Airat U. Ziganshin

PPADS selectively antagonizes P2X-purinoceptor-mediated responses in the rabbit urinary bladder.

1 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), an inhibitor of P2X‐purinoceptor‐mediated responses in rabbit vas deferens, was investigated for its ability to antagonize contractions evoked by α,β‐methylene ATP (α,β‐MeATP), carbachol and electrical field stimulation in the rabbit urinary bladder detrusor muscle. 2 PPADS (1–30 μm) caused concentration‐dependent inhibition of contractions to the stable P2X‐purinoceptor agonist, α,β‐MeATP, decreasing the maximum response to α,β‐MeATP (30 μm) at concentrations of 3–30 μm. The pD2 value for α,β‐MeATP in the absence of PPADS was 6.52 ± 0.10 (8). In the presence of PPADS at concentrations of 1, 3, 10 and 30 μm the negative log concentrations of α,β‐MeATP that cause the same contractile response as the pD2 value were significantly different from control, being respectively 6.17 ± 0.09 (8), 5.64 ± 0.12 (7), 5.15 ± 0.23 (7) and 4.78 ± 0.22 (5). 3 PPADS (1–30 μm) caused concentration‐dependent inhibition of contractions to stimulation of intramural purinergic nerves (1–32 Hz). There was a greater inhibition at lower frequencies (1–8 Hz) than at higher frequencies (16–32 Hz). PPADS, 30 μm, did not produce significantly greater antagonism than 10 μm. 4 PPADS (30 μm) had no significant influence on the contractile potency of carbachol: the pD2 values of carbachol in the absence and presence of PPADS were not significantly different being 6.42 ± 0.16 (5) and 6.33 ± 0.18 (5), respectively. However, PPADS caused a small, but significant, suppression of the maximal response of carbachol, reducing it by approximately 9%. 5 Radioligand binding studies carried out on rabbit bladder membranes with [3H]‐α,β‐methylene ATP ([3H]‐α,β‐MeATP) showed that PPADS concentration‐dependently inhibited the binding of [3H]‐α,β‐MeATP to P2X‐purinoceptors, while the binding of [3H]‐quinuclidinyl benzilate to muscarinic cholinoceptors was not affected. 6 Thus, PPADS (1–30 μm) antagonized responses mediated via P2X‐purinoceptors in the rabbit urinary bladder. It was selective for P2‐purinoceptor‐mediated contractions rather than those mediated via muscarinic receptors. Binding studies demonstrated that the antagonistic effect of PPADS is via a direct interaction with P2X‐purinoceptors.

Selective antagonism by PPADS at P2X‐purinoceptors in rabbit isolated blood vessels

1 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), a P2‐purinoceptor antagonist, was investigated for its ability to antagonize: (1) P2X‐purinoceptor‐mediated contractions of the rabbit central ear artery and saphenous artery evoked by either α,β‐methylene ATP (α,β‐MeATP) or electrical field stimulation (EFS); (2) P2Y‐purinoceptor‐mediated relaxations of the rabbit mesenteric artery; (3) endothelium‐dependent and endothelium‐independent, P2Y‐purinoceptor‐mediated relaxations of the rabbit aorta. 2 α,β‐MeATP (0.1–100 μm) caused concentration‐dependent contractions of the rabbit ear and saphenous arteries. The negative log[α,β‐MeATP] that produced a contraction equivalent to the EC25 for noradrenaline (ear artery) or histamine (saphenous artery) in the absence of PPADS was 6.60 ± 0.18 (9) and 6.18 ± 0.17 (9) in the ear artery and saphenous artery, respectively. These effects of exogenous α,β‐MeATP were concentration‐dependently inhibited by PPADS (1–30 μm). In the ear artery, the negative log[α,β‐MeATP] producing a contractile response equivalent to the EC25 of noradrenaline, in the presence of PPADS at 1, 3 and 10 μm was 6.16 ± 0.18 (8), 5.90 ± 0.18 (8) and 4.72 ± 0.36 (8), respectively (P < 0.01). In the saphenous artery, the negative log[α,β‐MeATP] values equivalent to the EC25 for histamine in the presence of PPADS at concentrations of 1, 3, 10 and 30 μm were 5.90 ± 0.19 (8), 5.73 ± 0.16 (8), 4.99 ± 0.14 (8) and 4.51 ± 0.13 (8), respectively (P < 0.01). 3 PPADS at a concentration of 1 μm had no effect on contractions of the ear artery evoked by EFS (4–64 Hz; 1 μm phentolamine present). At higher concentrations (3–30 μm) it caused concentration‐dependent inhibition of neurogenic contractions. In the saphenous artery, PPADS (1–30 μm) concentration‐dependently inhibited contractions evoked by EFS at frequencies of 4, 8 and 16 Hz. Contractions evoked by EFS at frequencies of 32 and 64 Hz were significantly inhibited by PPADS only at concentrations of 10 and 30 μm. 4 PPADS (30 μm) had no effect on relaxations to 2‐methylthio ATP (3 nm‐3 μm) in rabbit mesenteric artery and to ATP (1 μm − 1 mm) in rabbit aorta (with endothelium intact or removed). In addition, PPADS (30 μm) had no significant influence on the contractile potency of noradrenaline and histamine in rabbit ear and saphenous artery, respectively. 5 In conclusion, these results support the evidence that PPADS is a selective antagonist of P2X‐purinoceptor‐mediated responses.

