Efficacy Of ATP Research Articles

Structural characteristics that govern binding to, and modulation through, the cardiac ryanodine receptor nucleotide binding site.

Comparative molecular field analysis (CoMFA) predicts that the large electrostatic field around the phosphate groups of ATP plays a crucial role in stabilizing the open state of the cardiac ryanodine receptor (RyR) channel. We therefore investigated the effects of adenosine-5'-(beta,gamma-methylenetriphosphate) (AMP-PCP), an ATP analog with lower negative charge in this region, on the gating of the cardiac RyR channel. In the presence of 10 microM cytosolic Ca2+, AMP-PCP exhibited approximately 50% of the efficacy of ATP and optimal doses increased open probability (Po) to only 0.441 +/- 0.156 (n = 4), thus confirming the predictive ability of our preliminary CoMFA model. We also reveal that AMP-PCP has a higher affinity than ATP for the cardiac RyR, demonstrating that the structural properties required for tight binding to RyR differ from those necessary for recruiting long open states and high Po values. CoMFA identified very strong correlations between the structures of adenine-based ligands and their affinity for RyR and different (but also highly significant) correlations between structure and the ability to activate the channel. Analysis indicates that ATP may be more effective than other adenine nucleotides because it can convert the greatest amount of binding energy into conformational changes that stabilize the open channel state.

Glibenclamide binding to sulphonylurea receptor subtypes: dependence on adenine nucleotides.

1: ATP-sensitive K(+) channels are composed of pore-forming subunits Kir6.2 and of sulphonylurea receptors (SURs); the latter are the target of the hypoglycaemic sulphonylureas like glibenclamide. Here, we report on the negative allosteric modulation by MgATP and MgADP of glibenclamide binding to SUR1 and to SUR2 mutants with high glibenclamide affinity, SUR2A(Y1206S) and SUR2B(Y1206S). 2: ATP, in the presence of an ATP-regenerating system to oppose hydrolysis during incubation, inhibited glibenclamide binding to SUR1 and SUR2B(Y1206S) by approximately 60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC(50) values of 5-10 microM; the curve for SUR1 was biphasic (IC(50) values 4.7 and 1300 microM). 3: Glibenclamide inhibition curves for ADP, performed in the presence of an ATP-consuming system to oppose ATP formation from ADP, were generally shifted rightwards and showed positive cooperativity, in particular with the SUR2(Y1206S) isoforms. 4: In the absence of the coupled enzyme systems, inhibition curves of MgATP or MgADP were generally shifted leftwards. This indicated synergy of MgATP and MgATP in acting together. 5: Coexpression of SUR1 and SUR2B(Y1206S) with Kir6.2 reduced both potency and efficacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6: The data show (a) that the inhibitory effects of ATP and ADP on glibenclamide binding differ from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.

Prior exposure to high glucose augments depolarization-induced insulin release by mitigating the decline of ATP level in rat islets.

A brief exposure to elevated glucose augments the insulin secretory response of islets to subsequent stimulation. The site of this priming effect of glucose in the mechanism of the regulation of insulin secretion is not completely known, however. Insulin release triggered by a depolarizing concentration of K+ in the presence of basal glucose is markedly enhanced in primed rat islets. To clarify the role of priming on Ca(2+) and ATP efficacy in the exocytotic apparatus, islets were electrically permeabilized to vary the intracellular Ca(2+) and ATP concentrations according to the extracellular medium, and insulin release was evaluated. Ca(2+) and ATP efficacy in Ca(2+)- and ATP-dependent insulin secretion was not affected by priming, and alteration of the intracellular Ca(2+) concentration after depolarization cannot account for the phenomenon. There was no difference in ATP content before depolarization between nonprimed and primed islets. Moreover, the decline in ATP level after depolarization with basal glucose was observed in both primed and nonprimed islets. However, a reduced decline in ATP level in the early phase was observed in primed islets. In addition, oligomycin, a mitochondrial metabolism inhibitor, abolished the difference in ATP level between primed and nonprimed islets, suggesting that mitochondrial ATP production may be linked to the phenomenon.

