Actions of adenylyl compounds in the pedal disc of the cnidarian Actinia equina

1. The actions of adenylyl compounds were investigated in the circular muscle of the pedal disc of the sea anenome Actinia equina.2. Adenosine, adenosine 5'-diphosphate and adenosine 5'-triphosphate (ATP), but not adenosine 5'-monophosphate or the analogues of ATP, α,β-methylene ATP, and 2-methylthio ATP, caused concentration-dependent contractions.3. Neurogenic contractions in response to electrical field stimulation were not consistently affected by any of the adenylyl compounds and could be either potentiated or almost abolished by them.4. Reactive Blue 2, a vertebrate P₂-purinoceptor antagonist, caused concentration-dependent contractions which were mediated by nerves, being blocked by the anaesthetic chlorobutanol.5. The pharmacological profile of the adenylyl compounds in the pedal disc of Actinia equina is different from that observed in other invertebrate species and adds to the greater diversity of such profiles in invertebrates than in vertebrates.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Intracellular signalling by nucleotide receptors in PC12 pheochromocytoma cells

The effect of extracellular ATP was studied in PC12 cells, a neurosecretory line that releases ATP. The addition of micromolar concentrations of ATP to PC12 cells evoked a transient increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), as measured with the Ca²⁺-dye fura 2. AMP and adenosine were without effect, ruling out the involvement of P₁ receptors in mediating this response. The increase in [Ca²⁺]_i was reduced in calcium-free media and virtually eliminated by the addition of EGTA, suggesting that calcium influx was the primary response initiated by extracellular ATP. Nucleotide triphosphates such as UTP and, to a lesser degree, ITP also evoked an increase in [Ca²⁺]_i while GTP and CTP had little effect. In order to identify the receptor subtype mediating this response, the efficacy of ATP and ATP cogeners was assessed. The rank order potency was ATP > adenosine 5′-[γ-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) ～ adenosine 5′-[β-thio]diphosphate ? adenosine 5′-[αβ-methylene] triphosphate, adenosine 5′-[βγ-imido]triphosphate. This profile is not characteristic of either the P_2X or the conventional P_2Y receptors. The Ca²⁺ response exhibited desensitization to ATP that was dependent on the extracellular metabolism of ATP. UTP was equally effective in desensitizing the response. ATP, UTP, ITP, and to a much lesser extent 2MeSATP increased inositol phosphate production in a dose-dependent manner, suggesting receptor coupling to phosphatidylinositol-specific phospholipase C. These data are consistent with the view that PC12 cells express a class of non-P_2Y nucleotide receptors (P_2N) that mediate calcium influx and the accumulation of inositol phosphates. © 1993 Wiley-Liss, Inc.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP

Spinach chloroplasts were able to photophosphorylate the ADP analog α,β-methylene adenosine 5′-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and ³²P_i, the ³²P label was incorporated into α,β-methylene adenosine 5′-triphosphate (AOPCPOP).In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-P_i exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9.Photophosphorylation of AOPCP was inhibited by the α,β- and β,γ-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-P_i exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-P_i exchange was inhibited by the β,γ-methylene analog of ATP.Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Design,Syntheses and Pharmacology of ATP Analogues Selective for Subtypes of P2-Purinoceptors

Abstract

A series of analogues of adenine nucleotides have been synthesized and tested for Pharmacological potency and resistance to dephosphorylation at a variety of isolated tissue preparations where ATP is active. Structure-activity studies defined four subtypeso f purinoceptors, and enabled the design of specific agonists for P_2x and for P_2y purinoceptors to be undertaken. L-Adenosine 5′-β, γ-methylenetriphosphonate (L-AMP-PCP) is a specific P_2x purinoceptor agonist, and adenosine 5′-β-fluorodiphosphate (ADP-β-F) is a specific P_2y agonist.     

Selective suppression of blastogenesis induced by different mitogens: effect of noncyclic adenosine-containing compounds

Lymphocyte responses to the galactosyl binding lectins, SBA and PNA, and responses to Con A are suppressed by cAMP analogs and agents that increase intracellular cAMP to a greater extent than are responses to PHA. Adenosine shares with cAMP this selective inhibitory effect. ATP inhibits lymphocyte proliferation via generation of adenosine. This conclusion is based on the findings that (i) β,γ-methylene ATP (β,γ-met ATP) is similar to ATP in its inhibitory effects, whereas α,β-met ATP has little effect on lymphocyte proliferation, (ii) α,β-met ATP reverses both ATP and β,γ-met ATP induced suppression, but does not reverse adenosine-mediated suppression, and (iii) inhibition of adenosine deaminase potentiates ATP and β,γ met ATP-mediated suppression of blastogenesis. The relative potency of various noncyclic adenosine compounds in suppressing mitogen-induced blastogenesis is: 2-chloradenosine > ATP ≥ ADP > AMP > adenosine.     

