Ectonucleotidase Inhibitor Research Articles

ATP-stimulated ANP release through P1 receptor subtype

Extracellular ATP acts as a local regulator of physiological functions in the cardiovascular system via P1 and P2 receptors. However, little is known about the effect of ATP on the release of atrial natriuretic peptide (ANP) secretion. The purpose of this study was to investigate the effects of extracellular ATP on atrial hemodynamics and ANP release and to identify their receptor-mediated mechanism. ATP was infused into isolated perfused beating rat atria in the absence and presence of various receptor antagonists. ATP (from 0.1 to 30 μM) increased the ANP release with negative inotropism in a dose-dependent manner. ADP (30 μM ) also caused an increase in ANP release with similarity to ATP, but α,β-methylene ATP (α,β-MeATP, P2X1 receptor agonist) and 2-methylthioADP (2-MesADP, P2Y1 receptor agonist) did not. The rank order of potency for the increment of ANP release was adenosine>ATP=ADP>2-MesADP>α,β-MeATP. In contrast, UTP, an agonist for P2Y2,4,6 receptor, caused a decrease in ANP release without changes in contractility. Extracellular ATP-induced increase in ANP release and negative inotropism were completely blocked by the pretreatment of 8-cyclopentyl-1,3-dipropylxanthine (P1 receptor antagonist), but not by pyridoxal phosphate-6-azobenzene-2,4-disulfonic acid (P2X1 receptor antagonist) and suramin (P2XY receptor antagonist). Reactive Blue 2 (P2Y receptor antagonist) caused an augmentation of ATP-induced increase in ANP release without affecting negative inotropism. Adenosine 5′-(α,β-methylene) diphosphate, an ectonucleotidase inhibitor, did not affect ATP-induced augmentation of ANP release with negative inotropy. These results suggest that extracellular ATP-induced increase in ANP release and negative inotropism are mediated mainly by P1 receptor, and UTP decreases ANP release. Therefore, we suggest that extracellular ATP and UTP may have opposite actions on the regulation of ANP secretion.

Activation of ureter nociceptors by exogenous and endogenous ATP in guinea pig

This study was conducted to determine whether adenosine 5′-triphosphate (ATP) contributes to nociceptor activity induced by ureter distension. Multifibre recordings of ureter afferents were made using the guinea pig ureter preparation perfused in vitro. Distension of the ureter resulted in an initial rapid and later maintained increase in afferent nerve discharge. Intraluminal application of ATP (10–1000 μM, 0.1 ml/min for 3 min) or α,β-meATP (10–1000 μM) mimicked these increases in afferent activity. The afferent responses consisted of fast and slow components. Both agonists caused a sensitisation of the afferents to ureter distensions. TNP-ATP (30 μm), a P2X 3 receptor antagonist, and the non-specific P2 antagonist, PPADS (100 μm), blocked the rapid and reduced the slower response to ATP. The remaining responses were blocked by the selective A1 receptor antagonist, DPCPX. TNP-ATP and PPADS reduced distension-induced afferent activity. The selective ecto-ATPase inhibitor, ARL-67156 (100, 200 μM) and suramin (100, 200 μM), an ecto-nucleotidase inhibitor as well as a P2 receptor antagonist, produced an increase in baseline and distension-induced discharge. These results indicate that the ureter epithelium may tonically (at rest) as well as phasically (on distension) release ATP, which stimulates afferent terminals by interacting with multiple P2 and P1 receptors.

Extracellular ATP facilitates flow-induced vasodilatation in rat small mesenteric arteries.

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.

ATP modulates noradrenaline release by activation of inhibitory P2Y receptors and facilitatory P2X receptors in the rat vas deferens.