ATP stimulated cyclic AMP formation in bovine chromaffin cells is enhanced by neuropeptide Y

ATP increases cAMP formation in bovine chromaffin cells, EC 50 = 7.1 × 10 −6 M. NPY, EC 50 = 4.1 × 10 −8 M, increases the efficacy of ATP (1.5–2 fold). Inclusion of the selective Y1 receptor antagonist 1229U91 produced a decrease in NPY potency (EC 50 = 2.7 × 10 −7 M). PTX pretreatment did not abolish either the effect of ATP nor the enhancement by NPY. NPY could also enhance the ability of angiotensin and bradykinin to increase cAMP formation. The selective phospholipase C inhibitor, U73122, and the selective protein kinase C inhibitors, bisindolylmaleimide I and RO-31-8425, were effective inhibitors of the enhancing effect of NPY.

Differential coupling of the human P2Y(11) receptor to phospholipase C and adenylyl cyclase.

1. The human P2Y(11) (hP2Y(11)) receptor was stably expressed in two cell lines, 1321N1 human astrocytoma cells (1321N1-hP2Y(11)) and Chinese hamster ovary cells (CHO-hP2Y(11)), and its coupling to phospholipase C and adenylyl cyclase was assessed. 2. In 1321N1-hP2Y(11) cells, ATP promoted inositol phosphate (IP) accumulation with low microM potency (EC(50)=8.5+/-0.1 microM), whereas it was 15 fold less potent (130+/-10 microM) in evoking cyclic AMP production. 3. In CHO-hP2Y(11) cells, ATP promoted IP accumulation with slightly higher potency (EC(50)=3.6+/-1.3 microM) than in 1321N1-hP2Y(11) cells, but it was still 15 fold less potent in promoting cyclic AMP accumulation (EC(50)=62.4+/-15.6 microM) than for IP accumulation. Comparable differences in potencies for promoting the two second messenger responses were observed with other adenosine nucleotide analogues. 4. In 1321N1-hP2Y(11) and CHO-hP2Y(11) cells, down regulation of PKC by chronic treatment with phorbol ester decreased ATP-promoted cyclic AMP accumulation by 60--80% (P<0.001) with no change in its potency. Likewise, chelation of intracellular Ca(2+) decreased ATP-promoted cyclic AMP accumulation by approximately 45% in 1321N1-hP2Y(11) cells, whereas chelation had no effect on either the efficacy or potency of ATP in CHO-hP2Y(11) cells. 5. We conclude that coupling of hP2Y(11) receptors to adenylyl cyclase in these cell lines is much weaker than coupling to phospholipase C, and that activation of PKC and intracellular Ca(2+) mobilization as consequences of inositol lipid hydrolysis potentiates the capacity of ATP to increase cyclic AMP accumulation in both 1321N1-hP2Y(11) and CHO-hP2Y(11) cells.

Double-blind, Multicenter Comparative Study of Sertraline versus Amitriptyline in Outpatients with Major Depression

To compare the efficacy and safety of sertraline and amitriptyline in a German outpatient population. Patients with Major Depression (DSM-III-R) and HAM-D (21 items) > or = 21 in 19 German centers received double-blind treatment with sertraline (initial dose 50 mg, titration up to 100 mg) or amitriptyline (75 mg, up to 150 mg) over 6 weeks. HAM-D (21 items), HAM-D Bech, CGI, DSI and SDS were evaluated for the efficacy analysis. FSUCL (Fischer Somatic and Undesired Effects Check List) and spontaneously reported adverse events were used for safety analysis. Of the 240 patients enrolled in the study, 205 (100 sertraline; 105 amitriptyline) were evaluable for efficacy. No statistically significant differences were detected between the two groups in the ITT and ATP efficacy analyses. Response, defined as score 1 (very much improved) or 2 (much improved) of the CGI improvement score, was 76% in the sertraline and 81% in the amitriptyline group (efficacy evaluable patients = ATP population). In the structured FSUCL, the side-effect burden (FSUCL score >2 for drug related symptoms) was significantly higher in the amitriptyline group at all follow up visits (p<0.05). Both sertraline and amitriptyline are suitable for the treatment of Major Depression; sertraline is comparable to amitriptyline with regard to efficacy, and offers the additional benefit of a more favorable safety profile.

Structural factors that determine the ability of adenosine and related compounds to activate the cardiac ryanodine receptor.

