Hans G. Bäumert

P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.

P2X receptors are cation channels gated by extracellular ATP. The seven known P2X isoforms possess no sequence homology with other proteins. Here we studied the quaternary structure of P2X receptors by chemical cross‐linking and blue native PAGE. P2X1 and P2X3 were N‐terminally tagged with six histidine residues to allow for non‐denaturing receptor isolation from cRNA‐injected, [35S]methionine‐labeled oocytes. The His‐tag did not change the electrophysiological properties of the P2X1 receptor. His‐P2X1 was found to carry four N‐glycans per polypeptide chain, only one of which acquired Endo H resistance en route to the plasma membrane. 3,3′‐Dithiobis(sulfosuccinimidylpropionate) (DTSSP) and two of three bifunctional analogues of the P2X receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) cross‐linked digitonin‐solubilized His‐P2X1 and His‐P2X3 quantitatively to homo‐trimers. Likewise, when analyzed by blue native PAGE, P2X receptors purified in digitonin or dodecyl‐β‐D‐maltoside migrated entirely as non‐covalently linked homo‐trimers, whereas the α2βγδ nicotinic acetylcholine receptor (used as a positive control) migrated as the expected pentamer. P2X monomers remained undetected soon after synthesis, indicating that trimerization occurred in the endoplasmic reticulum. The plasma membrane form of His‐P2X1 was also identified as a homo‐trimer. If n‐octylglucoside was used for P2X receptor solubilization, homo‐hexamers were observed, suggesting that trimers can aggregate to form larger complexes. We conclude that trimers represent an essential element of P2X receptor structure.

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.

Vasoconstrictor and vasodilator responses to various agonists in the rat perfused mesenteric arterial bed: selective inhibition by PPADS of contractions mediated via P2x‐purinoceptors

1 The effect of pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) on vasoconstrictor and/or vasodilator responses to various agonists and electrical field stimulation was investigated in the rat mesenteric arterial bed at basal tone and at tone raised by methoxamine (15–50 μm). 2 At basal tone, nucleotides produced vasoconstriction with the following rank order of potency: α,β‐methylene ATP > > 2‐methylthio ATP ≥ ATP = UTP. PPADS (0.3–10 μm) concentration‐dependently antagonized α,β‐methylene ATP‐, 2‐methylthio ATP‐ and ATP‐induced responses. UTP‐, noradrenaline‐ and nerve‐mediated (4–32 Hz) increases in perfusion pressure remained unaffected by 10 μm PPADS. 3 In raised tone preparations, nucleotides produced vasodilations, their rank order of potency being 2‐methylthio ATP > ATP > UTP. Responses to 2‐methylthio ATP were slightly antagonized, whereas ATP‐ and UTP‐induced responses remained unaffected by 10μm PPADS. In addition, acetylcholine‐and adenosine‐elicited relaxations were not influenced by 10 μm PPADS. 4 The present results confirm the previously described selective P2x antagonism by PPADS, this compound being ineffective at muscarinic M3‐ and adenosine P1‐receptors as well as at α1‐adrenoceptors. There was some inhibition of P2y‐purinoceptors but at a much higher concentration than required for inhibition of P2x‐purinoceptors. 5 In addition, this study provides evidence for the ineffectiveness of PPADS at both vasoconstriction‐and vasodilatation‐mediating P2u‐purinoceptors.

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.

Investigation of the actions of PPADS, a novel P2X- purinoceptor antagonist, in the guinea-pig isolated vas deferens

1 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) was investigated for its ability to act as an antagonist at P2X‐purinoceptors which mediate neurogenic excitatory junction potentials (e.j.ps) and contractions in the guinea‐pig isolated vas deferens. 2 PPADS (10−7 m) caused a small potentiation of the phasic, predominantly purinergic component of contractions evoked by symapthetic nerve stimulation, but higher concentrations of PPADS (3 × 10−6–3 × 10−5 m) elicited a substantial and significant concentration‐dependent inhibition. In contrast, over the same concentration‐range, PPADS had no effect on the tonic, predominantly noradrenergic phase. 3 PPADS (3 × 10−;5 m) also inhibited contractile responses to exogenous α,β‐methyleneATP (10−8–10−3 m), a P2X‐purinoceptor agonist, without affecting the responses to exogenous noradrenaline (10−8−10−3 m), carbachol (10−5 m) or histamine (10−4 m). 4 PPADS (10−7–3 × 10−5 m) produced a concentration‐dependent reduction in e.j.p. magnitude and resting membrane potential. The maximum effect was seen at 10−5 m PPADS, which reduced e.j.p. magnitude from 13.7 ± 0.6 mV (n = 12) to 1.8 ± 0.7 mV (n = 12) and membrane potential from − 64.8 ± 0.6 mV (n = 51) to − 55.0 ± 1.8 mV (n = 12). 5 The PPADS‐induced depolarization was not inhibited by the P2X‐purinoceptor antagonist, suramin (10−4 m). This indicates that the depolarization was not due to an agonist action of PPADS at P2X‐purinoceptors. 6 The results support the proposal that PPADS is a selective antagonist at P2X purinoceptors as opposed to non‐P2‐purinoceptors in the guinea‐pig vas deferens, but its ability to cause membrane depolarization independently of P2X‐purinoceptors and also, at a low concentration, to potentiate the phasic component of the neurogenic contraction indicates that it has other actions.

Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens

The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-prefering antagonists.Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M1 receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M1(m1)-M4(m5) receptors in functional studies and binding experiments.The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the Al receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A1 and P2 purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC50): arecaidine propargyl ester (0.062 μM), 4-Cl-McN-A-343 (0.32 μM), 4-(4-fluorophenylcarbamoyloxy)-2-butynylN-methyl-pyrrolidinium tosylate (4-F-PyMcN+; 0.48 μM) and methacholine (0.86 μM). The affinity constants of most of the muscarinic antagonists [atropine: pKB = 9.47; (R)-trihexyphenidyl: pKB = 9.18; pirenzepine: pA2 = 7.68; methoctramine: pKB = 6.90] are consistent with estimates of these antagonists at M1(m1) receptors determined in various functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F-PyMcN+ argue for the involvement of M1, and against that of M2 and M3 receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pKB of 8.30 for himbacine is not in accordance with an M1 receptor; by contrast, it would be compatible with the presence of M2 or M4 receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of noradrenaline release that are probably of the same subtype as the putative M1 receptors inhibiting neurogenic twitch contractions, and are not of the M2, M3 or m5 subtype.

Design and pharmacological characterization of selective P2-purinoceptor antagonists

At least five distinct P2-purinoceptor subtypes have been characterized to date, based on the rank order of potency of several ATP analogues: P2X, P2Y, P2U, P2T and P2Z. However, the characterization of P2-purinoceptor subtypes is hampered by an unavailability of potent, highly selective, competitive antagonists. In the search for selective P2-purinoceptor antagonists, the structure-activity relationships for a series of analogues of pyridoxal-5-phosphate and suramin at P2-purinoceptor subtypes were investigated in our laboratories. Two of these compounds were the subject of a more detailed pharmacological characterization: pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and the symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulfonic acid (NF023). The results demonstrate that the two parent compounds, pyridoxal-5-phosphate and suramin, do not differentiate between P2X- and P2Y-purinoceptors. In contrast, PPADS and NF023 were found to be selective antagonists of P2X-purinoceptor-mediated responses in several smooth muscle preparations. In addition, PPADS and NF023 were shown to displace competitively the binding of [3H]α,β-methylene ATP to rabbit and rat bladder membranes, respectively, which indicates that these two compounds act directly on P2X-receptors. PPADS and NF023 were ineffective at P2U-purinoceptors in rat mesenteric arterial bed. P2T-purinoceptor-mediated platelet aggregation was only affected by PPADS in concentrations higher than 100 μM. Suramin and NF023 were inhibitors of ecto-ATPase activity in the same concentration range needed for P2-purinoceptor antagonism. In contrast, PPADS was only very weakly active in inhibiting ecto-ATPase activity. AT 100 μM, PPADS and NF023 did not interact with α1-adrenoceptors, adenosine A1- and A2-, histamine H1- and muscarinic M1-, M2- and M3-receptors. In conclusion, PPADS and NF023 are specific P2-purinoceptor antagonists showing a high selectivity for the P2X-subtype. These two compounds may prove to be useful starting points in the synthesis of novel, highly potent and selective antagonists at P2-purinoceptor subtypes.

Identification of a cross-linked double-peptide from the catalytic site of the Ca2+-ATPase of sarcoplasmic reticulum formed by the Ca2+- and pH-dependent reaction with ATP γP-imidazolidate

The Ca2+‐ATPase from sarcoplasmic reticulum can be inhibited by the Ca2+‐ and pH‐dependent reaction with ATP γP‐imidazolidate. The chemically and monofunctionally activated inhibitor introduces an intramolecular cross‐link between two neighbouring peptides of the active site. This can be followed by the reduced mobility of the ATPase upon SDS‐PAGE analysis which becomes even more pronounced after limited trypsinolysis. After cleavage of the cross‐linked ATPase molecule by cyanogen bromide and separation of the peptides a double‐peptide can be detected which upon sequencing can be identified as part of the phosphorylation and the nucleotide binding site, respectively.

Crosslinking studies in oligomycin-sensitive ATPase from beef heart mitochondria: Interrelations of subunits with an Mr 31 000 protein

A protein of Mr 3 1 000 in oligomycin-sensitive (OS)-ATPase is important for oligomycin sensitivity of the preparation [ 1,2]. Staining intensity of this protein band was decreased by the uncoupler FCCP, control values being restored by subsequent addition of a thiol reagent [3]. Interrelations should exist with the oligomycin-sensitivity conferring protein (OSCP) [4] of -20 kDa in our preparation. We have used crosslinking techniques to learn which interdependencies between the 3 land 20-kDa as well asbetween other subunits can be found. Using tartryl ditglycylazide) (TDGA) [5] we observed crosslinks between proteins of 20 and 34 kDa, between 25-28 and 31 kDa, and between 31 and 34 kDa. Crosslinking at similar sites was obtained with the SH-oxidizing reagent Cu-phenanthroline. It is thus established that the 3 1and 20-kDa proteins are connected via the 34kDa y-subunit of the OS-ATPase. We also have found evidence that the lo-, 15and 3 l-kDa proteins may exist as dimers. Interactions are also revealed between the 8-, 9-and 13-kDa proteins. Crosslinking with diepoxybutane (DEB) [6] resembled the results obtained with TDGA, and, moreover, showed a decrease in band intensity of the 3 1 -kDa protein band, stabilization of ATPase activity and a concomitant decrease of ATP-Pi exchange reaction.