The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [3H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5'-triphosphate (2-MeSATP), 2-methylthioadenosine 5'-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP > or = ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 microM) and by 2-MeSAMP (10 microM) but not by the selective P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179; 10 microM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP), beta,gamma-imidoadenosine 5'-triphosphate (beta,gamma-imidoATP), beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: alpha,beta-meATP > 2-MeSATP = ATP = beta,gamma-meATP > or = beta,gamma-imidoATP. The facilitatory effects of alpha,beta-MeATP and beta,gamma-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethylene 5'-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.

Glial Cell Inhibition of Neurons by Release of ATP

ATP is released by neurons and functions as a neurotransmitter and modulator in the CNS. Here I show that ATP released from glial cells can also serve as a potent neuromodulator, inhibiting neurons in the retina of the rat. Activation of glial cells by focal ejection of ATP, ATPgammaS, dopamine, thrombin, or lysophosphatidic acid or by mechanical stimulation evoked hyperpolarizing responses and outward currents in a subset of retinal ganglion cells by increasing a Ba(2+)-sensitive K(+) conductance in the neurons. This glia-evoked inhibition reduced the firing rate of those neurons that displayed spontaneous spike activity. The inhibition was abolished by the A(1) adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (10 nm) and was reduced by the ecto-ATPase inhibitor ARL-67156 (6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP) (50 microm) and by the ectonucleotidase inhibitor AOPCP [adenosine-5'-O-(alpha,beta-methylene)-diphosphonate] (250 microm). Selective activation of retinal glial cells demonstrated that Müller cells, but not astrocytes, mediate the inhibition. ATP release from Müller cells into the inner plexiform layer of the retina was shown using the luciferin-luciferase chemiluminescence assay. These findings demonstrate that activated glial cells can inhibit neurons in the retina by the release of ATP, which is converted to adenosine by ectoenzymes and subsequently activates neuronal adenosine receptors. The results lend support to the hypothesis that glial cells play an active role in information processing in the CNS.

Adenosine linking reduced O2 to arteriolar NO release in intestine is not formed from extracellular ATP

We have previously reported that adenosine formed in response to reduced arteriolar and/or tissue PO(2) preserves endothelial nitric oxide (NO) synthesis during sympathetic vasoconstriction in the rat intestine. To more precisely identify the site and mechanism of adenosine formation under these conditions, we tested the hypothesis that ATP released in response to reduced O(2) levels serves as a source of adenosine. Direct application of ATP to the wall of first-order arterioles elicited dose-dependent dilations of 15-33% above resting diameter that were reduced by 71-80% by the 5'-ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP, 4.5 x 10(-5) M) and completely abolished by N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M). Under control conditions, sympathetic nerve stimulation at 3 and 8 Hz induced arteriolar constrictions of 11 +/- 1 and 19 +/- 1 microm, respectively. These responses were enhanced by 58-69% in the presence of L-NMMA or when local PO(2) was maintained at resting levels. However, in the presence of AOPCP, the enhancing effects of L-NMMA and the high O(2) superfusate on sympathetic constriction were preserved. These results suggest that, although exogenously applied ATP can stimulate arteriolar NO release in the intestine largely through its sequential extracellular hydrolysis to adenosine, this process does not contribute to adenosine formation and sustained NO release during sympathetic constriction in this vascular bed.

Differential regulations between adenosine triphosphate (ATP)- and uridine triphosphate-induced Cl(-) secretion in bovine tracheal epithelium. Direct stimulation of P1-like receptor by ATP.