Inhibitory action of PPADS on relaxant responses to adenine nucleotides or electrical field stimulation in guinea-pig taenia coli and rat duodenum.

1 The effect of pyridoxarphosphate‐6‐azophenyl‐2′, 4′‐disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol‐contracted guinea‐pig taenia coli and rat duodenum, two tissues possessing P2y‐purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P2‐purinoceptor blockade and ecto‐ nucleotidase activity, its influence on ATP degradation was studied in guinea‐pig taenia coli. 2 The resulting rank order of potency for the adenine nucleotides in guinea‐pig taenia coli was: 2‐methylthio ATP≫ ATP>>α, β‐methylene ATP with the respective pD2‐values 7.96 ±0.08 (n = 23), 6.27 ±0.12 (n = 21) and 5.88 ±0.04 (n = 24). 3 In guinea‐pig taenia coli, PPADS (10–100 μm) caused a consistent dextral shift of the concentration‐response curve (CRC) of 2‐methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 μm PPADS, the respective pA2‐values at this particular concentration were 5.26 ±0.16 (n = 5) and 5.15 ±0.13 (n = 6). Lower concentrations of PPADS (3–30 μm) antagonized the relaxant effects to α, β‐methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 μm PPADS (pA2 = 5.97 ± 0.08, n = 6), and the Schild plot was again biphasic. 4 The relaxant responses to electrical field stimulation (80 V, 0.3 ms, 5 s, 0.5–16 Hz) in guinea‐pig taenia coli were concentration‐dependently inhibited by PPADS (10–100 μm). 5 In guinea‐pig taenia coli, the potency of ATP in inducing relaxation appeared to be independent of its rate of degradation by ecto‐nucleotidases, since the Km‐value (366 μm) obtained in the enzyme assay was much higher than the functional EC50‐value (0.45 μm) of ATP. PPADS (3–100 μm) was only weakly active in inhibiting ecto‐nucleotidase activity leaving a residual activity of 81.8 ±5.1% at 100 μm. Enzyme inhibition by PPADS was concentration‐independent and non‐competitive. 6 In rat duodenum, the rank order of potency was: 2‐methylthio ATP > ATP≫α, β‐methylene ATP, the respective pD2‐values being 6.98 ±0.04 (n = 76), 6.26 ±0.02 (n = 6) and 4.83 ±0.02 (n = 6). Among these agonists, 2‐methylthio ATP displayed the lowest apparent efficacy. 7 The CRC of 2‐methylthio ATP in rat duodenum was shifted to the right by PPADS (10–100 μm) in a concentration‐dependent manner, and Schild analysis gave a pA2‐value of 5.09 ±0.06 (slope =1.02, n=14). 8 PPADS was without any effect on the carbachol‐induced contraction in guinea‐pig taenia coli or rat duodenum and on the relaxation to noradrenaline or adenosine in guinea‐pig taenia coli. 9 In conclusion, the antagonistic properties of PPADS at the taenia coli and rat duodenum P2y‐ purinoceptors were different from those recently described at the P2x‐subtype: inhibition of P2y‐ purinoceptor‐mediated responses was observed at higher concentrations (3–100 μm vs. 1–10 (30) μm). Furthermore, we conclude that in addition to the classical P2y‐subtype, which is largely PPADS‐resistant, the guinea‐pig taenia coli may be endowed with a distinct relaxation‐mediating P2‐purinoceptor subtype which is sensitive to PPADS.

