Synthesis of a spin-labeled photoaffinity ATP analogue,and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers

A spin-labeled photoaffinity ATP analogue 3(2)-O-{4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benzoyl}benzoyl adenosine 5-triphosphate (SL-Bz₂ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3(2)-O-(4-benzoyl)benzoyl adenosine 5-triphosphate (Bz₂ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [-³²P]SL-Bz₂ATP was hydrolyzed and trapped as the diphosphate analogue with Co²⁺ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz₂ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.     

A volume-activated anion conductance in insulin-secreting cells

The whole-cell patch-clamp recording technique was used to measure volume-activated currents in K⁺-free solutions in RINm5F and HIT-T15 insulinoma cells and in dispersed rat islet cells. Cell swelling, induced by intracellular hypertonicity or extracellular hypotonicity, caused activation of an outwardly rectifying conductance which could be subsequently inactivated by hypertonic extracellular solutions. The conductance required adenosine 5-triphosphate (ATP) in the pipette solution but was Ca²⁺ independent. Na⁺ and Cl^– substitution studies suggested that the swelling-activated current is Cl^– selective with a halide permeability sequence of Br > Cl > 1. The conductance was reversibly inhibited by the anion channel inhibitors 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Further evidence for a volume-activated anion conductance was provided by studies of volume regulation in insulin-secreting cells. When RINm5F cells were exposed to a hypotonic medium, the initial cell swelling was followed by a regulatory volume decrease (RVD). This RVD response was also inhibited by DIDS and by NPPB. These data therefore provide evidence for a volume-activated anion conductance in insulin-secreting cells which could be involved in the RVD following osmotic stress. A possible role for the conductance in hypotonically induced insulin release is also discussed.     

Thyroid hormone action on intermediary metabolism

Summary The effect of thyroid hormones on mitochondrial respiration are summarized: T3 directly stimulates mitochondrial respiration and the synthesis of adenosine 5-triphosphate (ATP). Cytosolic ATP availability is increased by a thyroid hormone-induced increase in adenine nucleotide translocation across the mitochondrial membrane; the steady state ATP concentration and the cytosolic ATP/adenosine 5-diphosphate (ADP) ratio is even decreased in hyperthyroid tissues because of the simultaneous stimulation of the synthesis and consumption of ATP. With regard to the thyroid hormone-induced energy wasting processes, heart work, intra- and interorgan futile cycling and Na⁺/K⁺-ATPase are involved to varying degrees. As a consequence of the thyroid hormone-induced hydrolysis of ATP, thermogenesis is increased in hyper- and decreased in hypothyroidism. Despite an increased rate of glucose utilization, clinical and experimental hyperthyroidism is often characterized by an abnormal oral glucose tolerance test. This finding is due to the thyroid hormone-induced increase in intestinal glucose absorption as well as the still enhanced endogenous glucose production in the liver. Hypothyroid patients show a reduced glucose tolerance test because of a decrease in intestinal glucose absorption and a sometimes reduced glucose turnover. The thyroid hormone-induced alterations in glucose metabolism are most probably not due to alterations in serum insulin levels and/or to a peripheral insulin resistance at the receptor level.Thyroid hormones are defined as 3,5,3,5-tetraiodothyronine (thyroxine-T4) and its deiodinated or non-deiodinated metabolic products providing thyroimimetic activity, mainly 3,5,3-triiodothyronine (T3) [12]     

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19±3 pmol) and adenine nucleotides (101±7 pmol). Experiments performed in the presence of tubocurarine (5 M) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5-nucleotidase.Brief accounts of some of the results in this study have been published previously (refs. [6, 7]).     