Adenosine 5'-triphosphate (ATP) stimulates airway epithelial Cl(-) secretion in a complicated manner. We examined the difference between ATP- and uridine 5'-triphosphate (UTP)-induced responses of short-circuit current (Isc) in bovine tracheal epithelium treated with amiloride. Each nucleotide caused an increase in Isc composed of the first and second peaks, where the second peak induced by ATP was higher compared with UTP. The ATP-induced second peak was inhibited by the protein kinase (PK) A inhibitor H89, saturation of P1 receptor with adenosine, and the P1 receptor antagonist 8-p-sulfophenyltheophylline, but not by the Ca(2+) chelator ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus the endoplasmic reticulum Ca(2+)-pump inhibitor thapsigargin, the adenosine breakdown enzyme adenosine deaminase, the ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate, or saturation of P2Y2 receptor with UTP. Thus, the response is associated with PKA-dependent pathway via P1-like receptor but not with Ca(2+)-dependent pathway via P2Y2 receptor, and ATP degradation products do not contribute to this response. Further, stimulation of cells with ATP increased PKA activity. In addition, pretreatment with glybenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator, reduced the second peak of Isc induced by ATP but was without effect on that induced by UTP. Therefore, ATP stimulates glybenclamide-sensitive Cl(-) secretion, and this action is partly mediated by PKA-dependent pathway via P1-like receptor.

Potentiation of cytokine induction of group IIA phospholipase A2in rat mesangial cells by ATP and adenosineviathe A2A adenosine receptor

1. In rat mesangial cells extracellular nucleotides were found to increase arachidonic acid release by a cytosolic phospholipase A(2) through the P2Y(2) purinergic receptor. 2. In this study we investigated the effects of ATP and UTP on interleukin-1ss (IL-1ss)-induced mRNA expression and activity of group IIA phospholipase A(2) (sPLA(2)-IIA) in rat mesangial cells. 3. Treatment of cells for 24 h with extracellular ATP potentiated IL-1ss-stimulated sPLA(2)-IIA induction, whereas UTP had no effect. 4. We obtained the following evidence that the P2Y(2) receptor is not involved in the potentiation of sPLA(2)-IIA induction: (i) ATP-gamma-S had no enhancing effect; (ii) suramin, a P(2) receptor antagonist, did not inhibit ATP-mediated potentiation; (iii) inhibition of degradation of extracellular nucleotides by the 5'-ectonucleotidase inhibitor AOPCP did not enhance sPLA(2)-IIA induction and (iv) adenosine deaminase treatment completely abolished the ATP-mediated potentiation of sPLA(2)-IIA induction. 5. In contrast, treatment of mesangial cells with adenosine or the A2A receptor agonist CGS 21680 mimicked the effects of ATP in enhancing IL-1ss-stimulated sPLA(2)-IIA induction, whereas the specific A2A receptor antagonist ZM 241385 completely abolished the potentiating effect of ATP or adenosine. 6. The protein kinase A inhibitor Rp-8-Br-cyclic AMPS dose-dependently inhibited the enhancing effect of ATP or adenosine indicating the participation of an adenosine receptor-mediated cyclic AMP-dependent signalling pathway. 7. These data indicate that ATP mediates proinflammatory long-term effects in rat mesangial cells via its degradation product adenosine through the A2A receptor resulting in potentiation of sPLA(2)-IIA induction.

Effects of AMP derivatives on cyclic AMP levels in NG108-15 cells.

1. In NG108-15 neuroblastomaxglioma hybrid cells, ATP stimulates intracellular cyclic AMP formation, which is inhibited by both adenosine (P(1)) and P2 receptor antagonists. In the present study, we examined the effects of several AMP derivatives in NG108-15 cells and mouse neuroblastoma N18TG-2 cells. 2. Adenosine 2'-monophosphate (A2P), adenosine 3'-monophosphate (A3P) and adenosine 5'-phosphosulphate (A5PS) increased cyclic AMP levels with similar concentration-dependencies in NG108-15 cells. 3. Increases in cyclic AMP by AMP derivatives were inhibited by the P2 receptor antagonist PPADS, but not by suramin. Effects of AMP derivatives were also inhibited by P(1) receptor antagonists ZM241385, XAC, DPCPX and partially by alloxazine. The ecto-nucleotidase inhibitor alpha, beta-methyleneADP was without effect. 4. In contrast, AMP derivatives did not change cyclic AMP levels in N18TG-2 cells. Accumulation of cyclic AMP in N18TG-2 cells was stimulated by adenosine A(2) receptor agonists CGS21680 and NECA, but not by ATP or beta, gamma-methyleneATP, agonists for cyclic AMP production in NG108-15 cells. 5. Reverse transcription-coupled polymerase chain reaction (RT - PCR) analyses revealed that N18TG-2 cells express both A(2A) and A(2B) receptors, while NG108-15 cells express mainly A(2A) receptors. 6. AMP derivatives did not affect the P2X and P2Y receptors expressed in NG108-15 cells. 7. These results suggest that A2P, A3P and A5PS act as agonists for cyclic AMP production and that these compounds are valuable tools for determinating the mechanism of ATP-stimulated cyclic AMP response in NG108-15 cells.