The activation of P1- and P2-purinoceptors in the guinea-pig left atrium by diadenosine polyphosphates.

1 The effects of P1, P2‐di(adenosine) pyrophosphate (AP2A), P1, P3‐di(adenosine) triphosphate (AP3A), P1, P4‐di(adenosine) tetraphosphate (AP4A), P1, P5‐di(adenosine) pentaphosphate (AP5A), ATP, α, β‐methylene ADP and 2‐chloroadenosine (2‐ClAd) were examined in the guinea‐pig driven left atrium. 2 All these purine compounds except α, β‐methylene ADP produced a negative inotropic response with a rank order of potency of: 2‐ClAd> > AP2A ≥ ATP ≥ AP4A = AP3A = AP5A. The EC50 value for 2‐ClAd was approximately 1 μm, while those for the remaining compounds were in the range 10 μm‐100 μm, α, β‐Methylene ADP (10–300 μm), a selective P2Y‐purinoceptor agonist, produced a small positive inotropism. 3 The P1‐purinoceptor antagonist, 8‐para‐sulphophenyltheophylline (8‐pSPT, 20 μm) caused a rightward shift in the concentration‐response curves for 2‐ClAd, ATP and AP2A, but converted the responses of AP3A, AP4A, and AP5A into positive inotropisms. 4 The non‐selective P2‐purinoceptor antagonist, suramin (300 μm), had no significant effect on the concentration‐response curves for 2‐ClAd, ATP or AP2A, but significantly antagonized inhibitory responses to AP3A, AP4A and AP5A, and excitatory responses to α, β‐methylene ADP. 5 In the presence of 8‐pSPT (20 μm), suramin (300 μm) abolished the positive inotropic responses evoked by the dinucleotides. 6 ATP was degraded far more rapidly than any of the dinucleotides, and AP3A was the least stable of the diadenosine compounds. The relative order of stability was AP2A > AP4A = AP5A > AP3A > > ATP. Suramin (300 μm) reduced the rate of degradation of ATP and AP3A by approximately 30%. Suramin had no significant effect on the degradation of AP2A, AP4A or AP5A. 7 It is concluded that the diadenosine polyphosphates cause negative inotropic responses via P1‐purinoceptors and a hitherto undefined suramin‐sensitive P2‐purinoceptor, and that they appear to have positive inotropic effects mediated via another suramin‐sensitive P2‐purinoceptor.

Effects of P2-purinoceptor antagonists on degradation of adenine nucleotides by ecto-nucleotidases in folliculated oocytes of Xenopus laevis

The aim of the present study was to examine the effects of a number of P2-purinoceptor antagonists on degradation of adenine nucleotides by Xenopus laevis oocyte ecto-nucleotidase. Folliculated oocytes readily metabolize all three naturally-occurring nucleotides, the order of preferential substrates being ATP >ADP > AMP. The degradation of ATP and ADP was decreased significantly in the presence of several P2X- and P2Y-purinoceptor antagonists, including suramin, PPADS, Cibacron blue, Coomassie Brilliant blue, Evans blue, Trypan blue, Congo red, and PIT (each compound was used at 100 microM). All these compounds inhibited the degradation of ATP by up to 60%, whereas the hydrolysis of ADP was inhibited by Congo red and PIT by 75-80%. In addition, DIDS (100 microM) and TNP-ATP (100 microM) selectively inhibited the breakdown of ATP, and sodium azide (10 mM) selectively inhibited the breakdown of ADP. The enzymatic breakdown of either ATP or ADP was unaffected by 8-pSPT (100 microM), an antagonist of P1-purinoceptors, or by oxidized ATP (100 microM), an antagonist of P2Z-purinoceptors. The degradation of AMP was prevented completely by PIT (100 microM) and ingibited significantly by Congo red (100 microM). In conclusion, the present study shows that most of currently available antagonists of P2-purinoceptors inhibit the enzymatic breakdown of extracellular ATP and ADP. The inhibitory effect on ecto-nucleotidase activity should be taken into account when these antagonists are used in pharmacological experiments.