Pharmacological characterization of P2X7 receptors in rat peritoneal cells

Objective and design: P2X₇ receptor activation by ATP results in the release of IL-1 and IL-18. Prolonged stimulation can lead to pore formation and cell death. In this study we pharmacologically characterized P2X₇ receptors on rat peritoneal cells (RPC) and on 1321N1 cells transfected with rat P2X₇ receptor (1321rP2X₇-11).Materials and methods: RPC were isolated from rats by lavage. P2X₇ agonist induced pore formation in RPC was measured by EtBr uptake. P2X₇-stimulated pore formation and Ca⁺⁺ influx in 1321rP2X₇-11 cells were measured by a fluorometric imaging plate reader. The effects of pyridoxal phosphate-6-azo phenyl –2-4-disulfonic acid (PPADS) on pore formation and Ca⁺⁺ influx were examined in both RPC and 1321rP2X₇-11. P2X₇-mediated IL-1 release in RPC and the effect of PPADS were determined.Results: RPC express functional P2X₇ receptors that were activated by ATP analogs with a rank order of potency of 2- 3-O-(4-Benzoylbenzoyl) adenosine 5-triphosphate (BzATP) > ATP > ,-methylene ATP. Activation of P2X₇ receptors by BzATP was inhibited by PPADS. Similar results were also obtained in 1321rP2X₇-11 cells. Activation of P2X₇ receptors on RPC resulted in IL-1 secretion, which was inhibited by PPADS.Conclusions: RPC express functional P2X₇ receptors that form pores and mediate the release of IL-1.Received 20 July 2004; returned for revision 9 September 2004; accepted by N. Boughton-Smith 5 November 2004     

Structure of the cloned Locusta migratoria mitochondrial genome: restriction mapping and sequence of its ND-1 (URF-1) gene

Summary We have cloned the entire mitochondrial genome of Locusta migratoria in four fragments and characterised by restriction mapping. In addition, we have sequenced a 1,095 kb region containing the ND-1 (URF-1) gene. The inferred primary structure of the protein is highly homologous to its Drosophila counterpart (68%). The gene is flanked at the 5 end by the tRNA _CUN ^leu gene, interrupted by the sequence TTG. The 3 end is flanked by the tRNA _ser ^UCN gene, followed by a sequence homologous to the 3 end of D. yakuba cytochrome b. The relative position of the genes is conserved between Locusta and Drosophila, thus indicating conservation of mitochondrial gene order in insects.     

ATP and G proteins affect the runup of the Ca2+ current in bovine chromaffin cells

The Ca²⁺ current recorded by the whole-cell technique in chromaffin cells shows, before the often described rundown, a transient facilitation or runup. Initial current amplitude was 570±165 pA and then it increased by 49±23% (n=19, SD) over 2±1 min in the absence of adenosine 5-triphosphate (ATP). In the presence of ATP, this process occurred with the same magnitude but it was slowed in a dose-dependent manner, lasting 17±2 min with 2 mM ATP (n=8). Since adenosine 5-diphosphate (ADP) does not reproduce this ATP effect, a complex series of phosphorylations is likely to intervene and we show that, at least, a cAMP-dependent i.e., cyclic adenosine monophosphate) phosphorylation occurs. Pertussis toxin (PTX) pretreatment yielded an already maximal Ca²⁺ current (around 1000 pA) at the time of the patch rupture, which only slightly increased thereafter (10%, n=11). Also, guanosine 5-diphosphate (GDP) and guanosine 5-O-(2-thiodiphosphate) (GDP[s]), induced a fast runup, which was absent in the presence of GTP. Furthermore, we show that facilitation does not occur in the presence of dihydrophyridine (DHP) antagonists. Globally, our data suggest that an ATP-dependent phosphorylation stabilizes the inhibitory control exerted by a PTX-sensitive G protein and, as a result, slows down the facilitation of L-type Ca²⁺ channels. The recruitment of L-type channels can also be facilitated by the application of a DHP agonist or a depolarizing prepulse protocol. We show that these processes are only effective over a period which parallels the runup and are not additive to it. This suggests that a single process may underlie these various types of facilitation.     

