); ATP plus oligomycin, however, had no effect. The results are discussed in relation to the oxygen free radical theory of reperfusion injury.

Comparison between sympathetic adrenergic and purinergic transmission in the dog mesenteric artery.

1. Electrical transmural stimulation evoked a sympathetic contraction in the isolated dog mesenteric artery. This contraction consisted of adrenergic and purinergic components, which were separately observed under conditions where postjunctional P2 purinoceptors were desensitized with alpha, beta-methylene ATP or postjunctional alpha 1 adrenoceptors were blocked by prazosin, respectively. 2. The purinergic component was transient and developed immediately after the start of stimulation and declined rapidly after a peak. In contrast, the adrenergic contraction slowly developed and lasted longer than the purinergic component. Thus, the purinergic and adrenergic components predominantly contributed to the early and later phases of the total sympathetic response, respectively. 3. The peak amplitudes of contraction of both components were similar at 1 Hz, but the adrenergic component occurred more dominantly in the responses to higher frequency stimulation. 4. Cocaine, a neuronal uptake inhibitor of noradrenaline, potentiated the adrenergic component but attenuated the purinergic component. This attenuation was reversed by DG-5128, a prejunctional alpha 2 adrenoceptor antagonist. Treatment with DG-5128 alone augmented both adrenergic and purinergic components. 5. 8-Phenyltheophylline, a P1 purinoceptor antagonist, potentiated the purinergic component without affecting the adrenergic component. 6. Exogenous noradrenaline produced a sustained contraction, which was potentiated by cocaine and was competitively inhibited by prazosin. alpha, beta-Methylene ATP and 8-phenyltheophylline had no effect on the response to noradrenaline. Exogenous ATP produced a transient contraction, which was abolished under conditions where postjunctional P2 purinoceptors were desensitized with alpha, beta-methylene ATP. 8-Phenyltheophylline potentiated but cocaine or prazosin did not affect the ATP response. 7. Electrical stimulation produced an increase in 3H efflux from the sympathetic nerve terminals in the mesenteric arteries pre-incubated with [3H]noradrenaline. The evoked efflux was significantly augmented by cocaine or DG-5128 and was inhibited by guanethidine or tetrodotoxin. alpha, beta-Methylene ATP and 8-phenyltheophylline were without effect. Adenosine reduced the 3H efflux and the inhibition was suppressed by 8-phenyltheophylline. 8. These results suggest that sympathetic contraction of the dog mesenteric artery is caused through not only adrenergic but also purinergic mechanisms, and that both the sympathetic transmissions are predominantly modulated through prejunctional adrenergic mechanisms.

Epinephrine Induces Platelet Fibrinogen Receptor Expression, Fibrinogen Binding, and Aggregation in Whole Blood in the Absence of Other Excitatory Agonists

The exposure of fibrinogen receptors is an early event in agonist-induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC-PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.