PPADS and suramin as antagonists at cloned P2Y- and P2U-purinoceptors.

1. The effect of suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the stimulation of phospholipase C in 1321N1 cells transfected with the human P2U-purinoceptor (h-P2U-1321N1 cells) or with the turkey P2Y-purinoceptor (t-P2Y-1321N1 cells) was investigated. 2-Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t-P2Y-1321N1 cells and uridine triphosphate (UTP) at h-P2U-1321N1 cells. 2. Suramin caused a parallel shift to the right of the concentration-response curves for 2MeSATP in the t-P2Y-1321N1 cells, yielding a Schild plot with a slope of 1.16 +/- 0.08 and a pA2 value of 5.77 +/- 0.11. 3. Suramin also caused a shift to the right of concentration-response curves for UTP in the h-P2U-1321N1 cells, and on Schild plots gave a slope different from unity (1.57 +/- 0.19) and an apparent pA2 value of 4.32 +/- 0.13. Suramin was therefore a less potent antagonist at the P2U-purinoceptor than the P2Y-purinoceptor. 4. In the presence of the ectonucleotidase inhibitor, ARL 67156 (6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP) there was no significant difference in the EC50 or shapes of curves with either cell type, and no difference in pA2 values for suramin. 5. PPADS caused an increase in the EC50 for 2MeSATP in the t-P2Y-1321N1 cells. The Schild plot had a slope different from unity (0.55 +/- 0.15) and an X-intercept corresponding to an apparent pA2 of 5.98 +/- 0.65. 6. PPADS up to 30 microM had no effect on the concentration-response curve for UTP with the h-P2U-1321N1 cells. 7. In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y-purinoceptor than at the P2U-purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.

Forskolin evokes extracellular adenosine accumulation in rat cortical cultures

In this study, the effect on extracellular adenosine concentration of direct activation of adenylyl cyclase by forskolin was investigated using rat cortical cultures. Forskolin evoked intracellular and extracellular cAMP accumulation as well as extracellular adenosine accumulation. The accumulation of adenosine in response to forskolin could be blocked by the cyclic nucleotide phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 180 μM), but not isobutylmethylxanthine (100 μM). The accumulation of adenosine in response to forskolin could be blocked by the 5′-ectonucleotidase inhibitor guanosine 5′-monophosphate. These results demonstrate that forskolin can increase extracellular adenosine levels, and that this adenosine is ultimately derived from cAMP.

Atp receptor activation potentiates a voltage-dependent ca channel in hippocampal neurons

Activation of a purinergic P 2 receptor by adenosine 5′-triphosphate (ATP) has previously been shown to open a non-selective cation channel with a reversal potential of approximately 0 mV. We examined the effects of P 2 receptor activation on voltage-gated ionic currents in acutely isolated CA3 pyramidal neurons from guinea pig hippocampus using the whole-cell-patch technique. Under conditions designed to isolate current through voltage-dependent Ca channels ( I Ca), ATP (50 μM) potentiated I Ca by 36%. This increase in I Ca desensitized back to control levels within 4 min. In contrast to the non-selective cation channel, I Ca elicited from a holding potential (HP) of −100 mV showed significant potentiation in response to ATP when depolarized to a test potential (TP) of −10 mV but showed no effect on I Ca when the same neuron was alternately depolarized to TP = −70 mV. No change in holding current at HP = −100 mV occurred. Tail currents were unaffected by ATP exposure suggesting that I Ca potentiation was not due to modulation of L-type Ca channels. This potentiation was also observed either with ATP-γ-s, the slowly hydrolyzable ATP analog, or with ATP in the presence of α,β-methylene-ADP, an ectonucleotidase inhibitor, indicating that the effects observed were not due to activation of an adenosine receptor that required ATP hydrolysis. The potentiation of I Ca was not observed with the P 2X agonist, β,γ-methylene-ATP. These results suggest that ATP receptors can modulate voltageas well as ligand-gated channels permeable to calcium and may play an important role in the dynamics of intracellular Ca 2+ in these neurons.