Effects of uridine triphosphate on contractility,cyclic nucleotide levels and membrane potential in the isolated frog ventricle

A study has been made of the effects of uridine 5-triphosphate (UTP) on the isolated frog ventricle. Preparations were superfused with solutions containing different concentrations of UTP, and changes in contractility, cyclic nucleotide levels and membrane potential were measured. UTP produced a long lasting increase in isometric twitch tension, which was unaffected by adrenergic receptor antagonists (propranolol and phentolamine). The levels of adenosine 3,5-cyclic monophosphate (cyclic AMP) and guanosine 3,5-cyclic monophosphate (cyclic GMP) were measured at different times during exposure of the ventricle to 10^–4 M UTP. The increase in the force of contraction was found to be accompanied by a rise in intracellular cyclic AMP. Cyclic GMP levels were seen to fall initially, but then to increase later, as both twitch and cyclic AMP started to decline. UTP also produced marked changes in the shape of the action potential; its duration and positive overshoot were both increased. The effects of UTP on twitch tension, cyclic nucleotide levels and action potential parameters were all dose-related. The change in contractility was found to be linearly related to (a) the ratio of the amount of cyclic AMP: cyclic GMP present in the fibres, and (b) to the increase in duration of the action potential. These results suggest that cyclic AMP, cyclic GMP and the availability of Ca²⁺ may all be involved in mediating the response to UTP.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Calcium ion uptake induced by cholinergic and α-adrenergic stimulation in isolated cells of rat salivary glands

Summary Adrenaline (10^–5 M) and carbamylcholine (10^–4 M) stimulate⁴⁵Ca²⁺ uptake into isolated cells of rat submandibular gland and parotid glands. In the presence of the -adrenoreceptor blocking agent phentolamine, adrenaline stimulation of⁴⁵Ca²⁺ uptake is abolished. The -adrenergic stimulant isoproterenol has no effect on⁴⁵Ca²⁺ uptake. Carbamylcholine induced⁴⁵Ca²⁺ uptake is inhibited by atropine. The Ca²⁺ ionophore A23187 stimulates⁴⁵Ca²⁺ uptake, whereas dibutyryl cyclic adenosine 3,5-monophosphate and dibutyryl cyclic guanosine 3,5-monophosphate have no effect on⁴⁵Ca²⁺ uptake.A graphical analysis of the⁴⁵Ca²⁺ uptake curves reveals at least two phases: a fast phase and a slow phase, both of which are stimulated by adrenaline and carbamylcholine.The⁴⁵Ca-exchangeable pool size is increased by adrenaline and carbamylcholine in both the fast and the slow phases.These results suggest that -adrenergic and cholinergic agonists act by increasing the rate of Ca²⁺ transfer into the cells of the parotid and submandibular salivary glands most probably through an increase of the cell membrane permeability for Ca²⁺.Supported by Swiss National Science Foundation Grant No. 3.298.074     

Isolation and preliminary characterization of 9-β-d-arabinofuranosyladenine-resistant mutants of baby hamster cells

A large number of 9--d-arabinofuranosyladenine (araA) -resistant mutants of baby hamster kidney cells (BHK 21/Cl3) were isolated. These mutants can be grouped into three mechanistically distinct classes. All the mutants showed cross-resistance to deoxyadenosine (dAdo). The mechanism of resistance to araA and dAdo in the class I mutants can be attributed to a mutation to adenosine kinase (AK) deficiency. The class II mutants have normal levels of AK, adenosine deaminase, and deoxyadenosine kinase. These mutants also show resistance to 1--d-arabinofuranosylcytosine (araC), and the mechanism of resistance is probably due to a mutation in the ribonucleotide reductase gene producing an enzyme that has an increased resistance to the inhibition by 9--d-arabinofuranosyladenine 5-triphosphate (araATP) and 2-deoxyadenosine 5-triphosphate (dATP). The class III mutants, unlike those of classes I and II, show extreme adenosine (Ado) sensitivity. The Ado^s/araA^r/dAdo^r phenotypic properties can be attributed to a single mutation. Classes II and III are novel araA-resistant mutants.     

Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces

Summary Trehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.Abbreviations PMSF phenyl-methyl-sulfonyl fluoride - G-6-P glucose-6-phosphate - UDPG uridine-5-diphosphoglucose - PEP phosphoenol pyruvate - NAD⁺ -nicotinamine adenine dinueleotide - ATP adenonise 5-triphosphate - cAMP adenosine 2:3-cyclic monophosphate - MOPS 3 (N-morpholino) propanesulfonic acid     

Some characteristics of the ATP-induced histamine release from and permeabilization of rat mast cells

Extracellular adenosine 5-triphosphate (ATP) induced a characteristic, dose-dependent release of histamine and prostaglandin D₂ (PGD₂) from rat peritoneal mast cells. The process was relatively slow, non-cytotoxic, maximal at physiological pH and dependent on external calcium. Strontium and barium ions were able to substitute for calcium, although higher concentrations were required for maximal release. Cells stimulated in the absence of calcium progressively lost the ability to respond to subsequent reintroduction of the cation. The secretion of histamine induced by ATP was largely unaffected by the anti-asthmatic drugs disodium cromoglycate and nedocromil sodium but was inhibited by structurally related flavonoids and by cAMP-active drugs. Importantly, the non-hydrolysable guanosine 5-triphosphate (GTP) analogue, GTP--S, elicited a dose-dependent release of histamine when introduced into mast cells permeabilized with ATP in the absence of external calcium. ATP thus appears to be a useful cell permeabilizing tool with which to study the biochemical processes involved in mast cell activation.     

The Control of cell proliferation by preformed purines: A genetic study I. Isolation and preliminary characterization of Chinese hamster lines with single or multiple defects in purine “salvage” pathways

Sublines with single or multiple defects in purine salvage enzymes were isolated from the Chinese hamster fibroblastic line GMA32 through single or successive onestep selections for resistance to purine analogs. They were examined for their ability to incorporate purine bases and nucleosides into macromolecules, for their sensitivity to growth inhibitory purines, and for their rescue by exogenous purines from deprivation imposed by metabolic inhibitors of endogenous synthesis. The results show that a deficiency of either adenosine kinase (EC 2.7.1.20), adenine phosphoribosyltransferase (EC 2.4.2.7), or hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) abolishes the ability of adenine to cause cell death by interfering with pyrimidine synthesis;on the other hand, the pyrimidine starvation caused by adenosine is fully prevented only by a deficiency of adenosine kinase.Abbreviations WT wild-type line - AK adenosine kinase - APRT adenine phosphoribosyltransferase - HGPRT hypoxanthine guanine phosphoribosyltransferase - AD adenosine deaminase - A adenine - rA adenosine - I inosine - Hx hypoxanthine - dA 2-deoxyadenosine - dT 2-deoxythymidine - rU uridine - IMP inosine 5-monophosphate - AMP adenosine 5-monophosphate - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - PRPP phosphoribosylpyrophosphate - Amp aminopterin - TCA trichloracetic acid - ARA-A 9--darabinofuranosyladenine - Amp + dT medium normal (ERH) medium supplemented with Amp and dT     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Histamine release from the cultured mouse mast cell line PT 18 in response to immunologic and non-immunologic stimuli

Cultured mouse mast cells that are dependent on spleen-derived factors for their proliferation and maintenance have been shown rapidly to release histamine in response to immunologic stimuli, concanavalin A, and ionophores A23187 and Br-x537A. These cells did not, however, release histamine when treated with compound 48/80 or adenosine 5-triphosphate. Immunologically mediated histamine release was inhibited by theophylline and quercetin, but not by disodium cromoglycate. The results presented here are discussed in terms of the possible relationship of this cell line to mucosal mast cells.To whom correspondence should be addressed.     