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal‐5′‐phosphate‐6‐phenylazo‐2′,4′‐disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6‐phenylazo and 6‐naphthylazo derivatives were also evaluated. Among the 6‐phenylazo derivatives, 5′‐methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea‐pig taenia coli P2Y1 receptors, in guinea‐pig vas deferens and bladder P2X1 receptors, and in ion flux experiments by using recombinant rat P2X2 receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X1 receptors in differentiated HL‐60 cell membranes was carried out by using [35S]ATP‐γ‐S. A 2′‐chloro‐5′‐sulfo analog of PPADS (C14H12O9N3ClPSNa), a vinyl phosphonate derivative (C15H12O11N3PS2Na3), and a naphthylazo derivative (C18H14O12N3PS2Na2), were particularly potent in binding to human P2X1 receptors. The potencies of phosphate derivatives at P2Y1 receptors were generally similar to PPADS itself, except for the p‐carboxyphenylazo phosphate derivative C15H13O8N3PNa and its m‐chloro analog C15H12O8N3ClPNa, which were selective for P2X vs. P2Y1 receptors. C15H12O8N3ClPNa was very potent at rat P2X2 receptors with an IC50 value of 0.82 μM. Among the phosphonate derivatives, [4‐formyl‐3‐hydroxy‐2‐methyl‐6‐(2‐chloro‐5‐sulfonylphenylazo)‐pyrid‐5‐yl]methylphosphonic acid (C14H12O8N3ClPSNa) showed high potency at P2Y1 receptors with an IC50 of 7.23 μM. The corresponding 2,5‐disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y1 receptors, whereas at recombinant P2X2 receptors had an IC50 value of 1.1 μM. An ethyl phosphonate derivative (C15H15O11N3PS2Na3), whereas inactive at turkey erythrocyte P2Y1 receptors, was particularly potent at recombinant P2X2 receptors. Drug Dev. Res. 45:52–66, 1998. Published 1998 Wiley‐Liss, Inc.

Effects of cyclopiazonic acid on contractility and ecto-ATPase activity in guinea-pig urinary bladder and vas deferens.

1 Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic ATPase, was tested on guinea‐pig urinary bladder and vas deferens for its ability: (1) to modify contractile responses to electrical field stimulation (EFS), exogenous ATP, α,β‐methylene ATP (α,β‐meATP), carbachol, noradrenaline (NA), histamine, and KCl; (2) to affect ecto‐ATPase activity; (3) to modify the release of ATP evoked by EFS. 2 In the urinary bladder, CPA (10 μm) potentiated contractile responses to EFS, exogenous ATP (100 μm), α,β‐meATP (1 μm), carbachol (0.5 μm), histamine (30 μm) and KCl (30 μm). In the vas deferens, CPA (10 μm) potentiated responses to EFS, ATP, α,β‐meATP, NA (100 μm) and KCl. CPA at a concentration of 1 μm had no effect on ATP‐induced relaxation of carbachol‐precontracted guinea‐pig taenia coli, and at a concentration of 10 μm it markedly increased spontaneous contractile activity of taenia. 3 Ecto‐ATPase was estimated to have Vmax and Km values of 0.98 nmol Pi 30 min−1 mg−1 wet tissue and 881 μm ATP in the urinary bladder, and 0.75 nmol Pi 30 min−1 mg−1 wet tissue and 914 μm ATP in the vas deferens, respectively. CPA at a concentration of 10 μm significantly inhibited ecto‐ATPase activity by 18% in the urinary bladder and by 24% in the vas deferens. 4 In the guinea‐pig vas deferens, CPA significantly potentiated ATP release evoked by EFS from 2.2 ± 0.8 (6) pmol ATP min−1 g−1 wet tissue to 35.2 ± 4.8 (6) pmol ATP min−1 g−1 wet tissue (P < 0.01). 5 In conclusion, the potentiation of contractile responses of the guinea‐pig urinary bladder and vas deferens by CPA has a non‐specific character. CPA inhibited ecto‐ATPase activity and increased ATP release, but these effects do not appear to contribute to the potentiation of P2x‐purinoceptor‐mediated responses since the contractile actions of all the agonists studied were potentiated to the same extent.