Heterogeneity of Nucleotide Receptors in NG108-15 Neuroblastoma and C6 Glioma Cells for Mediating Phosphoinositide Turnover

Abstract: We have compared the characteristics of receptors for nucleotide analogues and the involvement of phospholipase C (PLC) in the effector mechanism in NG108-15 neuroblastoma and C6 glioma cells. The relative potency of these analogues to stimulate inositol phosphate (IP) formation is UTP > UDP ? 2-methylthio-ATP (2-MeSATP), GTP > ATP, CTP > ADP > UMP in NG108-15 cells and ATP > UTP > ADP > GTP > UDP ? 2Me-SATP, CTP, UMP in C6 glioma cells. α,β-Methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine had little or no effect in both types of cells. The EC₅₀ values were 3 and 106 µM for UTP in NG108-15 and C6 glioma cells, respectively. The EC₅₀ value for ATP in C6 glioma cells was 43 µM. 2-MeSATP was threefold more potent than ATP in NG108-15 cells but had little effect in C6 glioma cells at 1 mM. In NCB-20 cells, a similar rank order of potency to that found in NG108-15 cells, i.e., UTP ? GTP > ATP > CTP, was observed. In both NG108-15 and C6 glioma cells, preincubation with ATP or UTP caused a pronounced cross-desensitization of subsequent nucleotide-stimulated IP production. ATP and UTP displayed no additivity in terms of IP formation at maximally effective concentrations. In contrast, endothelin-1, bradykinin, and NaF interacted in an additive manner with either nucleotide in stimulating PI hydrolysis. Pretreatment with pertussis toxin did not affect ATP-, UTP-, and GTP-stimulated IP generation in these cells, indicating that nucleotide receptors coupled to PLC by a pertussis toxin-resistant G protein in both cell types. Short-term treatment of the cells with protein kinase C (PKC) activators [phorbol 12-myristate 13-acetate (PMA) and octylindolactam V] produced a dose-dependent inhibition of ATP- and UTP-induced IP formation with a greater extent and higher susceptibility in C6 glioma cells than in NG108-15 cells. Furthermore, a 24-h exposure of the cells to PMA resulted in an obvious attenuation of nucleotide-induced IP formation in C6 glioma cells but failed to change the response in NG108-15 cells. These results suggest that distinct nucleotide receptors that respond to ATP and UTP with different selectivity exist in NG108-15 and C6 glioma cells. These heterogeneous nucleotide receptors coupled to PLC undergo discriminative modulation by PKC. NG108-15 and NCB-20 neuroblastoma are two cell lines that showed the highest specificity to extracellular UTP rather than ATP among the nucleotide receptors so far studied in various cells, suggesting the presence of a pyrimidine receptor in these cells.     

Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K _m for p-Nph-5′-TMP hydrolysis of 61.8 ± 5.2 μM. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5′-TMP hydrolysis with K _i values ranging 13–43 μM. Nucleotide analogs, α,β-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas α,β-metADP induced mixed inhibition, with K _i ranging from 2 to 20 μM. Chromatographic analysis revealed that α,β-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and α,β-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

Dynamic affinity chromatography of heavy meromyosin subfragment-1

Heavy meromyosin subfragment-1 and its trinitrophenylated derivative 3ave been chromatographed on immobilized ATP, ADP and adenosine 5′-(β,γ-imino)triphosphate affinity chromatography columns, in the presence and in the absence of Mg²⁺ or Ca²⁺. Splitting of bound ATP was followed by using [γ-^{3 2}P]ATP columns. While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5′(β,γ-imino)triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins. The protein-elution and the P_i-release patterns were different for the native and the modified proteins. These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.     

