Inhibitory Action Of ATP Research Articles

ATP Reduces the Entry of Calcium Ions into the Nerve Ending by Blocking L-type Calcium Channels

At neuromuscular junctions, ATP inhibits both the evoked and spontaneous acetylcholine release and inward calcium current operating via presynaptic P2Y receptors. It was shown in the experiments with the frog neuromuscular synapse using specific calcium-sensitive dye Oregon Green Bapta 1 that exogenous ATP reduces the amplitude of calcium transient, which reflects the changes in the entry of calcium ions in response to the nerve pulse. The depressing effect of ATP on the transient was prevented by suramin, the blocker of P2 receptors. Nitrendipine, a specific blocker of L-type calcium channels, per se decreased the calcium transient amplitude and significantly attenuated the effect of ATP on the calcium signal. Contrariwise, the preliminary application of ATP to the neuromuscular junction completely eliminated the depressing effect of nitrendipine on the calcium response. The obtained data suggest that an essential component in the inhibitory action of ATP on the calcium transient amplitude is provided by reduction of the entry of calcium ions into a frog nerve ending via L-type voltage-gated calcium channels.

Effects of µ-Opioid Agonist on ATP-sensitive Potassium Channel Activity in Isolated Ventricular Cardiomyocytes

It is well known that opioid agonists are released from the myocardium during hypoxia or ischemia, and that ATP-sensitive potassium channels (KATP channels) exist in the cardiac cell membrane and function as a cardioprotector preventing the myocardium from ischemic damage. In the present study, therefore, to determine whether opioid agonists are involved in the regulation of ATP-sensitive potassium channel activities, effects of the μ-opioid agonist DAMGO were examined on KATP channel activities by using excised inside-out and cell-attached patch clamp techniques in enzymatically (collagenase and protease) isolated mouse ventricular cardiac myocytes. In the excised inside-out patches, DAMGO (1∼300 μmol/L) inhibited KATP channel activities in a dose-dependent manner. KATP channel activity, which had been attenuated by the addition of ATP (100 μmol/L) to the internal solution, was not reactivated by DAMGO. The fashion of the single-channel inhibition by DAMGO was that both channel opening frequency and mean open-time were decreased, but the amplitudes of single channel currents and channel conductances were not altered. The half-maximal inhibition concentration (IC50) for DAMGO was 18 μmol/L. In the cellattached patch configuration, however, DAMGO (1∼300 μmol/L) increased dinitrophenol (50 μmol/L)induced KATP channel activities. It was inferred that the μ-opioid agonist is involved in the regulation of ATP-sensitive potassium channel activity in cardiac myocardium, agonizing (through internal target) or antagonizing (through external target) the inhibitory action of ATP in a competitive manner, thereby attenuating or enhancing the channel openings.

The influence of ATP on poly(ADP-ribose) metabolism

ATP affects poly(ADP-ribose) metabolism at two distinct sites: it inhibits poly(ADP-ribose) polymerase-1 and activates the glycohydrolase directly. The inhibitory site of ATP on poly(ADP-ribose) polymerase-1 was identified by amino acid exchange mutation to be at the arginine 34 residue in the first Zn2+ finger. Mutation of 138 arginine residue of Zn2+ finger 2 had negligible influence on the inhibitory action of ATP, pinpointing arginine 34 of the first Zn2+ finger as the specific ATP site. The glycohydrolase protein was activated by ATP when the substrate was a long-chain ADP-ribose polymer, but not with a short-chain substrate. Isolated cell nuclei also responded to both inhibition of poly(ADP-ribose) polymerase by ATP and to poly(ADP-ribose) glycohydrolase activation by ATP, demonstrating that enzymological results can be extrapolated to cellular systems. The activation of poly(ADP-ribose) polymerase in nuclei by an alkylating drug was completely suppressed by ATP, demonstrating that the bioenergetic competence of cells can regulate the cytocidal action of DNA alkylating drugs. The potential significance of bioenergetic regulation of poly(ADP-ribose) metabolism is proposed.

Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2',7'-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

Effects of Glucocorticoids and Epinephrine on the Modulatory Action of Purines in the Frog Neuromuscular Junction

The effect of corticoids (hydrocortisone and dexamethasone) and epinephrine on the presynaptic action of purines was studied at the neuromuscular junction of the frog under two-electrode voltage-clamp conditions. Daily administration of hydrocortisone/dexamethasone (100 mg/kg into the lymphatic system) increased initially and later depressed the amplitude of multiquantum end-plate currents evoked by motor nerve stimulation. An initial facilitatory phase of the hormone action was accompanied by removal of the presynaptic action of ATP (for hydrocortisone only). Within the later phase (2 weeks of hydrocortisone administration), the inhibitory action of ATP was restored once again. The counteraction of ATP effect was reproduced under superfusion of the isolated muscle by a physiological solution containing hydrocortisone (not dexamethasone), indicating the nongenomic nature of the action of the hormone on presynaptic P2 receptors. This proved to be true in experiments on animals, which were stressed 30 min prior to the beginning of the experiment by electrical stimulation in a special cage. Independently of acute or chronic administration of hydrocortisone, the presynaptic action of another purine, adenosine, was preserved. Epinephrine only partially abolished the inhibitory effect of purines, which is indicative of the difference in the paths of incorporation of the biological effects of these agents. We suggest that prevention of the inhibitory action of ATP might be one of the components of a facilitatory acute stress reaction, while such an inhibitory feedback action is missing under chronic stress conditions.

The I182 Region of K ir6.2 Is Closely Associated with Ligand Binding in K ATP Channel Inhibition by ATP

The ATP-inhibited potassium (K ATP) channel is assembled from four inward rectifier potassium (K ir6.x) subunits and four sulfonylurea receptor (SURx) subunits. The inhibitory action of ATP is mediated by at least two distinct functional domains within the C-terminal cytoplasmic tail of K ir6.2. The G334D mutation of K ir6.2 virtually eliminates ATP-dependent gating with no effect on ligand-independent gating, suggesting a role in linkage of the site to the gate or in the ATP binding site, itself. The T171A mutation of K ir6.2 strongly disrupts both ATP-dependent and ligand-independent gating, suggesting a role for T171 in the gating step. A neighboring mutation, I182Q, virtually eliminates ATP inhibition, but its effect on ligand-independent gating remained unknown. We have now characterized both the K i values for inhibition by ATP and the ligand-independent gating kinetics of 15 substitutions at position 182. All substitutions decreased ATP-dependent inhibition gating as measured by the K i, many profoundly so, yet had little or no effect on ligand-independent gating kinetics. Thus, substitutions at position 182 are unlikely to act by disrupting inhibition gate movement. Our results indicate an indispensable role for I182 in a step of the ATP binding mechanism, the linkage mechanism coupling the ATP binding site to the inhibition gate, or both.

Inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes.

1. Modulation of intracellular free Ca2+ concentration ([Ca2+]i) by extracellular ATP was investigated in cultured adult rat brown adipocytes using the fluorescent Ca2+ indicator fura-2. 2. Bath application of ATP in micromolar concentrations caused a large increase in [Ca2+]i in cells previously stimulated with noradrenaline. This ATP-induced [Ca2+]i increase exhibited a monotonic decline to near the resting levels within approximately 2 min, even in the continued presence of the agonist. 3. The magnitude and time course of the [Ca2+]i increase in response to ATP were not significantly affected by removal of extracellular Ca2+, suggesting that a mobilization of intracellular Ca2+ primarily contributes to the increase. 4. The [Ca2+]i increase in response to ATP was sensitive to inhibition by suramin, suggesting the involvement of P2 purinoceptors in the response. 5. Thapsigargin (100 nM) evoked a gradual and irreversible increase in [Ca2+]i which was entirely dependent upon extracellular Ca2+, providing functional evidence for the expression of store-operated Ca2+ entry in these brown adipocytes. 6. Extracellular ATP at a concentration of 10 microM depressed this thapsigargin (100 nM)-induced [Ca2+]i increase by 92 +/- 3 % (n = 8 cells), strongly suggesting that ATP inhibits an influx of Ca2+ across the plasma membrane through the store-operated pathway. Bath application of phorbol 12-myristate 13-acetate (PMA, 5 microM) did not affect the thapsigargin-induced [Ca2+]i increase, indicating that the inhibitory action of ATP is not mediated by activation of protein kinase C (PKC). 7. These results indicate that extracellular ATP not only mobilizes Ca2+ from the intracellular stores but also exerts a potent inhibitory effect on the store-operated Ca2+ entry process in adult rat brown adipocytes.