The effects of adenosine and adenine on the gating of native sheep cardiac ryanodine receptor (RyR) channels were investigated. By examining the mechanisms underlying channel activation and by using comparative molecular field analysis (CoMFA) we have investigated the structural features of adenine-based ligands involved in channel activation. In the presence of 10 microM cytosolic Ca(2+), adenosine and adenine both activate the channel but only to a level approximately 10 and 20% respectively of that of ATP indicating that both are partial agonists of low efficacy. Adenosine was able to antagonize the ATP-induced increase in open probability (Po) as expected for a partial agonist of low efficacy at the ATP sites on the cardiac RyR. GTP (100 microM - 10 mM) had no effect on channel gating indicating that the adenine ring structure is important for agonist activity at the ATP-sites on RyR. CoMFA revealed an extremely strong correlation between the structural features of the five ATP analogues and the ability to increase (Po). Our model indicates that the high efficacy of ATP results primarily from the large electrostatic field established by the ionized phosphate groups. Reducing the number of phosphate groups lowers the strength of this field, leading to ligands with lower efficacy. In addition, steric interactions between the alpha-phosphate and ribose moieties and the RyR are correlated with low Po.

Long-term efficacy of antitachycardia pacing for treatment of ventricular tachycardia in patients with implantable cardioverter/defibrillator

Antitachycardia pacing techniques (ATP) have proved useful for termination of ventricular tachycardia (VT). However, little is known about the efficacy and safety off ATP during long-term follow-up in a larger study population. We analyzed the data of 80 ICD patients (pts) with spontaneous monormorphic VT, mean age 59 +/- 12 years, the mean follow-up was 26 +/- 17 months. 50 pts (62.5%) had coronary artery disease, 18 (22.5%) dilative cardiomyopathy (DCM), the remaining 12 pts (15%) had no or other cardiac diseases. 2926 episodes of ventricular tachycardia (cycle length 349 +/- 51 ms, 240-520 ms) occurred in 64/80 pts (80%), overall efficacy of ATP was 89.9%, acceleration occurred in 4.1% of VTs. Success of ATP did not correlate with positive ATP testing on induced arrhythmias, LVEF, NYHA class or aneurysm. Neither underlying heart disease nor antiarrhythmic medication had an impact on the ATP success rate. ATP efficacy was linked significantly to short VT cycle length (VTCL, 240-300 ms, p < 0.01) and long coupling intervals (91-97%), p < 0. 01). Acceleration occurred in 32% of pts and in 4.1% of VT episodes; it was also dependent on short VT cycle length (< 300 ms vs > 300 ms, p < 0.04) and short coupling intervals (< 81% vs >/= 81%, p </= 0. 01). First, ATP is a highly effective therapy, independent of patients characteristics. Second, short VT cycle length and long ATP coupling intervals were identified as predictors for successful ATP. Best conversation rates were obtained with coupling intervals between 90 and 97% of VTCL in tachycardias with a cycle length < 300 ms. Third, testing ATP on induced VTs before hospital discharge did not improve efficacy, empiric modes showed equal success rates. Fourth, acceleration rate of ATP depends on VT cycle length and coupling interval; therefore, aggressive ATP modes should be carefully applied in patients with VTs faster than 300 ms.

Modulation of ATP-responses at recombinant rP2X4 receptors by extracellular pH and zinc.