Purinergic modulation of spontaneous activity and of responses to high potassium and acetylcholine in rat ileal smooth muscle

1. In rat ileal smooth muscle both adenosine and ATP at 10⁻⁴ M significantly enhanced spontaneous mechanical activity. The excitatory actions of adenosine were blocked by the P₁ receptor antagonist 8-phenyltheophylline and the excitatory effects of ATP were significantly reduced by the P₂ receptor antagonist quinidine.2. The P₂ receptor desensitizer α,β-methylene-ATP was without effect on ACh responses nor did the stable analogue β,gg-methylene-ATP exert any effect on spontaneous mechanical activity.3. Pretreatment with adenosine caused a dose-dependent enhancement of K-induced contractures in the ileum. Low adenosine concentrations slightly inhibited and high concentrations slightly enhanced ACh-induced contractures in the ileum.4. ATP potentiated the phasic component of the ileal K-induced contracture but strongly inhibited tonic force at high concentrations. This agent slightly inhibited the phasic component of the ACh-induced contracture while strongly inhibiting ACh-induced tonic force.5. α,β-methylene-ATP inhibited ileal muscle ACh induced contractures while it potentiated both phasic and tonic K-induced contractures. β, γ-methylene ATP inhibited ACh-induced contractures but it enhanced K-induced phasic contractures while inhibiting K-induced tonic force.6. The results of this study suggest that rat ileum may contain the A₁ subtype of the P₁ receptor but the evidence for a P₂ receptor subtype is conflicting despite the inhibition of ATP actions by quinidine.7. The inhibition of K- and ACh-induced tonic force suggests that adenosine and ATP interactions with ileal smooth muscle may inactivate slow voltage-dependent calcium channels leading to EC uncoupling.     

Synthesis of modified tryptophanyl-adenylates and of modified adenosine-triphosphates and their use as tools for elucidation of the mechanism of tryptophanyl-tRNA synthetase from yeast

Six analogs of tryptophanyl-adenylate, which is an important intermediate in the enzymatic synthesis of Trp-tRNA^Trp, have been prepared. Four compounds, tryptophanyl-8-bromoadenylate, tryptophanyl-2-chloroadenylate, tryptophanyl-7-deazaadenylate and tryptophanyl-(N⁶-methyl)adenylate, contain modifications in the nucleobase moiety, while tryptophanyl-2′ deoxyadenylate and tryptophanyl-3′-deoxyadenylate were modified in the carbohydrate part of the molecule. Three of these analogs (2-chloro, 7-deaza, 2′-deoxy analogs) as well as ATP analogs with the same modifications were substrates in the aminoacylation reaction; three analogs (8-bromo, N⁶-methyl, 3′-deoxy analogs) were inactive as well as the corresponding ATP analogs. In contrast, in the $\text{ATP}\text{PP}{}_{\mathrm{<mtext>i</mtext>}}$ pyrophosphate exchange in the absence of tRNA all ATP analogs except 8-bromo-ATP were substrates. However, the presence of tRNA reduced the number of ATP analogs being substrates to that number of substrates observed in the aminoacylation. Therefore, it can be concluded that the presence of tRNA is responsible for an increase of specificity. The diastereomers of adenosine 5′-O-(3-thiotriphosphate) (ATPαS), adenosine 5′-O-(2-thiotriphosphate) (ATPβS), and adenosine 5′-O-(3-thiotriphosphate) (ATPγS) were tested with various divalent metals as substrates in the pyrophosphate exchange reaction. The S_p diastereomer of ATPαS is a substrate with Mg²⁺, whereas the R_p diastereomer is inactive. Both diastereomers are inactive in the presence of Zn²⁺. Since Zn²⁺ binds preferentially to the sulfur atom, an explanation of these results is that the Mg²⁺ ion is not bound to the α-phosphate. Only the S_p isomer of the diastereomers of ATPβS acts as substrate in the presence of Mg²⁺. The stereospecificity becomes reversed in the presence of Zn²⁺. ATPγS acts as substrate with both Mg²⁺ and Zn²⁺. These results suggest that the Δ isomer of the β,γ-bidentate ATP-Mg²⁺ complex is the substrate for this enzyme. From these results a molecular model of the ATP-Mg²⁺ complex in the active site can be derived in which the nucleotide is attached to the enzyme by interactions in which the 3′-OH and 6-NH₂ group, one oxygen atom of the α-phosphorus atom, and the coordinated magnesium cation are all involved.     