Multiple sites of purinergic control of insulin secretion in mouse pancreatic beta-cells.

In mouse pancreatic beta-cells, extracellular ATP (0.1 mmol/l) effectively reduced glucose-induced insulin secretion. This inhibitory action resulted from a direct interference with the secretory machinery, and ATP suppressed depolarization-induced exocytosis by 60% as revealed by high-resolution capacitance measurements. Suppression of Ca2+-dependent exocytosis was mediated via binding to P2Y1 purinoceptors but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. Inhibition of exocytosis by ATP resulted from G-protein-dependent activation of the serine/threonine protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. In contrast to the direct inhibitory action on exocytosis, ATP reduced the whole-cell ATP-sensitive K+ (K(ATP)) current by 30% (via activation of cytosolic phospholipase A2), leading to membrane depolarization and stimulation of electrical activity. The stimulatory effect of ATP also involved mobilization of Ca2+ from thapsigargin-sensitive intracellular stores. We propose that the inhibitory action of ATP, by interacting with the secretory machinery at a level downstream to an elevation in [Ca2+]i, is important for autocrine regulation of insulin secretion in mouse beta-cells.

Molecular biology of adenosine triphosphate-sensitive potassium channels.

KATP channels are a newly defined class of potassium channels based on the physical association of an ABC protein, the sulfonylurea receptor, and a K+ inward rectifier subunit. The beta-cell KATP channel is composed of SUR1, the high-affinity sulfonylurea receptor with multiple TMDs and two NBFs, and KIR6.2, a weak inward rectifier, in a 1:1 stoichiometry. The pore of the channel is formed by KIR6.2 in a tetrameric arrangement; the overall stoichiometry of active channels is (SUR1/KIR6.2)4. The two subunits form a tightly integrated whole. KIR6.2 can be expressed in the plasma membrane either by deletion of an ER retention signal at its C-terminal end or by high-level expression to overwhelm the retention mechanism. The single-channel conductance of the homomeric KIR6.2 channels is equivalent to SUR/KIR6.2 channels, but they differ in all other respects, including bursting behavior, pharmacological properties, sensitivity to ATP and ADP, and trafficking to the plasma membrane. Coexpression with SUR restores the normal channel properties. The key role KATP channel play in the regulation of insulin secretion in response to changes in glucose metabolism is underscored by the finding that a recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is caused by mutations in KATP channel subunits that result in the loss of channel activity. KATP channels set the resting membrane potential of beta-cells, and their loss results in a constitutive depolarization that allows voltage-gated Ca2+ channels to open spontaneously, increasing the cytosolic Ca2+ levels enough to trigger continuous release of insulin. The loss of KATP channels, in effect, uncouples the electrical activity of beta-cells from their metabolic activity. PHHI mutations have been informative on the function of SUR1 and regulation of KATP channels by adenine nucleotides. The results indicate that SUR1 is important in sensing nucleotide changes, as implied by its sequence similarity to other ABC proteins, in addition to being the drug sensor. An unexpected finding is that the inhibitory action of ATP appears to be through a site located on KIR6.2, whose affinity for ATP is modified by SUR1. A PHHI mutation, G1479R, in the second NBF of SUR1 forms active KATP channels that respond normally to ATP, but fail to activate with MgADP. The result implies that ATP tonically inhibits KATP channels, but that the ADP level in a fasting beta-cell antagonizes this inhibition. Decreases in the ADP level as glucose is metabolized result in KATP channel closure. Although KATP channels are the target for sulfonylureas used in the treatment of NIDDM, the available data suggest that the identified KATP channel mutations do not play a major role in diabetes. Understanding how KATP channels fit into the overall scheme of glucose homeostasis, on the other hand, promises insight into diabetes and other disorders of glucose metabolism, while understanding the structure and regulation of these channels offers potential for development of novel compounds to regulate cellular electrical activity.

MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit.

ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.

ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

Chloride channels in the sarcoplasmic reticulum (SR) are thought to play an essential role in excitation-contraction (E-C) coupling by balancing charge movement during calcium release and uptake. In this study the nucleotide-sensitivity of Cl- channels in the SR from rabbit skeletal muscle was investigated using the lipid bilayer technique. Two distinct ATP-sensitive Cl- channels that differ in their conductance and kinetic properties and in the mechanism of ATP-induced channel inhibition were observed. The first, a nonfrequent 150 pS channel was inhibited by trans (luminal) ATP, and the second, a common 75 pS small chloride (SCl) channel was inhibited by cis (cytoplasmic) ATP. In the case of the SCl channel the ATP-induced reversible decline in the values of current (maximal current amplitude, Imax and integral current, I') and kinetic parameters (frequency of opening FO, probability of the channel being open PO, mean open TO and closed Tc times) show a nonspecific block of the voltage- and Ca2+-dependent SCl channel. ATP was a more potent blocker from the cytoplasmic side than from the luminal side of the channel. The SCl channel block was not due to Ca2+ chelation by ATP, nor to phosphorylation of the channel protein. The inhibitory action of ATP was mimicked by the nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP) in the absence of Mg2+. The inhibitory potency of the adenine nucleotides was charge dependent in the following order ATP4- > ADP3- > > > AMP2-. The data suggest that ATP-induced effects are mediated via an open channel block mechanism. Modulation of the SCl channel by [ATP]cis and [Ca2+]cis indicates that (i) this channel senses the bioenergetic state of the muscle fiber and (ii) it is linked to the ATP-dependent cycling of the Ca2+ between the SR and the sarcoplasm.

Inhibition of voltage-dependent Ca2+influx by extracellular ATP in salivary cells of the leech Haementeria ghilianii

The effects of extracellular ATP on intracellular free Ca2+ concentration ([Ca2+]i) and depolarization-induced elevations of [Ca2+]i were investigated in salivary cells of the leech Haementeria ghilianii using the fluorescent Ca2+ indicator Fura-2. Simultaneously, the membrane potential was monitored or controlled by voltage-clamp. The cell membrane was depolarized either by transient elevations of the extracellular K+ concentration ([K+]o) to 90 mmol l-1 or by depolarizing steps under voltage-clamp. The resulting transient elevations of [Ca2+]i (Ca2+ transients) could be repeatedly elicited with little variability in amplitude. Ca2+ transients were completely inhibited by 2 mmol l-1 Ni2+ or in Ca2+-free saline. The transients are, therefore, dependent on Ca2+ influx from the external medium through voltage-gated Ca2+ channels. The Ca2+ influx was rapidly and reversibly inhibited by extracellular application of ATP. The effect was dose-dependent with a threshold concentration below 10(-7) mol l-1. A 50 % reduction in the amplitude of Ca2+ transients was obtained by application of 1­2 µmol l-1 ATP or ATP-gamma-S (apparent IC50, 1.6 µmol l-1 ATP) and Ca2+ transients were almost completely inhibited by 30­100 µmol l-1 ATP. Resting [Ca2+]i, the resting membrane potential and membrane potential changes induced by 90 mmol l-1 [K+]o were not affected by ATP. Adenosine (10 µmol l-1) did not affect resting [Ca2+]i, the resting membrane potential or membrane potential changes induced by 90 mmol l-1 [K+]o and had little effect on Ca2+ transients. Suramin, an antagonist of vertebrate P2 receptors, was without effect on the inhibitory actions of ATP. We conclude that activation of a suramin-insensitive purinoceptor by ATP inhibits Ca2+ influx through voltage-gated Ca2+ channels in the salivary cells of Haementeria ghilianii.

Cell-free production of the silkworm sex pheromone bombykol.