Isocratic high-performance liquid chromatographic determination of plasma adenosine

Due to manifold physiological and cardioprotective actions of adenosine, the demand for a simple but accurate method to determine its concentration in plasma is increasing. The aim of this study was firstly to develop a simple isocratic method instead of the gradient elution or peak-shifting techniques used earlier and secondly to check conflicting data on the composition of “stop-solution”, added to the sample in order to prevent changes in adenosine concentration. Isocratic elution improved signal to noise ratio and concentrations of 100 μmol L−1 dipyridamole and 2.5 μmol L−1 erythro-9(2-hydroxy-3-nonyl)adenine in the blood sample effectively prevented both adenosine formation and degradation, even without the use of a 5′-ecto-nucleotidase inhibitor. Lowering the concentration of dipyridamole to 25 μmol L−1 caused more than a tenfold increase of adenosine concentration in two out of five cases and even 100 μmol L−1 dipyridamole alone is not sufficient to inhibit adenosine deaminase in blood samples.

Contractile effects of uridine 5′‐triphosphate in the rat duodenum

1. Previous studies have shown that the rat duodenum relaxes to adenosine and adenosine 5'-triphosphate (ATP) via P1 and P2Y purinoceptors respectively, but in preliminary studies uridine 5'-triphosphate (UTP) was found to contract this tissue. The non-selective P2 antagonist suramin and a number of nucleotides were therefore used to investigate this response further. 2. ATP, UTP, adenosine 5'-diphosphate (ADP), adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), guanosine 5'-triphosphate (GTP) and uridine 5'-diphosphate (UDP) each relaxed the duodenum, with an agonist potency order of ATP = ADP > ATP-gamma-S >> GTP >> UTP = UDP, consistent with the presence of a P2Y purinoceptor mediating relaxation. 3. ATP-gamma-S, UTP and UDP each contracted the duodenum with an agonist potency order of ATP-gamma-S > UTP > UDP, although maximal responses to these agonists were not obtained at a concentration of 267 microM (ATP-gamma-S) and 300 microM (UTP and UDP). No contractions were observed with any of the other agonists at concentrations up to 300 microM. 4. Indomethacin (25 microM) did not inhibit the contractions induced by UTP, indicating that they were not mediated via production of prostaglandins. 5. Suramin (100 microM and 1 mM) inhibited relaxations induced by ATP, shifting the concentration-response curve to the right, with the maximal response to ATP being decreased by the higher concentration of suramin (1 mM). Suramin (1 mM) inhibited relaxations induced by ATP-gamma-S, shifting the concentration-response curve to the right, and completely abolished contractions induced by ATP-gamma-S. In contrast, suramin (100 JAM and 1 mM) had no effect on contractions induced by UTP.Contractions induced by UTP were, however, less sustained in the presence of suramin, which also affected the basal tone of some tissues when precontracted with carbachol (0.1 microM). In the presence of suramin (I mM), no contractions to ATP were observed.6. These results confirm that in the rat duodenum there is a P2Y purinoceptor that mediates relaxation in response to a number of purine nucleotides, and at which the pyrimidine nucleotides UTP and UDP are almost inactive. There are also receptors at which UTP and ATP-y-S act to cause contraction.Suramin discriminates between the contractile effects of these two agonists, which may indicate the presence of a suramin-insensitive pyrimidinoceptor as well as a suramin-sensitive receptor for ATP-y-S.An alternative explanation is that the differential effects of suramin are via its actions as an antagonist in addition to its action as an ectonucleotidase inhibitor.