The modulatory effects of extracellular H+ and Zn2+ were tested against ATP-responses at rat P2X4 (rP2X4) receptors expressed in Xenopus oocytes under voltage-clamp conditions. ATP (0.1-100 microM, at pH 7.5), evoked inward currents via rP2X4 receptors (EC50 value, 4.1+/-0.98 microM; nH, 1.2+/-0.1). ATP potency was reduced 2 fold, at pH 6.5, without altering maximal activity. ATP potency was reduced by a further 4 fold, at pH 5.5, and the maximal activity of ATP was also reduced. Alkaline conditions (pH 8.0) had no effect on ATP-responses. Zn2+ (100 nM - 10 microM) potentiated ATP-responses at the rP2X4 receptor by 2 fold, whereas higher concentrations (30 microM - 1 mM) inhibited ATP-responses. Zn2+ potentiation was due to an increase in ATP potency, whereas its inhibitory action was due to a reduction in ATP efficacy. Zn2+ modulation of ATP-responses was pH-dependent. At pH 6.5, the bell-shaped curve for Zn2+ was shifted to the right by 1 log unit. At pH 5.5, Zn2+ potentiation was abolished and its inhibitory effect reduced considerably. Suramin (50 microM) also potentiated ATP-responses at rP2X4 receptors. Neither H+ (pH 6.5 and 5.5), Zn2+ (10-100 microM) or a combination of both failed to reveal an inhibitory action of suramin at rP2X4 receptors. In conclusion, H+ and Zn2+ exerted opposite effects on the rP2X4 receptor by lowering and raising agonist potency, respectively. H+ (> or = 3 microM) and Zn2+ (> or = 30 microM) also reduces agonist efficacy by lowering the number of rP2X4 receptors available for activation. The striking differences between the modulatory actions of H+ and Zn2+ at rP2X4 and rP2X2 receptors are discussed.

Agonist Action of Adenosine Triphosphates at the Human P2Y1Receptor

The agonist selectivity for adenosine di- and triphosphates was determined for the human P2Y1 receptor stably expressed in human 1321N1 astrocytoma cells and was studied under conditions in which nucleotide metabolism was both minimized and assessed. Cells were grown at low density on glass coverslips, encased in a flow-through chamber, and continuously superfused with medium, and Ca2+ responses to nucleotides were quantified. Superfusion with high performance liquid chromatographically purified ADP, ATP, 2-methylthio-ADP, and 2-methylthio-ATP resulted in rapid Ca2+ responses, with EC50 values of 10 +/- 5, 304 +/- 51, 2 +/- 1, and 116 +/- 50 nM, respectively. Similar peak responses were observed with maximal concentrations of these four agonists and with the hydrolysis-resistant adenine nucleoside triphosphate adenosine-5'-O-(3-thiotriphosphate). No conversion of [3H]ATP to [3H]ADP occurred under these conditions. Similar full agonist activities of ATP, 2-methylthio-ATP, and ADP were observed in human embryonic kidney 293 cells, which natively express the P2Y1 receptor. In contrast to these results, Leon et al. [FEBS Lett 403:26-30 (1997)] and Hechler et al. [Mol Pharmacol 53:727-733 (1998)] recently reported that, whereas ADP and 2-methylthio-ADP were agonists, ATP and 2-methylthio-ATP were weak antagonists in studies of the human P2Y1 receptor expressed in human Jurkat cells. To assess whether differences in the degree of receptor reserve might explain this discrepancy of results, P2Y1 receptor-expressing 1321N1 cells were incubated for 24 hr with adenosine-5'-O-(2-thiodiphosphate), with the goal of down-regulating the level of functional receptors. Pretreatment with adenosine-5'-O-(2-thiodiphosphate) resulted in a 10-fold rightward shift in the concentration-effect curve for ADP; in contrast, the agonist activity of ATP was completely abolished. Taken together, our results indicate that adenosine di- and triphosphates are agonists at the human P2Y1 receptor. However, the intrinsic efficacy of ATP is less than that of ADP, and the capacity of ATP to activate second messenger responses through this receptor apparently depends on the degree of P2Y1 receptor reserve.

A novel phospholipase C- and cAMP-independent positive inotropic mechanism via a P2 purinoceptor.

Although ATP, acting through a P2 purinoceptor, can stimulate a pronounced positive inotropic effect in cardiac ventricular myocytes, the receptor-effector mechanism that underlies this stimulatory cardiac action is not well understood. The objectives of the present study were to develop the cultured chick embryo ventricular myocytes as a novel model for the cardiac P2 purinoceptor and to determine the mechanism underlying its positive inotropic effect. ATP caused an 89 +/- 8.9% (n = 14 cells) increase in the myocyte contractility, with an efficacy and potency order of ATP > ADP > AMP >> adenosine. 2-Methylthio-ATP (2-MeS-ATP) but not alpha,beta-methylene-ATP was able to stimulate myocyte contractility, with a maximal increase of 54 +/- 2.6% (n = 11 cells). Although UTP potently stimulates phosphoinositide hydrolysis, it had an only modest positive inotropic effect (27 +/- 7% maximal increase; n = 8 cells). In contrast to previous suggestions, the 2-MeS-ATP-stimulated positive inotropic response does not require the action of phospholipase C (PLC), such as that of the inositol phosphates; the UTP effect on contractility appears to be mediated via the 2-MeS-ATP-sensitive P2 receptor. The PLC inhibitor U-73122 had no effect on the 2-MeS-ATP-stimulated increase in contractility, providing further evidence against a role for PLC in the inotropic effect of 2-MeS-ATP. An adenosine 3',5'-cyclic monophosphate-independent Ca2+ entry-stimulating mechanism appears to underlie a direct coupling of the receptor to stimulation of the myocyte contractility. This new PLC- and adenosine 3',5'-cyclic monophosphate-independent positive inotropic mechanism represents a target for developing novel positive inotropic therapeutics.