Cell-free production of bombykol was done by incubating a pheromone gland homogenate in the presence of NADPH, ATP, and CoA. Addition of n-hexane to the reaction mixture stimulated bombykol production, resulting in production of 238 ng of bombykol from the homogenate equivalent to 2 pheromone glands after 23 h. Removal of either NADPH, ATP, or CoA resulted in no stimulation of bombykol production, suggesting that the final step of the bombykol biosynthetic pathway is done by acyl CoA synthetase and reductase, sequentially. Incubation first with ATP or high concentrations of ATP suppressed the production of bombykol. Since incubation with ATP also inhibited conversion of [1-14C]palmitoyl CoA into 1-hexadecanol, the inhibitory action of ATP seemed attributable to inactivation of the acyl CoA reductase by phosphorylation, as mediated by a protein kinase in the homogenate. Our results suggest that the activity of acyl CoA reductase in bombykol biosynthesis is regulated by phosphorylation/dephosphorylation, and that the activation occurs by dephosphorylation as mediated by phosphoprotein phosphatase.

ATP inhibits the hydrosmotic effect of AVP in rabbit CCT: evidence for a nucleotide P2u receptor.

In rabbit renal cortical collecting tubule (CCT), perfused in vitro at 38 degrees C, ATP in concentrations of 10(-7) M and greater inhibits arginine vasopressin (AVP)-stimulated osmotic water permeability (Pf). The P1-purinergic receptor antagonist 8-phenyltheophylline did not attenuate the inhibitory action of ATP, and the poorly hydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), mimicked the effect of ATP, arguing against an effect of ATP on a P1 receptor or the "P site." Purinergic receptor agonists inhibited AVP-stimulated Pf with the following rank order efficacy: ATP = ADP = UTP = AMP-PNP = alpha, beta-methylene-ATP > 2-methylthio-ATP >> AMP > adenosine, consistent with the pharmacology of a "nucleotide" receptor subtype. Pertussis toxin pretreatment attenuated the action of 10(-5) and 10(-6) MATP; however, 10(-4) MATP failed to inhibit the hydrosmotic action of forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate. Pretreatment with the phosphodiesterase inhibitor RO20-1724 or indomethacin did not inhibit the action of ATP. Staurosporin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester significantly attenuated the inhibition of Pf by lower concentrations of ATP. These data suggest that ATP activates nucleotide receptors on the CCT, mobilizing intracellular Ca2+, which inhibits the hydrosmotic action of AVP.

Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K + -channels in mouse pancreatic β-cells

The mechanisms by which nucleotides stimulate the activity of the ATP-regulated K(+)-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic beta-cells. ATP produces a concentration-dependent inhibition of channel activity with a Ki of 18 microns. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.

Extracellular Adenosine Triphosphate Completely Reverses the Thyrotropin-Induced Morphological Change in FRTL-5 Cells

We quantified the TSH-induced morphological change in FRTL-5 thyroid cells according to a morphological index corresponding to the mean cell area measured from microscopic photographs. Within 15 min, TSH induced, at 10 pM and higher concentrations, a decrease in morphological index together with a rise in cAMP levels in a TSH dose-dependent manner. Forskolin, 3-isobutyl-1-methylxanthine, and RO 20-1724, the latter two being phosphodiesterase inhibitors, mimicked these TSH effects, indicating that the rise in cAMP levels is responsible for the TSH effect. Extracellular ATP and its derivatives, known as purinergic receptor agonists, decreased cAMP levels and caused a complete reversal of the TSH morphological effect. Prior exposure of the cells to islet-activating protein (pertussis toxin), the depletion of extracellular Ca2+, or the addition of low doses of protein kinase-C inhibitors completely abolished the inhibitory action of ATP on the TSH effect, whereas phorbol 12-myristate 13-acetate, which activates protein kinase-C, mimicked the ATP action to some extent. Thus, although the TSH-induced change in cell morphology seems to be dependent on cAMP levels, the inhibition of TSH action by ATP seems to be mediated by at least two signal transduction pathways involving islet-activating protein substrate G-proteins: one inhibiting adenylate cyclase and the other involving Ca2+ and protein kinase-C.