Vasoconstrictor responses via P2X-receptors are selectively antagonized by NF023 in rabbit isolated aorta and saphenous artery

The effects of NF023, the symmetrical 3′‐urea of 8‐(benzamido)naphthalene‐1,3,5‐trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to α,β‐methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline‐precontracted rabbit isolated thoracic aorta. In rabbit isolated saphenous artery, α,β‐methylene ATP‐induced vasoconstrictor responses via P2X‐receptors were concentration‐dependently and competitively antagonised by NF023 (30–300 μm; pA2=5.69±0.04). Suramin (100–1000 μm) also competitively blocked vasoconstrictor responses to α,β‐methylene ATP, albeit with lower potency (pA2=4.79±0.05). In contrast, NF023 (100 μm) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. In noradrenaline‐precontracted rabbit isolated thoracic aorta preparations, ATP (3–3000 μm) concentration‐dependently induced relaxations via endothelium‐dependent or smooth muscle P2Y‐receptor subtypes. NF023 (30–300 μm) failed to block relaxant responses to ATP at endothelium‐dependent P2Y‐receptors, whereas suramin (100–1000 μm) did antagonise endothelium‐dependent vasodilator responses to ATP. Neither NF023 (100 μm) nor suramin (300 μm) influenced vasorelaxant responses to ATP via endothelium‐independent P2Y‐receptors. In conclusion, this study outlines the selectivity of NF023 as an effective P2X‐receptor antagonist in rabbit isolated blood vessels without affecting endothelium‐dependent or endothelium‐independent P2Y‐receptor subtypes, adrenoceptors or histamine receptors.

Molecular recognition in P2 receptors: ligand development aided by molecular modeling and mutagenesis.

Publisher Summary As molecular modeling of cloned G protein-coupled receptor (GPCR) sequences using a rhodopsin template has been refined, it has become possible to generate hypotheses for location of the binding sites that are consistent with mutagenesis results and ligand specificities. To obtain an energetically refined 3-D structure of the ligand–receptor complex, the chapter introduces a new computational approach, a “cross docking” procedure, which simulates the reorganization of the native receptor induced by the ligand. The molecular basis for recognition by human P2Y 1 receptors of the selective, competitive antagonist MRS 2179 is probed using site-directed mutagenesis and molecular modeling. The model was derived from primary sequence comparison, secondary structure predictions, and 3-D homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. A putative nucleotide binding site was localized, following a cross docking procedure to obtain energetically refined 3-D structures of the ligand–receptor complexes, and used to predict which residues are likely to be in proximity to agonists and antagonists. Molecular modeling using PowerFit has suggested a possible model of superimposition of two classes of antagonists, nucleotides related to MRS 2179, and non-nucleotides related to pyridoxal phosphate.

Potentiation by 2,2′‐pyridylisatogen tosylate of ATP‐responses at a recombinant P2Y1 purinoceptor

1 2,2′‐Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5′‐triphosphate (ATP) at metabotropic purinoceptors (of the P2Y family) in some smooth muscles. When a recombinant P2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2‐methylthioATP (2‐MeSATP) evoke calcium‐activated chloride currents (Icl,ca) in a concentration‐dependent manner. The effects of PIT on these agonist responses were examined at this cloned P2Y purinoceptor. 2 PIT (0.1–100 μm) failed to stimulate P2Y, purinoceptors directly but, over a narrow concentration range (0.1–3 μm), caused a time‐dependent potentiation (2–5 fold) of responses to ATP. The potentiation of ATP‐responses by PIT was not caused by inhibition of oocyte ecto‐ATPase. At high concentrations (3–100 μm), PIT irreversibly inhibited responses to ATP with a IC50 value of 13±9 μm (pKB = 4.88±0.22; n = 3). PIT failed to potentiate inward currents evoked by 2‐MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3 Known P2 purinoceptor antagonists were tested for their ability to potentiate ATP‐responses at the chick P2Y, purinoceptor. Suramin (IC50 = 230±80 nM; n = 5) and Reactive blue‐2 (IC50 = 580±130 nM; n = 6) reversibly inhibited but did not potentiate ATP‐responses. Coomassie brilliant blue‐G (0.1–3 μm) potentiated ATP‐responses in three experiments, while higher concentrations (3–100 μm) irreversibly inhibited ATP‐responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P2 purinoceptor antagonists. 4 In radioligand binding assays, PIT showed a low affinity (pKi<5) for a range of membrane receptors, including: α1, α2‐adrenoceptors, 5‐HT1A, 5‐HT1B, 5‐HT2, 5‐HT3, D1, D2, muscarinic, central benzodiazepine, H1, μ‐opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (pKi = 5.3) for an adenosine (A1) receptor. 5 In guinea‐pig isolated taenia caeci, PIT (12.5–50 μm) irreversibly antagonized relaxations to ATP (3–1000 μm); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10–100 μm), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 μm) and guanethidine (17 μm), were not affected by PIT (50 μm, for 25–60 min). 6 These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P2Y, purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue‐G) of ATP‐responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.