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 Ca(2+)-sensitive 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'-[gamma-thio]triphosphate > ADP > 2-methylthioadenosine triphosphate (2-MeSATP) approximately adenosine 5'-[beta-thio]diphosphate >> adenosine 5'-[alpha beta-methylene] triphosphate, adenosine 5'[beta gamma-imido]triphosphate. This profile is not characteristic of either the P2X or the conventional P2Y 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-P2Y nucleotide receptors (P2N) that mediate calcium influx and the accumulation of inositol phosphates.

ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

Regulation by cell metabolism and adenine nucleotides of a K channel in insulin-secreting B cells (RIN m5F).

A potassium channel in membranes of the insulin-secreting B-cell line, RIN m5F, was studied using the patch-clamp technique. In cell-attached patches, and for pipette solutions containing 140 mM KCl, the I-V curves exhibited a pronounced rectification, with the conductance being higher when the current flowed from the electrode into the cell (50 pS). Addition of glucose (5-7 mM) to the bath was sufficient to abolish the channel activity, while low doses of the metabolic inhibitor 2,4-dinitrophenyl enhanced it, indicating a link between channel activity and cell metabolism. In excised inside-out patches, the activity of a channel with similar conductive properties was inhibited by ATP in a dose-dependent fashion, the half-maximal concentration being approximately 70 microM. ADP also acted as an inhibitor, but with much lower potency. However, when ADP was present, the blocking effectiveness of ATP was reduced, suggesting a nonadditive interaction of the two nucleotides with the channel. In conclusion, complete closure of the K channel by glucose (5-7 mM) suggests that this channel may be responsible for the early phase of the B-cell depolarization, which is seen in normal cells for glucose increments up to similar values. In addition, since ATP and ADP are both present in the cells, the reduction by ADP of the blocking efficacy of ATP in excised patches may help one to understand why, in intact cells, the channel is still functional for intracellular ATP concentrations that in excised patches would completely suppress its activity.

Interactions of metal-nucleotide complexes with aspartate carbamoyltransferase in the crystalline state.

We report the results of crystallographic difference maps at 3.0-A resolution of complexes of metal-nucleoside triphosphates with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli. The complexes Gd3+-ATP, Al3+-ATP, and Gd3+-CTP bind to the allosteric effector domain of the enzyme in nearly the same orientation as the metal-free nucleotides. The result is consistent with kinetic observations of nearly identical allosteric efficacy of ATP and CTP and their complexes with cations. The effector Gd3+-GTP, however, binds in a distinctly different conformation and location than does 8-bromoguanosine 5'-triphosphate, reported in a separate investigation [Honzatko, R. B. & Lipscomb, W.N. (1982) J. Mol. Biol. 160, 265-286]. The difference in the binding modes of Gd3+-GTP and the bromo derivative suggests a possible mechanism for the relief of allosteric inhibition of GTP due to metal cations. We observe no binding of metal-nucleoside triphosphates in the region of the phosphate crevice of aspartate carbamoyltransferase, consistent with the reduced ability of metal nucleotides to compete with carbamoyl phosphate for the active site. However, a single Gd3+ ion binds in the region of the active site as evidenced by strong density. The binding of cations near the active site probably causes the inhibition of catalysis observed in kinetics experiments reported earlier [Honzatko, R.B., Lauritzen, A.M. & Lipscomb, W.N. (1981) Proc. Natl. Acad. Sci. USA 78, 898-902].