Competition between nucleoside diphosphates and triphosphates at the catalytic and allosteric sites of phosphorylase kinase.

The interactions of nucleotides at the allosteric and catalytic sites of phosphorylase kinase were examined. Binding of nucleoside triphosphates at the nucleoside diphosphate allosteric activation site inhibited enzymatic activity; this was observed with either ATP or GTP. Increasing concentrations of ADP caused a biphasic response: low concentrations activated and higher concentrations inhibited. Inhibition was due to the binding of ADP at the catalytic site, as opposed to an allosteric inhibitory site. GDP activated at low concentrations, but did not inhibit even at relatively high concentrations, and is therefore a specific probe for the allosteric site. Maximal activity of the nonactivated holoenzyme at pH 6.8 is achieved at an optimal ratio of ATP to ADP, such that the inhibitory actions of ATP at the allosteric site and of ADP at the catalytic site are balanced. Various potential molecular mechanisms to explain the allosteric activation by ADP were examined and ruled out, thus strengthening our previous conclusion that the activation is predominantly caused by a conformational transition in the beta subunits directly induced by the binding of ADP (Cheng, A., Fitzgerald, T. J., and Carlson, G. M. (1985) J. Biol. Chem. 260, 2535-2542; Trempe, M. R., and Carlson, G. M. (1987) J. Biol. Chem. 262, 4333-4340; Cheng, A., Fitzgerald, T. J., Bhatnager, D., Roskoski, R., Jr., and Carlson, G. M. (1988) J. Biol. Chem. 263, 5534-5542). The catalytic site exhibited high stereospecificity for inhibition by the Rp and Sp epimers of adenosine 5'-O-(1-thiodiphosphate), with the Rp epimer (Ki = 0.5 microM) being 136-fold more effective than its Sp counterpart. This can readily explain the inability of the Rp epimer to be an effective allosteric activator.

Blocking action of adenosine and ATP on synaptic transmission in isolated rat brain slices

The effect of ATP and adenosine on spontaneous activity and orthodromic responses of single neurons and on global evoked potentials was investigated in surviving slices of rat neocortex, hippocampus, dentate fascia, and cerebellumin vitro. ATP and adenosine, added to the incubation medium, had a twofold action on neurons: excitatory and inhibitory. Excitation was observed only if high concentrations of the substances (10−2, less frequently 10−3 M) were used, and in the case of adenosine it was very weak. The excitatory effect is evidently due to the direct depolarizing action of these substances on the cell membrane. The inhibitory action of both ATP and adenosine was manifested even in low concentrations (10−6–10−7 M) and was expressed as inhibition of postsynaptic responses of neurons at the presynaptic level and of their spontaneous activity. Hippocampal neurons were most sensitive to these substances, cerebellar neurons least. Apamine was found to have no effect on the inhibitory action of ATP. The results do not support the view that ATP and adenosine may be classed as CNS neurotransmitters. The possible role of these drugs as neuromodulators of synaptic transmission in the CNS is discussed.

Inhibitory action of exogenous adenosine-5'-triphosphate on glycolysis in isolated rat mast cells: significance for histamine release.

Abstract: Histamine release and lactate content were concomitantly determined in samples of isolated rat mast cells. Histamine release induced by exogenous ATP or compound 48/80 was inhibited by antimycin A (0.2 μM). Glucose (0.60 mM) restored the release induced by compound 48/80 but not that induced by ATP. ATP but not compound 48/80 inhibited the accumulation of lactate in suspensions of mast cells containing glucose (0.60 mM). ATP induced inhibition of lactate accumulation and release of histamine within the same concentration range. However, the time courses for the two processes were different. Antimycin A (0.2 μM) enhanced the accumulation of lactate, an effect which was counteracted by ATP. 0.05 mM ATP or more reduced the lactate accumulation to the same values as those found in the absence of antimycin A. The inhibitory action of ATP on glycolysis may explain the observed inability of glycolytic substrates to restore the ATP‐induced histamine release blocked by inhibitors of oxidative metabolism.