Suppression of human polymorphonuclear leukocyte phagocytosis by adenosine analogs

The effects of adenosine analogs on human polymorphonuclear leukocyte (PMN) phagocytosis to latex beads or sheep red blood cells were investigated in an in vitro experiment. The purine compounds such asN ⁶-phenylisopropyladenosine (PIA), 5-N-ethylcarboxamido-adenosine (NECA), an A₂ adenosine receptor agonist, and adenosine as high as 2 mM had no effect on PMN phagocytosis. In contrast, 2,5-dideoxyadenosine (DDA), a P-site adenosine receptor agonist, and 5-methylthioadenosine (MTA), a naturally occuring purine nucleoside, caused profound inhibition of phagocytic function of PMNs in a dose-dependent manner. Dipyridamole, which blocks purine nucleoside uptake, reversed the suppression due to MTA. Theophylline, an R-site receptor antagonist did not prevent the effect of MTA. These results suggested that phagocytosis of PMNs is suppressed by stimulation of the P-site adenosine receptors.     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

Adenine nucleotide degradation in the thoroughbred horse with increasing exercise duration

Summary Adenine nucleotide (AN) degradation has been shown to occur during intense exercise in the horse and in man, at or close to the point of fatigue. The aim of the study was to compare the concentrations of muscle inosine 5-monophosphate (IMP) and plasma ammonia (NH₃) during intense exercise with the concentrations of muscle and blood lactate. Seven trained thoroughbred horses were used in the study. Each exercised on a treadmill for periods of between 30 s and 150 s, at 11 and/or 12 m · s^–1. Blood and muscle samples were taken and analysed for lactate and NH₃ and adenosine 5-triphosphate (ATP), phosphorylcreatine (PCr), IMP, creatine, lactate and glycerol-3-phosphate respectively. Horses showed varying degrees of AN degradation as indicated by plasma [NH₃] and muscle [ATP] and [IMP]. Comparisons of [IMP] with muscle [lactate], and plasma [NH₃] with that of blood [lactate] indicated a threshold to the start of AN degradation. This threshold corresponded to a lactate content of around 80 mmol · kg^–1 dry muscle and 15 mmol · l^–1 in blood. We discuss the mechanisms which have been proposed to account for AN degradation and suggest that IMP formation occurs as a result of a sudden rise in the concentration of adenosine 5-diphosphate (ADP) and consequently the concentration of adenosine 5-monophosphate. The data suggest a critical pH below which there may be a substantial reduction in the kinetics of ADP rephosphorylation provided by PCr resulting in an increase in [ADP], which is the stimulus to AN degradation during intense exercise.     

Sensitivity to cyclic nucleotides and to Aspirin® of the kininogen-consuming system activated by adrenaline or carbamylcholine in rat blood

The activation by carbamylcholine of a kinin-forming system in rat blood in vivo or in vitro has been compared with that caused by adrenaline. After showing that free kinins were actually released into the circulation of the carbamylcholine-treated rat, experimentally induced changes in carbamylcholine or adrenaline activity were followed by measuring alterations in kinin precursor (kininogen) levels in plasma. Carbamylcholine was effective at 1.6–4.8 g/kg in the intact animal and optimal at 10^–8 M in oxalated rat blood in vitro. Aspirin^®, 10 mg/kg or 5×10^–4 M, inhibited the action of carbamylcholine and of adrenaline on kininogen. Salicylate or Indomethacin at similar levels were ineffective. N⁶-,2-O-dibutyryl-cyclic-3,5 adenosine monophosphate (diBu-cAMP) 2×10^–5 M, suppressed kininogen consumption in vitro; this inhibition could be overcome by 8-bromo-3–5-cyclic guanosine monophosphate (8-Br-cGMP), 10^–4 M. This compound potentiated the effect of adrenaline in the absence of diBu-cAMP. The inhibitory effect of Aspirin^® was decreased by 8-Br-cGMP. The action of autonomic effector drugs on kininogen consumption may be mediated by changes in the cAMP/cGMP ratio in circulatory target cells. The action of Aspirin^® may affect this system since it was partially overcome by 8-Br-cGMP. Since it was not reproduced by salicylate but could be mimicked by acetic anhydride, it most probably involves the transfer of the acetyl group of the drug to an as yet unknown acceptor cell protein. This moiety does not seem to be involved in prostaglandin-synthesis since Indomethacin, a very effective inhibitor of this process, failed to inhibit the action of either adrenaline or carbamylcholine on rat blood kininogen.