Origin of apparent negative cooperativity of F1-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F₁-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F₁-ATPase, α_(W463F)3β_(Y341W)3γ and α_{(K175A/T176A/W463F)3}β_(Y341W)3γ. For α_(W463F)3β_(Y341W)3γ, apparent K_m's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent K_m's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent K_m of ATPase activity (10 μM) roughly agreed with the highest K_m of fluorescence measurements (27 μM). The results indicate that in case of α_(W463F)3β_(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K_m of ATPase activity does not represent the ATP binding to a catalytic site. In case of α_{(K175A/T176A/W463F)3}β_(Y341W)3γ, the K_m of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α_{(K175A/T176A/W463F)3}β_(Y341W)3γ was rather linear compared with that of α_(W463F)3β_(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F₁-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K_m's of 100-300 μM and 1-30 μM range for wild-type F₁-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

Effects of purine compounds and forskolin on melanophores of the medaka,oryzias latipes: Evidence for an A2-adenosine receptor

1. The effects of purines on denervated melanophores of the medaka were studied under experimental conditions in which melanosomes were aggregated by norepinephrine or lithium ion beforehand.2. Adenosine and its derivatives caused melanosome dispersion; the order of potency for the series was; NECA > adenosine > ATP > 2-chloroadenosine > PIA > CHA > cyclic AMP.3. 8-Phenyltheophylline, a potent purinoceptor antagonist, blocked the effect of purines and caused a rightward shift of the adenosine and analog concentration-response curves.4. 8-Br cyclic AMP also caused melanosome dispersion but its action was not blocked by 8-phenyladenosine. Dibutyryl cyclic AMP, cyclic GMP, dibutyryl cyclic GMP, and 8-br cyclic GMP were all ineffective.5. The effect of adenosine was immediately eliminated by adenosine deaminase but, actions of NECA, AMP, ADP, ATP, and cyclic AMP were not.6. Forskolin, a potent activator of adenylate cyclase, mimicked the action of adenosine.7. It is concluded that adenosine and its derivatives mediate their melanosome-dispersing effect via a P₁-purinoceptor that displays characteristics of the A₂-subtype and that adenine nucleotides directly activate the A₂-receptor without conversion to adenosine.     

ATP-Activated Nonselective Cation Current in NG108-15 Cells

Abstract: ATP (1 mM) induced a biphasic increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), i.e., an initial transient increase decayed to a level of sustained increase, in NG108-15 cells. The transient increase was inhibited by a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), whereas the sustained increase was abolished by removal of external Ca²⁺. We examined the mechanism of the ATP-elicited sustained [Ca²⁺]_i increase using the fura-2 fluorescent method and the whole-cell patch clamp technique. ATP (1 mM) induced a membrane current with the reversal potential of 12.5 ± 0.8 mV (n = 10) in Tyrode external solution. The EC₅₀ of ATP was ～0.75 mM. The permeability ratio of various cations carrying this current was Na⁺ (defined as 1) > Li⁺ (0.92 ± 0.01; n = 5) > K⁺ (0.89 ± 0.03; n = 6) > Rb⁺ (0.55 ± 0.02; n = 6) > Cs⁺ (0.51 ± 0.01; n = 5) > Ca²⁺ (0.22 ± 0.03; n = 3) > N-methyl-d -glucamine (0.13 ± 0.01; n = 5), suggesting that ATP activated a nonselective cation current. The ATP-induced current was larger at lower concentrations of external Mg²⁺. ATP analogues that induced the current were 2-methylthio-ATP (2MeSATP), benzoylbenzoic-ATP, adenosine 5′-thiotriphosphate (ATPγS), and adenosine 5′-O-(2-thiodiphosphate), but not adenosine, ADP, α,β-methylene-ATP (AMPCPP), β,γ-methylene-ATP (AMPPCP), or UTP. Concomitant with the current data, 2MeSATP and ATPγS, but not AMPCPP or AMPPCP, increased the sustained [Ca²⁺]_i increase. We conclude that ATP activates a class of Ca²⁺-permeable nonselective cation channels via the P_2z receptor in NG108-15 cells.     

P2x-Receptor Characterisation in Rabbit Isolated Ear Artery

Abstract

The P_2x-receptor mediating contraction of the rabbit ear artery is characterised by the following agonist potency order: D-αβmethyleneATP < L-βγmethyleneATP < D-βγmethyleneATP ≥ 2-MeSATP < ATP.     