Metabolic characteristics of alpha-toxin-permeabilized smooth muscle.

Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydrolyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.

Potentiation of ATP Responses by α,β-Methyleneadp in the Rabbit Ear Artery

Abstract α,β-methyleneADP, the ectonucleotidase inhibitor, produced a potentiation and kinetic stabilization of ATP induced contractions of the rabbit ear artery. However, studies with stable and unstable agonists indicated that this effect was not due to inhibition of purine metabolism.

Catecholamines inhibit early steps of lymphocyte activation

In this study, the effect on extracellular adenosine concentration of direct activation of adenylyl cyclase by forskolin was investigated using rat cortical cultures. Forskolin evoked intracellular and extracellular cAMP accumulation as well as extracellular adenosine accumulation. The accumulation of adenosine in response to forskolin could be blocked by the cyclic nucleotide phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 180 μM), but not isobutylmethylxanthine (100 μM). The accumulation of adenosine in response to forskolin could be blocked by the 5′-ectonucleotidase inhibitor guanosine 5′-monophosphate. These results demonstrate that forskolin can increase extracellular adenosine levels, and that this adenosine is ultimately derived from cAMP.

Effects of purine nucleotides on the binding of [3H]cyclopentyladenosine to adenosine A-1 receptors in rat brain membranes.

Adenine nucleotides displace the binding of the selective adenosine A-1 receptor ligand [3H]cyclopentyladenosine (CPA) to rat brain membranes in a concentration-dependent manner, with the rank order of activity being ATP greater than ADP greater than AMP. Binding was also displaced by GTP, ITP, adenylylimidodiphosphate (AppNHp), 2-methylthioATP, and the beta-gamma-methylene isostere of ATP, but was unaffected by the alpha-beta-methylene isosteres of ADP and ATP, and UTP. At ATP concentrations greater than 100 microM, the inhibitory effects on CPA binding were reversed, until at 2 mM ATP, specific binding of CPA was identical to that seen in controls. Concentrations of ATP greater than 10 mM totally inhibited specific binding. Inclusion of the catabolic enzyme adenosine deaminase in the incubation medium abolished the inhibitory effects of ATP, indicating that these were due to adenosine formation, presumably due to ectonucleotidase activity. The inhibitory effects were also attenuated by the alpha-beta-methylene isostere of ATP, an ectonucleotidase inhibitor. Adenosine deaminase, alpha-beta-methylene ATP (100 microM), and beta-gamma-methylene ATP (100 microM) had no effect on the "stimulatory" phase of binding, although GTP (100 microM) slightly attenuated it. Comparison of the binding of [3H]CPA in the absence and presence of 2 mM ATP by saturation analysis showed that the KD and apparent Bmax values were identical. Examination of the pharmacology of the control and "ATP-dependent" CPA binding sites showed slight changes in binding of adenosine agonists and antagonists. The responses observed with high concentrations of ATP were not observed with GTP, AppNHp, the chelating agents EDTA and EGTA, or inorganic phosphate. The divalent cations Mg2+ and Ca2+ at 10 mM attenuated the stimulatory actions of high (2 mM) concentrations of ATP, whereas EGTA and EDTA (10 mM) enhanced the "stimulatory" actions of ATP. EDTA (10 mM) abolished the inhibitory effects of ATP, indicating a specific dependence on Mg2+ for the inhibitory response. The effects of ATP on [3H]CPA binding were reversible for antagonists but not agonists. The mechanism by which ATP reverses its own inhibitory action on adenosine A-1 radioligand binding is unclear, and from the observed actions of the divalent cations and chelating agents probably does not involve a phosphorylation-dependent process.