Structure of the active metal-adenosine 5′-triphosphate chelate complex used by light-triggered chloroplast ATPase

By employing phosphorothioate analogs of ATP in the presence of Mg²⁺ and Mn²⁺ as substrates in ATP hydrolysis, catalyzed by light and dithiothreitol-activated chloroplast ATPase, the structure of the reactive metal-nucleotide complex has been determined. Mg(S_P)-ATPαS and Mn(S_P)-ATPαS, in contrast to the corresponding R_P-isomers, are substrates in ATP hydrolysis. No metal-dependent change of specificity was observed. Mg(S_P)-ATPβS, having the Δ configuration, and Mn(S_P)-ATPβS and Mn(R_P)-ATPβS, consisting of a mixture of Δ and Λ configurations, were better substrates than Mg(R_P)-ATPβS, the isomer with almost exclusive Λ chelate structure. The same results were obtained when the competitive effect of the analogs on hydrolysis of ATP was studied. The competitive effect of the diastereomers on tight binding of ATP by membrane-associated CF₁, was investigated in the presence of Mg²⁺ and Cd²⁺. Mg(S_P)-ATPβS and Cd(R_P)-ATPβS, which both exhibit Δ structure, were more effective than Mg(R_P)-ATPβS and Cd(S_P)-ATPβS, showing the Λ configuration. No metal-dependent change of the preferred S_P-ATPαS specificity was detected. These results permit the conclusion that the actual substrate used by chloroplast ATPase is the β,γ-Δ-bidentate nucleotide chelate. Moreover, a stereospecific direct ionic interaction between the protein and α-phosphate is likely.     

Responses of the rectum and oesophagus of the snail Helix aspersa to purine nucleotides and nucleosides

1. Purine compounds were examined for pharmacological activity in the rectum and oesophagus of the garden snail Helix aspersa.2. In the rectum, adenosine, AMP, ADP and ATP (above 10μM) and acetylcholine (above 1 nM) consistently caused concentration-dependent contractions. The slope of the dose-response curve for ADP in the rectum was significantly steeper than for the other purine compounds. The contractile responses to the nucleotides and acetylcholine, but not adenosine, were selectively potentiated by physostigmine (1μM). Atropine (1 μM) and tubocurarine (30 μM) failed to block the responses to the purines or acetylcholine.3. In the oesophagus, adenosine, AMP, ADP and ATP (above 10 μM) and acetylcholine (above 1 nM) caused concentration-dependent contractions that were antagonised by atropine (l μM). Tubocurarine (30 μM) failed to block the responses to the purine compounds or acetylcholine. Physostigmine (1 μM) potentiated the responses to ADP and acetylcholine but not ATP, AMP or adenosine.4. In both the rectum and the oesophagus, the synthetic analogues of purine compounds inclucling 2-chloroadenosine, α, β -methylene ATP and 2-methylthio ATP were inactive up to a concentration of 100 μM.5. Electrical field stimulation of the rectum and oesophagus produced consistent contractions which were unaffected by atropine (1 μM), tubocurarine (30 μM) or physostigmine (1 μM). These responses were not modulated by any of the purine compounds or their stable analogues.6. The responses obtained appear novel even within known invertebrate purinergic systems, suggesting a differentiation of purinoceptor subtypes in this species. There is evidence in the rectum for AMP, ADP and ATP causing the release of acetylcholine; physostigmine potentiated responses to AMP, ADP and ATP, but not to adenosine. This indicates that activity may be mediated via different types of purinoceptors, perhaps equivalent to the P₁- and P₂-purinoceptors identified in vertebrates.     

Distribution and Roles of Purinoceptor Subtypes

Abstract

The basis of the established subdivision of receptors for purines into P₁-purinoceptors for adenosine and P₂-purinoceptors for ATP and ADP is considered, as well as the proposals for subdivision of P₁-purinoceptors into A₁ and A₂ subtypes and of ATP receptors into P_2x-, P_2y- P_2z- and P_2T-purinoceptor subtypes. The distribution and roles of these receptor subtypes in muscles, nerves and other tissues, including endothelial and epithelial cells, hepatocytes, blood cells, fibroblasts and astrocytes, are discussed.     

Fourier transform phosphorus magnetic resonance study of ATP--calcium--G-actin complex

³¹P-Fourier transform NMR spectroscopy (40.5 MHz) has been employed to investigate the mode of binding of adenosine 5′-triphosphate (ATP) to rabbit muscle G-actin in the presence of calcium in the pH range 6.5 to 10.5. Line width measurements reveal that the nucleotide binds tightest around pH=8.5. Spin lattice (T₁) and spin-spin (T₂) relaxation times of each of the three phosphorus atoms of bound ATP demonstrate the prime importance of P_β and P_γ in ATP binding to G-actin through a calcium bridge.